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Two new nonacosanetriols from Ginkgo biloba sarcotesta
Chemistry and Physics of Lipids 165 (2012) 731–736
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Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip
Two new nonacosanetriols from Ginkgo biloba sarcotesta Guisheng Zhou a , Xin Yao a , Yuping Tang a,∗ , Nianyun Yang a , Hanqing Pang a , Xuan Mo a , Shaoqing Zhu a , Shulan Su a , Dawei Qian a , Chun Jin b , Yong Qin b , Jin-ao Duan a,∗ a b
Jiangsu Key Laboratory for High Technology of TCM Formulae Research, Nanjing University of Chinese Medicine, Nanjing 210046, China Jiangsu Shenlong Pharmaceutical Co. Ltd., Yancheng 224200, China
a r t i c l e
i n f o
Article history: Received 9 June 2012 Received in revised form 29 August 2012 Accepted 31 August 2012 Available online 6 September 2012 Keywords: Ginkgo biloba Sarcotesta Fatty alcohol Anticoagulative activity Stereochemistry
a b s t r a c t Two new fatty alcohols named as (7S,8R,11S)-nonacosanetriol (1) and (10R,12R,15S)-nonacosanetriol (2), along with eight known compounds including ginkgolic acid (3), hydroginkgolic acid (4), sciadopitysin (5), ginkgetin (6), isoginkgetin (7), ginkgolide A (8), ginkgolide B (9) and ginkgolide C (10) have been isolated from the petroleum ether extract of Ginkgo biloba sarcotesta. Their structures were elucidated by means of chemical and extensive spectroscopic analysis. The absolute stereochemistry of compounds 1 and 2 was elucidated on the spectroscopic analysis of the R- and S-MTPA esters. Compounds 1 and 2 exhibited slight activity of antithrombin and moderate activity of antiplatelet aggregation in vitro. This was the first report regarding the anticoagulative activities of biflavonoids in G. biloba, and isoginkgetin (7) showed significant antithrombin and antiplatelet aggregation activity. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Ginkgo biloba is a deciduous and dioecious gymnosperm species (Major, 1967) originated in China and is the sole survivor of the ancient family of Ginkgoaceae (Carrier et al., 1998). Although G. biloba has been above 200 million years old, its true value has induced a range of attentions all around the world till the recent two decades. It contains a large number of active compounds, the most important of which are flavanol glycosides and terpene lactones. Its amazing vitality has attracted an increasing exploration into potential application in health, foods and supplements (van Beek, 2002; Singh et al., 2008). Male and female flowers are born on different plants and female plants bear a yellowish-green plumlike “fruit” (aril) (Choukchou-Braham et al., 1994) which is indeed the seed of ginkgo in gymnosperms. The outer malodorous fleshy layer is called sarcotesta. It surrounds an ovoid nut, which is called “Baiguo” in China and “Gin-nan” in Japan (Deng et al., 2011).
Abbreviations: ADP, adenosine diphosphate; COSY, correlation spectroscopy; DMAP, 4-dimethylaminopyridine; ESI, electrospray ionization; Gbs, Ginkgo biloba sarcotesta; HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear singular quantum correlation; HR, high resolution; MS, mass spectrometry; NMR, nuclear magnetic resonance; PPP, platelet poor plasma; PRP, platelet rich plasma; RMTPA, R-(−)-␣-methoxy-␣-(trifluoromethyl)phenylacetyl chloride; S-MTPA, S-(+)␣-methoxy-␣-trifluoromethylphenylacetyl chloride; TMS, tetramethylsilane; Tris, trishydroxymethylaminomethane; TT, thrombin time. ∗ Corresponding authors. Tel.: +86 25 85811916; fax: +86 25 85811916. E-mail addresses: [email protected] (Y. Tang), [email protected] (J.-a. Duan). 0009-3084/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemphyslip.2012.08.003
Ginkgo seeds have been used in traditional Chinese medicine (TCM) and as a foodstuff for centuries throughout Asia (Jin et al., 2008). However, Gbs, the epicarp of mature seeds which is peeled from G. biloba seeds, was treated as waste and discarded abundantly in soil and water, thus it polluted soil and poisoned fish in rivers and lakes (Pan et al., 2006). It has become an urgent problem to the local enterprises and governments of the planting area for its poison to the environment (Wu et al., 2011). Modern studies indicated that Gbs consists of ginkgolic acid and polysaccharide, which were reported to have many biological and pharmacological functions, including antitumor, anti-inflammatory, pesticidal and antibacterial properties (Chen et al., 2007). In Xingan (Guangxi