Natural Product Research

Natural Product Research Vol. 22, No. 6, 15 April 2008

[Formerly Known as Natural Product Letters] Best Wishes from ayush

Natural Product Research, Vol. 22, No. 6, 15 April 2008, 483–488

Two new phenolic water-soluble constituents from branch bark of Davidia involucrata ZU-JIAN WU*y, MING-AN OUYANGyz and SHI-BIN WANGz yInstitute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, P.R. China zDepartment of Bio-engineering & Technology, Huaqiao University, Quanzhou, Fujian 362011, China (Received 5 September 2005; in final form 11 May 2006) Two new phenolic water-soluble constituents, involcranoside A (1) and involcranoside B (2) have been isolated along with five known phenolic compounds: 3,4-dimethoxyphenyl-O- -Dgluco-pyranoside (3), picein (4), and 1,4-dihydroxy-3-methoxy-phenyl-4-O- -D-glucopyranoside (5), leonuriside A (6) and 4-hydroxy-3-methoxybenzoic acid (7) from the branch bark of Davidia involucrata. Identification of their structures was achieved by 1D and 2D NMR experiments, including 1H-1H COSY, NOESY, HMQC and HMBC methods and FAB mass spectral data. Keywords: Nyssaceae; Davidia involucrata; Branch bark; Phenolic compounds

1. Introduction More than 25 flavones, flavonoid glycosides, tannins, caffeoyl derivatives and neolignan glycosides have been isolated from the leaves and the branch bark of Davidia involucrata [1–4]. Our continued interest in the chemical constituents of the branch bark of Chinese Nyssaceae species has led to the isolation of seven phenolic glycosides. In this article, we describe the isolation and structural elucidation of two new phenolic water-soluble constituents, involcranoside A (1) and involcranoside B (2).

2. Results and discussion Chromatographic separation of a MeOH extract from the branch bark of D. involucrata yielded the new compounds 1 and 2 in addition to 3,4-dimethoxyphenyl-O- -D-glucopyranoside (3), picein (4) [5], and 1,4-dihydroxy-3-methoxy-phenyl4-O- -D-glucopyranoside (5) [6], leonuriside A (6) [7], and 4-hydroxy-3-methoxybenzoic *Corresponding author. Email: [email protected] Natural Product Research ISSN 1478-6419 print/ISSN 1029-2349 online ß 2008 Taylor & Francis http://www.tandf.co.uk/journals DOI: 10.1080/14786410600906426

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acid (7). HO 3″

O

HO

1″

H3CO O

C

O

O

5′ 4′

HO

O

6′

7′

HO

OCH3 3

OH

4

7

8

CH3

O

9

HO

3 OH

O

6′

4″

CH3

O

O

1″ 4′

3″

OH

1

O

C CH3 7

O

OH

C CH3

OH

8

4

1′ 2′

OH OH

4

O

HO

O

6″

OCH3

OH

1

OH

O

O

OH

6

1′ 2′

OH

C CH2

1

OH

2

OH

OH H3CO

H3CO HO

HO O

O

5 OH

O

O

O

OH HO

OH

OH OH

H3CO

OH

H3CO

OH

6

C

OH

7

OH

Compound 1 was obtained as a brown amorphous powder. The FAB-MS of 1 exhibited a quasi-molecular ion peak at m/z 493 [M
H]
. The HRFAB-MS indicated an m/z of 493.1342 for his peak, which was consistent with the molecular formula C23H26O12 for 1 and representing eleven elements of unsaturation. The absorption bands at max 3300, 1701, 1692, 1615, 1519 cm
1 in the IR spectrum were characteristic of hydroxyl, conjugated carbonyl and aromatic groups, respectively. Acid hydrolysis of 1 with 2 M TFA afforded a sugar, which was identified as glucose by TLC. The 1H NMR spectrum of 1 in CD3OD showed ABX spin system signals at H 7.71 (1H, d, J ¼ 1.7 Hz), 7.65 (1H, d, J ¼ 8.8 Hz), 7.93 (1H, dd, J ¼ 8.8, 1.7 Hz), an aromatic proton signal at H 7.91 (2H, s), an anomeric proton signal at H 5.66 (1H, d, J ¼ 7.2 Hz), a methoxy signal at H 3.62 (3H, s, –OCH3), and ethyl group signals at H 3.15 (1H, m), 3.02 (1H, m), 1.11 (3H, t, J ¼ 7.2 Hz). The 13C NMR spectrum of 1 gave two carbonyl signals at C 199.4 (ketone group) and 167.1 (ester group), 12 aromatic carbon signals, hexose signals, a methoxy signal and ethyl group signals (table 1). Evaluation of spin–spin coupling and chemical shifts of the hexose allowed the identification of one -glucopyranosyl unit. The 1H and 13C NMR spectra of 1 suggested a propiophenone moiety, and a galloyl group because of the signal at H 7.91 (2H, s) in the 1H NMR spectrum and the signals at C 167.1, 147.6  2, 141.1, 121.2, 110.4  2 in the 13C NMR spectrum. The cross-peaks in the HMBC experiments between signals H 7.91 (H-200 , H-600 ) and C 167.1 (C-700 ), 141.1 (C-400 ), 147.6 (C-300 , C-500 ), 121.2 (C-100 ), between H 5.27 (H-60 of Glc), 4.85 (H-60 of Glc) and 167.1 (C-700 ), between H 5.66 (H-10 of glc) and 151.7 (C-4), between H 7.71 (H-2), 7.92 (H-6), 1.11 (H-9) and C 199.4 (C-7) indicated that the gallic acid

Two new phenolic water-soluble constituents from branch bark of Davidia involucrata 485 Table 1.

1 2 3 4 5 6 7 8 9

10 20 30 40 50 60 100 200 300 400 500 600 700

13

C NMR spectral assignments of compounds 1–7 in CD3OD.

1

2

3

4

5

6

7

131.9 111.2 149.9 151.7 115.2 123.5 199.4 31.6 8.7 3-OCH3 55.6 4-Glc 102.0 74.7 78.6 71.7 75.9 64.8 60 -Galloyl121.2 110.4 147.6 141.1 147.6 110.4 167.1

162.9 131.7 117.3 132.6 117.3 131.7 199.7 17.9

151.5 103.1 148.0 141.7 114.8 108.8

132.5 117.2 131.6 162.9 131.6 117.2 199.3 26.7

145.9 104.7 151.0 153.8 109.3 113.9

155.9 106.2 154.6 129.5 154.6 106.2

108.5 113.9 148.4 152.8 125.0 116.2 169.2

3-OCH3 56.5 4-OCH3 57.3 1-Glc 102.6 73.9 77.0 70.5 77.1 61.6

4-Glc 101.4 74.7 77.9 71.4 77.1 67.8 60 -Rha102.1 72.1 72.4 73.9 69.9 18.1

3-OCH3 55.5 4-Glc 101.6 74.9 78.0 71.3 78.3 62.5

4-Glc 103.5 75.0 78.1 71.6 78.3 62.7

3-OCH3 56.9 5-OCH3 56.9 4-Glc 94.5 75.1 77.8 71.3 78.3 62.6

55.9

HO H3CO

O HO

C

O

O O O

HO

C

CH2 CH3

OH HO OH Figure 1.

HMBC

HMBC correlations for 1.

was linked at C-60 of the Glc and the sugar was linked at C-4 of the propiolphenone moiety. On the basis of above evidence, 1, named involcranoside A, was determined to be 4-O-(6-galloyl)- -D-glucopyranosyl propiophenone (figure 1). The negative FABMS of 2 showed a quasi-molecular ion peak at m/z 443 [M
H]
, concluding from the HRFAB-MS data was indicative of the formula C20H28O11 for 2 and representing seven elements of unsaturation. The absorption bands at max 3340, 1706, 1609, 1500 cm
1 in the IR spectrum were characteristic of hydroxyl, conjugated carbonyl, and aromatic group, respectively. Acid hydrolysis of 1 with 2 M TFA afforded a mixture of sugars, which were identified as glucose and rhamnose by TLC. The 1H NMR spectrum of 2 exhibited two aromatic proton signals at H 7.97 (2H, d,

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J ¼ 8.8 Hz), 7.14 (2H, d, J ¼ 8.8 Hz), two anomeric proton signals at H 5.00 (d, J ¼ 7.6 Hz), and at 4.68 (d, J ¼ 1.5 Hz), and a methyl signal at H 2.55 (3H, s). These signals suggested that compound 2 had a disubstituted phenyl group and an acetyl group. This evidence was also supported by the 13C NMR spectrum, which signals at C 199.7, 162.9, 132.6  2, 131.7, 117.3  2 and 17.9. Thus the genin was assigned as an acetophenone. From the 2D NMR experiments, the proton and carbon data of 2 was assigned. The correlations revealed the Rha-1 linking at Glc-6, and Glc-1 linking to the acetophenone. Thus, 2, named involcranoside B was identified as 4-O-L-rhamnopyranosyl (1 ! 6)- -D-glucopyranosyl acetophenone.

3. Experimental 3.1. General experimental procedures Melting points were determined on a Yanaco MP-S3 apparatus and are uncorrected. UV spectra were recorded with a Backman DU-64 spectrometer. Optical rotations were measured with a Jasco DIP-180 digital polarimeter spectrophotometer. The IR spectrum was recorded with a Perkin–Elmer 1750 FTIR spectrometer. 1H, 13C, DEPT, 1H–1H COSY, NOESY, HMQC and HMBC NMR spectra were obtained on a Varian Unity Plus 400 instrument. FAB mass spectra were recorded on a Jeol JMS-HX 110 instrument. Chromatographic stationary phases were RP-8 (40–60 m, Merck), silica gel (160–200 mesh), Sephadex LH-20 (25–100 m, Pharmacia Fine Chemical Co., Ltd) and MCI-gel CHP20P (75–150 m, Mitsubishi Chemical Industries, Ltd). The following solvent systems were (a) CHCl3–MeOH–H2O (80 : 20 : 3), CHCl3–MeOH– H2O (70 : 30 : 5) and MeOH–H2O (0–100%) for the glycosides; and (b) CHCl3–MeOH– H2O (7 : 3 : 1) lower-layer 9 mL þ 1 mL HOAc for sugars. Compounds on TLC were detected by spraying with 5% H2SO4, then by heating. Sugars were detected by spraying with aniline–phthalate reagent.

3.2. Plant material The branch bark of D. involucrata was collected on the Yun–Long Mountain, GongShan county of Yunnan province, China in 1995. The plant was identified by Dr Y.P. Yang, A voucher specimen (No. 12245) was deposited in the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China.

3.3. Extraction and isolation The dry branch bark of D. involucrata (10 kg) were extracted (2  10 L) with MeOH at room temperature (7 days  2). The extract was evaporated in vacuo to yield a residue, which was solubilised in water and then filtered. The water soluble fraction was passed through a Diaion resin column and was eluted with water and methanol. Evaporation of the methanol eluate yielded a brown fraction (A) (175 g). The fraction A was subjected to dry column chromatography (DCC) on silica gel (2.0 kg), eluted with CHCl3–MeOH–H2O (10 : 2 : 0.2) to afford thirteen fractions. Each fraction was purified by Sephadex LH-20, RP-8 gel column chromatography (solvent: MeOH–H2O,

Two new phenolic water-soluble constituents from branch bark of Davidia involucrata 487

10–70%) and finally purified by repeated chromatography on a silica gel column with CHCl3–MeOH–H2O (100 : 10 : 1–70 : 30 : 5) as eluent to yield 1 (21 mg), 2 (13 mg), 3 (11 mg), 4 (30 mg), 5 (15 mg), 6 (26 mg), 7 (38 mg). 3.3.1. Involcranoside A (1). Brown amorphous powder, m.p. 131–133 C, HRFABMS
1 m/z 493.1342 (Calcd for C23H25O12, 493.1346), ½21 D
11 (c 0.3, MeOH); IRmax cm :
1 3300, 1701, 1692, 1615, 1519; FAB-MS m/z 493 [M
H] ; H NMR  7.71 (1H, d, J ¼ 1.7 Hz, H-2), 7.65 (1H, d, J ¼ 8.8 Hz, H-5), 7.93 (1H, dd, J ¼ 8.8, 1.7 Hz, H-6), 3.15 (1H, dd, J ¼ 14.0, 7.2 Hz, H-8), 3.02 (1H, dd, J ¼ 14.0, 7.2 Hz, H-8), 1.11 (3H, t, J ¼ 7.2 Hz, H-9), 3.62 (3H, s, 3-OCH3), 5.66 (1H, d, J ¼ 7.2 Hz, H-10 ), 4.40 (1H, m, H20 ), 4.36 (1H, m, H-30 ), 4.15 (1H, m, H-40 ), 4.38 (1H, m, H-50 ), 5.27 (1H, dd, J ¼ 9.2, 1.3 Hz, H-60 ), 4.85 (1H, dd, J ¼ 9.2, 6.2 Hz, H-60 ), 7.91 (2H, s, H-200 , 600 ) and 13C NMR (table 1). 3.3.2. Involcranoside B (2). Colourless amorphous powder, m.p. 145–146 C, ½21 D
17 (c 0.16, MeOH); IRmax cm
1: 3340, 1706, 1609, 1500; FAB-MS m/z 443 [M
H]
, 297 [M
H-146]
, 135 [M
H-146–162]
; HR-FAB-MS m/z 443.1551 [M
H]
(Calcd for C20H27O11, 443.1553); 1H NMR  7.97 (2H, d, J ¼ 8.8 Hz, H-2, 6), 7.14 (2H, d, J ¼ 8.8 Hz, H-3, 5), 2.55 (3H, s, H-7), 5.00 (1H, d, J ¼ 7.6 Hz, H-10 ), 3.48 (1H, overlap, H-20 ), 3.49 (1H, overlap, H-30 ), 3.39 (1H, br d, H-40 ), 3.62 (1H, overlap, H-50 ), 4.02 (1H, dd, J ¼ 9.4, 4.6 Hz, H-60 ), 3.61 (1H, dd, J ¼ 4.6, 2.5 Hz H-60 ), 4.68 (1H, d, J ¼ 1.5 Hz, H100 ), 3.83 (1H, dd, J ¼ 3.3, 1.5 Hz, H-200 ), 3.69 (1H, dd, J ¼ 9.5, 3.3 Hz, H-300 ), 3.35 (1H, t, J ¼ 9.5 Hz, H-400 ), 3.63 (1H, m, H-500 ), 1.17 (3H, d, J ¼ 6.5 Hz, H-600 ) and 13C NMR (table 1). 3.3.3. 3,4-Dimethoxy-1-hydroxylphenyl-O-b-D-glucopyranoside (3). Colourless amorphous powder, FAB-MS m/z 315 [M
H]
; 1H NMR  6.81 (1H, d, J ¼ 2.4 Hz, H-2), 6.65 (1H, d, J ¼ 8.4 Hz, H-5), 6.83 (1H, dd, J ¼ 8.4, 2.4 Hz, H-6), 3.83 (3H, s, 3-OCH3), 3.77 (3H, s, 4-OCH3), 4.76 (1H, d, J ¼ 8.0 Hz, H-10 ), 3.18 (1H, dd, J ¼ 9.1, 8.0 Hz, H-20 ), 3.33 (1H, overlap, H-30 ), 3.28 (1H, t, J ¼ 9.1 Hz, H-40 ), 3.21 (1H, m H-50 ), 3.88 (1H, dd, J ¼ 12.0, 2.4 Hz, H-60 ), 3.66 (1H, dd, J ¼ 12.0, 2.45.6 Hz, H-60 ); and 13C NMR (table 1). 3.3.4. Picein (4). Colourless amorphous powder, FAB-MS m/z 301 [M
H]
; 1H NMR  7.96 (2H, d, J ¼ 8.8 Hz, H-2, 6), 7.15 (2H, d, J ¼ 8.8 Hz, H-3, 5), 2.55 (3H, s, H7), 5.20 (1H, d, J ¼ 7.6 Hz, H-10 ), 3.49 (1H, overlap, H-20 ), 3.48 (1H, overlap, H-30 ), 3.39 (1H, t, J ¼ 9.1 Hz H-40 ), 3.69 (1H, m, H-50 ), 3.91 (1H, dd, J ¼ 12.4, 2.0 Hz, H-60 ), 3.47 (1H, m, H-60 ); and 13C NMR (table 1). (5). Colourless 3.3.5. 1,4-Dihydroxyl-3-methoxyphenyl-4-O-b-D-glucopyranoside amorphous powder, FAB-MS m/z 301 [M
H]
, 239 [M
H-162]
; 1H NMR  6.79 (1H, d, J ¼ 2.2 Hz, H-2), 6.68 (1H, d, J ¼ 8.8 Hz, H-5), 6.57 (1H, dd, J ¼ 8.8, 2.2 Hz, H6), 3.82 (3H, s, 3-OMe), 4.73 (1H, d, J ¼ 7.0 Hz, H-7), 3.33–3.48 (4H, m, H-2  H-5), 3.89 (1H, dd, J ¼ 12.0, 2.0 Hz, H-6), 3.67 (1H, dd, J ¼ 12.0, 5.6 Hz, H-6) and 13C NMR (table 1).

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3.3.6. Leonuriside A (6). Colourless amorphous powder, FAB-MS m/z 331 [M
H]
; 1 H NMR  6.13 (2H, s, H-3, 5), 3.78 (6H, s, 2-OCH3, 6-OCH3), 4.65 (1H, d, J ¼ 7.2 Hz, H-10 ), 3.38–3.57 (4H, overlap, H-2  H-5), 3.85 (1H, dd, J ¼ 11.8, 2.1 Hz, H-60 ), 3.69 (1H, dd, J ¼ 11.8, 5.4 Hz, H-60 ); and 13C NMR (table 1).

3.3.7. 4-Hydroxy-3-methoxybenzoic acid (7). Colourless amorphous powder, FABMS m/z 167 [M
H]
; 1H NMR  8.07 (1H, d, J ¼ 2.0 Hz, H-2), 7.29 (1H, d, J ¼ 8.2 Hz, H-5), 8.15 (1H, dd, J ¼ 8.2, 2.0 Hz, H-6), 3.75 (3H, s, 3-OMe); and 13C NMR (table 1).

3.4. Acid hydrolysis A solution of each compound (8 mg) was heated at reflux at 100 C in 2 M aqueous CF3COOH (5 mL) on a water bath for 3 h. After this period, the reaction mixture was diluted with H2O (15 mL) and extracted with CH2Cl2 (3  5 mL). The combined CH2Cl2 extracts were washed with H2O and then evaporated to dryness in vacuo. After evaporation to dryness of the aqueous layer with MeOH until neutral, the sugars were analysed by comparison with authentic samples (solvent system b) on silica gel HPTLC.

Acknowledgements This research was supported by National Science Fundation of China (Grant No. 20272015). We thank the staff of the analytical group of Kunming Institute of Botany, Chinese Academy of Sciences for measurements of NMR and FAB-MS spectra.

References [1] [2] [3] [4] [5] [6] [7]

M.A. Ouyang, J. Huang, Q.W. Tan. Journal of Asian Natural Product Research, 9, 487–492 (2007). M.A. Ouyang, J.N. Zhou. Guihaia, 23, 568 (2003). M.A. Ouyang, J.N. Zhou, S.B. Wang. Journal of Natural Product Research, (in press) (2008). G.Q. Xiang, X.S. Lu. Acta Biotanica Sinca, 31, 540 (1989). M. Ushiyama, T. Furuya. Phytochemistry, 28, 3009 (1989). H. Pan, L.N. Lundgren. Phytochemistry, 39, 1423 (1995). H. Otsuka, M. Takeuchi, S. Inoshiri, T. Sato, K. Yamasaki. Phytochemistry, 28, 883 (1989).

Natural Product Research, Vol. 22, No. 6, 15 April 2008, 471–476

New caffeoyl derivatives from the leaves of Davidia involucrata MING-AN OUYANG*yz, JIAN-NIN ZHOUz and SHI-BIN WANGz yInstitute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, P.R. China zDepartment of Bio-engineering & Technology, Huaqiao University, Quanzhou, Fujian 362011, China (Received 18 April 2005; in final form 10 December 2005) Three new caffeoyl galactoic acid derivatives, davidiosides A–C (1–3) together with four known caffeoyl derivatives such as: caffeic acid (4), methyl caffeate (5), chlorogenic acid (6), and methyl chlorogenate (7) were isolated from the leaves of Davidia involucrate. The new structures were determined by spectroscopic data and chemical evidence. Keywords: Davidia involucrata; Nyssaceae; Leaves; Caffeoyl derivatives

1. Introduction Davidia involucrata (Nyssaceae) is a famous ornamental tree which only distributes the western China, such as Sichuan, Guizhou, western Hubei, and northern Yunnan [1–3]. To the chemical constituents of the Chinese plant species of the Nyssaceae, we have reported six flavones and their flavonoid glycosides, kaempferol, kaempferol-3-O[3-D-glucopyranoside, kaempferol-3-O-[3-D-galactopyranoside, quercetin, quercetin-3O-[3-D-arabinopyranoside, quercetin-3-O-[3-D-galactopyranoside from the leaves of D. involucrata collected from Yunnan province [4]. In this article, we described the isolation and structural elucidation of three new caffeoyl galactoic acid derivatives named as davidiosides A–C (1–3) along with four known caffeoyl derivatives such as, caffeic acid (4), methyl caffeate (5), chlorogenic acid (6), and methyl chlorogenate (7) [5] (figure 1).

2. Results and discussion
Compound 1 was obtained as colorless powder, m.p. 187–190
C, ½2H D þ 36 (c 0.6, MeOH). The FAB-MS of 1 exhibited a quasi-molecular ion peak at m/z 339 [M  H].

*Corresponding author. Email: [email protected] Natural Product Research ISSN 1478-6419 print/ISSN 1029-2349 online ß 2008 Taylor & Francis http://www.tandf.co.uk/journals DOI: 10.1080/14786410600898730

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Figure 1.

Structures of compounds 1–7.

The HRFAB-MS gave the molecular ion peak at m/z 339.0719, which was consistent with the molecular formula C15H16O9, representing eight elements of unsaturation. The absorption bands at vmax 3302, 1715, 1686, 1627, and 1602 cml in the IR spectrum were characteristic of hydroxyl, carbonyl, conjugated carbonyl, double bond and aromatic group, respectively. The 1H NMR spectrum of 1 in DMSO showed an ABX spin system at H 7.04 (1H, d, J ¼ 1.3 Hz, H-20 ), 6.99 (1H, dd, J ¼ 7.6, 1.3 Hz, H-60 ), 6.75 (1H, d, J ¼ 7.6 Hz, H-50 ), suggesting a 1, 3, 4-trisubstituted phenyl moiety, a trans-double bond signals attributed to two olefinic protons at H 7.47 (1H, d, J ¼ 15.7 Hz, H-70 ), 6.25 (1H, d, J ¼ 15.7 Hz, H-80 ), and six protons of bearing oxygen in the range H 4.25–3.41. The 13C NMR and DEPT spectra of 1 displayed 15 carbon signals including 9 sp2 carbons of a caffeoyl aglycone, 5 sp3 and 1 sp2 carbons (C 71.4, 70.1, 69.3, 67.0, 65.9, and 179.5) of a sugar-like moiety, which had no anomeric proton and carbon signal (table 1). The H–H COSY and TOCSY experiments assigned H1 to H5. The long-range HMBC gave the correlations from H-l to C-90 , H-2 to C-l, H-5 to C-6, and H-70 to C-90 , C-l0 , these correlations unequivocally established that a caffeoyl group was located at C-l of the sugar-like moiety. By the NOESY experiment, the relative configurations were exhibited H-l to H-2, H-2 to H-5, H-3 to H-4, and H-4 to H-5 (figure 2) and due to the molecular formula of 1 was eight elements of unsaturation, so the unsaturation of sugar-like moiety must be two, which structure suggested an ether ring. Above clues implied that the sugar-like moiety was a galactoic acid. Therefore, compound 1 was assigned as 1-caffeoyl galactoic ester, named davidioside A. Compound 2 was a methylated davidioside A at C-6 position, maybe an artifical product. Its IR spectrum showed absorption bands for hydroxyl (3318), ester (1735), conjugated carbonyl (1690), conjugated double bond (1613), and aromatic ring (1600). The FAB-MS spectrum exhibited a quasi-molecular ion peak at m/z 353 [M  H]. The 1 H and 13C NMR spectra of 2 displayed similar structure except for methoxyl group. Accordingly, the structure of 2 was determined as 1-caffeoyl-6-methyl galactoic ester, and named davidioside B. Compounds 3 and 1 were isomers having the same FABMS spectra, similar IR spectra, and similar lH and 13C NMR spectra except for ester linking position.

New caffeoyl derivatives from the leaves of Davidio involucrata

Figure 2.

473

HMBC and NOESY correlations of compound 1.

2D NMR experiments assigned all signals of the lH and 13C NMR data. In the HMBC spectrum the key long-range correlation from H-3 to C-90 was confirmed a caffeoyl linking at C-3 position of galactoic acid, so, 3 was determined as 3-caffeoyl galactoic acid, and named davidioside C.

3. Experimental 3.1. General experimental procedures Melting points were determined on a Yanaco MP-S3 apparatus and were uncorrected. IR spectra were recorded with a Perkin Elmer-377 infracord spectrophotometer using KBr pellets. Optical rotations were measured with a Jasco DIP-180 digital polarimeter spectrophotometer. 1H, 13C, DEPT, 1H–1H COSY, TOCSY, NOESY, HMQC and HMBC NMR spectra were obtained on a Varian Unity Plus 400 instrument. FAB mass spectra were recorded on a Jeol JMS-HX 110 instrument. Chromatographic stationary phase used RP-8 (40–60 mm, Merck), silica gel (160–200 mesh), Sephadex LH-20 (25–100 mm, Pharmacia Fine Chemical Co., Ltd.) and MCI-gel CHP20P (75–150 mm, Mitsubish Chemical Industries, Ltd.). The following solvent systems were used: CHCI3–MeOH–H2O (100 : 20 : 2), CHCI3–MeOH–H2O (80 : 20 : 3) and MeOH–H2O (0–100%) for each fraction; Spot of TLC was detected by spraying with 5% H2SO4 followed by heating.

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3.2. Plant material The leaves of D. involucrata Baill. were collected on the Yun-Long Mountain, Gong-Shan County of Yunnan province, China in 1995. The plant was identified by Dr Y. P. Yang, A voucher specimen (No. 12245) was deposited in the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China.

3.3. Extraction and isolation The fresh leaves of D. involucrate (3 kg) were extracted (5  5 L) with MeOH at room temperature (7 days  2). The extract was evaporated in vacuo to yield a residue, which was solubilized in water and then filtered. The water soluble fraction was passed through a D101 column and eluted with water and methanol. Evaporation of the methanol eluate yielded 25 g of a brown fraction (A). The fraction (A) was subjected to dry column chromatography (DCC) on silica gel (1.0 kg), eluted with CHCI3–MeOH– H2O (10 : 2 : 0.2) to get 13 fractions. Each fraction by Sephadex LH-20, RP-8 gel column chromatograph (solvent : MeOH–H2O, 5–70%) and finally chromatographed on silica gel with CHCI3–MeOH–H2O (100 : 10 : 1–80 : 20 : 2) as eluate to yield 1 (15 mg), 2 (21 mg), 3 (37 mg), 4 (64 mg), 5 (34 mg), 6 (11 mg), and 7 (35 mg). Davidioside A (1): Colorless amorphous powder, m.p. 187–190
C, HRFABMS m/z 339.0719 (Calcd for CI5HI5O9, 339.0716), ½2H D þ36 (c 0.6, MeOH); IR (KBr) vmax: 3302 (OH), 1715 (COOH), 1686 (CO), 1627 (C¼C), and 1602 (aromatic ring) cml; FAB-MS m/z 339 [M  H]; lH NMR and 13C NMR (table 1). Davidioside B (2): Amorphous powder, m.p. 132–133
C, HRFABMS m/z 353.0877 (Calcd for C16H17O9, 353.0872), ½2H D þ15 (c 0.9, MeOH); IR (KBr) vmax: 3318 (OH), 1735 (COOCH3), 1690 (CO), 1613 (C¼C), and 1600 (aromatic ring) cml; FAB-MS m/z 353 [M  H]; 1H NMR and 13C NMR (table 1). Davidioside C (3): Colorless amorphous powder, m.p. 159–163
C, HRFABMS m/z 339.0717 (Calcd for C15H1509, 339.0716), ½2H D þ16 (c 1.3, MeOH); IR (KBr) vmax: 3300 (OH), 1715 (COOH), 1683 (CO), 1624 (C¼C), and 1600 (aromatic ring) cm1, FAB-MS m/z 339 [M  H]1; lH NMR and 13DC NMR (table 1). Chi orogenic acid (6): Amorphous powder, C16H18O9, FAB-MS m/z 353 [MH]; 13C NMR (C5D5N) C 179.4 (C-7), 167.9 (C-90 ), 147.7 (C-70 ), 147.5 (C-30 ), 146.0 (C-40 ), 127.2 (C-10 ), 122.3 (C-60 ), 116.8 (C-20 ), 115.9 (C-50 ), 115.7 (C-80 ), 77.2 (C-1), 74.3 (C-4), 72.5 (C-5), 72.1 (C-3), 40.3 (C-2), and 39.0 (C-6). lH NMR H 7.44 (lH, d, J ¼ 15.8 Hz, H-70 ), 7.07(lH, d, J ¼ 2.0 Hz, H-20 ), 6.97 (lH, dd, J ¼ 2.0, 8.0 Hz, H-60 ), 6.77 (lH, d, J ¼ 8.0 Hz, H-50 ), 6.20 (lH, d, J ¼ 15.8 Hz, H-80 ), 5.14 (lH, br d, J ¼ 9.5 Hz, H-3), 3.95 (lH, br s, H-5), 3.52 (lH, br d, J ¼ 10.0 Hz, H-4), 2.00 (lH, d, J ¼ 15.1 Hz, H-6a), 1.89 (lH, br s, H-2), 1.75 (lH, d, J ¼ 15.1 Hz, H-6b). Methyl chlorogenate (7): White powder, C17H19O9; FAB-MS m/z 367 [M  H]; C NMR (CD3OD) C 175.4 (C-7), 168.3 (C-90 ), 149.7 (C-70 ), 147.2 (C-30 ), 146.8 (C-40 ), 127.7 (C-10 ), 122.8 (C-60 ), 116.5 (C-20 ), 115.3 (C-50 ), 115.2 (C-80 ), 75.9 (C-1), 72.6 (C-4), 72.1 (C-5), 70.4 (C-3), 53.0 (7-OCH3), 38.0 (C-2), and 37.8 (C-6). 1H NMR H 7.52 (lH, d, J ¼ 16.0 Hz, H-70 ), 7.05 (lH, d, J ¼ 2.0 Hz, H-20 ), 6.95 (lH, dd, J ¼ 2.0, 8.0 Hz, H-60 ),

13

CH2 CH CH CH CH C OCH3 C CH C C CH CH CH CH C

1 2 3 4 5 6

l0 20 30 40 50 60 70 80 90

DEPT

No.

115.8 121.4 143.4 114.1 166.6

125.6 114.8

65.9 67.0 69.3 71.4 70.1 179.5

1

H and

1

6.75 6.99 7.47 6.25

(1H, (1H, (1H, (1H, d, J ¼ 7.6 Hz) dd, J ¼ 7.6, 1.3 Hz) d, J ¼ 15.7 Hz) d, J ¼ 15.7 Hz)

65.8 66.9 69.0 71.4 70.5 175.5 51.5 125.6 114.4 145.6 148.4 115.8 121.4 145.5 114.1 166.4

2

6.75 6.99 7.48 6.25

(1H, (1H, (1H, (1H,

d, J ¼ 8.0 Hz) dd, J ¼ 8.0, 1.8 Hz) d, J ¼ 15.9 Hz) d, J ¼ 15.9 Hz)

7.04 (1H, d, J ¼ 1.8 Hz)

1

( Ha, m), 4.05 ( Hb, m) (1H, dd, J ¼ 16.0, 5.4 Hz) (1H, dd, J ¼ 16.0, 7.7 Hz) (1H, dd, J ¼ 14.5, 7.7 Hz) (1H, br s)

3.63 (3H, s)

4.16 3.97 3.40 3.84 4.27

1

125.6 114.7 145.6 148.5 115.8 121.5 145.1 114.1 166.6

64.4 65.5 79.5 73.9 71.5 179.3

3

C NMR spectral assignments of the davidiosides A–C (1–3) in DMSO.

13

( Ha, m), 4.06 ( Hb, m) (1H, dd, J ¼ 16.1, 5.2 Hz) (1H, dd, J ¼ 16.1, 7.8 Hz) (1H, dd,. J ¼ 14.2, 7.8 Hz) (1H, br s)

1

7.04 (1H, d, J ¼ 1.3 Hz)

4.14 3.96 3.41 3.81 4.25

1

Table 1.

(1Ha, m), 3.92 (1Hb, m) (1H, dd, J ¼ 15.8, 5.2 Hz) (1H, dd, J ¼ 15.8, 6.7 Hz) (1H, dd, J ¼ 15.0, 6.7 Hz) (1H, br s)

6.75 7.00 7.53 6.29

(1H, (1H, (1H, (1H,

d, J ¼ 7.9 Hz) dd, J ¼ 7.9, 2.0 Hz) d, J ¼ 16.0 Hz) d, J ¼ 16.0 Hz)

7.05 (1H, d, J ¼ 2.0 Hz)

4.02 3.83 4.61 3.78 4.22

New caffeoyl derivatives from the leaves of Davidio involucrata 475

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6.76 (1H, d, J ¼ 8.0 Hz, H-50 ), 6.20 (1H, d, J ¼ 16.0 Hz, H-80 ), 5.26 (1H, br d, J ¼ 5.5 Hz, H-3), 4.11 (1H, br s, H-5), 3.72 (1H, br d, J ¼ 7.0 Hz, H-4), 3.68 (3H, s), 2.20 (1H, m, H-6a), 2.20 (1H, m, H-2), 1.99 (1H, m, H-6b).

Acknowledgements We thank the staff of the analytical group of Department of Chemistry, Taiwan University for measuring the NMR and FAB-MS spectra and this research was supported by a Chinese National Science Fund (research project 20272015).

References [1] Institute of Botany, Chinese Academy of Sciences. In Iconographia Cormophytorum Sinicorum, Vol. II, p. 984, Science Publishing House, Beijing (2002). [2] J.S. He, H. Lin, W.L. Chen. Chinese Biodiversity, 3, 213 (1995). [3] L.Y. Li, D.Y. Zhang, F.W. Bai. J. Dalian. Nati. Univer., 3, 17 (1971). [4] M.A. Ouyang, J.N. Zhou. Guihaia, 23, 568 (2003). [5] C. Yang, J.G. Shi, S.Y. Mo, Y.C. Yang. J. Asian Nat. Prod. Res., 5, 143 (2003).

Natural Product Research, Vol. 22, No. 6, 15 April 2008, 477–482

Chemical composition and antioxidant activity of Campanula alliariifolia M. U. DUMLU*, E. GURKAN and E. TUZLACI Faculty of Pharmacy, Department of Pharmacognosy, University of Marmara, 34668 Istanbul, Turkey (Received 10 April 2006; in final form 17 September 2007) In this study, the chemical constituents of Campanula alliariifolia Willd. (Campanulaceae) are being investigated for the first time with the aid of this article. Five known compounds, which were quercetin-3-O-glucoside, quercetin-3-O-rutinoside, kaempferol-3-O-glucoside, lobetyolin (9-O- -D-glucopyranosyl-2,10-tetradecadien-4,6-diyne-8,14-diol) and lobetyol (2,10-tetradecadien-4,6-diyne-8,9,14-triol), were isolated from the methanol extract. The antioxidant activity of the methanol extract and the purified compounds of the plant was investigated with DPPH (1,1-diphenyl-picrilyhydrazyl) (free radical scavenging activity) and reducing power methods. The methanol extract has antioxidant capacity according to the mentioned methods. Lobetyol and lobetyolin showed significant antioxidant activity more than both methanol extract and other purified compounds. Keywords: Campanula alliariifolia; DPPH; Reducing power; Polyacetylene

1. Introduction Campanula alliariifolia synonyme of C. lamiifolia (Campanulaceae) is distributed naturally in northern Turkey. The plant is a densely pubescent perennial herb, height is 70 cm, growing in northern Turkey and nearly 95 species are found in the Turkish flora [1]. Campanula species are used for ear-pains in traditional folk medicine and known as ‘‘harebell’’ in the world. In Turkey, the species are used for wound healing and they are known as ‘‘c¸anc¸ic¸eg˘i’’ [2]. Campanulaceae and Campanula species contain a variety of chemical compounds such as polyphenols (flavonoid aglycones and their glycosides, phenolic acids and their esters), steroids, phenylpropanoid derivates, and polyacetylenes [3]. Although there are many scientific investigations about the antioxidant, chemical, ecological and cell culture studies [4–7] on Campanulaceae and some Campanula species, to our knowledge, no previous investigation has been done on C. alliariifolia. In this study, the antioxidant activity of the CHCl3 and MeOH extracts obtained from C. alliariifolia were evaluated with previously mentioned methods and compared *Corresponding author. Tel./Fax: 90216 4187739. Email: [email protected] Natural Product Research ISSN 1478-6419 print/ISSN 1029-2349 online ß 2008 Taylor & Francis http://www.tandf.co.uk/journals DOI: 10.1080/14786410701640429

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with L-ascorbic acid, buthylated hydroxyanisole (BHA), -tocopherol and the chemical constituents of the plant were investigated for the first time.

2. Experimental 2.1. Plant material The plant was collected from Northern Turkey in August 2000, and identified by Prof. Dr Ertan Tuzlacı, Department of Pharmaceutical Botany, Marmara University. The voucher specimens are deposited in the Marmara University, Faculty of Pharmacy Herbarium (Istanbul-Turkey) (MARE 6430).

2.2. Chemicals Column chromatography was carried on silica gel 60 (0.063–0.200 mm) (Merck, Germany), DPPH (Sigma, USA), -tocopherol (Vitamin E) (Merck, Germany), BHA (Sigma, USA), Kieselgel 60 F254 plates (Merck, Germany), Organic solvents (Merck, Germany) were used in this study.

2.3. Instruments 1

H NMR and 13C NMR spectra were recorded on Bruker AC-500, mass spectrums were run on Zabspec spectrometer, IR spectra were recorded on Perkin Elmer 1600 series FTIR, UV spectra were run on Shimatzu 2100.

2.4. Extraction and isolation The air-dried and powdered plant (500 g) was extracted at room temperature successively with CHCl3 (3
1500 mL) and MeOH (3
1500 mL). The extracts were evaporated to dryness to yield 14.7 g and 15.4 g, respectively. The MeOH extract was applied on silica gel column and eluted with CH2Cl2–MeOH (9 : 1–1 : 1). Total 15 fractions were obtained. Fraction 2 contains pure lobetyol (1, 80 mg). Fraction 6 contains lobetyolin (2, 10 mg). The compound 2 was purified with Sephadex LH-20 column (MeOH : CHCl3 5 : 2). Fraction 9 contains kaempferol-3-O-glucoside (3, 25 mg). The compound was purified with preperative TLC (MeOH : CHCl3 7 : 3). Fraction 12 contains quercetin-3-O-glucoside (4, 34 mg) and quercetin-3-O-rutinoside (5, 18 mg). The compounds were separated and purified with Sephadex LH-20 column (MeOH and MeOH–H2O 1 : 1).

2.5. DPPH method Various concentrations of the MeOH extract and the purified compounds (50, 100, 250, 500 mg L1) were mixed with 1 mL of a 1 mM methanolic solution of DPPH free radical. The mixtures were vigorously shaken and left to stand for 10 min in the dark,

Chemical composition and antioxidant activity of C. alliariifolia

479

and their absorbance were measured at 517 nm against blank sample. L-Ascorbic acid was used as the control [7].

2.6. Reducing power method Samples of 50, 100, 250, 500 mg L1 were mixed with 2.5 mL of phosphate buffer (pH ¼ 6.6) and 2.5 mL of 1% potassium ferricyanide, and the mixtures were incubated at 50 C for 30 min. After 2.5 mL of 10% tricloroacetic acid (w/v) was added, the mixtures were centrifuged at 3000 rpm for 10 min. The upper layers (5 mL) were mixed with 5 mL of deionized water and 1 mL of 0.1% ferric chloride, and the mixtures’ absorbance were measured at 700 nm against blank sample. BHA and -tocopherol (vitamin E) were used as controls [8].

3. Results and discussion 3.1. Scavenging of DPPH radicals MeOH extract showed variable DPPH radical-scavening activities. In particular, the MeOH extract showed strong antioxidant activity at 500 mg L1 concentrations, but its effect was less than that of L-ascorbic acid (see table 1). Compounds 1 and 2 have more scavenging activity than MeOH extract. Other purified compounds have a scavenging activity but less than other’s and MeOH extract. Compounds 1, 2 and MeOH extract have a relatively strong antioxidant effect at the same concentration (250 and 500 mg L1). The obtained antioxidant data are summarized in table 1.

3.2. Measurement of reducing power The reducing power of the extract exhibited reducing power activity within mentioned concentrations. The MeOH extract of the plant had the highest reducing power at 500 mg L1 concentration. Compounds 1, 2 have the highest reducing power activity in this study. Compounds 3, 4, and 5 have no significant reducing power activity (see table 2).

Table 1. Samples MeOH extract Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 Ascorbic acid

The obtained results from DPPH method.

50 mg L1

100 mg L1

250 mg L1

500 mg L1

75 85 82 60 65 70 0.98

79 86 85 65 65 71 0.98

86 89 87 67 67 75 0.98

93 98 94 69 68 78 0.98

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M. U. Dumlu et al.

3.3. Structure identification 1

H and 13C NMR data of the purified polyacetylene compounds 1 and 2 are indicated in tables 3 and 4, respectively. The structures of the isolated flavonoidal compounds were identified with UV spectral data [9,10]. IR spectra were recorded with authentic samples and compared with the flavonoidal compounds. All these authentic samples fingerprints were crossed with purified compounds in their IR spectra.

3.3.1. Lobetyol (1). An amorphous powder. UV (CHCl3, max, nm): 256.4, 270.4, 286.4. IR(KBr, , cm1): 3450 (OH), 2945 (Me), 2892 (CH2), 2242 (CC), 1650 (C¼C), 1373 (CH3), 964, 847, 716. FAB MS m/z (rel. int.): 257 [M þ Na]þ (47), 273 [M þ K]þ (32). 1H NMR see table 2. 13C NMR see table 4.

3.3.2. Lobetyolin (2). An amorphous powder. UV (H2O, max, nm): 207.4, 215.2, 241.4, 253.8, 267.6, 283.7. IR (KBr, , cm1): 3375 (OH), 2940 (Me), 2870 (CH2), 2240 (CC), 1675 (C¼C), 1390 (CH3), 1050 (CH2OH), 980. FAB MS m/z (rel. int.): 397 [M þ H]þ(27), 419 [M þ Na]þ (16), 435[M þ K]þ (9). 1H NMR see table 2. 13C NMR

Table 2.

The obtained results from reducing power method.

Samples

50 mg L1

100 mg L1

250 mg L1

500 mg L1

0.55 0.85 0.80 0.68 0.65 0.68 1.00 0.98

0.59 0.86 0.83 0.69 0.66 0.69 1.00 0.98

0.62 0.89 0.85 0.71 0.66 0.70 1.00 0.98

0.75 0.92 0.89 0.72 0.69 0.72 1.00 0.98

MeOH extract Compound 1 Compound 2 Compound 3 Compound 4 Compound 5 BHA -Tocopherol

Table 3.

1

H NMR spectral data of compunds 1 and 2.

H

1 (Me2CO-d6)

1 2 3 8 9 10 11 12 13 14 Glc.

1.73 6.29 5.55 4.40 4.20 5.40 5.86 2.09 1.56 3.49

0

10 20 30 40 50 6

4.32 3.24 3.24 3.34 3.24 3.57 and 3.77

2 (CDCl3) 1.82 6.34 5.52 4.29 4.13 5.57 5.88 2.18 1.68 3.67

Chemical composition and antioxidant activity of C. alliariifolia

481

see table 4. H

H H3C 1

3

C

C

4

C

6

5

C

C

7

C

8

9

CH

CH

OH

OR

2

H

11

C

C

10

CH2CH2CH2OH 12

13

14

H

R=H Lobetyol R=Glc. Lobetyolin

Table 4. C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Glc. 0

10 20 30 40 50 6

13

C NMR spectral data (compounds 1 and 2). 1 (Me2CO-d6)

2 (CDCl3)

19.2 145.8 110.4 82.4 78.2 72.9 70.8 66.5 81.8 126.4 138.6 29.7 33.0 61.9

18.9 144.6 109.4 79.1 78.1 71.4 71.4 66.9 75.5 127.5 135.2 28.9 31.8 62.3

100.8 74.7 77.9 71.5 77.8 62.7

In the present study, we found that C. alliariifolia shows antioxidant activity with DPPH and reducing power methods. These findings provide a pharmacological explanation for some of its uses in folk medicine. It has been reported that the antioxidant activity of plants is responsible for their therapeutic effect against cancer, cardiovascular diseases, and diabetes [11, 12]. The MeOH extracts of the plant showed variable radical scavenging activities. The MeOH extract, which contains polyphenolic components, showed strong antioxidant activity. Some steroids, aromatic acids, esters had been previously isolated from Campanula species [3, 13], but their extracts’ activity has not been previously reported. In our experiment, the purified compounds 1 and 2 showed relatively high DPPH radical scavenging activity. According to the reducing power method results, the MeOH extract showed significant antioxidant activity less than compounds 1, 2; more than compounds 3, 4, 5. Compounds 1 and 2 are the most active ones in our study. Two polyacetylene compounds were purified from the

482

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MeOH extract. Compound (2) gave specific absorptions in UV spectra (207.4, 241.4, 267.6) established that four quaternary carbons are parts of one conjugated dyne structure. The compound gave a prominent ion peak [M þ H]þ at m/z 397 in the FAB mass spectrum. 1H NMR spectrum data are shown (see table 3). The signals for an anomeric glucose proton [ 4.32 (1H, d, J ¼ 7.5 Hz)], one methyl group [ 1.73(3H, s)], three methylene groups [ 1.56 (1H, q), 2.09 (2H,br, dd, J ¼ 13.5, 6.8 Hz), 3.49 (2H, t, J ¼ 6.8 Hz)], two methine groups [ 4.20 (1H, t, J ¼ 6.8 Hz), 4.40 (1H, d, J ¼ 6.8 Hz)], olefinic protons [ 5.40 (1H, dd, J ¼ 16.2, 6.8 Hz), 5.86 (1H, dt, J ¼ 16.2, 6.8 Hz) and 5.55 (1H, dd, J ¼ 16.2, 2.3 Hz), 6.29 (1H, dq, J ¼ 16.2, 6.8 Hz)]. Each J ¼ 16.2 Hz shows two pairs of trans coupled. 13C NMR spectrum data are shown (see table 4). Glucose moiety (100.8, C1; 74.7, C2; 77.9, C3; 71.5, C4; 77.8, C5; 62.7, C6), one methyl (19.2, C-1), two methine (66.5, C-8; 81.8, C-9), three methylene (33.0, C-13; 29.7, C-12; 61.9, C-14) and four olefinic carbons (145.8, C-2; 110.4, C-3; 126.4, C-10; 138.6, C-11) were present in the spectrum and four quaternary carbon signals (70.8, C-7; 72.9, C-6; 78.2, C-5; 82.4, C-4) were also present in the spectrum. The spectra of compound (1) exhibited almost the same partial structure as lobetyolin. The compound gave a prominent ion peak [M þ Na]þ at m/z 257 in the FAB mass spectrum.1H and 13C NMR data of the compound, except for the absence of the glucose moiety signals, was similar as lobetyolin. According to all these obtained data and published literature compound 1 is ‘‘lobetyol’’ and compound 2 is ‘‘lobetyolin’’ [14–17]. In conclusion, the MeOH extract has antioxidant activity. Compound 1 and 2 which are polyacetylene structure show strong antioxidant capacity among the five purified compounds obtained from C. alliariifolia by DPPH and reducing power methods. Lobetyol and lobetyolin are isolated from Campanula species for the first time although they have been found out in some Campanulaceae plants. And, also this is the first investigation on C. alliariifolia from its chemical composition and antioxidant activity point of view.

References [1] P.H. Davis. Flora of Turkey and The East Aegean Islands, Vol 6, University Press, Edingburg (1977). [2] T. Baytop. Therapy with Medicinal Plants in Turkey (past and present), p. 371,, Nobelpress, Istanbul (1999). [3] M. Cuendet, O. Potterat, H. Kurt. Phytochemistry, 56, 631 (2001). [4] K. Ishimaru, M. Osabe, L. Yan, T. Fujioka, K. Mihaski, N. Tanaka. Phytochemistry, 62, 643 (2003). [5] J.Y. Lee, J.W. Yoon, C.T. Kim, S.T. Lim. Phytochemistry, 65, 3033 (2004). [6] J.Y. Lee, W.I. Hwang, S.T. Lim. J. Ethnopharma., 93, 409 (2004). [7] G.M. Wei, F. Nicola, S.L. Qıing, W. Jean-Luc, K. Hostettman. Phytochemistry, 45, 411 (1997). [8] L.M. McCune, T. Johns. J. Ethnopharma., 82, 197 (2002). [9] M.U. Dumlu, E. Gurkan. Z. Naturforsch C, 61, 44 (2006). [10] J.B. Harborne, T.J. Mabry. The Flavonoids: Advances in Research, Chapman and Hall, London (1982). [11] J.B. Harborne. The Flavonoids: Advances in Research Since 1986, Chapman and Hall, London (1994). [12] R.A. Anderson, C.L. Broadhurst, M.M. Polansky, W.F. Schmidt, A. Khan, V.P. Flanagan, N.W. Schoene, D.J. Graves. J. Agri. Food Chem., 52, 65 (2004). [13] S.A. Stanner, J. Hughes, C.N. Kelly. Burns, 27, 319 (2001). [14] N. Yaylı, N. Yıldırım, A. Usta, S. O¨zkurt, V. Akgu¨n. Turk. J. Chem., 27, 749 (2003). [15] T. Nakayama, S. Kawaagishi, T. Oosawa. CA 122:54685 JP06, 248–267 (1994). [16] K. Ishimaru, H. Yonemitsu, K. Shimomura. Phytochemistry, 30, 2255 (1991). [17] K. Ishimaru, S. Sadoshimo, S. Neera, K. Koyama, K. Tahahaski, K. Shimomura. Phytochemistry, 31, 1577 (1992).

Natural Product Research, Vol. 22, No. 6, 15 April 2008, 483–488

Two new phenolic water-soluble constituents from branch bark of Davidia involucrata ZU-JIAN WU*y, MING-AN OUYANGyz and SHI-BIN WANGz yInstitute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, P.R. China zDepartment of Bio-engineering & Technology, Huaqiao University, Quanzhou, Fujian 362011, China (Received 5 September 2005; in final form 11 May 2006) Two new phenolic water-soluble constituents, involcranoside A (1) and involcranoside B (2) have been isolated along with five known phenolic compounds: 3,4-dimethoxyphenyl-O- -Dgluco-pyranoside (3), picein (4), and 1,4-dihydroxy-3-methoxy-phenyl-4-O- -D-glucopyranoside (5), leonuriside A (6) and 4-hydroxy-3-methoxybenzoic acid (7) from the branch bark of Davidia involucrata. Identification of their structures was achieved by 1D and 2D NMR experiments, including 1H-1H COSY, NOESY, HMQC and HMBC methods and FAB mass spectral data. Keywords: Nyssaceae; Davidia involucrata; Branch bark; Phenolic compounds

1. Introduction More than 25 flavones, flavonoid glycosides, tannins, caffeoyl derivatives and neolignan glycosides have been isolated from the leaves and the branch bark of Davidia involucrata [1–4]. Our continued interest in the chemical constituents of the branch bark of Chinese Nyssaceae species has led to the isolation of seven phenolic glycosides. In this article, we describe the isolation and structural elucidation of two new phenolic water-soluble constituents, involcranoside A (1) and involcranoside B (2).

2. Results and discussion Chromatographic separation of a MeOH extract from the branch bark of D. involucrata yielded the new compounds 1 and 2 in addition to 3,4-dimethoxyphenyl-O- -D-glucopyranoside (3), picein (4) [5], and 1,4-dihydroxy-3-methoxy-phenyl4-O- -D-glucopyranoside (5) [6], leonuriside A (6) [7], and 4-hydroxy-3-methoxybenzoic *Corresponding author. Email: [email protected] Natural Product Research ISSN 1478-6419 print/ISSN 1029-2349 online ß 2008 Taylor & Francis http://www.tandf.co.uk/journals DOI: 10.1080/14786410600906426

484

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acid (7). HO 3″

O

HO

1″

H3CO O

C

O

O

5′ 4′

HO

O

6′

7′

HO

OCH3 3

OH

4

7

8

CH3

O

9

HO

3 OH

O

6′

4″

CH3

O

O

1″ 4′

3″

OH

1

O

C CH3 7

O

OH

C CH3

OH

8

4

1′ 2′

OH OH

4

O

HO

O

6″

OCH3

OH

1

OH

O

O

OH

6

1′ 2′

OH

C CH2

1

OH

2

OH

OH H3CO

H3CO HO

HO O

O

5 OH

O

O

O

OH HO

OH

OH OH

H3CO

OH

H3CO

OH

6

C

OH

7

OH

Compound 1 was obtained as a brown amorphous powder. The FAB-MS of 1 exhibited a quasi-molecular ion peak at m/z 493 [M
H]
. The HRFAB-MS indicated an m/z of 493.1342 for his peak, which was consistent with the molecular formula C23H26O12 for 1 and representing eleven elements of unsaturation. The absorption bands at max 3300, 1701, 1692, 1615, 1519 cm
1 in the IR spectrum were characteristic of hydroxyl, conjugated carbonyl and aromatic groups, respectively. Acid hydrolysis of 1 with 2 M TFA afforded a sugar, which was identified as glucose by TLC. The 1H NMR spectrum of 1 in CD3OD showed ABX spin system signals at H 7.71 (1H, d, J ¼ 1.7 Hz), 7.65 (1H, d, J ¼ 8.8 Hz), 7.93 (1H, dd, J ¼ 8.8, 1.7 Hz), an aromatic proton signal at H 7.91 (2H, s), an anomeric proton signal at H 5.66 (1H, d, J ¼ 7.2 Hz), a methoxy signal at H 3.62 (3H, s, –OCH3), and ethyl group signals at H 3.15 (1H, m), 3.02 (1H, m), 1.11 (3H, t, J ¼ 7.2 Hz). The 13C NMR spectrum of 1 gave two carbonyl signals at C 199.4 (ketone group) and 167.1 (ester group), 12 aromatic carbon signals, hexose signals, a methoxy signal and ethyl group signals (table 1). Evaluation of spin–spin coupling and chemical shifts of the hexose allowed the identification of one -glucopyranosyl unit. The 1H and 13C NMR spectra of 1 suggested a propiophenone moiety, and a galloyl group because of the signal at H 7.91 (2H, s) in the 1H NMR spectrum and the signals at C 167.1, 147.6  2, 141.1, 121.2, 110.4  2 in the 13C NMR spectrum. The cross-peaks in the HMBC experiments between signals H 7.91 (H-200 , H-600 ) and C 167.1 (C-700 ), 141.1 (C-400 ), 147.6 (C-300 , C-500 ), 121.2 (C-100 ), between H 5.27 (H-60 of Glc), 4.85 (H-60 of Glc) and 167.1 (C-700 ), between H 5.66 (H-10 of glc) and 151.7 (C-4), between H 7.71 (H-2), 7.92 (H-6), 1.11 (H-9) and C 199.4 (C-7) indicated that the gallic acid

Two new phenolic water-soluble constituents from branch bark of Davidia involucrata 485 Table 1.

1 2 3 4 5 6 7 8 9

10 20 30 40 50 60 100 200 300 400 500 600 700

13

C NMR spectral assignments of compounds 1–7 in CD3OD.

1

2

3

4

5

6

7

131.9 111.2 149.9 151.7 115.2 123.5 199.4 31.6 8.7 3-OCH3 55.6 4-Glc 102.0 74.7 78.6 71.7 75.9 64.8 60 -Galloyl121.2 110.4 147.6 141.1 147.6 110.4 167.1

162.9 131.7 117.3 132.6 117.3 131.7 199.7 17.9

151.5 103.1 148.0 141.7 114.8 108.8

132.5 117.2 131.6 162.9 131.6 117.2 199.3 26.7

145.9 104.7 151.0 153.8 109.3 113.9

155.9 106.2 154.6 129.5 154.6 106.2

108.5 113.9 148.4 152.8 125.0 116.2 169.2

3-OCH3 56.5 4-OCH3 57.3 1-Glc 102.6 73.9 77.0 70.5 77.1 61.6

4-Glc 101.4 74.7 77.9 71.4 77.1 67.8 60 -Rha102.1 72.1 72.4 73.9 69.9 18.1

3-OCH3 55.5 4-Glc 101.6 74.9 78.0 71.3 78.3 62.5

4-Glc 103.5 75.0 78.1 71.6 78.3 62.7

3-OCH3 56.9 5-OCH3 56.9 4-Glc 94.5 75.1 77.8 71.3 78.3 62.6

55.9

HO H3CO

O HO

C

O

O O O

HO

C

CH2 CH3

OH HO OH Figure 1.

HMBC

HMBC correlations for 1.

was linked at C-60 of the Glc and the sugar was linked at C-4 of the propiolphenone moiety. On the basis of above evidence, 1, named involcranoside A, was determined to be 4-O-(6-galloyl)- -D-glucopyranosyl propiophenone (figure 1). The negative FABMS of 2 showed a quasi-molecular ion peak at m/z 443 [M
H]
, concluding from the HRFAB-MS data was indicative of the formula C20H28O11 for 2 and representing seven elements of unsaturation. The absorption bands at max 3340, 1706, 1609, 1500 cm
1 in the IR spectrum were characteristic of hydroxyl, conjugated carbonyl, and aromatic group, respectively. Acid hydrolysis of 1 with 2 M TFA afforded a mixture of sugars, which were identified as glucose and rhamnose by TLC. The 1H NMR spectrum of 2 exhibited two aromatic proton signals at H 7.97 (2H, d,

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J ¼ 8.8 Hz), 7.14 (2H, d, J ¼ 8.8 Hz), two anomeric proton signals at H 5.00 (d, J ¼ 7.6 Hz), and at 4.68 (d, J ¼ 1.5 Hz), and a methyl signal at H 2.55 (3H, s). These signals suggested that compound 2 had a disubstituted phenyl group and an acetyl group. This evidence was also supported by the 13C NMR spectrum, which signals at C 199.7, 162.9, 132.6  2, 131.7, 117.3  2 and 17.9. Thus the genin was assigned as an acetophenone. From the 2D NMR experiments, the proton and carbon data of 2 was assigned. The correlations revealed the Rha-1 linking at Glc-6, and Glc-1 linking to the acetophenone. Thus, 2, named involcranoside B was identified as 4-O-L-rhamnopyranosyl (1 ! 6)- -D-glucopyranosyl acetophenone.

3. Experimental 3.1. General experimental procedures Melting points were determined on a Yanaco MP-S3 apparatus and are uncorrected. UV spectra were recorded with a Backman DU-64 spectrometer. Optical rotations were measured with a Jasco DIP-180 digital polarimeter spectrophotometer. The IR spectrum was recorded with a Perkin–Elmer 1750 FTIR spectrometer. 1H, 13C, DEPT, 1H–1H COSY, NOESY, HMQC and HMBC NMR spectra were obtained on a Varian Unity Plus 400 instrument. FAB mass spectra were recorded on a Jeol JMS-HX 110 instrument. Chromatographic stationary phases were RP-8 (40–60 m, Merck), silica gel (160–200 mesh), Sephadex LH-20 (25–100 m, Pharmacia Fine Chemical Co., Ltd) and MCI-gel CHP20P (75–150 m, Mitsubishi Chemical Industries, Ltd). The following solvent systems were (a) CHCl3–MeOH–H2O (80 : 20 : 3), CHCl3–MeOH– H2O (70 : 30 : 5) and MeOH–H2O (0–100%) for the glycosides; and (b) CHCl3–MeOH– H2O (7 : 3 : 1) lower-layer 9 mL þ 1 mL HOAc for sugars. Compounds on TLC were detected by spraying with 5% H2SO4, then by heating. Sugars were detected by spraying with aniline–phthalate reagent.

3.2. Plant material The branch bark of D. involucrata was collected on the Yun–Long Mountain, GongShan county of Yunnan province, China in 1995. The plant was identified by Dr Y.P. Yang, A voucher specimen (No. 12245) was deposited in the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China.

3.3. Extraction and isolation The dry branch bark of D. involucrata (10 kg) were extracted (2  10 L) with MeOH at room temperature (7 days  2). The extract was evaporated in vacuo to yield a residue, which was solubilised in water and then filtered. The water soluble fraction was passed through a Diaion resin column and was eluted with water and methanol. Evaporation of the methanol eluate yielded a brown fraction (A) (175 g). The fraction A was subjected to dry column chromatography (DCC) on silica gel (2.0 kg), eluted with CHCl3–MeOH–H2O (10 : 2 : 0.2) to afford thirteen fractions. Each fraction was purified by Sephadex LH-20, RP-8 gel column chromatography (solvent: MeOH–H2O,

Two new phenolic water-soluble constituents from branch bark of Davidia involucrata 487

10–70%) and finally purified by repeated chromatography on a silica gel column with CHCl3–MeOH–H2O (100 : 10 : 1–70 : 30 : 5) as eluent to yield 1 (21 mg), 2 (13 mg), 3 (11 mg), 4 (30 mg), 5 (15 mg), 6 (26 mg), 7 (38 mg). 3.3.1. Involcranoside A (1). Brown amorphous powder, m.p. 131–133 C, HRFABMS
1 m/z 493.1342 (Calcd for C23H25O12, 493.1346), ½21 D
11 (c 0.3, MeOH); IRmax cm :
1 3300, 1701, 1692, 1615, 1519; FAB-MS m/z 493 [M
H] ; H NMR  7.71 (1H, d, J ¼ 1.7 Hz, H-2), 7.65 (1H, d, J ¼ 8.8 Hz, H-5), 7.93 (1H, dd, J ¼ 8.8, 1.7 Hz, H-6), 3.15 (1H, dd, J ¼ 14.0, 7.2 Hz, H-8), 3.02 (1H, dd, J ¼ 14.0, 7.2 Hz, H-8), 1.11 (3H, t, J ¼ 7.2 Hz, H-9), 3.62 (3H, s, 3-OCH3), 5.66 (1H, d, J ¼ 7.2 Hz, H-10 ), 4.40 (1H, m, H20 ), 4.36 (1H, m, H-30 ), 4.15 (1H, m, H-40 ), 4.38 (1H, m, H-50 ), 5.27 (1H, dd, J ¼ 9.2, 1.3 Hz, H-60 ), 4.85 (1H, dd, J ¼ 9.2, 6.2 Hz, H-60 ), 7.91 (2H, s, H-200 , 600 ) and 13C NMR (table 1). 3.3.2. Involcranoside B (2). Colourless amorphous powder, m.p. 145–146 C, ½21 D
17 (c 0.16, MeOH); IRmax cm
1: 3340, 1706, 1609, 1500; FAB-MS m/z 443 [M
H]
, 297 [M
H-146]
, 135 [M
H-146–162]
; HR-FAB-MS m/z 443.1551 [M
H]
(Calcd for C20H27O11, 443.1553); 1H NMR  7.97 (2H, d, J ¼ 8.8 Hz, H-2, 6), 7.14 (2H, d, J ¼ 8.8 Hz, H-3, 5), 2.55 (3H, s, H-7), 5.00 (1H, d, J ¼ 7.6 Hz, H-10 ), 3.48 (1H, overlap, H-20 ), 3.49 (1H, overlap, H-30 ), 3.39 (1H, br d, H-40 ), 3.62 (1H, overlap, H-50 ), 4.02 (1H, dd, J ¼ 9.4, 4.6 Hz, H-60 ), 3.61 (1H, dd, J ¼ 4.6, 2.5 Hz H-60 ), 4.68 (1H, d, J ¼ 1.5 Hz, H100 ), 3.83 (1H, dd, J ¼ 3.3, 1.5 Hz, H-200 ), 3.69 (1H, dd, J ¼ 9.5, 3.3 Hz, H-300 ), 3.35 (1H, t, J ¼ 9.5 Hz, H-400 ), 3.63 (1H, m, H-500 ), 1.17 (3H, d, J ¼ 6.5 Hz, H-600 ) and 13C NMR (table 1). 3.3.3. 3,4-Dimethoxy-1-hydroxylphenyl-O-b-D-glucopyranoside (3). Colourless amorphous powder, FAB-MS m/z 315 [M
H]
; 1H NMR  6.81 (1H, d, J ¼ 2.4 Hz, H-2), 6.65 (1H, d, J ¼ 8.4 Hz, H-5), 6.83 (1H, dd, J ¼ 8.4, 2.4 Hz, H-6), 3.83 (3H, s, 3-OCH3), 3.77 (3H, s, 4-OCH3), 4.76 (1H, d, J ¼ 8.0 Hz, H-10 ), 3.18 (1H, dd, J ¼ 9.1, 8.0 Hz, H-20 ), 3.33 (1H, overlap, H-30 ), 3.28 (1H, t, J ¼ 9.1 Hz, H-40 ), 3.21 (1H, m H-50 ), 3.88 (1H, dd, J ¼ 12.0, 2.4 Hz, H-60 ), 3.66 (1H, dd, J ¼ 12.0, 2.45.6 Hz, H-60 ); and 13C NMR (table 1). 3.3.4. Picein (4). Colourless amorphous powder, FAB-MS m/z 301 [M
H]
; 1H NMR  7.96 (2H, d, J ¼ 8.8 Hz, H-2, 6), 7.15 (2H, d, J ¼ 8.8 Hz, H-3, 5), 2.55 (3H, s, H7), 5.20 (1H, d, J ¼ 7.6 Hz, H-10 ), 3.49 (1H, overlap, H-20 ), 3.48 (1H, overlap, H-30 ), 3.39 (1H, t, J ¼ 9.1 Hz H-40 ), 3.69 (1H, m, H-50 ), 3.91 (1H, dd, J ¼ 12.4, 2.0 Hz, H-60 ), 3.47 (1H, m, H-60 ); and 13C NMR (table 1). (5). Colourless 3.3.5. 1,4-Dihydroxyl-3-methoxyphenyl-4-O-b-D-glucopyranoside amorphous powder, FAB-MS m/z 301 [M
H]
, 239 [M
H-162]
; 1H NMR  6.79 (1H, d, J ¼ 2.2 Hz, H-2), 6.68 (1H, d, J ¼ 8.8 Hz, H-5), 6.57 (1H, dd, J ¼ 8.8, 2.2 Hz, H6), 3.82 (3H, s, 3-OMe), 4.73 (1H, d, J ¼ 7.0 Hz, H-7), 3.33–3.48 (4H, m, H-2  H-5), 3.89 (1H, dd, J ¼ 12.0, 2.0 Hz, H-6), 3.67 (1H, dd, J ¼ 12.0, 5.6 Hz, H-6) and 13C NMR (table 1).

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3.3.6. Leonuriside A (6). Colourless amorphous powder, FAB-MS m/z 331 [M
H]
; 1 H NMR  6.13 (2H, s, H-3, 5), 3.78 (6H, s, 2-OCH3, 6-OCH3), 4.65 (1H, d, J ¼ 7.2 Hz, H-10 ), 3.38–3.57 (4H, overlap, H-2  H-5), 3.85 (1H, dd, J ¼ 11.8, 2.1 Hz, H-60 ), 3.69 (1H, dd, J ¼ 11.8, 5.4 Hz, H-60 ); and 13C NMR (table 1).