province, China) the fruit juice produced during the peeling process was diluted with water and used as an insecticide by local peasants. Satisfactory results were achieved in the control of many kinds of insects, indicating the presence of some insecticidal constituents (Choi et al., 2009). In our preliminary experiment, 95% EtOH extract of Gbs showed anticoagulative activity. And then, in our present study, different solvent extracts of Gbs were tested, and the results showed that the petroleum ether extract had strong anticoagulative activity. Therefore, the petroleum ether extract of Gbs was chemically investigated, which resulted in the isolation of two new fatty alcohols, named as (7S,8R,11S)-nonacosanetriol (1) and (10R,12R,15S)-nonacosanetriol (2), together with eight known compounds including ginkgolic acid (3), hydroginkgolic acid (4), sciadopitysin (5), ginkgetin (6), isoginkgetin (7), ginkgolide A (8), ginkgolide B (9) and ginkgolide C (10). Some nonacosanetriols or similar compounds were isolated from nature, such as 7,8,10-nonacosanetriol and 7,9,10-nonacosanetriol from Typha
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angustifolia (Tao et al., 2010), 8,15-nonacosanediol and 6,13nonacosanediol from Trogopterus xanthipes (Yang et al., 2009a), and 9,10,11-trihydroxy-(12Z)-12-octadecenoic acid from Tuber indicum (Gao et al., 2001). The structures of the two new compounds were elucidated by means of chemical and extensive spectroscopic analysis. Furthermore, their anticoagulative activities were also evaluated. 2. Experimental 2.1. Materials and chemicals Gbs was collected on November 2010 from Taizhou, Jiangsu Province, China, and identified as the sarcotesta of Ginkgo biloba L. by Dr. Hui Yan (Department of Pharmacognosy, Nanjing University of Chinese Medicine, China). A voucher specimen (BG-20101120) was deposited at the Herbarium in Jiangsu Key Laboratory for TCM Formulae Research. Silica gel for column chromatography (CC) (60–80 m) and thin-layer chromatography plates (10–40 m) was purchased from Qingdao Marine Chemical (Qingdao, China). All solvents used were of analytical grade (Nanjing Chemical Plant, Nanjing, China). RMTPA and S-MTPA were obtained from Sigma Chemical Co. (St. Louis, MO). Thrombin was purchased from Xisen Sanhe (Leling, China). Tris was the product of Shanghai Jingxi Chemical Industry (Shanghai, China). Sodium citrate was purchased from Shanghai Lingfeng Chemical Reagent (Shanghai, China). Heparin sodium (biotech grade, 150 U mg−1 ) was purchased from Amresco (Solon, USA). ADP was purchased from Beijing Zhongqin Scientific Instrument Co. Ltd. (Beijing, China). A rabbit (3.8 kg) was supplied by Shanghai Sikelai Experimental Animal (Shanghai, China). 2.2. Apparatus Melting points were determined with a WRS-lB melting point apparatus (Shanghai Precision and Scientific Instrument, Shanghai, PR China) which was uncorrected. Optical rotation was measured on a Perkin-Elmer 341 polarimeter. NMR spectra were measured on a Bruker AV-500 MHz (500 MHz for 1 H NMR and 125 MHz for 13 C NMR), using TMS as internal standard; chemical shifts were recorded as ı (ppm) values. ESI-MS and HR-ESI-MS spectra were obtained on a Micromass Q/TOF mass spectrometer. Anticoagulative assay was performed on an LG-PABER-I coagulation-analysis instrument. The blood sample was treated on an Anke TDL-40B centrifuge. 2.3. Extraction and isolation Air-dried and powdered Gbs (25 kg) were extracted two times with 95% EtOH (2 × 250 L) under reflux for 2 h at 70–80 ◦ C each time. The extracts were concentrated under reduced pressure, and then they were suspended in H2 O and extracted successively with petroleum ether, ethyl acetate and n-butanol to give the respective extracts after solvent removal. The combined petroleum ether extracts were evaporated under reduced pressure to leave a residue (250 g) which was chromatographed on silica gel (2.5 kg) eluted with a petroleum ether–EtOAc stepwise gradient (100:0 → 1:1) and 5 fractions were collected. Fr. 4 (30 g) was separated by silica gel (petroleum ether–EtOAc, 5:1) to obtain compounds 1 (50 mg) and 2 (30 mg); Fr. 1 (60 g) was separated by silica gel (petroleum ether–EtOAc, 20:1) to obtain compounds 3 (100 mg) and 4 (60 mg); Fr. 3 (40 g) was separated by silica gel (petroleum ether–EtOAc, 8:1) to obtain compounds 5 (50 mg), 6 (10 mg) and 7 (10 mg), and Fr. 5 (40 g) was separated by silica gel (petroleum ether–EtOAc 3:1) to obtain compounds 8 (20 mg), 9 (30 mg) and 10 (10 mg).