3.3.7. 4-Hydroxy-3-methoxybenzoic acid (7). Colourless amorphous powder, FABMS m/z 167 [M
H]
; 1H NMR  8.07 (1H, d, J ¼ 2.0 Hz, H-2), 7.29 (1H, d, J ¼ 8.2 Hz, H-5), 8.15 (1H, dd, J ¼ 8.2, 2.0 Hz, H-6), 3.75 (3H, s, 3-OMe); and 13C NMR (table 1).

3.4. Acid hydrolysis A solution of each compound (8 mg) was heated at reflux at 100 C in 2 M aqueous CF3COOH (5 mL) on a water bath for 3 h. After this period, the reaction mixture was diluted with H2O (15 mL) and extracted with CH2Cl2 (3  5 mL). The combined CH2Cl2 extracts were washed with H2O and then evaporated to dryness in vacuo. After evaporation to dryness of the aqueous layer with MeOH until neutral, the sugars were analysed by comparison with authentic samples (solvent system b) on silica gel HPTLC.

Acknowledgements This research was supported by National Science Fundation of China (Grant No. 20272015). We thank the staff of the analytical group of Kunming Institute of Botany, Chinese Academy of Sciences for measurements of NMR and FAB-MS spectra.

References [1] [2] [3] [4] [5] [6] [7]

M.A. Ouyang, J. Huang, Q.W. Tan. Journal of Asian Natural Product Research, 9, 487–492 (2007). M.A. Ouyang, J.N. Zhou. Guihaia, 23, 568 (2003). M.A. Ouyang, J.N. Zhou, S.B. Wang. Journal of Natural Product Research, (in press) (2008). G.Q. Xiang, X.S. Lu. Acta Biotanica Sinca, 31, 540 (1989). M. Ushiyama, T. Furuya. Phytochemistry, 28, 3009 (1989). H. Pan, L.N. Lundgren. Phytochemistry, 39, 1423 (1995). H. Otsuka, M. Takeuchi, S. Inoshiri, T. Sato, K. Yamasaki. Phytochemistry, 28, 883 (1989).

Natural Product Research, Vol. 22, No. 6, 15 April 2008, 489–494

Microbial transformation of oleanolic acid by Fusarium lini and a-glucosidase inhibitory activity of its transformed products M. IQBAL CHOUDHARY*y, IFFAT BATOOLy, SHAMSUN NAHAR KHANy, NIGHAT SULTANAz, S. ADNAN ALI SHAHy and ATTA-UR-RAHMANy yHEJ Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Karachi 75270, Pakistan zPharmaceutical Research Center, PCSIR Laboratories Complex, University Road, Karachi 75270, Pakistan (Received 15 March 2006; in final form 25 October 2006) The biotransformation of a pentacyclic triterpene, oleanolic acid (1), with Fusarium lini afforded two oxidative metabolites, 2,3 -dihydroxyolean-12-en-28-oic acid (2), and 2,3 , 11 -trihydroxyolean-12-en-28-oic acid (3). Metabolite 3 was found to be a new compound. The structures were characterized on the basis of spectroscopic studies. These metabolites exhibited a potent inhibition of -glucosidase enzyme. Keywords: Oleanolic acid; Microbial transformation; Fusarium lini; Enzyme inhibition; -Glucosidase

1. Introduction Oleanolic acid (1), one of most extensively studied pentacyclic triterpenoids, is widely present in food and medicinal plants [1–4]. It is reported to possess various biological properties such as antitumor [5,6], hepatoprotective [7–9], anti-HIV [10–12], skinprotective [13–15], etc. We have reported the -glucosidase inhibitory activity of 1 for the first time [16]. -Glucosidase (EC 3.2.1.20) catalyzes the final step in the digestive process of carbohydrates [17]. Diabetes is a metabolic disorder which causes an elevated fasting and postprandial blood glucose levels. Among the therapeutic drugs to prevent high blood glucose levels, the inhibitors of -glucosidase are effective by delaying the glucose absorption [18]. The inhibitors of this enzyme delay carbohydrate digestion, and cause a reduction in the rate of glucose absorption into blood. Therefore, inhibition of -glucosidase is an important intervention in the management of non-insulin dependent diabetes [16]. In order to obtain new structural analogues with -glucosidase inhibitory activities, compound 1 was subjected to a fungal transformation with filamentous fungus *Corresponding author. Email: [email protected] Natural Product Research ISSN 1478-6419 print/ISSN 1029-2349 online ß 2008 Taylor & Francis http://www.tandf.co.uk/journals DOI: 10.1080/14786410601083787

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M. I. Choudhary et al. 30

29

CH3

H3C

21

19

22 11 26

25

CH3

9

CH3

13

H

17

3

5

H

7

OH

O

15

1

28

CH3 27

HO H 3C

23

H CH3

1

24

H3C H3C

CH3

CH3 HO OH OH

CH3

HO

CH3

H

CH3

HO

CH3

HO H3C

H CH3

2 Figure 1.

H O

O H

CH3 CH3

H HO H3C

H CH3

3

Transformation of oleanolic acid (1) by F. lini.

Furarium lini, resulting into two oxidative metabolites 2 and 3 (figure 1). These metabolites exhibited a potent inhibition of the -glucosidase.

2. Results and discussion Metabolism of oleanolic acid (1), C30H48O3 (Mþ 456.3258 in HREI MS), with F. lini (NRRL 68751) for 12 days afforded two oxidative metabolites 2 and 3. The metabolite 2 was obtained as a white solid. The HREI MS of metabolite 2 exhibited an Mþ at m/z 472.3490 (C30H48O4, Calcd 472.3563), which indicated an increment of 16 a.m.u. as compared to compound 1. The 1H-NMR spectrum (500 MHz, CD3OD) of 2 showed an additional hydroxyl-bearing methine proton, resonated at  3.60 (ddd, J(2a,3a) ¼ 14.4 Hz, J(2a,1a) ¼ 9.1 Hz, J(2a,1e) ¼ 3.9 Hz), which showed couplings with the C-3 ( 2.90) and C-1 protons ( 2.21, 1.94). The 13C-NMR spectrum (CD3OD, 100 MHz) also showed an additional signal for a hydroxyl-bearing methine carbon at  69.5. Hydroxylation at C-2 position was finally deduced from the HMBC correlations of H-2 ( 3.60) with C-3 ( 85.4). The configuration of the C-2 methine proton was deduced to be (axial)-oriented on the basis of multiplicity of H-2 signal ( 3.60, ddd, J(2a,3a) ¼ 14.4 Hz, J(2a,1a) ¼ 9.1 Hz, J(2a,1e) ¼ 3.9 Hz), and through NOESY cross peaks between H-2 and -oriented C-10 methyl group ( 0.89). Thus, the structure of metabolite 2 was finally deduced as 2,3 -dihydroxyolean-12-en-28-oic-acid.

Microbial transformation of oleanolic acid Table 1.

491

-Glucosidase inhibitory activity of compounds 1–3.

Compound 1 2 3 Deoxynojirimycinb Acarboseb

a

IC50 (mM)  SEM 12.8  0.00 444.0  8 666.0  20 425.6  8.14 780.0  0.28

a

IC50 values are the mean  SEM of three assays. Standard -glucosidase inhibitors.

b

This compound was previously obtained as a secondary metabolite from Hyptis capitata, and many other plants [19,20]. The HRFAB MS of 3 exhibited a [M þ Na]þ at m/z 487.3780 (C30H48O5, Calcd 487.3832) which indicated the introduction of 32 a.m.u. as compared to compound 1. In the 1H-NMR spectrum (500 MHz, CD3OD), two new hydroxyl-bearing methine protons were appeared at  3.62 (ddd, J(2a,3a) ¼ 14.2 Hz, J(2a,1a) ¼ 9.3 Hz, J(2a,1e) ¼ 4.1 Hz), and 4.04 (dd, J(11e,9a) ¼ 7.1 Hz, J(11e,12) ¼ 3.6 Hz), with corresponding carbons at  69.7 and 65.0, respectively, in the HMQC spectrum. The signal resonating at  3.62, was assigned to the H-2 on the basis of its homonuclear couplings with H-3 ( 2.91) and H2-1 ( 2.03, 1.92), and HMBC correlation with C-3 ( 85.0). The axial-orientation of H-2 was deduced on the basis of multiplicity of H-2 signal. A signal appeared at  4.04 (dd, J(11e,9a) ¼ 7.1 Hz, J(11e, 12) ¼ 3.6 Hz), was assigned to the H-11 on the basis of its COSY 45
coupling with H-12 ( 5.23). The hydroxylation at C-11 position was further inferred from the HMBC correlations of Me-27 protons ( 1.32) with C-11 ( 65.0). Stereochemistry of C-11 hydroxyl group was deduced to be (axial) on the basis of coupling constant of H-11 signal (J11eq,9ax ¼ 7.1 Hz). The structure of this new trihydroxylated metabolite 3 was deduced to be 2,3 , 11 -trihydroxyolean-12-en-28-oic-acid. The -glucosidase inhibitory activity of the oleanolic acid (1) was first time reported by our group [16]. However, compound 1 and 3-O-glucuronic acid moiety of oleanolic acid were also reported as an oral hypoglycemic agent for type II diabetes [21,22]. Our study, thus, indirectly confirms the mechanism of antidiabetic activity of 1 through an -glucosidase inhibition. As a follow-up, derivatives of compound 1 were also screened. During this study, both the transformed products 2 and 3 of oleanolic acid showed more potent enzyme inhibitory activities than acarbose, a clinically used drug. Their inhibitory activities were also found to be comparable to an another standard drug, deoxynojirimycin. The transformed product 2, with a hydroxyl group at C-2, was found to be more potent than the compound 3, which has hydroxyl groups at C-2 and C-11 (table 1). This indicated that polar hydroxyl groups cause a decrease in activity.

3. Experimental section 3.1. General experiment procedures The 1H-NMR spectra ( in ppm, J in Hz) were obtained in deuterated solvent (CD3OD) at 500 MHz on Bruker Avance-500 NMR spectrometer, while 13C-NMR spectra were

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recorded in CD3OD on the same instrument at 125 MHz. A Varian MAT 311A mass spectrometer was used to record the mass spectra. The HREI MS spectra were recorded on a Jeol JMS-600H mass spectrometer. The FT–IR was recorded on Shimadzu FTIR-8900 spectrophotometer, and UV spectra were recorded on Advance UV spectrophotometer. Optical rotations were measured on a Jasco DIP 360 digital polarimeter. Melting points were recorded on a Buchi 535 apparatus. The purity of the samples was checked on precoated plates (silica gel 60, F254 0.2 mm, E. Merck). The metabolites were purified by column chromatography on flash silica gel and by precoated TLC plates (silica gel 60, F254 0.2 mm, E. Merck).

3.2. Fungi and culture conditions Culture of F. lini (NRRL-68751) was grown on Sabouraud-4% glucose-agar (Merck) at 25
C and stored at 4
C. Furarium lini broth media was prepared by mixing glucose (30.0 g), glycerol (30.0 mL), peptone (15.0 g), yeast extract (15.0 g), KH2PO4 (15.0 g), and NaCl (15.0 g), in 3.0 L distilled water.

3.3. General fermentation and extraction conditions The fungal media was transferred into conical flasks (100 mL each) and autoclaved at 121
C. Seed flasks were prepared from 3 days old slant and fermentation was allowed for 2 days on a shaker at 25
C. The remaining flasks were inoculated from seed flasks. After 2 days, oleanolic acid (1) (300 mg) was dissolved in acetone and distributed to flasks (10 mg mL1) and all flasks were placed on a rotatory shaker (128 rpm) at 25
C for fermentation for 12 days. The culture media were filtrated and extracted with CH2Cl2 and ethyl acetate. The extract was dried over anhydrous Na2SO4, evaporated under reduced pressures to a gummy crude (1.02 g) and analyzed by thin layer chromatography. Metabolites 2 and 3 were isolated by using column chromatography, i.e. 2 (12.0 mg) was eluted with petroleum ether–EtOAc (50 : 50), and 3 (9.0 mg) with petroleum ether–EtOAc (60 : 40). 3.3.1. 2a,3b-Dihydroxyolean-12-en-28-oic acid (2). Obtained as a white solid (4.0%); m.p. 285–295
C; ½25 D þ40 (c 0.4, MeOH); UV (MeOH) max 218 nm; IR (CHCl3) max 3424, 2912, 1668, 1408 cm1; 1H-NMR (CD3OD, 500 MHz)  3.60 (ddd, J(2a,3a) ¼ 14.4 Hz, J(2a,1a) ¼ 9.1 Hz, J(2a,1e) ¼ 3.9 Hz, H-2), 2.90 (1H, d, J(2a,3a) ¼ 14.2 Hz, H-3), 2.86 (1H, dd, J(18a,19a) ¼ 14.0 Hz, J(18a,19e) ¼ 4.1 Hz, H-18 ), 5.23 (1H, t, J(12,11) ¼ 3.5 Hz, H-12), and 0.79, 0.89, 0.95, 0.99, 1.00, 1.10, 1.15 (7  CH3); 13C-NMR (CD3OD, 100 MHz) data (table 2); EI MS m/z (rel. int., %) 472 Mþ (4), 454 (13), 426 (15), 408 (58), 248 (100), 233 (37), 203 (34); HREI MS: m/z 472.3490 (C30H48O4, Calcd 472.3563). 3.3.2. 2a,3b,11b-Trihydroxyolean-12-en-28-oic acid (3). Obtained as a white powder (3.0%); m.p. 290–298
C; ½25 D þ128 (c 0.35, MeOH); UV (MeOH) max 208 nm; IR (CHCI3) max 3410, 2928, 1687, 1450 cm1; 1H-NMR (CD3OD, 500 MHz)  3.62 (ddd, J(2a,3a) ¼ 14.2 Hz, J(2a,1a) ¼ 9.3 Hz, J(2a,1e) ¼ 4.1 Hz, H-2), 2.91 (1H, bd, J ¼ 14.1 Hz, H-3), 2.86 (1H, dd, J(18a,19a) ¼ 14.0 Hz, J(18a,19e) ¼ 4.1 Hz,

493

Microbial transformation of oleanolic acid Table 2. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

13

C-NMR data of compounds 1–3 (100 MHz; CD3OD). 1a C

2 C

3 C

40.9 28.2 78.0 39.4 55.8 18.8 33.3 39.8 48.1 37.4 23.8 122.5 144.8 42.0 28.3 23.8 46.7 42.0 46.7 31.0 34.3 33.3 28.7 16.5 15.5 17.5 26.2 180.2 30.3 32.8

48.1 69.5 84.4 39.3 55.6 19.5 34.2 40.8 54.5 39.2 24.4 123.1 145.7 40.5 28.5 24.2 43.3 42.9 47.2 31.6 35.0 33.9 24.1 17.2 17.5 17.9 26.4 180.0 29.3 33.6

48.7 69.7 84.0 39.5 54.6 19.9 35.2 38.8 58.6 39.1 65.0 125.0 145.0 38.4 28.5 24.5 43.6 43.9 47.6 31.8 35.2 33.7 27.1 17.5 16.5 17.0 25.4 181.3 29.2 28.8

a

Reported in literature [1–4].

H-18 ), 4.04 (dd, J(11e,9a) ¼ 7.1 Hz, J(11e,12) ¼ 3.6 Hz, H-11), 5.28 (1H, d, J(11e,12) ¼ 3.6 Hz, H-12), 0.78, 0.80, 0.92, 0.99, 1.00, 1.18, 1.30 (7  CH3); 13C-NMR (CD3OD, 100 MHz); data (table 2); HRFAB MS; m/z 487.3780 (C30H48O5, Calcd 487.3832). 3.3.3. a-Glucosidase enzyme inhibition assay. The inhibitory activity of the compounds was determined against -glucosidase (EC 3.2.1.20) from Saccharomyces sp., purchased from Wako Pure Chemicals Industries Ltd. (Wako 076-02841). The inhibition was measured spectrophotometrically at pH 6.9 and at 37
C using 0.7 mM p-nitrophenyl -D-glucopyranoside (PNP-G) as a substrate and 250 m units mL1 enzyme, in 50 mM sodium phosphate buffer containing 100 mM NaCl. 1-Deoxynojirimycin (425.6  8.14 mM) and acarbose (780.0  0.28 mM) were used as positive controls [16]. The increment in absorption at 400 nm, due to the hydrolysis of PNP-G by -glucosidase, was monitored continuously with a spectrophotometer at 400 nm (Spectra Max, Molecular Devices, USA) [16]. 3.3.4. Estimation of IC50 values. The concentration of the test compounds, which inhibited the hydrolysis of PNP-G by -glucosidase by 50% (IC50), were determined

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by monitoring the effect of increasing concentrations of these compounds on the inhibition values. The IC50 values were then calculated using EZ-Fit enzyme kinetics program (Perrella Scientific Inc., Amherst, MA, USA) [16].

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22]

C.J. Shao, R. Kasai, J.Da. Xu, O. Tanaka. Chem. Pharm. Bull., 37, 42 (1989). D. Lontsi, B.L. Sondengam, J.F. Ayafor. J. Nat. Prod., 52, 52 (1989). A. Ikuta, H. Itokawa. Phytochemistry, 27, 2813 (1988). M. Bruno, G. Savona, J.A.H. Rodriguez, C. Pascual, B. Rodriguez. Phytochemistry, 26, 497 (1987). N. Banno, T. Akihisa, H. Tokuda, K. Yasukawa, H. Higashihara, M. Ukiya, K. Watanabe, Y. Kamura, J. Hasegawa, H. Nishino. Biosci. Biotech. Biochem., 68, 85 (2004). S.G. Bang, J.H. Lee, G.Y. Song, D.H. Kim, M.Y. Yoon, B.Z. Ahn. Chem. Pharm. Bull., 53, 1451 (2005). Y. Chen, J. Liu, X. Yang, X. Zhao, H. Xu. J. Pharm. Pharmacol., 57, 259 (2005). N.Y. Kim, M.K. Lee, M.J. Park, S.J. Kim, H.J. Park, J.W. Choi, S.H. Kim, S.Y. Cho, J.S. Lee. J. Med. Food, 8, 177 (2005). M.A. Mohamed, M.S. Marzouk, F.A. Moharram, M.M. El-Sayed, A.R. Baiuomy. Phytochemistry, 66, 2780 (2005). F. Mengoni, M. Lichtner, L. Battinelli, M. Marzi, C.M. Mastroianni, V. Vullo, G. Mazzanti. Planta Med., 68, 111 (2002). N. Nakamura. Yakugaku Zasshi, 124, 519 (2004). Z. Ovesna, A. Vachalkova, K. Horvathova, D. Tothova. Neoplasma, 51, 327 (2004). R. Tanaka, K. Shanmugasundararam, C. Yamaguchi, Y. Ishikawa, H. Tokuda, K. Nishide, M. Node. Cancer Lett., 214, 149 (2004). L. Zaprutko, D. Partyka, B. Bednarczyk-Cwynar. Bioorg. Med. Chem. Lett., 14, 4723 (2004). T. Oguro, J. Liu, C.D. Klaassen, T. Yoshida. Toxicol. Sci., 45, 88 (1998). M.S. Ali, M. Jahangir, S. Shazad-ul-Hussan, M.I. Choudhary. Phytochemistry, 60, 295 (2002). S. Sou, S. Mayumi, H. Takahashi, R. Yamasaki, S. Kadoya, M. Sodeoka, Y. Hashimoto. Bioorg. Med. Chem. Lett., 10, 1081 (2000). T. Matsui, C. Yoshimoto, K. Osajima, T. Oki, Y. Osajima. Biosci. Biotech. Biochem., 60, 2019 (1996). T. Furuya, Y. Orihara, C. Hayashi. Phytochemistry, 26(3), 715 (1987). R. Caputo, L. Mangoni, P. Monaco, L. Previtera. Phytochemistry, 13, 2825 (1974). W.L. Li, H.C. Zheng, J. Bukuru, N. De Kimpe. J. Ethnopharmacol., 92, 1 (2004). A.K. Tiwari, J.M. Rao. Curr. Sci., 83, 30 (2002).

Natural Product Research, Vol. 22, No. 6, 15 April 2008, 495–498

Reaction mechanism of direct episulfidation of caryophyllene and humulene TATSUYA ASHITANI*yz, ANNA-KARIN BORG-KARLSONz, KOKI FUJITAx and SHIZUO NAGAHAMA{ yInstitute of Wood Technology, Akita Prefectural University, 11-1 Kaieizaka, Noshiro 016-0876, Japan zDepartment of Chemistry, Royal Institute of Technology, Teknikringen 30, Stockholm, SE 100 44, Sweden xFaculty of Agriculture, Department of Forest and Forest Products Sciences, Kyushu University, 6-10-1, Hakozaki, Fukuoka 812-8581, Japan {Department of Applied chemistry, Faculty of Engineering, Sojo University, 4-22-1, Ikeda, Kumamoto 860-0082, Japan (Received 12 February 2007; in final form 25 July 2007) Direct episulfidations of caryophyllene or humulene with elemental sulfur were examined by means of gas chromatography. Caryophyllene-6,7-episulfide was formed at an early stage in a reaction of the caryophyllene and elemental sulfur at 120
C. Caryophyllene-3,6-sulfide and polymer compounds were formed after the episulfidation. Formations of the these compounds were related to the disappearance of the caryophyllene-6,7-episulfide. Isomerization from the caryophyllene to isocaryophyllene was also observed during the reaction. In the reaction of humulene with elemental sulfur, humulene-6,7-episulfide was initially produced and then converted to humulene-9,10-episulfide. It was assumed that the polymer compound in the reaction of humulene with sulfur was related to the disappearance of the both humulene episulfides. Keywords: Episulfidation; Elemental sulfur; Caryophyllene; Humulene; Medium ring olefin

Natural existence of episulfides of caryophyllene (1) and humulene (2) was found in hop oil [1]. On the other hand, we reported [2] that the episulfides were obtained easily through a reaction between original terpene and sulfur without solvent and catalysis at 120
C. In addition, it was suggested that the direct episulfidation occurred on medium ring olefin and cis-trans isomerization of original olefin was observed during the reaction. Similarly, the synthesis of episulfide was noticed in organic chemistry field [3]. Since, episulfidation by elemental sulfur has been known only in a few cases, for example, norbornene [4,5], bibenzonorbornenylidene [6], and adamantylideneadamantane [7], *Corresponding author. Tel.: þ81-185-52-6984. Fax: þ81-185-52-6976. Email: [email protected] Natural Product Research ISSN 1478-6419 print/ISSN 1029-2349 online ß 2008 Taylor & Francis http://www.tandf.co.uk/journals DOI: 10.1080/14786410701591903

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therefore, in this article, the reaction mechanism of episulfidations of 1 and 2 with elemental sulfur was investigated. One gram of 1 or 2 was mixed with same molar amounts of sulfur and half molar amounts of tetradecane as an internal standard in a 10 mL -round bottom flask. The mixture was stirred at 120
C in oil bath (1 and 2 were separated from lauan oil. Tetradecane as internal standard was commercial product (Wako Pure Chemical Industries, Osaka, Japan)). GC analyses were carried out using gas chromatograph (Shimadzu GC-14A) with FID detector. The analyses employed a TC-WAX capillary column (15 m  0.25 mm i.d.; film thickness 0.25 mm), column temperature 70
C (3 min), 8
C min1 to 24
C (hold), injector temperature 180
C, detector temperature 230
C, nitrogen carrier gas at 1.5 mL min1. (Identifications and/or NMR and MS data for compounds 3–7 were already described in Ref. [2]). The result of examination for the direct episulfidation of 1 analyzed by GC is shown in figure 1. Caryophyllene-6,7-episulfide (3) was formed initially followed by isomerization from caryophyllene to isocaryophyllene (4) in the next stage. It was considered that the isomerization was caused by action of sulfur as catalyst during the reaction. Moreover, formation of polymer compounds was observed. Caryophyllene-3,6-sulfide (5) was formed by transannular reaction at a later stage. The polymer compound and 5 were related to the disappearance of the episulfide. From the above result, reaction of 1 and sulfur is summarized in scheme 1. Figure 2 shows the change of substrate and product in a reaction of 2 with sulfur. In the reaction of 2 with sulfur, humulene-6,7-episulfide (6) was initially produced. Humulene-9,10-episulfide (7) was formed after the formation of 6. Polymerization was

Caryophyllene (1) Caryophyllene-6,7-episulfide (3)

GC %

Caryophyllene-3,6-episulfide (5) Isocaryophyllene (4) Polymer compounds

Time (h) Figure 1. Amounts of products in reaction of caryophyllene with sulfur. Amount of polymer compound was calculated following equation. Amount of polymer compound (GC%) ¼ 100(1 þ 3 þ 5 þ 4).

497

Episulfidation of caryophyllene and humulene S S

H

S

3

1

5

S Polymerization

4


Scheme 1.

Reaction of caryophyllene with sulfur at 120 C.

GC %

Humulene (2) Humulene-6,7-episulfide (6) Humulene-9,10-episulfide (7) Polymer compounds

Time (h) Figure 2.

Amount of product in reaction of humulene with sulfur.

S S S

2

+

6

7

Polymerization Scheme 2.




Reaction of humulene with sulfur at 120 C.

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(CH2)n−4

(CH2)n−4

S −S

(CH2)n−4 S

S

Sx

S Polymerization

n = 8−11 (CH2)n−2

S

(CH2)n−2

(CH2)n−2

−S S Scheme 3.

Sx

S

S

Reaction mechanism of medium-ring olefin with sulfur.

observed also in episulfidation of 2. However, isomerization and transannular reaction were not observed in the case of 2. It is known that the trans-isomer is stable in 11-membered ring olefin and cis-isomer is stable in 8–10-membered olefin [9,10]. Thus, the cis/trans isomerization did not occur in the case of 2 as it occurred in 11-membered ring olefin with sulfur. The transannular reaction also did not occur in case of reaction of 2 with sulfur, since the distance of carbon to carbon in 11-membered ring was so far compared with 9-membered ring. From the above result, reaction of 2 and sulfur is summarized in scheme 2. The reaction mechanism of medium ring olefin with sulfur is summarized in from former article [2] and this study (Scheme 3). It was considered from the previous study [6,11] about thermal reaction of elemental   sulfur that initial stage of the reaction was the formation of SSxS . biradical. Therefore, reaction proceeded through radical mechanism. Cis/trans isomerization occurred by sulfur acting as catalyst and the episulfide was polymerized. The final composition of products might be decided by thermal stabilities.

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]

T.L. Peppared, F.R. Sharpe, J.A. Elvidge. J. Chem. Soc., Parkin Trans. 1, 311 (1980). T. Ashitani, S. Nagahama. Nat. Prod. Lett., 13, 163 (1999). W. Adam, R.M. Bargon. Chem. Rev., 104, 251 (2004). P.D. Bartlett, T.J. Ghosh. J. Org. Chem., 52, 4937 (1987). J. Emsley, D.W. Giffiths, G.J.J. Jayne. J. Chem. Soc., Parkin Trans. 1, 228 (1979). Y. Sugihara, K. Noda, J. Nakayama. Tetrahedron Lett., 41, 8913 (2000). J. Nakayama, Y. Ito, A. Mizumura. Sulfur Lett., 14, 247 (1992). A.C.C. Cope, P.T. Moore, W.R. Moore. J. Am. Chem. Soc., 81, 3153 (1959). J.A. Marshall. Acc. Chem. Res., 13, 213 (1980). S. Oae, R. Sato. In Yuki Iou Kagaku (Gousei Hannou Hen), S. Oae (Ed.), Chapter 1.6.1, Kagakudojin, Kyoto (1982).

Natural Product Research, Vol. 22, No. 6, 15 April 2008, 499–506

Biotransformation of tetrahydro-a-santonins by Absidia coerulea LIN YANGyz and JUNGUI DAI*z yCollege of Life and Environmental Sciences, The Central University for Nationalities, 27 South Zhongguancun Street, Beijing, 100081, P.R. China zInstitute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College (Key Laboratory of Bioactive Substances and Resources Utilization, Ministry of Education), 1 Xian Nong Tan Street, Beijing, 100050, P.R. China (Received 9 December 2006; in final form 30 April 2007) The fungus, Absidia coerulea was employed to bioconvert tetrahydro--santonins, 1,2,4,5-tetrahydro--santonin (1), and its 4-epimer (2), from which 10 products (3–12) were obtained. Furthermore, their structures were determined, based on their chemical and spectroscopic data analyses. Among them, 3–5, 7, 9, 11 and 12 were observed to be seven new compounds. The reactions mainly involved in these bio-process included hydroxylation(s) (C-4, C-11, and C-1), reduction (C-3 ketone to alcohol). Keywords: Tetrahydro--santonin; Biotransformation; Absidia coerulea

1. Introduction Eudesmanolide compounds are biogenetic and chemical precursors of a range of sesquiterpene lactones, among which eudesman-6, 12-olides are the most abundant compounds, which have been studied chemically and biogenetically. Moreover, these compounds have attracted a great deal of interest from chemists on account of their wide range of biological activities, such as cytotoxic, antitumor, immunosuppressive, insecticidal and anti-HIV activities [1]. Bioconversion of sesquiterpenes is actually accepted as a preferable method, in combination with chemical reactions, for the semi-synthesis of products of interest [2–4]. In this context, in order to obtain useful intermediates conveniently for the synthesis of these types of bioactive compounds and diversify the structures of this type compounds, the studies on the bioconversions of -santonin and 6 -santonin by a variety of fungi and plant cell cultures have been extensively carried out in our laboratory [5–7]. As the part of ongoing investigation, in the present article we report the biotransformations of tetrahydro--santonins,

*Corresponding author. Tel.: þ86-10-63165195. Fax: þ86-10-63017757. Email: [email protected] Natural Product Research ISSN 1478-6419 print/ISSN 1029-2349 online ß 2008 Taylor & Francis http://www.tandf.co.uk/journals DOI: 10.1080/14786410701592018

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O

O H

O

O

H

O O

1 1,2,4α,5α-tetrahydro-α-santonin Figure 1.

O

O

α-santonin

O

2 1, 2, 4β, 5α-tetrahydro-α-santonin

The structures of -santonin and tetrahydro--santonins.

1,2,4, 5-tetrahydro--santonin and its 4-epimer, two synthetic derivatives from -santonin (figure 1) by filamentous fungus Absidia coerulea.

2. Results and discussion 2.1. Biotransformation of 1 with A. coerulea Compound 1 was added to 2-day-old cell cultures of fungus A. coerulea and incubated for additional 7 days to give five products. On the basis of the chemical and physical data, their structures were identified as 2-oxo-1, 5,6 ,11 -H-eudesman-3(4)-en6,12-olide (3, ca 4%), 4-hydroxy-3-oxo-5,6 ,11 -H-eudesman-6,12-olide (4, 18%), 3 ,11 -dihydroxy-1,2,4,5,6 -H-eudesman-6,12-olide (5, ca 12%), 3 -hydroxy1,2,4,5,6 ,11 -H-eudesman-6,12-olide (6, ca 6%), and 1 -hydroxy-3-oxo4,5,6 ,11 -H-eudesman-6,12-olide (7, ca 4%), respectively (figure 2). Among them, 6 was a known compound, which was synthesized through both chemical and microbial conversion [4, 8], with its chemical and spectral data being in good agreement with those reported. The other four products were new compounds. Compound 3 gave an HREIMS molecular ion peak at m/z 248.1410 [M]þ, consistent with the molecular formula C15H20O3, suggesting dehydrogenation of 1. It was confirmed by the observation of olefinic signals [C  144.36 (d) and related H  6.08 (s), C  129.07 (s)] in its 1H and 13C NMR spectra, and its IR absorption at 1640 cm
1. By combination of its 1D and 2D NMR spectral analyses, the carbonyl group existed at C-2 position instead of at C-3 position and double bond existed at C-3(4), thus, the structure of 3 was determined as 2-oxo-1,5,6 ,11 -H-eudesman-3(4)-en-6,12-olide. HREIMS of compounds 4 and 7 exhibited molecular ion peaks at m/z 266.1524 and 266.1506 [M]þ, respectively, consistent with the molecular formula C15H22O4, indicating one hydroxyl group introduction in both 4 and 7 compared with 1. It was further confirmed by IR absorption at 3604 and 3624 cm
1, respectively. The position and configuration of introduced hydroxyl group were determined by 1D and 2D NMR spectral data and their NOE difference spectra (Material and methods), followed by the determination of their structures as 4-hydroxy-3-oxo-5,6 ,11 -H-eudesman6,12-olide (4) and 1 -hydroxy-3-oxo-4,5,6 ,11 -H-eudesman-6,12-olide (7), respectively. Compound 5 was deduced to obtain the molecular formula C15H24O4 from the HREIMS and NMR spectral data analyses. Its 1H and 13C NMR closely resembled those of compound 6, except for the disappearance of H-11 signal and the singlet of H-13 instead of doublet. All of these indicated the existence of an additional hydroxyl group at C-11 position in 5 compared with 6. Therefore, the structure of 5 was identified as 3 ,11 -dihydroxy-1,2,4,5,6 -H-eudesman-6,12-olide. In fact, this compound had

Biotransformation of tetrahydro--santonins by A. coerulea

501 HO

O O HO

H O O

O HO

H O

Figure 2.

OH H O

HO HO

10 (11%)

7 (2%)

OH

HO H O

H O

O

O

O O

6 (3%)

HO

9 (3%)

H

O O

5 (6%)

H O

O

8 (11%)

H O

O

3 (2%)

O

HO

H O

4 (9%)

HO

OH

HO H O

11 (8%)

O O

12 (16%)

The structures of the biotransformed products of tetrahydro--santonins by A. coerulea.

been obtained by microbial transformation of 3 -hydroxy-1,2,4,5,6 ,11 H-eudesman-6,12-olide, although it was reported as its 3 -acetylated derivative [8].

2.2. Biotransformation of 2 with A. coerulea Moreover, five products (8–12, figure 2) were obtained from microbial conversion of 2 and were determined to be 3 -hydroxy-1,2,4 ,5,6 ,11 -H-eudesman-6,12-olide (8, ca 11%) [10], 4 -hydroxy-3-oxo-1,2, 5,6 ,11 -H-eudesman-6,12-olide (9, ca 3%), 3 ,11 -dihydroxy-1,2,4 ,5,6 -H-eudesman-6,12-olide (10, ca 11%) [4,8], 3 ,4 -dihydroxy-1,2,5,6 ,11 -H-eudesman-6,12-olide (11, ca 8%), 3,11 -dihydroxy-1,2,4 ,5,6 -H-eudesman-6,12-olide (12, ca 16%). Among them, products 8 and 10 were two known compounds synthesized through chemical and/or enzymatic approaches [4, 8, 9], with their chemical and spectral data being in good agreement with those reported. The products 9, 11 and 12 were the three new compounds. HREIMS of 9 displayed a molecular ion peak at m/z 266.1520 [M]þ. Consistent with the molecular formula C15H22O4, the 1H and 13C NMR were very similar to those of compound 2 except for the disappearance of H-4 signal and the appearance of an oxygenated carbon signal ( 76.60, s) instead of the carbon signal ( 44.5, d), and the change of the doublet of H-15 ( 1.28, 3H, d, J ¼ 6.8 Hz) to a singlet ( 1.45, 3H, s). All of these suggested the presence of a hydroxyl group at C-4 position in 9, in comparison with 2 and it was confirmed by its IR absorption at 3512 cm
1. Thus, the structure of 9 was determined to be 4 -hydroxy-3-oxo-1,2,5,6 ,11 -H-eudesman-6,12-olide. HREIMS of 11 gave a molecular ion peak at m/z 268.1680 [M]þ, consistent with the molecular formula C15H24O4. Its 1H and 13C NMR were very similar to those of compound 9 except for the upfield shift of C-3 signal from  211.91 (s) to  81.83 (d), and the observation of an oxymethine proton signal at  3.84 (t, J ¼ 4.5 Hz), all of which suggested the reduction of C-3 ketone to alcohol. The stereochemistry of C-3 OH group was determined as configuration by NOE difference spectra. So, the structure of 11 was identified as 3 ,4 -dihydroxy-1,2,5,6 ,11 -H-eudesman-6,12-olide, as it might be formed from 9 via reduction of C-3 ketone. The molecular formula of 12 was deduced to be C15H24O4 by HREIMS, in which a molecular ion peak at m/z 268.1664 [M]þ was observed. Its 1H and 13C NMR closely resembled those of compound 10 except for the differences between C-3 and H-3 signals, which were observed as C 76.06 and H 3.14 for 10, while C 76.55 and

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H 3.56 for 12. Further, NOE difference spectra (Materials and method) indicated that H-3 in 10 was  configuration, and H-3 in 12 was configuration. Therefore, the structure of 12 was identified to be 3,11 -dihydroxy-1,2,4 ,5,6 -H-eudesman-6,12olide. 3. Conclusion In summary, several products were obtained from the biotransformations of tetrahydro-santonins by A. coerulea and seven of them were observed to be new compounds. Furthermore, the reactions mainly occurred that included hydroxylation(s) (C-4, C-11, and C-1), reduction (C-3 ketone to alcohol). In our previous report [7], only two reactions (C-11 and C-8 hydroxylations) occurred when -santonin was incubated with this fungus, and C-11 hydroxylation was the major reaction, however, C-11 hydroxylation did not occur when 6 -santonin was used as the substrate. In the present report, C-11 hydroxylations of this pair of isomers also occurred as one of the major reactions, indicating that the fungus A. coerulea also possesses the similar specific hydroxylation ability to these two 6,12-eudesmanolides, further confirming that the configuration (6 or 6 ) of the lactone exerts substantial influence on the C-11 hydroxylation of these type of eudesmanolides. Furthermore, the other major reactions in the two biotransformations of C-4 hydroxylation and reduction of ketone to alcohol at C-3 position(s), the minor reaction of hydroxylation of 4 -Me isomer (1) were observed. These results implied that: (1) the structure (including stereochemistry) of the substrate had significant effects on the biotransformation modes; and (2) biotransformation was a powerful tool to structural diversification of one compound, being even greater by the combination of chemical and enzymatic approaches. 4. Experimental 4.1. Apparatus Optical rotations were obtained using a Horiba SEPA-200 polarimeter. 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were recorded with a Varian Unity-PS instrument using CDCl3 as solvent and internal reference. 1H and 13C NMR assignments were determined by 1H–1H COSY, DEPT, HMQC and HMBC experiments. HREIMS were carried out on a JEOL-HX 110 instrument. IR spectra were taken on a Hitachi 270–30 spectrometer in CHCl3. Semi-preparative HPLC was performed on a Hitachi L-6200 HPLC instrument with an Inertsil Prep-sil (GL Science, 25 cm  10 mm i.d.) stainless steel column and a YRU-883 RI/UV bi-detector, with the flow rate being 5.0 mL min
1. Silica gel (230–300 mesh) was employed for column chromatography, Analytical TLC plates (silica gel 60 F254, Merck) were visualized at UV254 by spraying 5% H2SO4 (in EtOH), followed by heating.

4.2. Substrates Compounds 1 and 2 were synthesized in our laboratory according to the reference [10] and dissolved in EtOH before use, with the final concentration added being 66.7 mg L
1.

Biotransformation of tetrahydro--santonins by A. coerulea

503

4.3. Organism and cultural conditions The fungus, A. coerulea IFO4011 was purchased from the Institute for Fermentation, Osaka, Japan (IFO). The seed cultures were prepared in 500 mL flask with 150 mL of PDA liquid medium and incubated for 2 days. Then 5 mL of the seed cultures were added to each flask and cultivated on a rotary shaker at 110 rpm and (25  2) C in the dark for the use of biotransformation.

4.4. Biotransformation of 1 with A. coerulea Compound 1 (50 mg) was dissolved in EtOH (2.0 mL), distributed among five flasks of 2-day-old cultures and incubated for 7 days, after which the cultures were filtered and thoroughly washed with water. The filtrate was saturated with NaCl and extracted 5 times with ethyl acetate. All the extracts were pooled, dried over anhydrous Na2SO4, and concentrated under vacuum at 40 C to give 206 mg of residue and separated by combination of open silica gel chromatography and normal phase semi-prep. HPLC to afford substrate (1, 15.0 mg, 30%; analyzed by TLC and 1H NMR), 3 (2.2 mg, ca 4%), 4 (9.0 mg, ca 18%), 5 (6.1 mg, ca 12%), 6 (2.9 mg, ca 6%) and 7 (2.2 mg, ca 4%). Their structures and yields are shown in figure 2. The chemical and physical data of the new compounds are shown as follows. 4.4.1. 2-oxo-1,5a,6b,11b-H-eudesman-3(4)-en-6,12-olide (3). White powder; []20D þ 29.6 (c 0.08, CHCl3); IR (CHCl3) max 2948, 1780, 1714, 1630, 1458, 1386, 1362, 1230, 1222, 1118, 1008 cm
1; 1H NMR (CDCl3, 500 MHz)  2.43 (2H, brs, H-1), 6.08 (1H, s, H-3), 2.77 (1H, dd, J ¼ 2.0, 11.2 Hz, H-5), 3.97 (1H, dd, J ¼ 10.5, 10.7 Hz, H-6), 1.66 (1H, m, H-7), 1.64 (1H, m, H-8a), 1.57 (1H, m, H-8b), 1.501.64 (2H, m, H-9), 2.33 (1H, dq, J ¼ 6.8, 7.1 Hz, H-11), 1.25 (3H, d, J ¼ 7.1 Hz, H-13), 1.04 (3H, s, H-14), 2.07 (3H, d, J ¼ 2.0 Hz H-15); 13C NMR (CDCl3, 125 MHz)  52.38 (t, C-1), 192.31 (s, C-2), 144.36 (d, C-3), 129.07 (s, C-4), 50.45 (d, C-5), 80.37 (d, C-6), 53.69 (d, C-7), 22.41 (t, C8), 39.19 (t, C-9), 40.81 (s, C-10), 40.41 (d, C-11), 178.76 (s, C-12), 12.41 (q, C-13), 18.74 (q, C-14), 15.22 (q, C-15); HREIMS m/z 248.1410 [M]þ (Calcd for C15H20O3, 248.1412). 4.4.2. 4-hydroxy-3-oxo-5a,6b,11b-H-eudesman-6,12-olide (4). White powder; []20D þ 75.8 (c 0.51, CHCl3); IR (CHCl3) max 3604, 3036, 2948, 1780, 1712, 1458, 1292, 1230, 1222, 1210, 1122, 1000 cm
1; 1H NMR (CDCl3, 500 MHz)  1.76 (1H, ddd, J ¼ 2.5, 6.8, 13.4 Hz, H-1a), 1.70 (1H, m, H-1b), 2.55 (1H, m, H-2a), 2.43 (1H, ddd, J ¼ 2.4, 5.4, 15.4 Hz, H-2b), 1.58 (1H, d, J ¼ 10.2 Hz, H-5), 4.38 (1H, dd, J ¼ 10.5, 11.7 Hz, H-6), 1.53 (1H, dd, J ¼ 6.4, 11.3 Hz, H-7), 1.83 (1H, ddd, J ¼ 4.4, 12.9, 13.0 Hz, H-8a), 1.68 (1H, m, H-8b), 1.70 (1H, m, H-9a), 1.32 (1H, ddd, J ¼ 3.8, 12.4, 13.0 Hz, H-9b), 2.52 (1H, m, H-11), 1.24 (3H, d, J ¼ 6.6 Hz, H-13), 1.20 (3H, s, H-14), 1.45 (3H, s, H-15); 13C NMR (CDCl3, 125 MHz)  17.71 (t, C-1), 37.36 (t, C-2), 211.48 (s, C-3), 72.68 (s, C-4), 55.25 (d, C-5), 81.75 (d, C-6), 53.82 (d, C-7), 40.78 (t, C-8), 39.98 (t, C-9), 36.62 (s, C-10), 45.01 (d, C-11), 177.35 (s, C-12), 13.79 (q, C-13), 18.25 (q, C-14), 21.44 (q, C-15); NOE: (1) H-6 (
100%), H-11 (þ1.48%), H-14 (þ1.72%), H-15 (þ1.40%); (2) H-14 (
100%), H-6 (þ4.42%), H-11 (þ 6.95%), H-15 (þ0.71%);

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(3) H-15 (
100%), H-6(þ 0.44%), H-14 (þ 0.21%); HREIMS m/z [M]þ 266.1524 (Calcd for C15H22O4, 266.1518). 4.4.3. 3b,11b-dihydroxy-1,2,4a,5a,6b-H-eudesman-6,12-olide (5). White powder; []20D þ65.1 (c 0.41, CHCl3); IR (CHCl3) max 3620, 2948, 1778, 1460, 1218, 1208, 1100, 1022 cm
1; 1H NMR (CDCl3, 500 MHz)  1.40 (1H, m, H-1a), 1.22 (1H, m, H-1b), 1.90 (1H, m, H-2 ), 1.80 (1H, m, H-2), 3.99 (1H, ddd, J ¼ 4.9, 4.9, 12.0 Hz, H-3), 2.69 (1H, m, H-4), 1.53 (1H, dd, J ¼ 4.4, 11.7 Hz, H-5), 4.81 (1H, dd, J ¼ 10.0, 11.5 Hz, H-6), 1.62 (1H, m, H-7), 1.96 (1H, m, H-8a), 1.65 (1H, m, H-8b), 1.35 (1H, m, H-9a), 1.11 (1H, ddd, J ¼ 4.4, 14.0, 14.1 Hz, H-9b), 1.64 (3H, s, H-13), 0.98 (3H, s, H-14), 1.23 (3H, d, J ¼ 7.6 Hz, H-15); 13C NMR (CDCl3, 125 MHz)  40.65 (t, C-1), 26.80 (t, C-2), 72.43 (d, C-3), 35.64 (d, C-4), 50.11 (d, C-5), 78.57 (d, C-6), 56.86 (d, C-7), 18.88 (t, C-8), 43.46 (t, C-9), 36.04 (s, C-10), 73.77 (s, C-11), 179.15 (s, C-12), 21.73 (q, C-13), 21.02 (q, C-14), 9.43 (q, C-15); NOE: (1) H-6 (
100%), H-4 (þ1.44%), H-13 (þ2.26%), H-14 (þ6.53%), H-15 (þ6.27%); (2) H-3 (
100%), H-4 (þ13.85%), H-5 (þ9.56%), H-2 (þ5.70%); HREIMS m/z 268.1680 [M]þ (Calcd for C15H24O4, 268.1675). 4.4.4. 1b-hydroxy-3-oxo-4a,5a,6b,11b-H-eudesman-6,12-olide (7). White powder; []20D þ17.0 (c 0.2, CHCl3); IR (CHCl3) max 3632, 3032, 2944, 1776, 1714, 1458, 1386, 1316, 1220, 1146, 1120, 1056, 1012 cm
1; 1H NMR (CDCl3, 500 MHz)  3.66 (1H, dd, J ¼ 5.6, 11.5 Hz, H-1), 2.73 (1H, dd, J ¼ 5.6, 15.1 Hz, H-2a), 2.55 (1H, ddd, J ¼ 2.7, 11.6, 15.1 Hz, H-2b), 2.50 (1H, dq, J ¼ 6.6, 12.0 Hz, H-4), 1.40 (1H, dd, J ¼ 11.0, 11.5 Hz, H-5), 3.99 (1H, dd, J ¼ 10.3, 10.4 Hz, H-6), 1.63 (1H, m, H-7), 1.94 (1H, ddd, J ¼ 3.4, 6.9, 12.9 Hz, H-8a), 1.55 (1H, ddd, J ¼ 3.2, 13.4 Hz, H-8b), 2.14 (1H, dt, J ¼ 3.2, 13.7 Hz, H-9a), 1.28 (1H, m, H-9b), 2.28 (1H, dq, J ¼ 6.9, 7.1 Hz, H-11), 1.23 (3H, d, J ¼ 7.0 Hz, H-13), 1.14 (3H, s, H-14), 1.25 (3H, d, J ¼ 6.8 Hz, H-15); 13C NMR (CDCl3, 125 MHz)  76.32 (d, C-1), 46.51 (t, C-2), 208.08 (s, C-3), 44.63 (d, C-4), 44.99 (d, C-5), 82.49 (d, C-6), 53.30 (d, C-7), 22.88 (t, C-8), 36.50 (t, C-9), 41.94 (s, C-10), 40.66 (d, C-11), 178.77 (s, C-12), 12.45 (q, C-13), 11.90 (q, C-14), 13.65 (q, C-15); NOE: (1) H-1 (
100%), H-2 (þ6.61%), H-4 (þ0.34%), H-5 (þ10.14%); (2) H-6 (
100%), H-4 (þ3.12%), H-11 (þ7.10%), H-14 (þ7.37%); (3) H-5 (
100%), H-1 (þ9.12%); HREIMS m/z 266.1506 [M]þ (Calcd for C15H22O4, 266.1518).

4.5. Biotransformation of 2 with A. coerulea In this experiment, also 50 mg of 2 was administered. The procedure of incubation, extraction and separation was carried out as described in section 2.4., and afforded substrate (2, 11.0 mg, 22%; analyzed by TLC and 1H NMR), 8 (5.4 mg, ca 11%), 11 (4.3 mg, ca 8%), 10 (5.6 mg, ca 11%), 9 (1.5 mg, ca 3%) and 12 (8.2 mg, ca 16%). Their structures and yields are shown in figure 2. The chemical and physical data of the new compounds shown is as follows. 4.5.1. 4b-hydroxy-3-oxo-1,2,5a,6b,11b-H-eudesman-6,12-olide (9). white powder; []20D
4.9 (c 0.09, CHCl3); IR (CHCl3) max 3512, 2992, 2944, 1782, 1716, 1608, 1456, 1386, 1336, 1318, 1220, 1210, 1144, 1114, 1076, 1022 cm
1; 1H NMR

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(CDCl3, 500 MHz)  1.82 (1H, ddd, J ¼ 2.4, 5.9, 13.4 Hz, H-1a), 1.68 (1H, m, H-1b), 2.76 (1H, ddd, J ¼ 5.9, 15.9, 15.9 Hz, H-2a), 2.51 (1H, ddd, J ¼ 2.4, 3.9, 15.2 Hz, H-2b), 1.97 (1H, d, J ¼ 10.2 Hz, H-5), 4.13 (1H, dd, J ¼ 10.7, 10.7 Hz, H-6), 1.68 (1H, m, H-7), 1.90 (1H, m, H-8a), 1.62 (1H, m, H-8b), 1.60 (1H, m, H-9a), 1.38 (1H, m, H-9b), 2.32 (1H, dq, J ¼ 6.9, 7.0 Hz, H-11), 1.24 (3H, d, J ¼ 7.0 Hz, H-13), 1.26 (3H, s, H-14), 1.45 (3H, s, H-15); 13C NMR (CDCl3, 125 MHz)  40.56 (t, C-1), 34.26 (t, C-2), 211.91 (s, C3), 76.60 (s, C-4), 57.47 (d, C-5), 79.04 (d, C-6), 52.84 (d, C-7), 23.39 (t, C-8), 42.36 (t, C9), 36.91 (s, C-10), 40.76 (d, C-11), 178.41 (s, C-12), 12.61 (q, C-13), 19.34 (q, C-14), 22.95 (q, C-15); NOE: (1) H-6 (
100%), H-5 (þ3.16%), H-11 (þ5.57%), H-14 (þ8.52%), H-15 (þ0.42%); HREIMS m/z 266.1520 [M]þ (Calcd for C15H22O4, 266.1518). 4.5.2. 3b,4b-dihydroxy-1,2,5a,6b,11b-H-eudesman-6,12-olide (11). White powder; []20D þ73.5 (c 0.15, CHCl3); IR (CHCl3) max 3608, 2944, 2880, 1776, 1460, 1382, 1334, 1228, 1214, 1168, 1134, 1098, 1080 cm
1; 1H NMR (CDCl3, 500 MHz)  1.58 (1H, ddd, J ¼ 2.4, 5.9, 13.4 Hz, H-1a), 1.28 (1H, m, H-1b), 1.87 (2H, m, H-2a), 3.84 (1H, t, J ¼ 4.5 Hz, H-3), 2.36 (1H, d, J ¼ 6.3 Hz, H-5), 4.34 (1H, dd, J ¼ 6.4, 10.7 Hz, H-6), 2.12 (1H, ddd, J ¼ 3.9, 6.4, 12.0 Hz, H-7), 1.88 (1H, m, H-8a), 1.46 (1H, m, H-8b), 1.76 (1H, m, H-9a), 1.26 (1H, m, H-9b), 2.17 (1H, dq, J ¼ 6.4, 6.6 Hz, H-11), 1.24 (3H, d, J ¼ 6.8 Hz, H-13), 1.12 (3H, s, H-14), 1.42 (3H, s, H-15); 13C NMR (CDCl3, 125 MHz)  36.61 (t, C-1), 22.35 (t, C-2), 81.83 (d, C-3), 80.83 (s, C-4), 48.25 (d, C-5), 81.60 (d, C-6), 46.37 (d, C-7), 23.91 (t, C-8), 34.07 (t, C-9), 35.81 (s, C-10), 41.81 (d, C-11), 178.99 (s, C-12), 12.60 (q, C-13), 29.93 (q, C-14), 26.80 (q, C-15); NOE: (1) H-5 (
100%), H-6 (þ12.31%), H-14 (þ3.96%); (2) H-6 (
100%), H-5 (þ13.05%), H-11 (þ10.51%), H-14 (þ7.91%); HREIMS m/z 268.1680 [M]þ (Calcd for C15H22O4, 268.1675). 4.5.3. 3a,11b-dihydroxy-1,2,4b,5a,6b-H-eudesman-6,12-olide (12). white powder; []20D þ20.8 (c 0.55, CHCl3); IR (CHCl3) max 3600, 3024, 2944, 2884, 1778, 1606, 1458, 1396, 1310, 1266, 1236, 1220, 1154, 1122, 1068, 1018 cm
1; 1H NMR (CDCl3, 500 MHz)  1.46 (1H, m, H-1a), 1.33 (1H, m, H-1b), 1.79 (1H, m, H-2a), 1.51 (1H, m, H-2b), 3.56 (1H, dd, J ¼ 4.6, 12.0 Hz, H-3), 2.29 (1H, dq, J ¼ 7.0, 6.8 Hz, H-4), 1.69 (1H, m, H-5), 4.08 (1H, dd, J ¼ 10.5, 11.2 Hz, H-6), 1.66 (1H, m, H-7), 1.83 (1H, m, H-8a), 1.46 (1H, m, H-8b), 1.55 (1H, m, H-9a), 1.25 (1H, m, H-9b), 1.28 (3H, s, H-13), 1.00 (3H, s, H-14), 1.22 (3H, d, J ¼ 6.8 Hz, H-15); 13C NMR (CDCl3, 125 MHz)  39.25 (t, C-1), 26.11 (t, C-2), 76.55 (d, C-3), 40.53 (d, C-4), 53.30 (d, C-5), 80.73 (d, C-6), 55.68 (d, C-7), 23.48 (t, C-8), 43.01 (t, C-9), 37.73 (s, C-10), 75.15 (s, C-11), 178.45 (s, C-12), 17.92 (q, C-13), 20.08 (q, C-14), 12.51 (q, C-15); NOE: (1) H-3 (
100%), H-7 (þ7.77%); (2) H-6 (
100%), H-4 (þ9.55%), H-13 (þ6.06%), H-14 (þ8.50%); 3) H-15 (
100%), H-14 (þ1.87%), H-6 (2.10%), H-7 (2.31%); HREIMS m/z 268.1664 [M]þ (Calcd for C15H24O4, 268.1675).

Acknowledgements This work was supported by the ‘‘211’’ and ‘‘985’’ Projects of The Central University for Nationalities.

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References [1] Y. Tu, L. Sun. Tetrahedron Lett., 39, 7935 (1998). [2] Y. Amate, J.L. Breto´n, A. Garcı´ a-Granados, A. Martı´ nez, M.E. Onorato, A. Sae´nz de Buruaga. Tetrahedron, 46, 6939 (1990). [3] Y. Amate, A. Garcı´ a-Granados, A. Martı´ nez, A. Sae´nz de Bumaga, J.L. Breto´n, M.E. Onorato, J.M. Arias. Tetrahedron, 47, 5811 (1991). [4] T. Hashimoto, Y. Noma, Y. Asakawa. Heterocycles, 54, 529 (2001). [5] L. Yang, J. Dai. Acta Pharma. Sin., 40, 834 (2005). [6] L. Yang, J. Dai, J. Sakai, M. Ando. Biotech. Lett., 27, 793 (2005). [7] L. Yang, J. Dai, J. Sakai, M. Ando. J. Asian Nat. Prod. Res., 8, 317 (2006). [8] Y. Garcı´ a, A. Garcı´ a-Granados, A. Martı´ nez, A. Parra, F. Rivas. J. Nat. Prod., 58, 1498 (1995). [9] L. Cardona, B. Garcı´ a, J.E. Gime´nez, J. Pedro. Tetrahedron, 48, 851 (1992). [10] M. Ando, T. Wada, H. Kusaka, K. Takase, N. Hirata, Y. Yanagi. J. Org. Chem., 52, 4792 (1987).

Natural Product Research, Vol. 22, No. 6, 15 April 2008, 507–515

Antioxidant activities of protein-enriched fraction from the larvae of housefly, Musca domestica HUI AI, FURONG WANG and CHAOLIANG LEI* College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, P. R. China (Received 17 December 2006; in final form 18 August 2007) The protein-enriched fraction (PEF) was isolated and purified from the larvae of housefly, Musca domestica. This study was designed to investigate amino acid compositions, antioxidative effects and protective effects of PEF on red blood cell (RBC) hemolysis, lipid peroxidation. The effects of PEF treatment were studied on aged mice liver lipid peroxidation and antioxidant enzyme activities, which included superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Results: PEF not only inhibited H2O2 stimulated oxidative hemolysis of erythrocytes of mice, but also depressed malondialdehyde (MDA) production in mice liver homogenate by auto-oxidation and hepatic mitochondria expanded induced by Fe2þ-ascorbic acid system. Compared to control group, treatments of PEF significantly increases SOD and GSH-Px activity of serum and liver homogenate in aged mice. MDA level of serum and liver homogenate decreased significantly in aged mice. In conclusion, this study demonstrates that PEF possesses antioxidative activity and might be a valuable source of natural antioxidative agents. Keywords: Larva of housefly; Hemolysis; MDA; SOD; GSH-Px; Antibiooxidation

1. Introduction Over the past several decades, much attention has been paid on the nutritional value of the larvae of housefly. The larvae of housefly contained 678 g crude protein (CP) kg21 on a dry matter basis [1]. They are an excellent source of high-quality protein, polyunsaturated fats, vitamins, minerals, and other nutrients for both human food and animal feed [2]. The larvae of housefly have been used for centuries and are still used to help debride and heal wounds [3–6]. It has also been recorded in traditional Chinese pharmacopeia, Compendium of Materia Medica during the Ming Dynasty (1596, Li) that maggots could be used as a medicinal agent to treat diseases of spleen and stomach [7]. We have reported earlier from our laboratory that homogenate of the larva of housefly possessed hydroxyl radical scavenging activity and antiviral activity against influenza virus in vitro [8,9]. It is obvious that the larvae of housefly might be considered as a rich source of natural antioxidants. *Corresponding author. Tel.: þ86 27 87287207. Fax: þ86 27 87287207. Email: [email protected] Natural Product Research ISSN 1478-6419 print/ISSN 1029-2349 online ß 2008 Taylor & Francis http://www.tandf.co.uk/journals DOI: 10.1080/14786410701592034

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At the present time, the use of natural antioxidants has the advantage that the consumer considers them to be safe because of no chemical contamination, readily accepts them and no safety tests are required for them by the legislation if the food component is generally recognized as safe (GRAS) [10]. Therefore, current attention is focusing on natural antioxidants. There are various antioxidative constituents that were found in plants and animals, and without related report, also in insects. The aims of the present study were; first, to isolate protein-enriched fraction (PEF) from the maggots; second, to assess their protein quality from amino acid compositional data according to Food and Agricultural Organization (FAO)/World Health Organization (WHO)[11], and the US Food and Drug Administration (USFDA, 1993); third, to investigate the effect of PEF on red blood cell (RBC) hemolysis, lipid peroxidation, and antioxidative enzyme activities in aged mice following PEF treatment.

2. Materials and methods 2.1. Materials Thiobarbituric acid (TBA), oxidized glutathione-reductase (GSSG-reductase), nicotinamide adenine dinucleotide phosphate (NADPH) and glutathione (GSH) were purchased from Sigma Chemical Co. (USA). All other chemicals and reagents used were of analytical reagent grade.

2.2. Animals Specific pathogen free (SPF) Kunming aged mice (11–12 month) and Wistar mice (8 week, 130–150 g) supplied by Experimental Animal Centre of Hubei Centre for Diseases Prevention and Control (CDC), P.R. China, were used in this study. All mice were kept at the animal facilities under specific pathogen-free conditions until used. Sterile food and water were supplied. Wistar mice were used to determine the in vitro antioxidative activities. To determine the in vivo antioxidative activities, the aged mice were pretreated, by force feeding using a stomach tube, with the PEF suspension, at the doses of 50, 100, and 200 mg kg
1 of mouse’s body weight for 20 days. Control group mice were treated with equal volume of normal saline. Numbers of mice were 10 for each group. Body weights of mice were measured.

2.3. Maintenance of larvae and preparation of PEF Experiments were performed with a laboratory strain of the housefly as described [12]. Adult flies were maintained in gauze-covered cages at 28  2 C and fed a diet of sugar beet and dry milk powder. Husks of wheat seeds were provided as substrate for egg laying. Husks with eggs were placed in plastic containers and maintained at 28  2 C under photoperiod of 12 light: 12 dark. After the eggs hatched, the larvae were transferred to moistened (relative humidity (RH) 75  5%) husks for feeding and development. Third-instar larvae were collected, washed with distilled water, frozen, and lyophilized. The lyophilized maggots were extracted with petroleum ether

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(b.p. 30–60 C) in a Soxhlet apparatus for 50 h. The defatted maggots were pulverized at low temperature and treated using three sequences of cold (4 C) protein buffer (0.1 M citrate–Na2HPO4, 0.18 M NaCl dilution, pH adjust to 7.0) for 0.5 h. The extracts were centrifuged at 1800 g for 15 min at 4 C. The supernatant was transferred to a new container and acidified to pH 5.8 with 0.01 M HCl. The solution was fractionated between 65% saturation of (NH4)2SO4 and the precipitate dialyzed against a low-ionicstrength pH 7.5 buffer. All the dialyzed extracts were centrifuged and the supernatant was concentrated and lyophilized. The lyophilized supernatant was PEF. It was stored at
20 C until needed.

2.4. Amino acid analysis An amino acid analysis system for the quantification of PEF was programmed on a Hitachi High Speed Amino Acid Analyzer Model 835-50. Namely, a ninhydrin post-column detecting system with a Hitachi 2619-F column (2.6  150 mm) was programmed. Areas of amino acid standards were used to calculate quantity of each amino acid in samples with the amino acid composition of protein.

2.5. H2O2-induced red cell oxidation hemolysis Rat RBC was washed three times with normal saline and made into 0.5% suspension. RBC suspension (1 mL) was incubated with H2O2 (100 mM) at 37 C for 1 h after adding test compounds or vehicle. After diluting five times with normal amine, the mixture was centrifuged at 1000 g for 10 min. The absorbance (A) of supernatant was measured at 415 nm under 0.5 cm optical path. The A of control tube was defined as 100%. The hemolysis extent was calculated by referring to control tubes [13]. Hemolysis inhibitory rate ð%Þ ¼

ODcontrol
ODsample  100 ODcontrol

2.6. Determination of lipid peroxidation Blood samples were collected from rats under light ether anesthesia by ocular vein puncture and serum was separated by centrifugation. Livers of the rats were taken out and perfused with cold 50 mM TrisHCl buffer (pH 7.4) containing 0.15 M KCl and 2 mM EDTA. Liver samples were then homogenized in 50 mM TrisHCl buffer (pH 7.4), containing 0.15 M KCl and 0.25 M sucrose to give a final tissue concentration of 250 mg ml21. The homogenates were centrifuged at 10,000 g for 20 min at 4 C. Lipid peroxidation was estimated in homogenates by the method of Buege and Aust [14]. The formation of TBA-reactive metabolites of lipids such as malondialdehyde (MDA) in the presence of FeSO4 (1 mM) and ascorbic acid was measured as the pink-colored chromophore that gives maximum absorption at 532 nm.

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2.7. Preparation of liver mitochondria for quantitative estimation of lipid peroxidation Liver tissues of the animals were collected, washed in 0.9% saline, soaked in filter paper, weighed, and a 400 mg of liver was homogenized in 0.15 M KCl. The homogenates were then centrifuged at 12,000 g at 4 C for 10 min, and the supernatants were collected and again centrifuged at 10,000 g at 4 C for 10 min. This supernatant was then collected for assay of lipid peroxidation. Lipid peroxidation was measured by the method of Buege and Aust [14].

2.8. Assays of MDA and antioxidant enzyme activity in aged mice According to section 2.6, liver homogenates and serum samples were prepared and stored in
70 C for enzyme assays. MDA was measured in homogenates by the method of Buege and Aust [14]. Glutathione peroxidase (GSH-Px) was assayed by the method of Flohe and Gunzler [15]. The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.5), 0.5 mM EDTA, 1.0 mM NaN3, 0.24 U of GSSG-reductase, 1.0 mM GSH, 0.15 mM NADPH, 0.15 mM H2O2 and supernatant. The decrease in absorbance due to NADPH oxidation was monitored at 340 nm. Estimation of superoxide dismutase (SOD) was performed by the method of Kono [16]. The inhibitory effect of SOD on the reduction of nitroblue tetrazolium dye (96 mM) by superoxide anions that are generated by the photooxidation of hydroxylamine hydrochloride (20 mM) was monitored at 560 nm.

2.9. Protein determination Protein concentration was determined in each sample by the method of Lowry et al. [17]

2.10. Statistical analysis Results were expressed as mean  deviation (SD). The statistical significance of the differences between PEF-treated groups and control group was evaluated by the variance analysis, followed by Student’s t-test. Significant differences were set at p < 0.05 and p < 0.01.

3. Results 3.1. Amino acid analysis The result of essential amino acid contents in PEF are summarized in the tables 1 and 2. According to these tables, PEF contains various amino acid compositions and possess preferable protein value. The content of human essential amino acid (41.5%) in PEF is higher than FAO/WHO standard.

Antioxidant activities of protein-enriched fraction from M. domestica Table 1.

The content of amino acids in PEF (%).

Amino acid

PEF

Amino acid

PEF

Asp Thr Ser Glu Gly Ala Cys Val Met

8.78 3.53 2.84 11.35 2.90 3.27 0.73 3.94 2.89

Ile Leu Tyr Phe Lys His Arg Pro Trp

3.25 5.69 7.54 6.51 6.30 1.87 3.61 2.77 1.15

Table 2.

511

Comparison of the content of essential amino acids (mg g
1 N) in PEF and WHO/FAO standard.

Amino acid Thr Val Met þ Cys Ile Leu Phe þ Tyr Lys Trp Total Percentage in total (%)

PEF

WHO/FAO standard

221 246 226 203 356 878 394 72 2596 41.5

250 310 220 250 440 380 340 60 2190 35

3.2. H2O2-induced red cell oxidation hemolysis The protective effect of PEF on RBC hemolysis was evaluated by oxidative stress induced experimentally by using H2O2. Under the given conditions, PEF can inhibit H2O2, which significantly causes oxidative RBC hemolysis in a dose-dependant manner (figure 1). It is worthy to note that PEF exerted the strong protective effect, which IC50 of inhibition of RBC hemolysis is 0.871 mg mL
1.

3.3. Liver homogenate lipid peroxidation level Protein-enriched fraction could significantly improve the normal liver function. It was interestingly noticed from table 3 that PEF could inhibit dose-dependently the lipid peroxidation-induced MDA formation in normal control liver homogenate.

3.4. Liver mitochondrial lipid peroxidation level The level of liver mitochondrial lipid peroxidation decreased in all the treated groups in comparison to control group. Lipid peroxidation decreased significantly (p < 0.01) by 19.64, 23.81, 25.60, and 32.74% compared to control group (table 4).

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Inhibitory rate

50 40 30 20 10 0 0

0.2

0.4 0.6 0.8 Concentration (mg/ml)

1.0

1.2

Figure 1. H2O2-induced hemolysis of red blood cells (RBCs). Hemolysis was evaluated as described in Materials and methods. Values are means  SD.

Table 3. Treatment Control PEF PEF PEF PEF

Inhibitory effect of PEF on lipid peroxidation in liver homogenate. Concentration (mg mL
1)

MDA (nmol mg protein
1)

Inhibition rate (%)

0(CK) 0.25 1.0 2.5 10

2.95  0.13 2.77  0.17 2.34  0.10* 2.10  0.31* 1.08  0.18**

– 6.10 20.68 28.81 63.39

Values are expressed as mean  SD. *p < 0.05, **p < 0.01 (compared to control group).

Table 4. Treatment Control PEF PEF PEF PEF

Inhibitory effect of PEF on lipid peroxidation in liver mitochondrial. Concentration (mg mL
1)

MDA (nmol mg protein
1)

Inhibition rate (%)

0 0.25 1.00 2.50 10.00

0.504 0.405 0.384 0.375* 0.339*

– 19.64 23.81 25.60 32.74

Values are expressed as mean  SD. *p < 0.05, **p < 0.01 (compared to control group).

3.5. Assays of MDA and antioxidant enzyme activity in aged mice The effects of PEF on MDA, SOD, and GSH-Px activity of serum and liver homogenate in aged mice are shown in tables 5 and 6. Compared to control group, treatments of PEF significantly increase SOD (25.41, 30.29, and 35.16%) and GSH-Px activity (24.65, 36.86, and 39.42%) of liver homogenate in aged mice. Treatments of PEF significantly increase SOD (24.07, 78.01, and 65.98%) and GSH-Px activity (31.52, 61.96, and 52.90%) of serum in aged mice. MDA level of Serum and liver homogenate decreased significantly as compared to control group.

Antioxidant activities of protein-enriched fraction from M. domestica Table 5. Treatment Control PEF (50 mg kg
1bw.) PEF (100 mg kg
1bw.) PEF (200 mg kg
1bw.)

513

Effect of PEF on MDA, SOD and GSH-Px in liver of aged mice. MDA (nmol mg protein
1)

SOD (U mg protein
1)

GSH-Px (U mg protein
1)

2.70  0.32 1.48  0.36* 1.38  0.31* 1.08  0.14**

199.1  6.2 249.7  16.9* 259.4  18.9* 269.1  13.1*

86.0  4.0 107.2  1.9 117.7  3.3* 119.9  2.0*

Values are expressed as mean  SD. *p < 0.05, **p < 0.01 (compared to control group).

Table 6. Treatment Control PEF (50 mg kg
1bw.) PEF (100 mg kg
1bw.) PEF (200 mg kg
1bw.)

Effect of PEF on MDA, SOD and GSH-Px in serum of aged mice. MDA (nmol mg protein
1)

SOD (U mg protein
1)

GSH-Px (U mg protein
1)

6.17  1.52 5.78  0.26 5.58  0.40 3.52  0.26**

48.2  4.0 59.8  4.75* 85.8  6.09** 80.0  7.12**

27.6  2.9 36.3  3.2 44.7  2.8* 42.2  2.1*

Values are expressed as mean  SD. *p < 0.05, **p < 0.01 (compared to control group).

4. Discussion In the present study, the essential amino acid compositions of PEF are compared to those of the FAO/WHO reference pattern [11]. PEF contains various amino acid compositions in which the content of human essential amino acids (41.5%) is more preferable than (compared to) FAO/WHO standard (35%). Contents of Tryptophan, lysine and phenylalanine are higher than FAO/WHO standard. Tryptophan, lysine, and phenylalanine are important biological constituents of numerous proteins in animals and plants, which are essential amino acid in human and animal diets. These results indicate that PEF provides sufficient amount and proper balance of the essential amino acids required for the body to function. Therefore, PEF possesses great commercial value, which might be a valuable source of high quality protein. Oxidants are involved in many human diseases and aging processes. In chronic damage associated with the development of ageing, destructive oxidants and oxygen free radicals can be generated, which are very toxic to tissues and may result in further tissue necrosis and cellular damage [18]. The antioxidants constitute a major cell defense against acute oxygen toxicity and protect membrane components against damage caused by free radicals [19]. In the past years some authors claim that proteins possess antioxidant properties [20,21]. They demonstrated that protein insufficiency aggravates the enhanced lipid peroxidation and reduces the activities of antioxidative enzymes in rats. Larson et al. have also found that proteins affect lipid metabolism in laboratory animals [22]. The present study was carried out to investigate the effect of PEF on RBC hemolysis, lipid peroxidation and antioxidative enzyme activities in aged mice following the PEF treatment. In our experiment, it is suggested that PEF can inhibit H2O2 that causes oxidative RBC hemolysis and the lipid peroxidation, which induces MDA formation in normal mice liver homogenate and mitochondrial. A strong protective effect against

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H2O2-generated hemolysis was observed. A number of investigators have demonstrated that H2O2 causing hemolysis mainly reflects the extent of lipoperoxidation of RBC membrane [23]. Furthermore, MDA generation was used as an indicator of OH-caused lipid peroxidation and indirectly reflected the amount of.OH formation in tissue homogenate stimulated by Fe2þ-ascorbic acid [24]. The level of liver homogenate and mitochondrial lipid peroxidation significantly decreased in all the treated groups in comparison to control group. In the present study, we have also examined several enzymes and MDA level of serum and liver homogenate to evaluate the effects of PEF treatment in aged mice. Particular importance was placed on SOD and GSH-Px for well-known parameters as the antioxidative enzymes. Compared to control group, treatments of PEF showed reduced MDA level and increased SOD and GSH-Px activity in the serum and liver tissue of aged mice. Each of these antioxidative enzymes has a specific and irreplaceable role in cell defence and an alteration in any of these might trigger a response that may disturb the oxidant defence system against the free radicals [25], such as SOD detoxifying superoxide free radicals and GSH-Px detoxifying hydrogen peroxide [26]. SOD inhibits HO radical production by scavenging O22 and leads to a decrease in the initiation of lipid peroxidation [27]. Therefore, the observed decrease in lipid peroxidation in the PEF group may be associated with increased SOD activity. During examinations, we found that PEF significantly augmented SOD and GSH-Px activity of serum and liver homogenate in aged mice. In the past investigation, Wang et al. found that the homogenate from larvae of housefly provided with natural ingredients possess hydroxyl radical scavenging activity in vitro [8,9]. It is obvious that PEF possesses antioxidative activity, which might be valuable sources of natural antioxidative agents. In conclusion, PEF has strong antioxidative effects, which decrease lipid peroxidation and increase antioxidant enzyme (SOD and GSH-Px) activities. This study shows that PEF may be a protective agent for free radical generating compounds, although at present, we cannot explain the effects of PEF in detail. The present observations appear to give some support to the traditional use of the maggots in Chinese traditional medicine. We are now in progress to isolate the active constituent and investigate the pharmacological mechanism.

Acknowledgements This research was financially supported by the Ningbo Science and Technology Program from Ningbo City, China. We are grateful to Dr Huangwen and Dr Qianchun Deng for their technical support and collaboration. We are also thankful to the Director, Experimental Animal Centre of Hubei Centre for Diseases Prevention and Control (CDC), P.R. China, for providing the necessary facilities and technical assistance to carry out this work. References [1] M.J. Zuidhof, C.L. Molnar. Anim. Feed Sci. Technol., 105, 225 (2003). [2] G.D. Ren, A.M. Shi. Entomol. Knowledge, 10, 103 (2002). [3] R.A. Sherman, J.M. Tran, R. Sullivan. Arch Dermatol., 132, 254 (1996).

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[4] S. Thomas, M. Jones, S. Shutler, S. Jones. J. Wound Care, 5, 60 (1996). [5] K.Y. Mumcuoglu, A. Ingber, L. Gilead, J. Stessman, R. Friedmann, H. Schulman, H. Bichucher, I. Ioffe-Uspensky, J. Miller, R. Galun, I. Raz. Int. J. Dermatol., 38, 623 (1999). [6] R.A. Sherman. Int. J. Low Extrem. Wounds, 1, 135 (2002). [7] L. Shizhen. Compendium of Materia Medica, p. 2289, People’s Medical Publishing House, Beijing, China (1981). [8] F.R. Wang, H. Ai, C.L. Lei, W. Huang. Chin. Bull. Entomol., 43, 82 (2006). [9] F.R. Wang, W. Huang, Y.L. Wang, C.L. Lei. Chin. Bull. Entomol., 42, 546 (2005). [10] Ji-Cheon Ieonga, Cheol-Ho Yoona, Woo-Hun Lee. J. Ethnopharmacol., 3, 259 (2005). [11] FAO/WHO. Expert Consultation. Protein Quality Evaluation, FAO/WHO Nutrition Meetings, Report Series 51. Rome, Italy: Food and Agriculture Organization/World Health Organization. (1991). [12] C.L. Lei, Y. Jiang, C.Y. Niu, G.H. Wu, X.Y. Yu, Z.W. Fu. J. Huazhong Agri. Univ., 29, 25 (1999). [13] Y.J. Wu, W.G. Li, Z.M. Zhang, X. Tian. Acta Pharmacol. Sinica., 18, 150 (1997). [14] J.A. Buege, S.D. Aust. Methods Enzymol., 52, 302 (1978). [15] L. Flohe, W.A. Gunzler. Assay of glutathione peroxidase. Methods in Enzymology, pp. 114–126, Academic Press, New York (1984). [16] Y. Kono. Arch. Biochem. Biophys., 186, 189 (1978). [17] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall. J. Biol. Chem., 193, 265 (1951). [18] Xiang-Yu Cui, Jin-Hwa Kim, Xin Zhao. J. Ethnopharmacol., 2, 223 (2006). [19] C.P. Reddy Avula, G. Fernandes. J. Clin. Immunol., 1, 35 (1999). [20] C.J. Huang, M.L. Fwu. J. Nutr., 122, 1182 (1992). [21] M. Heinonen, D. Rein, M.T. Satue-Gracia, S.W. Huang, J.B. German, E.N. Frankel. J. Agric. Food Chem., 46, 917 (1998). [22] M.R. Larson, S. Donovan, S. Potter. Nutr. Res., 16, 1563 (1996). [23] L.M. Feng, F.B. Lin, H.Z. Pan, Z.N. Zhang, F. Huang, X.F. Liang. Chin. Pharmacol. Bull., 6, 224 (1990). [24] X.Y. Lu. Biochem. Biophys., 16, 372 (1989). [25] C. Michiels, M. Raes, O. Toussaint, J. Remacle. Free Radical Biol. Med., 17, 235 (1994). [26] C.K. Chow. In Cellular Antioxidant Defense Mechanisms, pp. 217–237, CRC Press, Boca Raton, FL (1988). [27] I. Fridovich. Annu. Rev. Biochem., 64, 97 (1995).

Natural Product Research, Vol. 22, No. 6, 15 April 2008, 516–520

Chemical composition and antibacterial activity of essential oils from leaves, stems and flowers of Salvia reuterana Boiss. grown in Iran AKBAR ESMAEILI*y, ABDOLHOSSEIN RUSTAIYANz, MARJAN NADIMIz, KAMBIZ LARIJANIx, FARZAD NADJAFI{, LILA TABRIZI{, FIROZEH CHALABIAN> and HAMZEH AMIRI? yDepartment of Chemical Engineering, North Tehran Branch, Islamic Azad University, Tehran, Iran zMarine Science and Technology, North Tehran Branch, I.A.University, Tehran, Iran xDepartment of Chemistry, Science & Research Campus, I.A.University, P.O.Box 14515-775 Tehran, Iran {Department of Agronomy, Faculty of Agriculture, Ferdowsi University of Mashhad, Iran >Department of Biology, Islamic Azad University, North Tehran Branch, Tehran, Iran ?Department of Plant Biology, Science & Research Campus, Islamic Azad University, Tehran, Iran (Received 7 February 2007; in final form 25 July 2007) The essential oils obtained by hydrodistillation of the leaves, stems and flowers of Salvia reuterana (Lamiaceae) were analysed by GC and GC/MS. Germacrene D and -caryophyllene were the major constituents in all the three oils: (28.5, 27.7 and 32.5%) and (15.5, 11.4 and 16.6%), respectively. Bicyclogermacrene (10.2 and 13.2%) was also prodominated in the stem and flower oils. The composition of the oils was mostly quantitativel rather than qualitatively different. All the oils consisted mainly of sesquiterpenes and a small percentage of nonterpenoid compounds. In all the three oils, monoterpenes were in a concentration less than 0.5%. Antibacterial activity was determined by the measurement of growth inhibitory zones. Keywords: Salvia reuterana; Lamiaceae; Essential oil -Caryophyllene; Bicyclogermacrene; Antibacterial activity

composition;

Germacrene

D;

1. Introduction The genus Salvia (Lamiaceae) comprises about 700 herbs and shrubs, growing in the temperate and warmer zones of the world [1]. Some of these species are used as medicinal, aromatic and ornamental plants. Salvia officinalis is one of the most widespread species and since ancient times it has been used in the treatment of various disorders, such as tuberculosis [2], psoriasis and seborrhoeic eczemas [3]. It has shown strong antibacterial and antifungal activities [4]. The oil of S. sclarea *Corresponding author. Email: [email protected] Natural Product Research ISSN 1478-6419 print/ISSN 1029-2349 online ß 2008 Taylor & Francis http://www.tandf.co.uk/journals DOI: 10.1080/14786410701592067

Chemical composition and antibacterial activity of S. reuterana essential oils

517

showed significant anti-inflammatory and peripheral analgestic properties [5]. The rhizomes of S. miltiorrhiza have been widely used to treat coronary heart diseases, particularly angina pectoris and myocardial infarction [6]. Previous chemical investigations on different species of Salvia have shown the presence of flavonoids [7], diterpenoids [8,9] and sesterterpenes [10–13]. Many other reports on the phytochemical analysis of this species-rich genus, including essential oil analyses, are also found in the literature [14–17]. Previously, we reported the oil composition of S. leriifolia Benth. [18], S. aethiopis L., S. hypoleuca Benth., S. multicaulis Vahl. [19], S. sahandica Boiss. [20] and S. hydranjea DC. ex. Benth [21]. The essential oil of S. reuterana has been the subject of considerable work in Iran. Twenty-one components were identified with (E)- -ocimene (32.3%), -gurjunene (14.1%) and germacrene D (11.2%) as the major constituents [22]. However, to the best of our knowledge, no report on the oils from leaves, stems and flowers of this plant exists, which led us to the present work. In this article, we describe the analyses of the oils from leaves, stems and flowers of S. reuterana Boiss. and their antibacterial activity for the first time.

2. Materials and methods 2.1. Plant material Leaves, stems and flowers of S. reuterana were collected in July and August of 2003 from Dolat-Abad, the Province of Khorassan, Iran. Voucher specimens were deposited at the Herbarium (Voucher No. 6597) of the Research Institute of Forests and Rangelands (TARI), Tehran, Iran.

2.2. Oil isolation Fresh leaves (80 g), stems (90 g) and flowers (70 g) of S. reuterana were subjected to separate hydrodistillation for 3 h using a Clevenger-type apparatus. After decanting and drying over anhydrous sodium sulphate, the corresponding yellowish coloured oils were recovered from the leaves, stems and flowers in yields of 0.9, 0.8 and 1.1% (w/w), respectively.

2.3. Analysis GC analysis of the oils was performed on a Shimadzu 15A gas chromatograph equipped with a split/splitless injector (250
C). N2 was used as carrier gas (1 mL min1) and the capillary column used was DB-5 (50 m  0.2 mm, film thickness 0.32 mm). The column temperature was maintained at 60
C for 3 min and then heated to 220
C with a 5
C min1 rate and kept constant at 220
C for 5 min. GC/MS analysis was performed using a Hewlett-Packard 6890/5973 with a HP-5MS column (30 m  0.25 mm, film thickness 0.25 mm). The column temperature was maintained at 60
C for 3 min and programmed to 220
C at a rate of 5
C min1, and kept constant at 220
C for 5 min. The flow rate of Helium as carrier gas was (1 mL min1). MS was taken at 70 eV.

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Identification of the constituents of each oil was made by comparison of their mass spectra and retention indices (RI) with those given in the literature and the authentic samples [23–25]. Relative percentage amounts were calculated from peak area using Shimadzu C-R4A chromatopac without using correction factors.

2.4. Antibacterial activity A collection of seven microorganisms was used, including the Gram-(þ) bacteria, Staphylococcus aureus (PTCC 1113), Staphylococcus epidermidis (PTCC 1349) and Staphylococcus saprophyticus (PTCC 1379), the Gram-() bacteria, Salmonella typbi (PTCC 1185), Shigella flexneri (PTCC 1234), Escherichia coli (PTCC 1330) and Pseudomonas aeruginosa (PTCC 1310) identified by Research Center of Science and Industry, Tehran, Iran. Microorganisms (obtained from enrichment culture of the microorganisms in 1 mL of Mueller–Hinton broth, incubated at 37
C for 12 h) were cultured on Mueller–Hinton agar medium. After drilling, 450 mL of oils dissolved in 450 mL n-hexane and 50 mL from solutions in each well were poured. After incubation at 37
C during 24 h, the inhibition zone diameter was measured. Table 2 shows the results of the oils of leaves, stems and flowers of S. reuterana by measurement of growth inhibitory zone (well method).

3. Results and discussion Chemical components identified in the three oils of S. reuterana and their percentage compositions are listed in table 1. The leaf oil consisted of 21 identified compounds representing 98.4% of the oil composition. The main compounds were germacrene D (28.2%), -caryophyllene (15.5%) and bicyclogermacrene (13%). Other notable constituent was 14-hydroxy muurolene (10.8%). In the stem oil, 18 compounds were identified representing 94.0% of the oil composition. The main compounds were also germacrene D (22.7%), -caryophyllene (12.5%) and bicyclogermacrene (10.3%) while cis-calamene (11.6%) was found in large amounts. Germacrene D (32.5%), -caryophyllene (16.6%) and bicyclogermacrene (13.3%) were the main components among the 14 constituents characterised in the flower oil representing 91% of the total components detected. Dehydro-aromadendrane (11%) was present in a considerable amount in the flower oil. Sesquiterpenes represented the most abundant constituents of the oil of leaves, stems and flowers (96.0, 74 and 82.3%, respectively). Germacrene D was the major constituent of these oils (28.2, 22.7 and 32.5%, respectively). Another notable constituent was -caryophyllene (15.5, 12.4 and 16.7%, respectively). The antibacterial assays showed that the oils of leaves, stems and flowers of S. reuterana inhibited the growth of the all bacteria. The results are shown in table 2.

Chemical composition and antibacterial activity of S. reuterana essential oils Table 1.

Percentage composition of the leaf, stem and flower oils of Salvia reuterana.

Compound Camphene p-Cymene Isobornyl formate -Elemene -Cubebene -Copaene -Bourbonene -Cubebene -Elemene -Caryophyllene Aromadendrane -Humulene -Cadinene Germacrene D Indipone Bicyclogermacrene (Z)-calamene -Cadinene -Vetivenene (E)-calamene Occidentalol Germacrene B Spathulenol -Copaen-4--ol Khusimone -Eudesmol Bulnesol Cadalene Kusinol n-Heptadecane Cedroxyde Iso-lungifolol (Z)-dihydro occidentalol acetate -14-Hydroxy muurolene -Methyl-pipitzol Methyl hexadecanoate Hexadecanoic acid Tricosane

Table 2.

519

RI

Leaf oil

Stem oil

Flower oil

953 1026 1239 1338 1351 1376 1384 1390 1391 1404 1459 1454 1470 1480 1492 1494 1521 1524 1526 1532 1548 1556 1576 1584 1593 1648 1666 1673 1674 1700 1704 1726 1738 1775 1833 1927 1972 2300

– – 0.3 0.7 – 4.8 0.5 0.3 2.6 15.5 – 0.8 – 28.1 – 13.0 – 2.1 0.5 – – 0.8 1.2 – 0.6 – 8.1 – 0.6 3.3 – 0.7 – 10.87 – – 3.0 –

0.4 – –

– 0.4 –

– 2.9 0.6 0.7 0.9 11.4 0.5 – 22.7 9.3 10.3 11.6 1.2 – 0.6 0.4 – – – – 6.9 – 9.7 – – 1.7 – –

4.4 – – 0.7 – 16.6 11.0 – 1.7 32.5 – 13.3 – – – 0.7 0.7 – – 0.4 – – – – – – – – 0.5

2.6 – – –

– 4.2 – 4.6

Antibacterial activity of leaves, stems and flowers of Salvia reuterana oils.

Bacterial species

Gram (þ/)

Leaf oil

Stem oil

Flower oil

n-Hexane

þ þ þ    

35 20 45 30 37 20 16

20 15 25 24 28 17 13

18 14 20 20 22 15 13

– – – – – – –

Staphylococcus aureus PTCC 1113 Staphylococcus epidermidis PTCC 1349 Staphylococcus saprophyticus PTCC 1376 Salmonella typbi PTCC 1185 Shigella flexneri PTCC 1234 Escherichia coli PTCC 1330 Pseudomonas aeruginosa PTCC 1310 Values are the mean diameter of inhibitory zones (mm).

Acknowledgements We are grateful to Dr V. Mozaffarian (Research Institute of Forest and rangelands, Tehran) for his helpful assistance in the botanical identification.

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References [1] M. Chadefaud, L. Emberger. Traite de Botanique (Systematique), p. 839, Masson et Compagnie Editeurs, Paris (1960). [2] V.N. Dobrynin, M.N. Kolsovo, B.K. Chernov, N.A. Derbntseva. Khim. Prir. Soedin, 5, 686 (1976). [3] I. Janosik. Czechoslovakian Patet, 185, 262 (1980); Chem. Abstr., 95, 68027f (1981). [4] H. Hitokoto, S. Morozumi, T. Wauke, S. Sakai, H. Kurata. Appl. Environ. Microbiol., 39, 818 (1980). [5] A. Peana, M. Satta. Pharmacol. Res., 27, 25 (1993). [6] H.M. Chang, P.P. But. Pharmacology and Applications of Chinese Material Medica, Vol. 1, p. 255, World Science Publishing Co., Singapore (1986). [7] A. Ulubelen, N. Evren, E. Tuzlaci, C. Johansson. J. Nat. Prod., 51, 1178 (1988). [8] L. Rodriguez-Hahn, B. Esquival, J. Cardenas, T.P. Ramamoorthy. In Advances in Labiataea Science, R.M. Harley, T. Reynolds (Eds), p. 335, Royal Botanic Gardens: Kew, Richmond (1992). [9] Z. Habibi, F. Eftekhar, K. Samiee, A. Rustaiyan. J. Nat. Prod., 63, 270 (2000). [10] A. Rustaiyan, A. Niknejad, L. Nazarians, J. Jakupovic, F. Bohlmann. Phytochem., 21, 1812 (1982). [11] A. Rustaiyan, S. Koussari. Phytochem., 27, 1767 (1988). [12] A. Rustaiyan, A. Sadjadi. Phytochem., 26, 3078 (1987). [13] M.S. Gonzalez, J.M. Sansegundo, M.C. Grande, M. Medarde, I.S. Bellido. Tetrahed., 45, 3575 (1989). [14] B. Demirci, K.H.C. Baser, B. Yildiz, Z. Bahcecioglu. Flavour Fragr. J., 18, 116 (2003). [15] M.M. Endeshaw, O.R. Gautun, N. Asfaw, A.J. Aasen. Flavour Fragr. J., 15, 27 (2000). [16] M. Kurkcuogiu, K.H.C. Baser, H. Duman. J. Essent. Oil Res., 14, 241 (2002). [17] A.O. Tucker, M.J. Maciarello. J. Essent. Oil Res., 8, 669 (1996). [18] A. Rustaiyan, S. Masoudi, M. Yari, M. Rabbani, H. Motifar, K. Larijani. J. Essent. Oil Res., 12, 601 (2000). [19] A. Rustaiyan, S. Masoudi, A. Monfared, H. Komeilizadeh. Flavour Fragr. J., 14, 276 (1999). [20] A. Rustaiyan, H. Komeilizadeh, S. Masoudi, A.R. Jassbi. J. Essent. Oil Res., 9, 713 (1997). [21] A. Rustaiyan, S. Masoudi, A.R. Jassbi. J. Essent. Oil Res., 9, 599 (1997). [22] M. Mirza, F. Sefidkon. Flavour Fragr. J., 14, 230 (1999). [23] Eight Peak Index of Mass Spectra, Mass Spectrometry Data Centre (ed.), Unwin Brothers Ltd, Surrey (1991). [24] R.P. Adams. Identification of Essential Oil Components by Gas Chromatoghraphy/Mass Spectroscopy, Allured Publ. Corp. Carol Stream, USA (1995). [25] D. Joulain, A.W. Konig. The Atlas of Spectral Data of Sesquiterpene Hydrocarbons, Hamburg, Germany (1998).

Natural Product Research, Vol. 22, No. 6, 15 April 2008, 521–524

A new labdane diterpenoid arabinoside from Conyza blinii YANFANG SU*y, LEI CHENy, DAWEI WANGy, XIN CHAIy and DEAN GUOz ySchool of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, P.R. China zSchool of Pharmaceutical Sciences, Peking University, Beijing 100083, P.R. China (Received 8 January 2007; in final form 26 April 2007) A new diterpenoid glycoside, 14,15-dinor-labdan-13-one-8-O--L-arabinopyranoside (1), was isolated from Conyza blinii. The structure of this new arabinoside was elucidated on the basis of extensive NMR and HRMS spectroscopy. In addition, the presence of 3R-hydroxyoctadecanoic acid in C. blinii was reported for the first time. Keywords: Conyza blinii; Diterpenoid; Labdane; Arabinoside

1. Introduction The genus Conyza (Asteraceae) consists of about 80 species. Labdane and clerodane diterpenoids are regarded as characteristic constituents in Conyza plants [1]. The aerial parts of Conyza blinii Le´vl., a native herbaceous plant of southwest China, are used in Chinese folk medicine for the treatment of gastroenteritis, chronic bronchitis and other inflammatory diseases. Our chemical investigation of this species have led to the identification of 17 new triterpenoid saponins, 3 new labdane diterpenoids, 1 new clerodane diterpenoid and one new phenolic glucoside [2–6]. Further investigation led to the identification of a new labdane diterpenoid, 14,15-dinor-labdan-13-one-8-O-L-arabinopyranoside (compound 1, figure 1), as well as a free fatty acid, 3R-hydroxyoctadecanoic acid.

2. Results and discussion Compound 1 was obtained as a syrup. Its molecular formula was determined to be C23H40O6 according to its 13C and DEPT NMR spectroscopic data and a

*Corresponding author. Tel.: þ86-22-27402885. Fax: þ86-22-27892025. Email: [email protected] Natural Product Research ISSN 1478-6419 print/ISSN 1029-2349 online ß 2008 Taylor & Francis http://www.tandf.co.uk/journals DOI: 10.1080/14786410701592232

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12

13

16

O 9 1

17

O

8 7

4

OH O

OH

HO

O

5′

4′

1′

3′ 2′

OH

1 Figure 1.

2 Structures of compounds 1 and 2.

quasi-molecular ion peak of m/z 435.2708 [M þ Na]þ in a high resolution MALDI-MS spectrum. IR spectroscopy revealed the presence of a hydroxyl group (3427 cm
1) and a carbonyl group (1710 cm
1). The signals of five tertiary methyl groups ( 0.79, 0.83, 0.87, 1.26 and 2.16) observed in the 1H NMR spectrum of compound 1, coupled with information from its 13C and DEPT NMR spectra (five primary carbons at  15.5, 21.4, 21.8, 29.9 and 33.3, one oxygen-bearing quaternary carbon at  81.7 and one carbonyl carbon at  211.7), pointed to the presence of a labdane-type diterpenoid [5]. The 1H and 13C NMR spectra of compound 1 returned a set of resonance signals for one -L-arabinopyranoside unit (table 1). Of the 23 carbon signals observed in the 13C NMR spectrum of compound 1, five were attributed to the sugar moiety, and the remaining eighteen were attributed to a diterpenoid moiety with two carbon atoms less than in labdane. More detailed study of 1D and 2D NMR spectra of compound 1 revealed that the NMR resonance signals of the aglycone moiety were similar to those of a synthetic labdane diterpenoid [7], 8-hydroxy-14,15-dinor-labdan-13-one (compound 2, figure 1). The downfield shifted resonance signal of C-8 at  81.7 ppm and the upfield shifted resonance signals of C-7 at  39.7 ppm, C-9 at  58.5 ppm and C-17 at  21.8 ppm in the 13C NMR spectrum of compound 1, compared with those of compound 2 (table 1), revealed that the hydroxy group at C-8 of the diterpenoid moiety was substituted by a sugar unit. The correlation point between the H-10 ( 4.44 ppm) peak of arabinose and the C-8 ( 81.7 ppm) peak of the aglycone moiety in HMBC spectrum confirmed the above deduction. Consequently, the structure of compound 1 was elucidated as 14,15-dinor-labdan-13-one-8-O--L-arabinopyranoside (figure 1). 3R-Hydroxyoctadecanoic acid was detected and identified by analysis and comparison of its NMR and MS spectroscopic data, as well as optical rotation, with those reported in the literature [5,8]. This was the first time that 3R-hydroxyoctadecanoic acid was isolated from Conyza blinii.

New labdane diterpenoid arabinoside from C. blinii Table 1.

1

H (500 MHz) and 13C (125 MHz) NMR spectroscopic data for compounds 1 and 2 [7] in CDCl3. 1

No. 1 2 3 4 5 6 7 8 9 10 11 12 13 16 17 18 19 20 10 20 30 40 50 a

523

13

C

39.9 18.3 41.8 33.2 55.8 19.9 39.7 81.7 58.5 39.2 20.0 46.5 211.7 29.9 21.8 33.3 21.4 15.5 96.3 71.7 73.0 67.9 65.0

2

a

(t) (t) (t) (s) (d) (t) (t) (s) (d) (s) (t) (t) (s) (q) (q) (q) (q) (q) (d) (d) (d) (d) (t)

1

a,b

H (ppm)

0.94, 1.63 1.42, 1.60 1.37, 1.14 (m) – 0.93 1.24, 1.70 (m) 1.44, 2.00 (m) – 1.24 – 1.60, 1.82 (m) 2.50 (m), 2.60 (m) – 2.16 (s) 1.26 (s) 0.87 (s) 0.79 (s) 0.83 (s) 4.44 (d, 6.5) 3.55 (dd, 7.0, 8.5) 3.64 (dd, 9.0, 3.0) 3.91 3.50 (br d, 11.5), 3.92

13

C

40.2 18.5 42.1 33.4 56.6 19.0 44.8 73.8 60.9 39.2 20.6 46.3 210.6 29.6 24.2 33.4 21.5 15.2

Multiplicity and coupling constants (J in hertz) are in parentheses. Signals without multiplicity are overlapped.

b

3. Experimental 3.1. General experimental procedures Optical rotations were measured on a Perkin–Elmer Model 341 polarimeter. IR spectrum was recorded by a BIO-RAD 3000 FTIR spectrometer. NMR spectra were obtained on a Varian INOVA 500 FT NMR spectrometer in CDCl3 and CD3OD. High resolution mass spectrum was returned by an IonSpec ProMALDI mass spectrometer. Solvents were of analytical grade. Silica gel GF254 (Qingdao Marine Chemical Co.) and silica gel (100–200 mesh and 200–300 mesh, Qingdao Marine Chemical Co.) were used for TLC and open-column chromatography.

3.2. Plant material The aerial parts of C. blinii were collected in August 1999 in Chengdu, Sichuan Province, People’s Republic of China, and the specimen was kept in the School of Pharmaceutical Science and Technology, Tianjin University.

3.3. Extraction and isolation The air-dried plant material (17 kg) was twice refluxed with 95% ethanol and then once with 60% ethanol. The ethanol extract was concentrated and then suspended in water

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and partitioned with petroleum ether, chloroform and ethyl acetate. The chloroform extract (177 g) was fractionated through silica gel column chromatography, using a gradient of petroleum ether and acetone. Fractions 73–76 were then passed into a silica gel column with chloroform-acetone 6 : 4, a Sephadex LH-20 column with methanol and finally an ODS column with methanol-water 8 : 2, to yield 15 mg of compound 1. The ethyl acetate extract was subjected to silica gel column chromatography and eluted with gradient proportions of chloroform in methanol. Resulting fractions 30–40 were then purified through a silica gel column with chloroform-methanol 97 : 3 and a Sephadex LH-20 column with methanol, to obtain 200 mg of 3R-hydroxyoctadecanoic acid. 20
19.0 14,15-Dinor-labdan-13-one-8-O--L-arabinopyranoside (1), syrup, []D
1 1 13 (c ¼ 0.62, CHCl3); IR (KBr) max: 3427, 2947, 1710, 1070 cm ; H and C NMR spectroscopic data, see table 1; HRMALDIMS found: m/z 435.2708 [M þ Na]þ, calculated for [C23H40O6Na]þ 435.2717.

Acknowledgements We thank the National Natural Science Foundation of China (NSFC, Grant No. 30200024) and the Natural Science Foundation of Tianjin City (Grant No. 033607711) for their financial support. We also thank Mr Philippe Andre for checking the English language.

References [1] [2] [3] [4] [5] [6] [7]

Y. Su, Y. Luo, L. Chen, F. Yang. Nat. Prod. Res. Dev., 18, 878 (2006). Y. Su, D. Guo, H. Guo, J. Liu, J. Zheng, K. Koike, T. Nikaido. J. Nat. Prod., 64, 32 (2001). Y. Su, K. Koike, D. Guo, T. Satou, J. Liu, J. Zheng, T. Nikaido. Tetrahedron, 57, 6721 (2001). Y. Su, K. Koike, T. Nikaido, J. Liu, J. Zheng, D. Guo. J. Nat. Prod., 66, 1593 (2003). Y. Su, K. Koike, D. Guo, J. Jia, J. Liu, J. Zheng, T. Nikaido. Heterocycles, 56, 265 (2002). Y. Su, D. Guo, Y. Cui, J. Liu, J. Zheng. J. Asian Nat. Prod. Res., 3, 229 (2001). I.S. Marcos, P. Basabe, M. Laderas, D. Dı´ ez, A. Jorge, J.M. Rodilla, R.F. Moro, A.M. Lithgow, I.G. Barata, J.G. Urones. Tetrahedron, 59, 2333 (2003). [8] L. Negelmann, S. Pisch, U. Bornscheuer, R.D. Schmid. Chem. Phys. Lipids, 90, 117 (1997).

Natural Product Research, Vol. 22, No. 6, 15 April 2008, 525–532

Oil composition and some morphological characters of Crambe orientalis var. orientalis and Crambe tataria var. tataria from Turkey N. COMLEKCIOGLU, S. KARAMAN* and A. ILCIM Faculty of Science and Letters, Department of Biology, Kahramanmaras Sutcu Imam University, Avsar Campus Kahramanmaras 46100, Turkey (Received 29 March 2007; in final form 25 June 2007) Native Crambe orientalis var. orientalis and Crambe tataria var. tataria collected from Kahramanmaras flora were morphologically examined and seed oil composition was determined. Volatile acid and fatty acid composition of seeds were examined with GC and GC/MS and the ratio of volatile acids to total oil was 3.49% in C. orientalis and 17.49% in C. tataria. The ratio of fatty acids to total oil was 92.03 and 67.28% in C. orientalis, and C. Tataria, respectively. The amount of erucic acid was 39.29% in C. orientalis and 29.87% in C. tataria. High linolenic acid (21.21%) and linoleic acid (12.42%) was found in C. orientalis oil, and high linolenic acid (15.01%) and linoleic acid (9.00%) was also found in C. tataria oil. Keywords: Crambe orientalis; Crambe tataria; Morphology; Volatile oils; Fatty acids; Erucic acid

1. Introduction The Brassicaceae family consists of 13–19 tribes, 350 genera and about 3500 species in the world [1] and 85 genera and 515 species in Turkey [2]. The Brassicaceae tribe is very important economically, containing several species with edible seed oils, such as Brassica, Sinapis and Crambe [1]. Crambe is native to the Mediterranean region and erects annual, biennial or perennial herbs with large, pinnately lobed leaves. Plant height varies from 30 to 120 cm depending on the season and the population density [2,3]. The white or yellow flowers are clustered in racemes and the fruits are spherical, almost always one seeded, indehiscent, and remain attached to the plant at maturity, unless heavy rain and wind cause extensive shedding. Three Crambe L. species and five taxons were naturally found in Turkey. C. tataria Sebeok grows in Central Anatolia and one taxon of this species is endemic for South Turkey. C. orientalis L. founds a wide distribution in Center Anatolia, East Anatolia and South East Anatolia [2]. C. maritima L. took part in Linne’s ‘Species Plantarum’ *Corresponding author. Tel.: 344 2191041. Fax: 344 2191012. Email: [email protected] Natural Product Research ISSN 1478-6419 print/ISSN 1029-2349 online
2008 Taylor & Francis http://www.tandf.co.uk/journals DOI: 10.1080/14786410701592349

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and distributed in North-Northeast Anatolia. According to IUCN category, C. maritima can be vulnurable (V), C. tataria var. tataria and C. orientalis var. orientalis are rather rare [4]. Crambe L. has an industrial interest because its seed oil has high erucic acid which is used mainly as an erucamide and the most important industrial application is being a lubricant in plastics fabrication. Seed oil contains high amounts of erucic acid. Erucic acid is a hydrocarbon with 22-carbon and a double bond (C22 : 1). This property gives high boiling and evaporating point (229
C) to erucic acid rich oils. Ability to resist high temperatures and maintain liquid form in low temperatures makes the oil a good lubricant and transfer oil [5]. Erucamide also has many applications in pharmaceutical detergents, cosmetics, perfume, pesticide, textile industries, foam suppressants and adhesive [3]. Recently, public concern about the environment has renewed market demand for more rapidly biodegradable lubricants from vegetable oils. Concomitantly the specific properties of high erucic acid oil and of derivatives of erucic acid are leading to their increasing use in oleochemical industry [6]. Crambe oil is much more biodegradable than mineral oils, so it may be used alone as additives for the textile, steel and shipping industries [7]. In addition to these properties, crambe oil has an advantage of being a renewable natural product with a lower environmental impact [8]. Crambe seed is mainly composed from oil, protein and an extract without nitrogen. Besides the wide usage of Crambe oil in industry, it was also evaluated according to its nutritional characteristics. Culture studies showed that dehulled Crambe seed contains 46% oil and 27% protein. However, the hull has considerably high fibril content. Protein has an important nutritional value because of its amino acid composition, so it has a potential usage in animal nutrition. But thioglucosinolates and anti-growth factors, which constitutes 8–10% of crambe meal, posses a problem, therefore chemical and enzymatic modifications were needed [9]. All the literature reports about Crambe spp. were focused on C abyssinica cultures. There were no reports on C. orientalis and C. tataria oil yield and composition. The objective of this study was to determine morphological characteristics, seed oil and composition of C. orientalis var. orientalis and C. tataria var. tataria, which are grown naturally in South East Mediterranean Region in Turkey and to investigate their economic possibility.

2. Material and methods 2.1. Collection of specimens from the native flora Crambe spp. were collected from native stands Kahramanmaras (Turkey) in June–July 2004. C. orientalis var. orientalis was collected from Ah|rdag| Mountain (latitude: 37.36
; longitude: 36.56
) at an altitude of 700–800 m. C. tataria var. tataria was collected from Kahramanmaras-Elbistan (latitude: 38.132
; longitude: 37.12
) at an altitude of 1200–1300 m. The plant collecting areas, Kahramanmaras Ah|rdag| and Elbistan have a Mediterranean climate, rainfall and mean temperature go all the year round, were obtained from the nearest meteorological stations and given in table 1. Ecological conditions vary in these areas because of latitude, longitude, altitude and climatic differences. It was noted that the plants had mature seeds when they

Oil composition and some morphological characters of Crambe sp. Table 1.

527

Climatic data of the weather stations in Kahramanmaras and Elbistan.

Locations Kahramanmaras (2004) Elbistan (2004)

TAR (mm)

MAT (
C)

MAX. T (
C)

MIN. T (
C)

721.50 357.20

17.20 11.20

41.40 37.20

9.00 17.40

TAR ¼ Total annual rainfall; MAT ¼ Mean annual temperature; MAX. T ¼ Maximum temperature; MIN. T ¼ Minimum temperature.

were picked. Plants were identified according to Flora of Turkey and East Aegean Islands [2].

2.2. Determination of morphological characteristics Plant height (cm), number of branches, plant dry weight (g), pod size and seed size (mm), pod dry weight (g) and thousand seed dry weight (g) of C. orientalis and C. tataria were determined. These morphological characteristics were measured using 20 plants for each species. Average values of these plants were recorded.

2.3. Lipid extraction Seeds were dried overnight at 50
C and ground into powder in a Moulinex coffee grinder. Five grams of powder was mixed with 100 mL petroleum ether and the lipid fraction was extracted in a Soxhalet apparatus for 8 h at 60
C. The solvent was evaporated, and the lipid fraction residues were weighed. Three accessions were evaluated for each species and average values of these plants were recorded.

2.4. Methylation of Crambe oil Two normal KOH with methanol (2 g KOH/100 mL MeOH) and 0.1 g Crambe oil were added in a 50 mL flask and boiled in a water bath with a condensator for 25 min. Two millilitres of Pentane was added on the mixture and transferred into a separatory funnel. Then pentane was removed under nitrogen stream [10].

2.5. Gas chromatography of methylated fatty acids Gas Chromatography (GC) and Gas Chromatography-Mass Spectrofotometry (GC/ MS) analyses were conducted in Chemistry Laboratory of Munich Technical University. Qualification of the oil was analyzed on a Finnegan-MAT 8200 Mass Spectrometer (low resolution) coupled with a Hewlett-Packard GC-5890II series GC. and, a SE-54 fused silica capillary column (30 m  0.25 mm i.d.; 0.25 mm film thickness) and Helium was used as a carrier gas with flow rate of 1.15 mL min1. One mL of the oil was injected into the column. The GC oven temperature was kept at 60
C for 5 min and programed to 260
C at a rate of 2
C min1 and then kept at 260
C. The injector temperature was 250
C and the amount of injection was 1 ml. The carrier gas was delivered at a constant pressure of 5 kg cm2. MS spectra were taken at EI ion source of 70 eV. Split ratio was 1 : 5. Retention indices for all components were determined

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according to Van Den Dool and Kratz method [11] using n-alkanes as standard. The components of the oil were identified by comparing their mass spectra with that of internal (computer) library, NIST libraries reference compounds and those described by Adams [12] and confirmed by comparison of their retention indices with those of reference compounds. Quantification of the essential oil was conducted by a GC with flame ionization detector (GC-FID) on a Hewlett-Packard GC-5890II series GC. One ml of the oil was injected into the same column under the same GC conditions as described for GC-MS study. However, split ratio was 1 : 14. The following fatty acids were identified by comparison with known standards: -pinene, -pinene,
-3-carene, limonene, 1,8-cineole, linalool, -bourbenene, -caryophyllene, -humulene, Germacrene-D, S cadinene, palmitic (C16 : 0), palmitoleic (C16 : 1), stearic (C18 : 0), oleic (C18 : 1), linoleic (C18 : 2), linolenic (18 : 3), arachidic (C20 : 0), c-11 eicosenoic (C20 : 1), t-11 eicosenoic (C20 : 1), behenic (C22 : 0), erucic (C22 : 1), lignoseric (C24 : 0), and nervonic acids (24 : 1).

3. Result and discussion 3.1. Morphological characteristics Some morphological characteristics of C. orientalis and C. tataria were recorded and minimum-maximum and average values are given in table 2. While plant height was measured at 71–120 cm of C. orientalis, it was determined as 40–120 cm and 35–110 cm by Davis [2] and Keskiner [14], respectively, in this regard our results agree with the reports. Davis [2] reported that plant height of C. tataria was 30–100 cm, but our data (73–116 cm) was higher than Davis’s report. According to our results, numbers of branches of C. tataria and C. orientalis were 2–15 and 1–10, respectively. Pod size of C. orientalis and C. tataria was found as 2.9–5.22 mm and 2.81–4.21 mm, respectively. However these values in Davis [2] and Kurs at [4] were 3.5–4 mm and 3.1–5 mm in C. orientalis; 5.5–6 mm and 4.4–7 mm in C. tataria, respectively. Our data was lower than Davis [2] and Kurs at [4] reports. Plant length and a thousand seed weights were the important factors that affect the product yield of Crambe [15]. Pod and a thousand seed weights of C. orientalis were 10.0–29.0 gm and 7.0–14.60 gm, respectively. For C. tataria, these values were 9.0–18.0 gm and 7.10–11.0 gm. Thousand seed weight of different Crambe species, C. abyssinica culture was found 6.5–8.1 gm by Mastebroek and Lange [16] and 5.5–6.5 gm by Wang et al. [15]. Thousand seed weights of C. orientalis and C. tataria were higher than C. abyssinica.

3.2. Oil composition The results of the oil analysis were obtained from three replicates for each species of C. orientalis and C. tataria collected from the native flora in K. Maras, which are summarized in tables 3 and 4. The figures represented amounts of the volatile acids in order of elution from silica capillary column and fatty acids percentage of the total oil. C. orientalis oil had 10 volatile acids constituting 3.49% in oil and presented high levels of Germacrene D (0.76%), linalool (0.57%), bourbene (0.55%) and 1,8

Oil composition and some morphological characters of Crambe sp. Table 2.

529

Minimum-maximum and average values of morphological characteristics of C. orientalis and C. tataria. C. orientalis Min.

Max.

C. tataria

Ave.

Min.

Max.

Ave.

Plant height (cm) 71.00 120.00 91.40  4.26 73.00 116.00 95.70  4.71 Plant weight (g) 50.00 615.00 295.90  52.33 135.00 1070.00 485.50  100.91 Number of branch 1.00 10.00 5.73  0.79 2.00 15.00 8.60  1.36 Pod size (mm) 2.90 5.22 3.83  0.06 2.81 4.21 3.47  0.04 Seed size (mm) 2.83 3.58 3.34  0.06 2.40 3.27 2.78  0.04 Pod weight (g) 10.00 29.00 17.50  4.09 9.00 18.00 12.50  2.02 Thousand seed weight (g) 7.00 14.60 9.70  1.682 7.00 11.00 8.95  0.80

Table 3.

Composition of volatile acids of the oils of C. orientalis and C. tataria.

Volatile acids -Pinene -Pinene
-3-carene Limonene 1,8-cineole Linalool -Bourbonene -Caryophyllene -Humulene Germacrene-D S cadinene Total

Table 4.

C. tataria (%)

0.15 0.29 0.16 – 0.46 0.57 0.55 0.18 0.10 0.76 0.27 3.49

1.85 0.85 2.21 0.23 0.29 0.21 0.31 2.16 0.45 8.45 0.48 17.49

Composition of fatty acids of the oils of C. orientalis and C. tataria.

Oil Content (%) Fatty acid Palmitoleic acid Palmitic acid Linoleic acid Linolenic acid Oleic acid Stearic acid c-11 Eicosenoic acid t-11 Eicosenoic acid Arachidic acid Erucic acid Behenic acid Nervonic acid Lignoseric acid SAFA MUFA PUFA Total

C. orientalis (%)

Whole seed Dehulled seed Number of carbon 16 : 1 16 : 0 18 : 2 18 : 3 18 : 1 18 : 0 20 : 1 20 : 1 20 : 0 22 : 1 22 : 0 24 : 1 24 : 0

C. orientalis (%)

C. tataria (%)

11.00 26.00

15.00 25.00

0.20 3.27 12.42 21.21 1.61 0.53 9.95 1.39 0.42 39.39 0.45 0.99 0.20 4.87 53.53 33.63

0.41 2.30 9.00 15.01 1.41 0.32 6.48 1.22 0.25 29.87 0.27 0.68 0.08 3.22 40.07 24.01

92.03

67.28

All values given are means of three determinations. SAFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids.

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cineole (0.46%). C. tataria oil had 11 volatile acids constituting 17.49% in oil and presented high levels of Germacrene D (8.45%),
-3-carene (2.21%), -caryophyllene (2.16%), -pinene (1.85%) and -pinene (0.85%). Related knowledge with volatile acid content of Crambe species was not found in literature. Oil content of the whole seeds of C. orientalis and C. tataria was 11 and 15%, respectively. Oil content in the dehulled seed of C. orientalis and C. tataria was 26 and 25%, respectively. Our results confirmed Knights [8] who reported that hull remains attached to the Crambe seed during harvest, but it can be removed easily, thus raising the oil content. The ratio of fatty acids to total oil was found 92.03% in C. orientalis and 67.28% in C. tataria. The percentage content of the fatty acids is summarized in table 4. In the oil of C. orientalis, 13 fatty acids were identified. The major constituents of the oil were erucic acid (39.39%), linolenic acid (21.21%), linoleic acid (12.42%) and c-11 eicosenoic acid (9.95%). Other components were present in lesser amount than 9.5%. C. tataria oil has 13 fatty acids and presented high levels of erucic acid (29.87%), linolenic acid (15.01%), linoleic acid (9.00%) and c-11 eicosenoic acid (6.48%). Other components were present in lesser amount than 7%. The ratio of palmitic acid (3.27% for C. orientalis and 2.3% C. tataria) was higher according to other fatty acids. Oleic acid, which is one of the most common monounsaturated fatty acids, had low ratios 1.61% in C. orientalis and 1.41% in C. tataria, respectively. Palmitoleic, stearic, arachidic, behenic, nervonic and lignoseric acids ratios of these two species were less than 1%. The health benefits of n-3 polyunsaturated fatty acids (PUFA) have been documented by numerous epidemiological and clinical studies. Recent recommendations for human diets suggest increasing n-3 PUFA consumption and decreasing the n-6 : n-3 ratio to 6 : 1 [17] PUFA contents of C. orientalis were 33.63% and C. tataria was 24.01%. Linoleic acid is related to cardiovascular disease, prostate cancer, macular degeneration, ischemia and KA-induced epilepsy and linolenic acid has anti-inflammatory properties, autoimmune disorders, arthiritis, eczema, PMS, anti-cancer agent, anticonvulsant medication, tumor growth and metastasis. Diets high in saturated fatty acids (SAFA) increased incidence of atherosclerosis and coronary heart disease. Monounsaturated fatty acids (MUFA) were protective against cardiovascular disease [18]. SAFA contents of C. orientalis and C. tataria were 4.87 and 3.22%, respectively. MUFA contents of C. orientalis and C. tataria were 53.53 and 40.07%, respectively. Because of its high content of erucic acid, the Crambe oil has a promising range of applications in the oleochemical industry,therefore a lot of work has been performed onto fatty acid content of C. abyssinica [19–23]. Because there was no literature studied concerning C. orientalis and C. tataria, we compared the fatty acid results with different Crambe species, C. abyssinica. Generally our results about erucic acid were lower than different Crambe, C. abyssinica results [19–21,23], these differences explain the variability between species and genetic. Also, on the contrary to C. abyssinica culture, our Crambe species were native plants, this could be the reason for obtaining poor fatty acid. Moreover, although oil content in seed of C. orientalis (11–26%) and C. tataria (15–25%) was lower than C. abyssinica (35–45%) [15], thousand seed weights of C. orientalis (7–14 g) and C. tataria (7–11 g) were higher than C. abyssinica (5.5–6.5 g). So it is possible that high oil and erucic acid yield could be obtained from C. orientalis and C. tataria in per

Oil composition and some morphological characters of Crambe sp.

531

plant and area. So it is possible that high erucic acid content with studied Crambe spp. according to cultivation is the result. There are various factors that influence the amounts of effective plant secondary metabolites. Ceylan [24] reported that oil ratio and chemical compounds can exchange according to species, plant segment, growth period, ecological factors and genetic structure. Mastebroek et al. [25] reported that, large variation was found in earliness, length of the top branch, thousand seed weight and fatty acid composition and this characteristic showed a high correlation between the years tested besides seed yield and oil content that were considerably affected by environmental conditions. C. oriantalis grows in open spaces, sides of the road, stony soil, among vineyards and foot path. It also grows on the sides of filled agricultural land, sandy soil and unseated agricultural land. C. orientalis and C. tataria are able to grow in variety of growing locations, in terms of sunny, yard, temperature, altitude and drought. Also the variation of growth habitat of plant may be possibily related to moisture, sunny and age of plant. To confirm the potential interest of Crambe oil in some niches of the lubricating, emulsifying and refrigerating fields, some characteristics of these plants such as some components of unsaponifiable water (sterols and aliphatic alcohols) and main physical properties (smoke point, saponification number, viscosity) must be determined. As a conclusion we suggest that cultivation of these two species and improvements could affect high oil content and erucic acid ratio. Also C. orientalis and C. tataria plants can be evaluated in animal breeding because of high linoleic and linolenic acid contents.

Acknowledgement We would like to thank Prof. Dr Harun PARLAR who is chair of Chemical Technical Analysis and Food Technology Institute, Munich Technical University, and Food Chemistry Laboratory personals in Germany.

References [1] J. Onyilagho, A. Bala, R. Hallett, M. Gruber, J. Soroka, N. Westcott. Biochem. System. Ecol., 31, 1309 (2003). [2] P.H. Davis. Flora of Turkey and East Eagen Islands, Vol. 1, pp. 272–73, Edinburgh at the University Press (1982). [3] D.B. Erickson, P. Bassin. Rapeseed and Crambe: Alternative Crops with Potential Industrial Uses, Agricultural Experiment Station, Kansas State University, Manhattan, Bulletin 656 (1990). [4] Z. Kurs at. Morphological, anatomical and polinological investigation on some Crambe L. species. Master Thesis, University of Osmangazi, Institute of Natural And Applied Sc|ences, Department of Biology, Eskis ehir (1999). [5] T. Ekim, M. Koyuncu, S. Erik, R. Ilarslan. Turkiye’nin Tehlike Alt|ndaki Nadir ve Endemik Bitkileri, Tu¨rkiye Tabiatini Koruma Derneg˘i, Yayi|n No. 18, Ankara (1989). [6] A. Grombacher, L. Nelson, D. Baltensperger. Crambe Production. Field Crops: Miscellaneous Crops, Institute of Agriculture and Natural Resources, Nebraska, USA (1993). [7] W.J.M. Meijer, E.W.J.M. Mathijssen, A.D. Kreuzer. Ind. Crop. Prod., 19, 221 (1999). [8] S.E. Knights. Crambe: A North Dakota Case Study, A Report for the rural industries research and development corporation, RIRDC Publication No. W02/005, RIRDC Project No TA001-55 (2002). [9] P. Daubos, V. Grumel, R. Iori, O. Leoni, S. Palmieri, P. Rolin. Ind. Crop. Prod., 7, 187 (1998). [10] G. Gastaldi, G. Capretti, B. Focher, C. Cosentino. Ind. Crop. Prod., 8, 205 (1998).

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[11] B. Kalender. Extraction of opium (Papaver somniferum L.) seed oil and determination of its composition. Master Thesis, University of Anadolu, Institute of Natural and Applied Sc|ences, Department of Biology, Eskis ehir (2002). [12] H. Van Den Dool, D.J. Kratz. J. Chromatogr., 11, 463 (1963). [13] R.P. Adams. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy, Allured Publishing Co., Carol Stream, IL (1995). [14] S. Keskiner. Crambe orientalis L. Uzerinde Morfolojik, Anatomik, Karyolojik ve Doku Kulturu (Embriyo Kuturu) Calismalari. Master Thesis, University of Anadolu, Institute of Natural and Applied Sc|ences, Department of Biology, Eskis ehir (1992). [15] Y.P. Wang, J.S. Tang, C.Q. Chu, J. Tian. Ind. Crop. Prod., 12, 47 (2000). [16] H.D. Mastebroek, W. Lange. Ind. Crop. Prod., 6, 221 (1997). [17] A.D. Basco, C. Castellini, L. Bianchi, C. Mugnai. Meat Sci., 66, 407 (2004). [18] Anonymous, 2006. http://en.wikipedia.org/wiki/Linoleic_acid; http://en.wikipedia.org/wiki/Saturated_ fatty_acid; http://en.wikipedia.org/wiki/Monounsaturated_fat [19] L.H. Pr|ncen, J.A. Rothfus. J. Am. Oil Chem. Soc., 61, 281 (1984). [20] K.D. Carlson, D.L. Van Dyne, Industrial feedstocks and products from high erucic acid oil: crambe and industrial rapeseed, University of Missouri, Columbia (1992). [21] C. Leonard. President, Hume Company, Inc., Memphis, Tennessee. Personal Communication (1996). [22] P. Bondioli, L. Folegatti, L. Lazzeri, S. Palmieri. Ind. Crop. Prod., 7, 231 (1998). [23] Z. Yaniv, E. Shabelsky, D. Schafferman, I. Granot, T. Kipnis. Ind. Crop. Prod., 9, 1 (1998). _ [24] A. Ceylan. T|bbi Bitkiler I, No. 312, Ege U¨niversitesi Ziraat Faku¨ltesi Yay|nlar|, Izmir (1994). [25] H.D. Mastebroek, S.C. Wallenburg, L.J.M. Soest. Ind. Crop. Prod., 2, 129 (1994).

Natural Product Research, Vol. 22, No. 6, 15 April 2008, 533–536

Fuyuziphine, a new alkaloid from Fumaria indica M. B. PANDEYy, A. K. SINGH*z, J. P. SINGHy, V. P. SINGHz and V. B. PANDEYz yDepartment of Chemistry, S.G.R. Post Graduate College, Dobhi-222149, Purvanchal University, Jaunpur, India zDepartment of Medicinal Chemistry, Institute of Medical Sciences, Banaras Hindu University, Varanasi-221005, India (Received 4 February 2007; in final form 18 August 2007) A new alkaloid, fuyuziphine together with (
)-a-hydrastine has been isolated from the whole plant of Fumaria indica. The structures of these alkaloids have been established by spectral and chemical evidences. Keywords: Fumaria (
)-a-Hydrastine

indica;

Fumariaceae;

Isoquinoline

alkaloids;

Fuyuziphine;

1. Introduction Fumaria indica commonly found in most part of India and used in Indian System of Medicine as laxative, diuretic is also beneficial in dyspepsia and liver complaints [1]. In continuation of our work on F. indica, we now report the isolation of a new isoquinoline alkaloid designated fuyuziphine (1) together with (
)-a-hydrastine (2).

2. Results and discussion The alkaloid 1 (figure 1), C19H23NO3 (HRMS, [M]þ, 313.1678) was recognised as benzylisoquinoline alkaloid from its colour reaction and UV spectral data [2]. It showed absorption band in its IR spectrum at 2815 cm1 for methoxyl and at 1605 and 1560 cm1 for aromatic double bond. 1H NMR spectrum (table 1) exhibited six proton multiplets (
2.66, 2.86 and 3.24) for three methylene protons, two ortho (
6.63 and 6.76), four ortho-meta (
6.89 and 7.20) coupled aromatic protons, three aromatic methoxyl groups (
3.75, 3.82 and 3.88) together with one proton double doublet (
4.34) which favoured the structure 1 for the alkaloid. The strong peaks at m/z 192 and 121 appeared in the mass spectrum due to cleavage of C-1 to C-a bond characteristic of benzylisoquinoline alkaloid suggested the presence of two methoxy groups in ring *Corresponding author. Email: [email protected] Natural Product Research ISSN 1478-6419 print/ISSN 1029-2349 online ß 2008 Taylor & Francis http://www.tandf.co.uk/journals DOI: 10.1080/14786410701592596

534

M. B. Pandey et al. 5 6

4a

3 2

7

CH3O

4

8

8a

OCH3

1

N R

α

1′

2′ 3′ 4′

6′ 5′

OCH3

1: R=H 3 : R = CH3 Figure 1.

Table 1.

500 MHz 1H NMR and

No. 1 2 3 4 4a 5 6 7 8 8a a 10 20 30 40 50 60 40 -OMe 7-OMe 8-OMe

13

The structures of 1 and 3.

C NMR (CDCl3 þ 10%CD3OD) spectroscopic data of alkaloid 1.

1

H NMR,
H (multi., J in Hz) 4.34 2.66 2.66 3.24 2.86 6.63 6.76

2.86 3.24 7.20 6.89 6.89 7.20 3.75 3.82 3.88

(1H, dd, 3, 10) (1H, m) (1H, m) (1H, m) (2H, m) – (1H, d, 8.3) (1H, d, 8.3) – – – (1H, m) (1H, m) – (1H, d, 8.6) (1H, d, 8.6) – (1H, d, 8.6) (1H, d, 8.6) (3H, s) (3H, s) (3H, s)

13

C NMR,
C 56.2 – 37.3 28.4 124.8 142.2 144.6 119.7 109.5 127.7 37.1 131.8 130.4 114.2 158.3 114.2 130.4 52.9 54.0 55.0

A and one methoxy group in ring C. The structure 1 for fuyuziphine was further supported by the methylation of 1 which gave N-methylated product of 1, identical to the reported synthetic compound 7,8-dimethoxy-2-methyl-1-(4-methoxy benzyl)1,2,3,4-tetrahydroisoquinoline (3) [3] (figure 1). 13CNMR data fits well with structure 1 for fuyuziphine (table 1).

3. Experimental 3.1. General experimental procedures Melting points were determined on a Toshniwal apparatus and are uncorrected. IR were recorded on Perkin–Elmer spectrophotometer model 720 in KBr pellet and UV on

Fuyuziphine, a new alkaloid from F. indica

535

Carry-14 spectrophotometer using spectral MeOH. 1H and 13C NMR were taken on 100, 400 and 500 MHz NMR on Bruker HX-90 in CDCl3 and CD3OD with TMS as internal standard. MS were performed on Kratos MS-30 and MS-50 mass spectrometer operation at 70 eV with evaporation of sample in the ion source at 200 . [a]D in CHCl3 at 25 was carried out on Perkin–Elmer polarimeter 141. CC: silica gel column (BDH, 60–120 mesh); TLC: silica gel G (Merck); solvents for TLC: CHCl3–MeOH (9 : 1). Analytical samples were dried routinely over P2O5 for 24 h in vacuo.

3.2. Plant material The plant material, Fumaria indica was collected from Mirzapur district, Uttar Pradesh, India and identified by Prof. N.K. Dubey, Department of Botany, Banaras Hindu University, Varanasi, India. Specimen sample is kept in the department.

3.3. Extraction and isolation Air dried powdered whole plant of F. indica (3.5 kg) was extracted with MeOH in a soxhlet extractor which on evaporation of the solvent gave brown coloured semi-solid (150 g). The MeOH extract was extracted with 7% aqueous citric acid. The acidic solution on basification with NH4OH and extraction with CHCl3 furnished the crude base fraction (13.5 g). It was chromatographed over SiO2 gel column eluting with solvents of increasing polarity. The eluants collected from CHCl3–MeOH (9 : 1) and (8 : 2) furnished respectively, the alkaloids, fuyuziphine (22 mg) (1) and (
)-a-hydrastine (32 mg) (2) [4].

3.3.1. Fuyuziphine (1). Compound 1 was crystallised from MeOH as colourless  amorphous powder; ½25 D –95 (c, 0.26, CHCl3); Rf 0.50 (CHCl3–MeOH, 9 : 1). It exhibited UV (MeOH) max (log "): 240 (1.82), 275 (1.65), 282 (1.55) nm; IR (KBr) max: 2815 (–OMe), 1605 and 1560 (Ar-double bond) cm1; 1H NMR data, see table 1; 13C NMR data, see table 1; HRMS, m/z (relative intensity%): 313.1678 ([M]þ) (5), 192 (100), 177 (35), 162 (25), 121 (24), 67 (22).

3.3.2. Methylation of fuyuziphine (1). Compound 1 (9 mg) was dissolved in methanol and stirred magnetically with formaldehyde (5 drops) and small quantity of sodium borohydride for 8 h. The reaction mixture was poured over crushed ice, stirred for about an hour and then extracted with chloroform. The chloroform extract was evaporated to dryness to give TLC single spot semi-solid as compound 3. It could not be crystallised with different organic solvents. The ethanolic solution of compound 3 was treated with equal volume of ethanolic HCl which gave hydrochloride salt of compound 3, m.p. 177–78 C. Mass spectrum of the free base exhibited molecular ion peak at m/z 327 ([M]þ) and other significant peaks at m/z 206, 191, 176, 121 were identical to 7,8-dimethoxy-2-methyl-1-(4-methoxybenzyl)-1,2,3,4-tetrahydroisoquinoline alkaloid (3) in MS data and melting point.

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3.3.3. ()-a-Hydrastine (2). Compound 2 was crystallised from MeOH as colourless  shining granules, m.p. 134–36 C; ½25 D
0 ; Rf 0.35 (CHCl3–MeOH, 9 : 1). It exhibited UV (MeOH) max: 218, 240 sh, 294, 320 nm; IR (KBr) max: 1740 (a,-unsaturated-lactone) cm1; 400 MHz 1H NMR (CDCl3):
2.30–3.03 (4H, m, Ar–CH2–CH2–N), 2.46 (3H, s, N–Me), 3.76 (3H, s, 40 -OMe), 3.88 (3H, s, 50 -OMe), 3.91 (1H, d, J ¼ 3 Hz, 1-H), 5.46 (1H, d, J ¼ 3 Hz, 9-H), 5.68 (1H, d, Jgem ¼ 1.2 Hz, 1-H of 6,7-O–CH2–O), 5.74 (1H, d, Jgem ¼ 1.2 Hz, 1-H of 6,7-O–CH2–O), 6.28 (1H, s, 5-H), 6.57 (1H, s, 8-H), 7.00 (1H, d, J ¼ 8 Hz, 30 -H), 7.25 (2H, d, J ¼ 8 Hz, 20 -H); 100 MHz 13C NMR (CDCl3):
66.0 (C-1), 51.0 (C-3), 29.0 (C-4), 125.0 (C-4a), 108.0 (C-5), 146.0 (C-6), 145.4 (C-7), 107.0 (C-8), 129.8 (C-8a), 80.8 (C-9), 140.7 (C-10 ), 118.0 (C-20 ), 118.0 (C-30 ), 152.0 (C-40 ), 147.2 (C-50 ), 118.9 (C-60 ), 100.5 (6,7-O–CH2–O), 56.0 (C-40 -OMe), 62.0 (C-50 -OMe), 44.5 (–NMe), 168.0 (>C¼O); HRMS, m/z (relative intensity %): 383.1345 ([M]þ, C21H21NO6) (4), 193 (5), 190 (100), 188 (8), 175 (5), 165 (3).

Acknowledgements The authors are thankful to Dr. H.C. Jha, Physiologisch Chemisches Institut and Prof. G. Ru¨cker, Pharmazentisches Institut, University of Bonn, Germany for spectral analysis and supply of authentic samples.

References [1] R.N. Chopra, S.L. Nayar, I.C. Chopra. Glossary of Indian Medicinal plants, p. 122, CSIR, New Delhi (1956). [2] M. Shamma. The Isoquinoline Alkaloids, p. 83, Academic Press, New York and London (1972). [3] D. Dwurma-Badu, J.S.K. Ayim, T.T. Dabra, M.M. El-Azizi, P.L. Schiff Jr., D.J. Slatkin, J.E. Knapp. J. Nat. Prod., 46, 342 (1983). [4] G. Blasko, D.J. Gula, M. Shamma. J. Nat. Prod., 45, 105 (1982).

Natural Product Research, Vol. 22, No. 6, 15 April 2008, 537–546

Gynecological efficacy and chemical investigation of Vitex agnus-castus L. fruits growing in Egypt N. A. IBRAHIMy, A. S. SHALABYz, R. S. FARAGx, G. S. ELBAROTYx, S. M. NOFAL{ and E. M. HASSAN*z yPharmacognosy Department, National Research Centre, Cairo, Egypt zMedicinal and Aromatic Plants Department, National Research Centre, Cairo, Egypt xFaculty of Agriculture, Biochemistry Department, Cairo University, Cairo, Egypt {Pharmacology Department, National Research Centre, Cairo, Egypt (Received 3 December 2006; in final form 25 July 2007) Flavonoid glycosides, orientin and apigenin 3, 8-di-C-glycosides in addition to, iridoid compound, aucubin were isolated from the ethanolic extract of Vitex agnus-castus fruits. Their structures were identified on the basis of the spectroscopic data. The estrogenic activity of the ethanolic extract in two dose levels 0.6 and 1.2 g kg
1 per body weight (b.w.) was studied by the vaginal smear, and uterine weight methods for normal and ovariectomized female rats. The extract induced significant increase in the uterine weight of ovariectomized rats at two dose levels comparable to that of control group. The percentages of the total average number of scores were increased significantly too. Significant increases in plasma progesterone and total estrogens levels were shown at the two dose levels when compared to that of control group. On the other side, the extract induced significant reduction in lutinizing and plasma prolactin hormones. Keywords: Vitex agnus-castus fruits; Verbinaceae; Orientin; Apigenin 3; 8-di-C-Glycoside; Aucubin; Gynecological efficacy

1. Introduction Chaste tree (Vitex agnus-castus Linn.) Verbenaceae is an ornamental shrub or small tree widely distributed in Mediterranean costal region and central Asia [1,2]. Several authors reported that different parts of chaste tree are traditionally used for various medicinal purposes, as carminative, antispasmodic, antiseptic, diuretic, stomachic and used for treatment of headache. The most interesting reported effect for this tree fruit was its ability to balance the female sex hormones and counteracts estrogen reduction and in the treatment of hyperprolactinemia [3,4]. Various flavonoids were isolated from the root bark of V. agnus-castus and Vitex rotundifolia [5–8]. Labdan diterpene alkaloid was isolated from the fruits of V. agnus-castus by Li et al. [9]. Ketosteroid hormones were isolated from the flowers and the leaves of V. agnus-castus [10]. *Corresponding author. Tel.: þ20127152706;. Fax: þ2023370931. Email: [email protected]. Natural Product Research ISSN 1478-6419 print/ISSN 1029-2349 online ß 2008 Taylor & Francis http://www.tandf.co.uk/journals DOI: 10.1080/14786410701592612

538

N. A. Ibrahim et al.

As replacement estrogenic therapies, which may be needed in some cases of menopause, have a risk of developing malignancy. The positive correlation between endogenous estrogen hormone levels and risk of breast cancer, has been supported this concept [11]. This directed us towards the natural estrogenic substances hoping to avoid this risk.

2. Materials and methods 2.1. Plant materials Plant samples of chaste tree (V. agnus-castus) were collected from El-Sadat city, Menofyia governorate. The fruits were collected from the trees early in the morning, at the end of flowering stage which took place in November. Samples of this plant were subjected to botanical identification in the Orman Botanical Garden, Giza. The collected samples (mainly the fruiting tops) were air-dried at 27  3 C, powdered (30 meshes) and kept in paper bags in a desiccator’s over anhydrous calcium chloride till chemical analyses. 2.2. Drug . Biochemical kits from Sigma chemical company (St. Louis, USA). . Estradiol benzoate (Folone 0.1 mg 0.1 mL
1 in oily solution), from Miser Co. for pharm.IND.A.ACairo-Egypt. . Olive oil. 2.3. Animals Female albino rats, weighing 150–200 g and mice weighing 25–30 g were obtained from the animal house of the National Research Centre. Standard diet was provided and water available adlibitum. All animal procedures were performed after approval from the ethics committe of the National Research Centre, and in accordance with the recommendation for the proper care and use of laboratory animals (NIH publication No. 85–23/revised1985). 2.4. Apparatus UV spectrophotometer, UV lamp 336 and 254 nm, Shimadzu at wave length ranged between 190 and 900 nm, connected with IBM computer. 1H NMR analysis using Joel GLM, EX 300 MHz, Germany using dimethyl sulfoxide (DMSO). Mass spectra were carried out on Finnigan SS Q 700 spectrometer, 70 eV. Thin Layer Chromatography (TLC) silica gel plate (F254, Fluka) and polyamide ready made plates (Merck). Diagnostic shift reagents using, sodium methoxide, aluminum chloride, hydrochloric acid, sodium acetate and boric acid. 2.4.1. Solvent systems. 1-Butanol : acetic acid : water (4 : 2 : 1, v/v/v). 2-Ethyl acetate : methanol : H2O (77 : 15 : 8, v/v/v). 3-chloroform : methanol : water (15 : 7 : 0.5, v/v/v).

Gynecological efficacy and chemical investigation of V. agnus-castus

539

3. Experimental 3.1. Isolation of the flavonoids The air-dried powdered fruits of chaste tree (500 g) were exhaustively extracted with ethanol; the dried crude extract was dissolved in distilled water (250 mL). The aqueous solution was successively extracted with chloroform, ethyl acetate and finally with butanol. The butanol extract was evaporated to dryness under vacuum, and then the residue was dissolved in a small quantity of methanol, and examined by polyamide ready made plates (Merck) using solvent systems 1 and 2, respectively, and examined by UV light at 366 nm before and after exposure to NH3 vapour, Rf value was calculated. A preparative polyamide TLC plates were used for isolation of the major flavonoids, using the same two different solvent systems. Each band was eluted with methanol, and purified by Sephadex LH-20. The identification of the isolated compound was carried out using the UV spectrophotometer, by the addition of certain diagnostic shift reagents (table 1), and confirmed by carrying out 1H NMR analysis.

3.2. Isolation of iridoid The air-dried fruit powder of chaste tree (250 g) was exhaustively extracted with ethanol (70%), evaporated to a small volume. Then, the extract was successively extracted with chloroform, ethyl acetate and finally, with n-butanol [12]. The butanolic extract was concentrated under vacuum to a small volume, and chromatographed on a silica gel plate using solvent system (2). Detection was carried out by spraying the plates with 10% H2SO4. One major compound was detected and isolated by preparative TLC using the previously mentioned solvent system and confirmed its purity using solvent system 3. The isolated compound was identified by spectral analysis.

3.3. Determination of LD50 of ethanolic extract of chaste tree fruits The LD50 of the extract was determined using mice, weighing 25–30 g of both sexes, were picked randomly and divided into equal groups, each of ten mice. Groups were orally administered successive doses of the tested extract. Twenty four hours later, the mortality in each group was observed. Deaths within this period were recorded and the LD50 was determined according to Laurence and Bacharach [13].

3.4. Determination of estrogenic activity Ovarictomy in rats was performed through lateral incision in both flanks under ether anesthesia by inhalation, and then the ovariectomized rat were left for 3 weeks before treatment [14]. Female albino rats were kept under balanced diet, and exposed to light for a constant time every day to ensure that light factor would not alter plasma levels of gonadohormones. Animals were divided into two major groups, normal and ovariectomized female rats, both normal and ovariectomiezed rats were subdivided into four subgroups (eight rats each); the 1st subgroup acted as control group and was treated with saline (1 mL rat
1), 2nd and 3rd subgroups were treated with ethanol

540

N. A. Ibrahim et al.

extract of chaste tree fruits, in dose of 0.6 and 1.2 g kg
1 and the 4th was treated with Estradiol 0.1 mg 0.1 mL
1 per rat per day. All groups were treated orally twice daily for 5 days. For vaginal smear, all the ovariectomized rats were smeared on the evening of the third day, the morning and evening of the fourth day and in the morning of the fifth day. Smears were fixed on slides by gentle heat and stained with 1% aqueous methylene blue and examined microscopically. The method stated by Sharaf [15] in which numerical values are given to the cells in each smear was used to estimate the results. After the period of the experiment, blood samples were collected from the retroorbital venous plexus through the eye canthus under ether anesthesia [16] then centrifuged to obtain serum, total serum estrogens, progesterone, follicle stimulating hormone (FSH), latinizing hormone (LH), and prolactin hormones were estimated according to Abraham [17] using biochemical kits, Then female rats were weighed, sacrificed and the uteri and ovaries were dissected, dried and weighed to be registered relatively per 100 g body weight. Any deviation from the normal weight of the negative control was recorded as the effect of the tested substance [18,19].

3.5. Statistical analysis Results are expressed as mean  SE. Differences between vehicle control and treatments groups were tested using one-way-analysis of variance (ANOVA) followed by the least significant differences (LSD). Methods of statistical analysis were done according to Armitage [20].

4. Results and discussions 4.1. Identification of the ethanolic extracts constituents Two flavonoids were isolated by TLC and identified by UV spectra at the region 200–400 nm in absolute spectroscopic methanol and after adding chemical shift reagents. The identification of the isolated compounds was confirmed by 1H NMR analysis and by comparison with the published data. 4.1.1. Compound 1. Orientin (luteolin 8-C-D-glucopyranoside). It is a bright yellow amorphous powder (12 mg), Rf values 0.40 and 0.60 using solvent systems 1 and 2, respectively. It was soluble in methanol and water, and insoluble in petroleum ether, chloroform and benzene. The UV spectrum (table 1) showed a typical orientin spectrum. max in MeOH showed two absorption maxima at 350 nm for band I and 258 nm for band II. A large bathochromic shift (up to 50 nm), in band I with increased intensity with NaOMe was observed referring to the presence of free 40 -OH. Also, an orthodihydroxy B-ring and 5,7-dihydroxy A-ring was expected from the AlCl3 and AlCl3/HCl UV spectra. A free 7-OH group was existed in small bathochromic shift (14 nm) in band II on addition of NaOAc reagent [21]. 1H NMR (300 MHz, DMSO, d6): ppm 7.5 (1H, dd, J ¼ 8.1 and 2.4 Hz, H-60 ), 7.23 (1H, d, J ¼ 2.4 Hz, H-20 ), 6.31 (1H, S, H-3), 6.08 (1H, S, H-6), 4.54 (1H, d, J ¼ 9.6 Hz, H-100 ), 4.09 (1H, t-like, J ¼ 9.4 Hz, H-200 ), and 3.9–3.0 m (remaining sugar hidden by H2O-signal). From the above

Gynecological efficacy and chemical investigation of V. agnus-castus Table 1.

541

UV spectra of isolated flavonoids of chaste tree fruits with diagnostic shift reagents. Compounds

Reagents MeOH NaOMe AlCl3 AlCl3/HCl NaOAc NaOAc/H3BO3

mentioned data compound glucopyranoside).

I

II

350, 270sh, 258, 242 4400, 267, 208 4400, 351, 276 388, 367, 278 379, 325,272 375, 363, 211

331, 309sh, 272 4400, 329, 279 382, 350, 303,278 381, 350, 303,278 365, 303, 277 336, 303, 272

1

was

identified

as

orientin

(luteolin

8-C-D-

4.1.2. Compound 2. (Apigenin 3, 8-di-C–glycoside) Amorphous yellow powder (9 mg) from methanol, soluble in MeOH and H2O, Rf values of 0.51 and 0.82 using two different solvent systems 1 and 2. UV spectrum with the different diagnostic shift reagents represented in table 1; two absorption maxima at (331 nm) for band I and 272 nm for band II. A high bathochromic shift (up to 70 nm) in band I with increased intensity with NaOMe indicating the presence of free 40 -OH. A bathochromic shift (18 nm) in band I with AlCl3 and did not change with HCl, referring the presence of free 5-OH. Small bathochromic shifts (5 nm) in band II with NaOAc, indicating the presence of free 7-OH [21]. 1H NMR analysis in DMSO at ppm 7.73 (2H, d, J ¼ 8.7 Hz, H-20 /60 ), 6.84 (2H, d, J ¼ 8.7 Hz, H-30 /50 ), 6.29 (1H, S, H-6), 4.49 (1H, br S, H-1 Rh), 4.47 (1H, d, hidden by 1-H Rh signal, H-1 Glc), and 0.85 (3H, d, J ¼ 6 Hz, CH3 Rh). On the basis of its chromatographic properties, UV and 1H NMR spectral data, compound 2 was tentatively identified as Apigenin 3, 8-di-C-glucoside and its glycoside moieties are rhamnose and glucose. Based on all the above mentioned data, it could be concluded that chaste tree fruits contained two flavonoidal compounds identified as orientin (letulin 8-C-D-glucopyranoside) and apigenin 3, 8-di-C-glycoside, respectively. It is noting that the compounds 1 and 2 were identified for the first time. Ono [22] and Chawla et al. [23] isolated artemetin from the unsaponifiable matter of V. negundo oil. Four flavonoids were isolated from chaste tree by Hirobe et al. [24], which were identified as letulin 6-C-(400 methyl-600 -O-trans-caffeoyl glucoside), letulin 6-C-(600 -O-transpenduletin and 5,30 -dihydroxy-6,7,40 -triethoxyflavone were isolated from V. rotundifolia [24]. Leitao and Monache [25] isolated 200 -O-caffeoyl orientin from Vitex. polygama leaf. Recently, Ono et al. [22] isolated letulin 6-C-(200 -O-trans-caffeoyl glucoside), and letulin 7-O-(600 -benzoyl glucoside) from V. rotundifolia. Also, mentioned letulin, crysosplenol D, Ono et al. [26] isolated artemetin from V. rotundifolia. The flavonoid compounds such casticin, orientin and apigenin are potential pharmaceutical values. For instance, You et al. [27] reported that cacticin may be a general inhibitor of lymphocyte proliferation and its effect due to the inhibition of T-mitogen which induced lymphocyte proliferation. Also, apigenin induced the same effect. So, these compounds could be used for treating inflammatory/immunorelated disease or lymphomas in vivo. Ko et al. [8] mentioned that polymethoxy flavonoid compounds, such as casticin (vitexicarpin) isolated from V. rotundifolia, had an antitumor activity, which inhibit cell proliferation of many tumor cell lines in vitro.

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4.1.3. Compound 3. (aucubin)White amorphous powder (12 mg), freely soluble in methanol and water. Mass spectrum shows a molecular ion (Mþ) at m/z 346, which corresponds to the molecular formula C15H22O9 and the base peak was at m/z 91 (100%) Also, several peaks were present at m/z 166, 148, 131, 120, 96, 85, 73 and 57. 1 H NMR spectrum shows the main expected signals of aucubin. The spectrum shows signals at 2.62 – 2.67 (1H, m, H-5), 2.89 (1H, t-like, J ¼ 7.3 Hz, H-9), 3.64 (1H, dd, J ¼ 5.3 and 11.9 Hz, H-60 ), 3.85 (1H, dd, J ¼ 1.8 and 11.9, H-10 ), 4.20 (1H, d, J ¼ 15.4 Hz, H-10), 4.32–4.43 (1H, m, H-6), 4.67 (1H, d, J ¼ 8.1 Hz, H-10 ), 4.94 (1H, d, J ¼ 7.0 Hz, H-1), 5.09 (1H, dd, J ¼ 3.9 and 6.1 Hz, H-4), 5.77 (1H, br s, H-7) and 6.30 (1H, dd, J ¼ 1.8 and 5.9 Hz, H-3). IR spectrum (KBr, cm
1), shows the following bands at 3440 for (OH), 1460 for (CH3) and 1230 for (CH). From the previous spectral data, it could be concluded that the compound 1 was identified as aucubin. These results are in line with the data of Kuruuzum et al. [28], who isolated aucubin from the fruits of chaste tree. Kooiman [29] mentioned that agnuside and aucuboside were found in several species of Vitex. For instance, Isao et al. [30] isolated eurostoside and agnuside from V. rotundifolia leaves. Suksamrarn et al. [31] isolated, limoniside and agnuside from the dry bark of V. limonifolia. While, Suksamrarn et al. [32] reported that pedunculariside and agunside were found in Vitex peduncularis bark. On the other hand, Iwagawa et al. [12] reported that the leaves of Vitex Cannabifolia, contained aucubin and this result agreed with that reported in this study.

5.1. Determination of LD50 of ethanolic extract of chaste tree fruits The LD50 of the tested extract was found to be 12.5 g kg
1 per b.w. On the other hand, oral administration of the tested extract to mice induced no obvious toxic effects, and all the treated animals were still alive after 24 h.

5.2. Endocrinological efficacy of ethanolic extract of chaste tree fruits Premenstrual syndrome (PMS) is a complex combination of symptoms characterized not only by psychological changes including irritability, aggression, tension, anxiety and depression but also somatic changes such as a fluid retention, breast tenderness, headache and feeling of bloating weight which is associated with hyper secretion of prolactin. Traditionally, PMS is treated with extract prepared from fruits of chaste tree [33]. Studies were carried out to evaluate, the optimum hormonal characteristics of chaste tree extracts especially 70% ethanolic extracts on the following parameters.

5.3. A-estrogenic effect of chaste tree fruits 5.3.1. Vaginal smears. The results in table 2 revealed that the administration of chaste tree extract in two dose level 0.6 and 1.2 g kg
1 for 5 days to ovariectomized rats induced estrogenic like effect. The percentage of the total average number of scores of cornified cells were increased with 332 and 432%, respectively, referred to that of untreated ovariectomized rats. Standard estradiol hormone increased this score by 625%, referred to the control rats. The responsiveness of rat vagina to exogenous estrogens was mainly regulated by the presence of estrogen binding receptors in the

Gynecological efficacy and chemical investigation of V. agnus-castus Table 2.

543

The estrogenic effect of the ethanol extract of chaste tree fruits comparable to that of estradiol on ovariectomized rat’s vagina. Average number of scores*

Animal groups
1

Control (1 mL saline kg b.w.) Ethanol ext. (0.6 g kg
1 b.w) Change (%) Ethanolic ext. (1.2 g kg
1 b.w.) Change (%) Estradiol (1 mg kg
1 b.w.) Change (%)

1st Smear

2nd Smear

3rd Smear

4th Smear

Total average

0.79  0.01 1.83  0.02

0.73  0.01 3.00  0.03

0.63  0.02 2.60  0.10

0.60  0.12 4.50  0.20

2.66  0.11

4.50  0.15

2.70  0.32

4.80  0.10

4.20  0.21

5.00  0.22

5.20  0.15

5.60  0.23

0.69a  0.09 2.98b  0.11 þ332 3.67b  0.11 þ432 5.00c  0.59 þ625

The value in the column followed by the same letter is not significantly different at LSD50.05 ¼ 0.9. *The value was representing the average of eight rats (SD). Percentage of change was calculated as regard the value of mean effect of ovariectomized control group.

cystosol of rat vagina [34]. Estrogen receptors are derived from two different gene resources referred to estrogen receptor alpha (ER-) and estrogen receptor beta (ER- ). Both of them bind to a number of estrogen response elements present in endogenous hormone mainly, and display different patterns of affinities from naturally occurring hormones [35]. These results agreed with those obtained by Jarry et al. [34], who reported that the flavonoids prevented hot flushes and vaginal dryness in postmenopausal women. Thus, the phytoestrogen flavonoids may be functioning in a manner similar to estradiol and produce a typical and predictable estrogenic response, when administrated to animal causing vaginal cornification and uterine hypertrophy in intact immature rats. One has to recall that; apigenin glycoside was isolated and identified in this study. In this respect, Jarry et al. [34] stated that the flavonoid apigenin was the most active ERSS-selective phytoestrogen in chaste tree. 5.3.2. Effect of chaste tree fruits extracts on body, uterine and ovaries weights. Data obtained from this experiment indicated that the administration of ethanol extract at two dose levels 0.6 and 1.2 g kg
1 b.w. in normal rats exhibited a significant increase in the ovarian weight by 40% referred to that of untreated rats. It was also observed that the administration of the extract caused a significant increase in the uterine weights of ovariectomized rats by 229 and 243%, respectively, when compared to ovariectomized control group, no effect was observed on the uterine weight of normal rats (table 3). Yet on contrary to the expected results no body weight gain was observed which considered a good sign. The significant increase in uterine weight is presumably due to the estrogen like activity in chaste tree extract is confirmed by the results obtained by Russell et al. [36].

5.4. B-Hormonal levels of Estrogen, Progesterone, FSH and LH in treated groups As shown in tables 4 and 5, the tested doses (0.6 and 1.2 g kg
1 per b.w.) of extracts induced a significant increase in the plasma levels of total estrogens of normal and ovariectomized rats compared to that of untreated group (control) by 24.34, 115.57% and 11.99, 23.62%, respectively. Results showed significant increase in plasma

544 Table 3.

N. A. Ibrahim et al. Effect of the ethanolic extract of chaste tree fruits on the body, uterine and ovaries weights (g)* of normal and ovariectomized rats. Normal rats

Animal groups Control (1 mL saline kg
1 b.w.) Ethanolic ext. (0.6 g kg
1 b.w.) Change (%) Ethanolic ext. (1.2 g kg
1 b.w.) Change (%) Estradiol (1 mg kg
1 b.w.) Change (%) LSD

Ovariectomized rats

Body weight

Uterine weight

Ovaries weight

Body weight

Uterine weight

152a  8.34 146a  11.70
3.95 160a  15.69 þ5.26 – – 14.92

0.21a  0.03 0.20a  0.07
4.76 0.21a  0.06 0.0 – – 0.083

0.05a  0.01 0.07b  0.01 þ40.0 0.07b  0.01 þ40.00 – – 0.014

167.67a  8.8 174.17a  7.9 þ3.88 148.17b  128
11.63 167.83a  23.4 þ0.10 14.92

0.07a  0.02 0.23b  0.09 þ229 0.24b  0.07 þ243 0.48c  0.13 þ586 0.083

The value in the column followed by the same letter is not significantly different at LSD (0.05). Percentage of change was calculated as regard the value of mean effect of normal and ovariectomized control group. *Each value refers to the average of eight rats (SD).

Table 4.

Effects of ethanol extract of chaste tree fruits on the plasma levels of estrogen, progesterone, FSH, LH and prolactin in normal rats.* Ethanolic extract

Hormones Estrogen Change (%) Progesterone (ng mL
1) Change (%) FSH (IU L
1) Change (%) LH (mIU mL
1) Change (%) Prolactin (ng mL
1) Change (%)

Control (10 mL saline kg
1 b.w.)

Estradiol benzoate (0.1 mg kg
1 b.w.)

(0.6 g kg
1 b.w.)

(1.2 g kg
1 b.w.)

45.60a  10.85

162.50b  23.61 þ256.36 6.90a  1.44 þ23.21 0.88a  0.08
14.56 0.80a  0.24
13.98 2.10a  0.54 þ16.67

56.70a  8.16 þ24.34 7.40a  1.87 þ32.14 1.10b  0.20 þ6.80 0.87a  0.23
6.45 2.10a  0.45 þ16.67

98.30c  13.29 þ115.57 9.90b  2.84 þ76.79 1.15b  0.18 þ11.65 0.77a  0.23
17.20 2.20a  0.19 þ22.22

5.60a  0.20 1.03ab  0.14 0.93a  0.80 1.80a  0.22

Small letters indicates significant difference of effects between the tested groups. Any two means sharing the same letters is not significantly different at p50.05 (LSD ¼ 7.55, 2.45, 0.18, 0.24 and 0.47, respectively). Percentage of change was calculated as regard the value of mean effect of normal control group. *Each value refers to the average of eight rats (SD).

progesterone level in normal and ovariectomized-rats treated with 1.2 g kg
1 per b.w. (76.79 and 113.64%), Results also revealed that the tested extract had no significant change on plasma level of FSH of normal or ovariectomized rats, and also on plasma LH level in normal rats. However, the plasma LH level of ovariectomized-rats treated with the ethanolic extract at 0.6 and 1.2 g kg
1 per b.w. significantly decreased by 15.79, 17.90 respectively, in comparison to that of ovariectomized control rats. The estrogenic effect of chaste tree was confirmed by, the plasma levels of endogenous estrogens, progesterone, FSH, LH and prolactin. The increase in the plasma levels of estrogen and progesterone obtained in the present work indicates that the tested extracts secrete substantial amounts of androgen. These androgens are converted to estrogen [37,38].

Gynecological efficacy and chemical investigation of V. agnus-castus

545

Table 5. Effects of ethanolic extract of chaste tree fruits on the plasma levels of estrogen, progesterone, follicle stimulating hormone (FSH), luteinizing hormone (LH) and prolactin in ovariectomized rats.* Ethanolic extract Hormone

Ovariectomized control Estradiol benzoate (10 mL olive oil kg
1 b.w.) (0.1 g kg
1 b.w.) (0.6 g kg
1 b.w.) (1.2 g kg
1 b.w.)

Estrogen Change (%) Progesterone (ng mL
1) Change (%) FSH (IU L
1) Change (%) LH (mIU mL
1) Change (%) Prolactin (ng mL
1) Change (%)

54.20a  12.01 Reference 4.40a  2.88 Reference 1.01a  0.16 Reference 0.95a  0.18 Reference 2.30a  0.20 Reference

81.70b  22.29 þ50.74 10.80b  2.83 þ145.46 0.95a  0.14
5.94 0.58b  0.12
38.95 1.70b  0.39
26.09

60.70ac  13.14 þ11.99 5.80a  1.99 þ31.82 0.98a  0.13
2.97 0.80abc  0.24
15.79 1.60b  0.20
30.44

67.00ac  12.2 þ23.62 9.40b  2.28 þ113.64 1.03a  0.14 þ1.98 0.78abc  0.23
17.90 1.43b  0.05
37.83

Small letters indicates significant difference of effects between the tested groups. Any two means sharing the same letters is not significantly different at p50.05 (LSD ¼ 7.55, 2.45, 0.18, 0.24 and 0.47, respectively). Percentage of change was calculated as regard the value of mean effect of ovariectomized control group. *Each value refers to the average of eight rats (SD).

5.5. Prolactin hormone The effect of oral administration of ethanolic extract on prolactin level of normal and ovariectomized rats, are shown in tables 4 and 5. Significant decrease in plasma prolactin level by (
30.44,
37.83%) of ovariectomized rats in comparison to control group. While, no significant changes in plasma prolactin level of normal rats. This peripheral conversion of androgen accounts for this circulating estrogen, as only free estrogen, which can stimulate the target organs of the reproductive tract (uterus and vagina) in women and which can exert feedback effect on the CNS, hypothalamic pituitary unit to influence its gonadotropins secretion [39]. Here again, the hormonal effect was confirmed by examining the effect of chaste tree extracts on the plasma FSH, LH and prolactin. The present results agreed with that obtained by Jarry et al. [40] who stated that subcutaneous injection of chaste tree extract for 4 days gave indirect signs of putative inhibition of LH and prolactin levels in serum. Prolactin secretion from the anterior pituitary gland is under the dual control of hypothalamic factor, which stimulates prolactin release and the catecholamine dopamine (DA), which acts as a prolactin inhibitory factor. Prolactin has numerous targets in the body, among them the mammary gland and corpus luteum. While a hyper secretion of prolactin causes fertility disorders [41], premenstrual syndrome (PMS), like mastodynia is associated with the latent hyper secretion of prolactin and misalign [42]. Latent hyperprolactinemia is often manifested during the time of decreasing progesterone, and estradiol values prior to menstruation [43,44]. The mode of action of chaste tree extracts in the treatment of menstrual disorders was attributed to its dopaminergic effect. Constituents of the plant occupy, the D2-receptor in lactotropic pituitary cells and thus, inhibit stimulation to release prolactin [33,45]. Lucks et al. [46] mentioned that the essential oil of chaste tree showed a medicinal action on menopausal symptoms. Jarry et al. [34] reported that apigenin was the most active phytoestrogen, which binds to the estrogen receptor. Liu et al. [47] mentioned that linoleic acid of V. agnus-castus can bind to estrogen receptors and induce certain

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estrogen inducible genes. Hence, it is concluded that dopaminergic compounds present in chaste tree are clinically the important compound, which improve premenstrual mastodynia and possibly other symptoms.

References [1] L.H. Bailey. The Standard Cyclopedia of Horticulture, Vol. III-P-Z, p. 3480. The MacMillan Co., New York (1947). [2] R.K. Brummitt. Vascular Plants: Families and Genera, P. 690, Royal Botanic Gardens, Kew (1992). [3] J.M. Sorensen, S.T Katsiotis. J. Essent. Oil Res., 11, 599 (1999). [4] E. Mancho. Adv Nurse Pract., 13, 43 (2005). [5] C. Hirobe, Z. Qiao, K. Takeya, H. Itokawa. Phytochem., 46, 521 (1997). [6] E. Okuyama, S. Fujmori, M. Yamazaki, T. Deyama. Chem. Pharm. Bull., 46, 655 (1998). [7] K.M. You, Y.G. Jong, H.P Kim. Arch. Pharm. Res., 22, 18 (1999). [8] W.G. Ko, T.H. Kang, S.J. Lee, N.Y. Kim, D.H. Sohn, B.H. Lee. Food Chem. Toxcol., 38, 86 (2000). [9] S. Li, H. Zhang, S. Qui, X. Niu, B.O. Santarsiero, A.D. Mesecar, H.H. Fong, N. Farnsworth, H. Sun. Tetrahydron Lett., 43, 5131 (2002). [10] M. Saden-Krehula, D. Kustrak, N. Blazevic. Planta Med., 56, 547 (1990). [11] G.A. Colditz. J. Women Health, 8, 357 (1999). [12] T. Iwagawa, A. Nakahara, A. Miyauchi, H. Okamura, M. Nakatani. Rep. Fac. Sci., 26, 61 (1993). [13] D.R. Laurence, A.L. Bacharach. Peget and Barnes; Evaluation of Drug Activities Pharmacometrics, Vol. 1, P. 135, Academic Press, London and New York (1974). [14] P. De Moor, M. Rdam-Heykn, H. Van Baelen, G. Verhoeven. J. Endocrinol., 67, 71 (1975). [15] A. Sharaf, S. Negm. Qual. Plant Mater. Veg., XXII(3–4), 249 (1973). [16] O.W. Schlam. Veterinary Hematology, 1st Edn, Lec and Febiger, Philadelphia. (1961). [17] G.E. Abraham. Radioimmunoassay of Estrogen, Progesterone, FSH, and LH. Hand book of Radioimmunoassay System in Clinical Endocrinology, 2nd Edn, P. 475, Marcel Dakkar, New York (1981). [18] J.S. Evans, R.F. Varney, F.C. Koch. Endocrinology, 8, 747 (1941). [19] S.A. El-Batran. Farmacology, 140, 119 (2001). [20] P. Armitage. Statistical Methods in Medical, 1st Edn, P. 147, Blackwell Scientific Puplications, London (1971). [21] T.J. Mabry, K.R. Markham, M.B. Thomas. The Systematic Identification of Flavonoids, Springer-Verlag, Berlin, Heidelberg, New York (1970). [22] M. Ono, H. Sawamura, Y. Ito, K. Mizuki, K. Nohara. Phytochemistry, 55, 873 (2000). [23] A.S. Chawla, A.K. Sharma, S.S. Handa, K.L. Dhar. Indian J. Chem., 30, 773 (1991). [24] E. Okuyama, K. Suzumura, M. Yamazaki. Nat. Med., 52, 218 (1998). [25] S.G. Leitao, F.D. Monache. Phytomedicine, 49, 2167 (1998). [26] M. Ono, Yanaka, M. Yamamoto, Y. Ito, T. Norhara. J. Nat. Prod., 65, 537 (2002). [27] K.M. You, K.H. Son, H.W. Chang, S.S. Kang, H.P. Kim. Planta Med., 64, 546 (1998). [28] A. Kuruuzum, K. Stroch, O. Demirezer, A. Zeeck. Phytochemistry, 63, 959 (2004). [29] P. Kooiman. Acta Botan. Neerl., 24, 459 (1975). [30] K. Isao, I. Masaaki, O. Yoshiko, F. Tamayo, K. Nobusuke. Phytochemistry, 27, 611 (1988). [31] S. Suksamrarn, S. Kumcharoen, A. Suksamrarn. Planta Med., 65, 392 (1999). [32] A. Suksamrarn, S. Kumpun, K. Kirtikra, B. Yingyong, S. Suksamrarn. Planta Med., 68, 72 (2002). [33] D.A. Roeder. Zeitschr Phytother., 15, 157 (1994). [34] H. Jarry, B. Spengler, A. Porzel, J. Schmidt, W. Wuttke, V. Christoffel. Planta Med., 69, 945 (2003). [35] S.M. Hyder, C. Chiapetta, G.M. Staneel. Biochem. Pharmacol., 57, 597 (1999). [36] H. Russell, G.S. Hicks, A. Low, J.M. Shepherd, C.A. Brown. Am. J. Med. Sci., 324, 185 (2002). [37] A. Schindler, A. Epert, E. Friedrich. J. Clin. Endocr. Metab., 35, 627 (1972). [38] P.C. MacDonald, J.M. Grodin, C.D. Edman, F. Vellios, P.K. Suteri. Obstet Gynac., 47, 644 (1976). [39] Y. Nakai, T. Plant, D. Hess. Endocrine, 102, 1008 (1978). [40] H. Jarry, S. Leaonhardt, W. Wuttke, B. Behr, C. Gorkow. Zeitsch. Phytother., 12, 77 (1991). [41] H. Jarry, S. Leonhard, C. Gorkow, W. Wuttke. Exp. Clin. Endocrin., 102, 448 (1994). [42] W. Wuttke, H. Jarry, V. Christoffel, B. Splenger, D. Seidlova-Wuttke. Phytomedicine, 10, 348 (2003). [43] R.M. Maclood. Endocrine, 85, 916 (1969). [44] U. Halbreich, M. Assad, M. Ben-David, R. Bornstein. Lancet, 15, 654 (1976). [45] W. Wuttke, C. Gorkow, H. Jarry. Phtopharmaka Forsch. Klinischer Anwendung., 16, 82 (1995). [46] B.C. Lucks, J. Sorensen, L. Veal. Complementary Ther. Nurs. Midwifery, 8, 148 (2002). [47] J. Liu, E. Burdette, Y. Sun, S.M. Deng, M. Schlecht, W. Zheng, D. Nikolic, G. Mahady, R.B. Van Breemen, H.H. Fong, J.M. Pezzzuto, L. Bolton, N.R. Farnsworth. Phytomedicine, 11, 18 (2004).

Natural Product Research, Vol. 22, No. 6, 15 April 2008, 547–553

Ionophoric properties of atropine: complexation and transport of Na1, K1, Mg21 and Ca21 ions across a liquid membrane LATIFA RABI, ADNANE MOUTAOUAKKIL* and MOHAMED BLAGHEN Unit of Bio-industry and Molecular Toxicology, Laboratory of Microbiology, Biotechnology and Environment, Faculty of Sciences Aı¨ n Chock, University Hassan II – Aı¨ n Chock, Km 8 route d’El Jadida, BP. 5366 Maˆarif 20100 Casablanca, Morocco (Received 5 February 2007; in final form 25 July 2007) The activity of atropine on the complexation and transport of Naþ, Kþ, Mg2þ and Ca2þ ions across a liquid membrane was investigated using a spectrophotometric method. Atropine is a natural drug that blocks muscarinic receptors. It is a competitive antagonist of the action of acetylcholine and other muscarinic agonists. Atropine is shown to extract Naþ, Kþ, Mg2þ and Ca2þ ions from an aqueous phase into an organic one with a preference for Ca2þ ions. According to a kinetic study, divalent cations (Mg2þ and Ca2þ) are more rapidly transported than monovalent ones (Naþ and Kþ). In both complexation and transport, the flux of the ions increases with the increase of atropine concentration. Atropine might act on the membrane permeability; its complexation and ionophoric properties shed new lights on its therapeutic proprieties. Keywords: Atropine; Complexation; Transport; Ionophore; Mono- and divalent cations

1. Introduction The study of cation–ionophore binding is one of the fundamental subjects for understanding molecular recognition [1]. It is well known that some kinds of peptides and natural ionophores, such as monensin and valinomycin, play important roles as ion carriers in the typical metal ion transport system [2]. This later is one of the fundamental mechanisms for accumulation of energy, and the medium by which muscle is controlled and information passed on in living systems [2]. Atropine is an anti-cholinergic drug that is derived from the plant Atropa belladonna [3]. It is a widely used as a competitive antagonist against the activation by acetylcholine of the cholinergic muscarinic receptors [4] and also shown to have a parasympatholytic effect [5]. In vitro, for effective pharmacological blockade of muscarinic receptors, atropine is usually used within the range of 10 nM to 10 M. It is also used therapeutically, often by subcutaneous route, in doses of 0.5–10 mg [6]. Atropine can also be applied topically for ophthalmic purposes, intravenously in the treatment of food poisoning due to ingestion of anti-cholinergic plant alkaloids [7], and as an *Corresponding author. Tel.: þ212-22-230680/84. Fax: þ212-22-230674. Email: [email protected] Natural Product Research ISSN 1478-6419 print/ISSN 1029-2349 online ß 2008 Taylor & Francis http://www.tandf.co.uk/journals DOI: 10.1080/14786410701592620

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antidote for organophosphate poisoning [8]. Other investigations have shown atropine to demonstrate a wide range of biological activities including vagolytic effects, relaxation action of vascular smooth muscle [4], suppression of gland and mucous secretions [9] and treatment of cyanide-induced neurotoxicity in the presence of Ca2þ and thiosulfate [10]. It has also been used to treat peptic ulcer by reducing the production of stomach acid. The biochemical mechanism through which atropine exerts its activity is not yet clearly established. Therefore, in the light of its wide therapeutic applications, if not properly administered, overdose of atropine can result in adverse effects associated with the suppression of cholinergic systems, manifested as dry mouth, blurred vision, respiratory failure, convulsions, urinary retention, mydriasis, tachycardia and flushing [7]. In the present article, we show atropine to be able to complex Naþ, Kþ, Mg2þ and Ca2þ ions from aqueous picrate solution into an organic medium and to facilitate the transport of these metal cations through a chloroform liquid membrane model.

2. Results and discussion The mobile carrier theory for ionophore-facilitated cation transport through membranes has been supported strongly by the finding of a correlation between the ability of the ionophores to induce transport from an aqueous medium into an organic phase via the mechanism of complexation [11]. Extensive research has been conducted to discover natural compounds that are able to complex and to transport mono- and divalent ions [12–15]. In general, two design features must be incorporated into the ionophore to achieve high selectivity for a particular guest; (i) the pocket of the ionophore must be an appropriate size to bind the guest, and (ii) a pre-organized structure is needed to reduce the entropic and enthalpic costs of complexation. Structure examination of atropine, an anti-cholinergic drug considered as one of the most biologically active molecules, reveals that atropine is a simple piperidine alkaloid, because it consists of carbon ring into which a nitrogen atom is inserted. Moreover, atropine contains different oxygenated groups, able, in precise conformation, to complex mono-and/or divalent ions. The ability of atropine to complex Naþ, Kþ, Mg2þ and Ca2þ ions was studied using two solutions in equilibrium: a chloroform solution containing atropine and an aqueous picrate solution. The metal picrate salt, insoluble in chloroform, is extracted as an atropine complex. The decrease in absorption of picrate in the aqueous phase allows the evaluation of cations complexing efficiency of atropine. Indeed, complexation experiments showed that atropine had remarkable activities with all of the metal ions tested, but with different affinities (figure 1). The reactivity was in the order Ca2þ > Mg2þ > Kþ > Naþ. Thus, 10.14
105 M of Ca2þ was complexed by atropine within 20 min, whereas only 5.65
105 M of Mg2þ, 4.07
105 M of Kþ and 2.47
105 M of Naþ were complexed by atropine within the same period of time (figure 1). The rate of complex formation depends on atropine concentration (figure 2). In control experiments without atropine no complexation occurred. In order to study ionophoric properties of atropine, we tested its ability to act as a carrier in the transport of Naþ, Kþ, Mg2þ and Ca2þ ions through a liquid membrane. In the system used, the metal picrate in aqueous phase I moved, as a complex, through

549

Ionophoric properties of atropine

Ions complexed (10−5 M)

12 10 8 6 4 2 0 0

5

10

15

20

25

30

Time (min) Figure 1. Kinetics of complexation of Naþ, Kþ, Mg2þ and Ca2þ ions by atropine. Transfer of sodium picrate (-*-), potassium picrate (-
-), magnesium picrate (-g-) and calcium picrate (-˙-) by atropine (2.10
105 M) from an aqueous phase to a chloroform phase as a function of time.

Ions complexed (10−5 M)

12 10 8 6 4 2 0 0

5

10

15

20

25

30

Time (min) Figure 2. Effect of atropine concentration on complexation kinetics of Ca2þ ions. Concentration of calcium picrate complexed as a function of time at different concentrations of atropine (-s-; 1.05
105 M), (-˙-; 2.10
105 M).

the chloroform solution containing atropine and was released into aqueous phase II. Ions tested were all transported by atropine but at different rates, in the order Mg2þ > Ca2þ > Kþ > Naþ (figure 3). Divalent cations (Mg2þ and Ca2þ) were transported faster than monovalent ones (Kþ and Naþ). Indeed, within 30 min at atropine concentration of 2.10
105 M, 29.94
106 M of Mg2þ, 25.47
106 M of Ca2þ, 9.15
106 M of Kþ and 5.19
106 M of Naþ were transported (figure 3). As for the complexation, the transport rate depends also on atropine concentration (figure 4(a) and (b)). For example after 15 min, the concentrations of Mg2þ and Ca2þ transported were, respectively, 17.69
106 M and 15.45
106 M at atropine concentration of 2.10
105 M. Whereas at the same time period, only 8.09
106 M of Mg2þ and 8.06
106 M of Ca2þ were transported at a lower atropine concentration

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Ions transported (10−6 M)

30 25 20 15 10 5 0 0

5

10

15 Time (min)

20

25

30

Figure 3. Kinetics of transport of Naþ, Kþ, Mg2þ and Ca2þ ions by atropine. The concentration of Naþ (-*-), Kþ (-
-), Mg2þ (-g-) and Ca2þ (-˙-) ions transported through a chloroform barrier containing atropine at 2.10
105 M.

(1.05
105 M) (figure 4(a) and (b)). In control experiments without atropine no transport occurred. The essential conclusions which can be drawn is that all ions tested form complexes and are transported across chloroform by atropine, with a preference for divalent cations, Mg2þ and Ca2þ. This difference in the affinities is probably in part responsible of therapeutic effects of atropine. Furthermore, atropine has an important potential for complexing metal ions, particularly Ca2þ and this may suggest additional uses as a tool for studying cellular functions mediated by changes in Ca2þ. In the other hand, Ca2þ shift is still the primary factor mediating cellular responses, although other factors may also play significant roles in atropine pharmacology. Moreover, the higher affinity of atropine for Ca2þ ions reinforce the investigations reported by Choi et al. [16], that may provide an indication of mechanism through which atropine exerts its effect, especially a relaxation effect, on isolated rabbit corpus carvernosal smooth muscle. This relaxation may be mediated by decreasing intracellular calcium sequestration, and probably by direct change of calcium transport via voltage dependent calcium channel or sacroplasmic reticulum. This observation may be a tie between a relaxation effect and ionophoretic activity of atropine. Potential pharmacological application of drugs is in the area of nuclear medicine. Ionophores may favourably modify the systemic distribution of the radionucleotide 201 Ti, which is used for the radioimagining of myocardial infarcts [17]. The same principle should be extended to improve the resolution of organ imaging by radionucleotides for the diagnosis of tumours and pathological conditions. Increasing membrane permeability by ionophore administration may also be effective in treating heavy-metal toxicity. Since the balance between Naþ, Kþ, Mg2þ and Ca2þ ions plays a crucial role in biology, including the involvement of potassium and sodium in nerve impulse transmission which depends upon the efficient transport of cations across cellular membranes. It would be of interest to study the rate of transport of these ions through an intact cell membrane and the effect of atropine on cellular functions. Studies in

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Ionophoric properties of atropine (a)

30 Ca2+

Ions transported (10−6 M)

25 20 15 10 5 0 0

5

10

15

20

25

30

20

25

30

Time (min) (b)

30 Mg2+

Ions transported (10−6 M)

25 20 15 10 5 0 0

5

10

15 Time (min)

Figure 4. Effect of atropine concentration on transport kinetics of Ca2þ (A) and Mg2þ (B) ions. Concentration of Ca2þ and Mg2þ ions as a function of time at different concentrations of atropine (-*-; 1.05
105 M), (-f-; 2.10
105 M).

biological systems in the presence of these ions may provide a better model for atropine mediated selective complex-formation and transport. It may also help to explain its biological activity.

3. Experiment 3.1. Kinetics of complex formation The interaction between atropine and Naþ, Kþ, Mg2þ and Ca2þ ions using the extraction procedure described by Frensdorff [18] was studied: the aqueous solutions

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were prepared from standard stock solutions of NaOH, KOH, Mg(OH)2, Ca(OH)2 and picric acid. Atropine was dissolved in the organic phase. Equal volumes of the two solutions in screw-cap beaker flasks were thoroughly stirred on a vortex junior mixer. All extractions were conducted in a constant room temperature at 25  0.5 C. Five millilitres of an aqueous solution, consisting of metal picrate (103 M) and metal nitrate (5
103 M), and 5 mL of a chloroform solution of atropine at the concentration of 1.05
105 M or 2.10
105 M were placed in a beaker, and the organic layer was stirred at constant speed. The rate of complex-formation was monitored by measuring the decrease of the adsorption at 355 nm of the metal picrate in the aqueous solution. Each experiment was carried out in triplicate.

3.2. Transport kinetics Experiments on the transport of Naþ, Kþ, Mg2þ and Ca2þ ions were carried out in a cylindrical glass cell (3.2 cm i.d.) containing a cylindrical glass-walled tube (2.0 cm i.d.), separating two aqueous phases (phase I and phase II) [19]. The aqueous phase I contained metal picrate (103 M), metal nitrate (0.05 M) and metal hydroxide (5
105 M) in 5 mL of double-distilled water, while the aqueous phase II consisted of 5 mL of double-distilled water. The two phases were separated by 5 mL of chloroform containing atropine at the concentration of 1.05
105 M or 2.10
105 M. The absorption at 355 nm of the metal picrate transported into aqueous phase II was measured at regular time intervals. To confirm the results obtained by the spectrophotometric procedure, we have used the atomic absorption technique. At the end of each experiment, the aqueous phase II was recuperated for the purpose to dose the ion tested using a flame photometer (Jenway) type PFP7 with specific filters for Naþ, Kþ, Mg2þ and Ca2þ.

Acknowledgements This work was supported by the Moroccan CNRST and the urban community of Casablanca. The authors would like to thank Dr Nourrddine Chafik (Sothema Laboratories) for helpful corrections of the text.

References [1] C. Cui, S.J. Cho, K.S. Kim. J. Phys. Chem. A, 102, 1119 (1998). [2] M. Shizuma, Y. Takai, M. Kawamura, T. Takeda, M. Sawada. J. Chem. Soc., Perkin Trans., 2, 1306 (2001). [3] C. Kirchhoff, Y. Bitar, S. Ebel, U. Holzgrabe. J. Chromatography A, 1046, 115 (2004). [4] C.Y. Kwan, Z. Wen-Bo, T.K. Kwan, Y. Sakai. Naunyn-Schmiedeberg’s Arch. Pharmacol., 368, 1 (2003). [5] W.J. Brady, A.D. Perron. Am. J. Emergency Med., 19, 81 (2001). [6] B.F. Katzung. In Basic and Clinical Pharmacology, B.F. Katzung (Ed.), Appleton and Lange, Norwalk (1987). [7] D.B. Hoover. In Modern Pharmacology, C.R. Craig, R.E. Stitzel (Eds), Little Brown, Boston (1994). [8] F.D. Carvalho, I. Machado, M.S. Martinez, A. Soares, L. Guilhermino. Ecotoxicology and Environmental Safety, 54, 43 (2003). [9] O. Karadi, Z. Nagy, B. Bodis, G. Mozsik. J. Physiology – Paris, 95, 29 (2001).

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[10] H. Yamamoto. Toxicol. Lett., 80, 29 (1995). [11] D.H. Haynes (Ed.). The kinetics of potassium ion complexation by ionophores, North-Holland Publishing Company, Amsterdam (1971). [12] A. Agtarap, J.W. Chamberlin, M. Pinkerton, L. Steinrauf. J. Am. Chem. Soc., 89, 5737 (1967). [13] M.E. Haney, M.M. Hoehn, J.M. Guire. Thereof. U.S. Patent, 3, 501 (1970). [14] P. Gachon, P. Chaput, G. Jeminet, J. Juillaud, J.P. Morel. J. Am. Chem. Soc., 907 (1975). [15] M. Blaghen, A. Bouhallaoui, H. Taleb, H. Idrissi, F. Tagmouti, M. Talbi, K.F. Zarrouck. Toxicon, 35, 843 (1997). [16] Y.D. Choi, W.S. Chung, Y.K. Choi. The J. Urol., 161, 1976 (1999). [17] D.J. Cook, I. Bailey, H.W. Dtrauss, J. Rouleau, H.N. Wagner, B. Pitt. J. Nucl. Med., 17, 583 (1976). [18] H.K. Frensdorff. J. Am. Chem. Soc., 93, 4684 (1971). [19] J.P. Behr, J.M. Lehn. J. Am. Chem. Soc., 95, 6108 (1973).

Natural Product Research, Vol. 22, No. 6, 15 April 2008, 554–563

Isolation and characterization of ACTX-6: a cytotoxic L-amino acid oxidase from Agkistrodon acutus snake venom L. ZHANGyz and W. T. WU*y ySchool of Lifesciences and Biotechnology, China Pharmaceutical University, Nanjing, 215009, PR China zSchool of Pharmacy, Soochow University, Suzhou, 215023, PR China (Received 26 March 2007; in final form 28 May 2007) The characterization of an L-amino acid oxidase purified from Agkistrodon acutus snake venom was investigated. An L-amino acid oxidase (LAAO) was purified from A. acutus snake venom through DEAE Sepharose F.F. and Source 30 S chromatography. The molecular mass of this enzyme was determined by SDS-PAGE, size exclusion chromatography, and mass spectrometry. Substrate specificity, cytotoxicity, antitumor activity in vivo, and apoptosisinducing activity were assayed. The LAAO purified from A. acutus snake venom was designated as ACTX-6. It is a covalently bound homodimer and its molecular mass is about 96 kDa. This enzyme preferred to oxidize hydrophobic L-amino acids; the best substrates were L-Met, L-Leu, L-Trp, and L-Phe. ACTX-6 demonstrated cytotoxicity in vitro and could inhibit tumor growth in vivo. Flow cytometry analysis showed that it could markedly increase accumulation of sub-G1 phase, which suggested that this enzyme could induce apoptosis. ACTX-6 could effectively inhibit tumor growth and it is a potential substance to develop into an antitumor drug. Keywords: L-amino acid oxidase (LAAO); Cytotoxin; Snake venom; ACTX-6

1. Introduction Snake venoms have been widely investigated and many (LAAO) have been isolated and characterized [1–3]. These LAAOs demonstrated various biological activities such as induction of apoptosis, hemorrhagic effect, and antibacterial activity [4–6]. LAAO can catalyze the oxidative deamination of L-amino acids to the corresponding -keto acids with liberation of hydrogen peroxide and ammonia. It is deduced that LAAO can induce cell death through generation of hydrogen peroxide [4,7]. Agkistrodon acutus snake is widely distributed in South China and Taiwan province. Considerable studies on this snake venom in recent years have revealed that A. acutus snake contains a variety of proteins and polypeptides that affect thrombosis and hemostasis [8–10]. *Corresponding author. Tel.: þ86-25-8322-0372. Fax: þ86-025-8322-0372. Email: [email protected] Natural Product Research ISSN 1478-6419 print/ISSN 1029-2349 online ß 2008 Taylor & Francis http://www.tandf.co.uk/journals DOI: 10.1080/14786410701592679

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In this study a cytotoxic protein with LAAO activity was isolated and its biological characterization was described.

2. Materials and methods 2.1. Materials Lyophilized powder of A. acutus snake venom was obtained from a local merchant. DEAE Sepharose FF, Source 30S, and AKTATM explorer was produced by Amersham Biosciences (Uppsala, Sweden). Standard proteins for molecular weight estimation were produced by Shanghai Donngfeng Biochemical Technology Co. (Shanghai, China). MTT was purchased from Amresco (USA). A549 human lung cancer cell line was purchased from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Carcinomas of Heps (Hepatoma 22), S180 (Sarcoma 180) and EAC (Ehrlich ascites carcinoma) were obtained from Tumor Hospital of Jiangsu Province, Nanjing, China.

2.2. Purification procedure Lyophilized A. acutus venom (1 g) was dissolved in 20 mL of 30 mM phosphate-buffered saline (PBS) buffer, pH 7.0 and then centrifuged at 7000 rpm
15 min under 4 C. The supernatant was dialyzed against 30 mM PBS buffer, pH 7.0 for 24 h and loaded on a DEAE Sepharose FF column (1.6 cm
10 cm) equilibrated with the same buffer. Elution was achieved with a linear gradient of 0–0.5 mol L1 NaCl for 200 mL at the flowrate of 8 mL min1. The crude venom and fractions were pooled and assayed for LAAO activity. The peak showed the strongest LAAO activity and was dialyzed against 30 mmol L1 PBS buffer, pH 6.4 for 24 h. Then the sample was loaded on a Source 30 S column (2.6 cm
10 cm) and eluted with the same buffer (30 mM PBS buffer, pH 6.4) with a linear gradient of 0–0.2 mol L1 NaCl for 400 mL at the flowrate of 5 mL min1. The elution was collected 8 mL per tube and each fraction was assayed for LAAO activity. The peak 7 showed the strongest LAAO activity and was designated as ACTX-6. We pooled it for further study.

2.3. Molecular mass Reducing and non-reducing SDS-PAGE was carried out in 12% gels at pH 8.3 by the method of Laemmli. Molecular mass standards were rabbit myoglobin-200 kDa, calmodulin binding protein-130 kDa, rabbit muscle phosphorylase b-97.4 kDa, bovine serum albumin-66.2 kDa, and the rabbit actin-43 kDa. Protein bands were detected by Coomassie blue staining. The molecular mass of the native enzyme was estimated by size exclusion chromatography. Five hundred microliters of ACTX-6 (1 mg mL1) was injected into the Sephacryl S-200 column and was eluted with 20 mM Tris-HCl, 0.5 M NaCl, and pH 7.4 at the flowrate of 0.5 mL min1. Molecular mass standards were -globulin160 kDa, bovine serum albumin (fraction V)-88 kDa, bovine serum albumin-66 kDa, chicken egg ovalbumin-43 kDa, and bovine trypsinogen-24 kDa.

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The molecular mass determination by MALDI-TOF was performed at the Monash Biomedical proteomics facility (Monash University, Victoria 3800, Australia) on Applied Biosystems 4700 MALDI-ToF ToF (Foster City, CA) using alpha cyano-4hydroycinnamic acid as matrix. The spectra were acquired in linear mode collecting 1500 shots per spectrum.

2.4. LAAO activity LAAO activity was determined spectrophotometrically [11]. The reaction mixtures (1 mL) contained 0.1 M Tris-HCl buffer, pH 8.5, 1 mM L-leucine as a substrate, 0.26 mM o-dianisidine, 20 mg horseradish peroxidase (7U) and the known amount of the eluted fractions or purified LAAO as well as the whole venom. One unit of the enzymatic activity was defined as the oxidation of 1 mmol of L-leucine per minute. Molar absorption coefficient of the reaction product was 8.31
103 M1 cm1. To test the enzymatic specificity of the purified LAAO, L-leucine was replaced with other L-amino acids under identical assay conditions. The amount of purified LAAO in the reaction mixture was 1.0 mg.

2.5. Cell culture The human cervical cancer A549 cell line was maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum, 100 U mL1 penicillin and 100 U mL1 streptomycin at 37 C in a humidified atmosphere containing 5% CO2/95% air. ACTX-6 was dissolved in culture medium and in all experiments negative control cells were treated with medium without ACTX-6.

2.6. Cytotoxic activity The cellular toxicity of the crude venom and its fractions on A549 cells was assessed by MTT colorimetric assay as described before [12]. Briefly, 150 mL of 5
104 cells were seeded on a 96-well-microtiter plate and after 6 h the 50 mL of various concentrations of sample was added. The plate was incubated at 37 C in a humidified atmosphere of 5% CO2 for 24 h. After discarding the 100 mL supernatant, 20 mL MTT reagent was added to the well and the plate was incubated for 6 h. Following aspiration of the medium, 150 mL of DMSO was added to solubilize the MTT-formazan product. After keeping for 15 min at room temperature, the plate was read on an enzyme-linked immunosorbent assay (ELISA) reader at 570 nm. All the measurements were performed in quadruplicate. According to the adopted method the fraction that caused IC50 in excess of 200 mg mL1 was considered non-cytotoxic.

2.7. In vivo antitumor activity Fifty male Kun-Ming mice (20  3 g body weight) were divided into five groups. Tumor cells were routinely implanted s.c. in the right axillary region. Three different concentrations of ACTX-6 dissolved in phosphate-buffered saliva were administered in the vein (1.5, 3, and 6 mg kg1 body weight, 0.2 mL
10 g1 body weight) for five

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times on 2, 4, 6, 8, and 10 days from the tumor implantation day. The control group was treated with the same amount of vehicle solution PBS and the standard reference group was treated with cyclophosphamide i.v. injections (20 mg kg1, 0.2 mL
10 g1 body weight). At Day 11 the animals were sacrificed, the tumors were removed and their weights determined [13]. Three carcinomas of Heps, S180, and EAC were assayed.

2.8. Flow cytometry analysis Cells were treated with 20 mg mL ACTX-6 for 24 h. In control group ACTX-6 was substituted by PBS buffer. Approximately 1
106 A549 cells were suspended in 100 mL PBS and 200 mL 95% ethanol was added while vortex-mixing. Cells were incubated at 4 C for 1 h and washed once with PBS. Cells were resuspended in 250 mL 1.12% (w/v) sodium citrate buffer (pH 8.4) together with 12.5 mg RNase. Incubation was continued at 37 C for 30 min. Cellular DNA was then stained by applying 250 mL of propidium iodide (50 mg mL1) for 30 min at room temperature. The stained cells were analyzed by the fluorescent activated cell sorting (FACS) on a FACScan flow cytometer for a relative DNA content based on red fluorescence.

2.9. Statistical analysis The statistical significance of the experimental results was determined by the Student’st-test. For all analyses, p < 0.05 was accepted as a significant probability level.

3. Results 3.1. Isolation of ACTX-6 First the crude snake venom was loaded on DEAE Sepharose F.F. column and six fractions were obtained (figure 1a). Peak I displayed strong LAAO activity and was chosen for further purification. A cation exchange chromatography step was adapted successively. Peak I was loaded on Source 30 S column and seven major peaks were obtained. Peak 4 demonstrated strong LAAO activity (figure 1b) and it was homogeneity that was verified by RP-HPLC (result not shown). Then the component was designated as ACTX-6. The results of each step are summarized in table 1.

3.2. Molecular mass The molecular mass of ACTX-6 estimated by reducing SDS-PAGE was about 49 kDa and in non-reducing SDS-PAGE the molecular mass was about 97 kDa (figure 2a), which indicated that ACTX-6 contained two covalently bound subunits. The molecular mass determined on size exclusion chromatography was about 100 kDa (figure 2b). In MALDI-TOF mass spectrometry the reversed purified enzyme gave a molecular mass of 96,168 Da. These results suggested that ACTX-6 is a covalently bound homodimer.

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Figure 1. (a) Separation of A. acutus venom by anion-exchange chromatography on DEAE Sepharose FF (1.6
10 cm column). Samples (1 g) of crude venom dissolved in 20 mL of 30 mM PBS, pH 7.0, were applied to the column, which was equilibrated with the same buffer. The column was eluted with a linear gradient of 0–0.5 M NaCl for 200 mL at the flowrate of 8 mL min1, and tubes of 8 mL were collected. All the fractions were studied for LAAO activity. Peak I (arrow shows) demonstrated the strongest LAAO activity and was pooled for next purification. (b) Separation of Peak I by cation-exchange chromatography on Source 30 S (2.6
cm column). Samples (100 mg) of Peak I dissolved in 20 mL of 30 mM PBS, pH 6.4, were applied to the column, which was equilibrated with the same buffer. The column was eluted with a linear gradient of 0–0.2 mol L NaCl for 400 mL at the flowrate of 5 mL min1, and tubes of 8 mL were collected. All the fractions were studied for LAAO activity. Peak 4 (arrow shows) displayed strong LAAO activity and was pooled for further study.

Table 1. Results of each chromatography step. Step Crude venom DEAE Sepharose F.F. Source 30S

Total protein

Specific activity (U mg1)

Total activity (U)

Yield (activity%)

Purification (n-fold)

1000 240 20

0.66 1.78 10.69

660 427 214

100 64.7 32.42

1 2.7 16.2

3.3. Substrate specificity The LAAO activity was tested using different amino acids as substrates in the concentration of 1 mM. In our study, ACTX-6 preferentially oxidizes L-Met, L-Leu, L-Phe, L-Trp, and L-Arg (table 2).

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Figure 2. Molecular mass of ACTX-6 determined by SDS-PAGE and gel filtration on a Sephacryl S-200 column. (a) SDS-PAGE of ACTX-6. Lane 1 – molecular mass marker; Lane 2 – ACTX-6 (reducing); Lane 3 – ACTX-6 (non-reducing). (B) Molecular mass determined by gel filtration. Protein standards and their molecular weights are: 1, -globulin (160 kDa); 2, ACTX-6(100 kDa); 3, bovine serum albumin (fraction V) (88 kDa); 4, bovine serum albumin(66 kDa);5, chicken egg ovalbumin (43 kDa). 4, bovine trypsinogen (24 kDa).

Table 2. L-Amino

Met Leu Trp Phe Arg His Ile Lys Glu Tyr Gln Asn Gly Pro Ser Thr Val Ala Thr Cys

acid

Substrate specificity of ACTX-6. Specific activity (U mg1) 13.2 10.8 9.6 6.1 3.6 1.5 0.7 0.5 0.5 0.2 0 0 0 0 0 0 0 0 0 0

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Figure 3. ACTX-6 induced cell death in A549 cells. The percentage of cell viability of treated cells was determined by MTT assay. (a) A549 cells were treated with various concentrations of ACTX-6 (0–40 mg mL1) for 24 h. (b) Cells were treated with 10 mg mL1 of ACTX-6 for 12, 24, and 48 h. The data represent a mean of three independent experiments. The results are shown as mean  SEM (n ¼ 3). The error bars indicate the SD from three independent experiments. The significance of differences between the experimental and control groups was evaluated using t-test: *p < 0.05 and **p < 0.01.

3.4. Cytotoxicity studies The result of MTT (figure 3a and b) showed that cytotoxicity of ACTX-6 on A549 cells was elevated in a dose and time dependent manner. When cells were treated with ACTX-6 for 24 h, the IC50 was about 20 mg mL. When 40 mg mL1 of ACTX-6 was treated for 24 h, almost all the cells were killed (figure 3a). When cells treated with 10 mg mL1 ACTX-6 for 12, 24, and 48 h, the cell viability decreased with the time (figure 3b).

3.5. Antitumor activity in vivo Treatment of tumor-bearing mice with i.v. injection of ACTX-6 demonstrated that at the dose of 6 mg kg1 ACTX-6 could effectively inhibited tumor growth in vivo (Heps, 40.5%; S180, 42.4%; EAC, 43.4%) (table 3).

3.6. Apoptosis-inducing activity Cells were treated with 10 mg mL1 ACTX-6 for 24 h and flow cytometry was used to assess the proportion of cells found in the G0/G1, S, G2/M, and sub-G1 cell cycle

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Isolation and characterization of ACTX-6 Table 3. Drug 1.5 mg kg1 ACTX-6 3 mg kg1 ACTX-6 6 mg kg1 ACTX-6 20 mg kg1 cyclophosphamide

In vivo antitumor activity of ACTX-6.

Inhibitionon Heps (%)

Inhibition on S180 (%)

Inhibition on EAC (%)

21.6  3.5 24.6  4.6 40.5  4.7 37.4  3.8

12.9  2.9 27.4  5.1 42.4  3.9 41.7  2.7

14.2  3.1 32.7  4.3 43.4  4.2 41.6  3.4

Figure 4. ACTX-6 induced apoptosis in A549 cells. FACS analysis of apoptotic cells. A549 cells were treated with 10 mg mL1 ACTX-6 for 24 h and their DNA content was measured after propidium iodine staining. Control group cells were treated with PBS buffer instead of ACTX-6. The results represent three independent experiments.

phases (figure 4). In control group about 8% cells were found in sub-G1 phase while in drug-treated group the proportion increased to 23%. Then it could be concluded that ACTX-6 induced a markedly increased accumulation of sub-G1 phase, which suggested that ACTX-6 could effectively trigger cell apoptosis.

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4. Discussion An LAAO named ACTX-6 has been isolated form A. acutus snake venom through two-step chromatography. The molecular mass of ACTX-6 is about 96 kDa and it is a covalently bound homodimer. In earlier studies the effect of LAAO is reduced to its main function, catalysis of the oxidative deamination of L-amino acids to form the corresponding -keto acids and ammonia accompanied with the reduction of FAD. However until now it is found that LAAOs display biological effects on cancer cells, platelets, microbial organisms, etc. The LAAO obtained in this study demonstrates cytotoxic and apoptosis-inducing activity. Snake venom LAAOs are usually homodimeric protein with a molecular mass about 110–150 kDa under non-denaturing conditions. Results of SDS-PAGE under reduced or non-reduced conditions showed that ACTX-6 was a covalently bound homodimeric protein with molecular mass of 97 kDa. It is reported that generally hydrophobic amino acids are better substrates for snake venom LAAOs [14,15]. The substrate specificity of ACTX-6 is similar to other snake venom LAAOs. L-Met, L-Leu, L-Trp, and L-Phe are specific substrates for ACTX-6 while L-Ser, L-Pro, L-Thr, and L-Cys etc. can not be oxidized by ACTX-6. The catalytic preference can be explained by differences of side-chain binding sites in the enzyme responsible for substrate specificity. Tan and coworkers have investigated the mechanism of oxidation and supposed that these enzymes contained an amino acid side chain binding site comprising 3–4 hydrophobic subsites. In our study we found that ACTX-6 could induce A549 cell death in vitro and inhibit tumor growth in vivo. Flow cytometry analysis showed that ACTX-6 could induce apoptosis. Many LAAOs demonstrate apoptosis-inducing activity [16–19] and it is partially due to the generation of hydrogen peroxide. Hydrogen peroxide belongs to reactive oxygen species (ROS) and it is widely accepted that mitochondrial perturbation has been associated with the increased production of ROS [20,21]. The mitochondrial perturbation will lead to cell apoptosis [22–24], so the ability of ACTX-6 to induce cell apoptosis is perhaps related to its catalytic activity.

5. Conclusion In this study we isolated an LAAO designated ACTX-6 from A. acutus snake venom and it is a covalently bound homodimer with molecular mass of 96 kDa. L-Met, L-Leu, L-Trp, and L-Phe are specific substrates for ACTX-6. This LAAO displays cytotoxic activity on A549 cells in vitro and antitumor activity in vivo. Flow cytometry analysis showed that ACTX-6 could induce cell apoptosis.

Acknowledgements The authors thank Dr Guo Q.L. for his support in antitumor experiment in vivo and Du R.H. for her help in pharmacological experiment.

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