(7S,8R,11S)-nonacosanetriol (1) white powder, [␣]D 20 + 3.0◦ (c = 0.045, MeOH), m.p. 160–162 ◦ C, 1 H NMR (CD3 OD, 500 MHz) ı 3.80 (1H, m, H-11), 3.58 (1H, m, H-8), 3.40 (1H, m, H7), 1.76 (1H, t, J = 3.5 Hz, H-10a), 1.73 (1H, t, J = 3.5 Hz, H-9a), 1.53 (1H, m, H-9b), 1.51 (1H, m, H-10b), 1.45–1.50 (4H, br. m, H-6, 12), 1.24–1.39 (40H, br. m, H-2–5, 13–28), 0.89 (6H, t, J = 7.0 Hz, H-1, 29); 13 C NMR (CD3 OD, 125 MHz) ı 76.4 (C7), 76.0 (C-8), 72.4 (C-11), 40.4 (C-9, 10), 39.1 (C-6, 12), 34.1 (C-3, 27), 30.9–31.3 (C-4, 14–26), 27.4 (C-5), 26.9 (C-13), 24.1 (C-2, 28), 14.8 (C-1, 29). ESI-MS: m/z 455 [M−H]− , 457 [M+H]+ , 479 [M+Na]+ ; HR-ESI-MS: m/z 479.7761 [M+Na]+ (C29 H60 O3 Na, calc. 479.7747). C(7),C(8),C(11)-tris-(S)-MTPA ester of compound 1 [(S)-MTPA1] white powder, m.p. 175–180 ◦ C, 1 H NMR (CD3 OD, 500 MHz) ı 7.58 (6H, m, MTPA-ArH-3, 5), 7.41 (9H, m, MTPA-ArH-2, 4, 6), 3.83 (1H, m, H-11), 3.60 (1H, m, H-8), 3.56 (9H, s, MTPA-OCH3 ), 3.42 (1H, m, H-7), 1.75 (1H, m, H-9a), 1.70 (1H, m, H-10a), 1.56 (1H, m, H-9b), 1.52 (1H, m, H-10b), 1.38–1.43 (4H, m, H-6, 12), 1.27–1.38 (40H, m, H-2–5 and H-13–28), 0.90 (6H, t, J = 7.0 Hz, H-1, 29). ESI-MS: m/z 1105 [M+H]+ . C(7),C(8),C(11)-tris-(R)-MTPA ester of compound 1 [(R)-MTPA1] white powder, m.p. 169–173 ◦ C, 1 H NMR (CD3 OD, 500 MHz) ı 7.44 (6H, m, MTPA-ArH-3, 5), 7.36 (9H, m, MTPA-ArH-2, 4, 6), 3.81 (1H, m, H-11), 3.64 (1H, m, H-8), 3.55 (9H, s, MTPA-OCH3 ), 3.40 (1H, m, H-7), 1.76 (1H, m, H-9a), 1.73 (1H, m, H-10a), 1.57 (1H, m, H-9b), 1.55 (1H, m, H-10b), 1.35–1.40 (4H, br. m, H-6, 12), 1.30–1.35 (40H, br. m, H-2–5, 13–28), 0.89 (6H, t, J = 7.0 Hz, H-1, 29). ESI-MS: m/z 1105 [M+H]+ . (10R,12R,15S)-nonacosanetriol (2) white powder, [␣]D 20 − 8.0◦ (c = 0.015, MeOH), m.p. 185–187 ◦ C, 1 H NMR (CDCl3 , 300 MHz) ı 3.88 (3H, m, H-10, 12, 15), 1.61 (1H, m, H-11a), 1.58 (1H, m, H-11b), 1.42–1.48 (8H, br. m, H-9, 13, 14, 16), 1.26–1.29 (38 H, br. m, H-2–8, 17–28), 0.88 (6H, t, J = 6.6 Hz, H-1, 29); 13 C NMR (CDCl3 , 75 MHz) ı 75.4 (C-12), 74.5 (C-15), 72.8 (C-10), 38.6 (C-11), 38.0 (C-13, 14), 36.8 (C-9, 16), 32.0 (C-3, 27), 29.4–29.7 (C-4–7, 18–26), 26.0 (C-8), 25.4 (C-17), 22.6 (C-2, 28), 14.0 (C-1, 29). ESI-MS: m/z 457 [M+H]+ , 479 [M+Na]+ ; HR-ESI-MS: m/z 479.7706 [M+Na]+ (C29 H60 O3 Na, calc. 479.7747). C(10),C(12),C(15)-tris-(S)-MTPA ester of compound 2 [(S)MTPA-2] white powder, m.p. 209–213 ◦ C, 1 H NMR (CDCl3 , 500 MHz) ı 7.58 (6H, m, MTPA-ArH-3, 5), 7.42 (9H, m, MTPA-ArH2, 4, 6), ı 3.90 (3H, m, H-10, 12, 15), 3.56 (9H, s, MTPA-OCH3 ), 1.73 (1H, m, H-11a), 1.70 (1H, m, H-11b), 1.52 (4H, m, H-13, 14), 1.45 (4H, m, H-9, 16), 1.25–1.31 (38 H, m, H-2–8 and H-17–28), 0.88 (6H, t, J = 6.6 Hz, H-1, 29). ESI-MS: m/z 1105 [M+H]+ . C(10),C(12),C(15)-tris-(R)-MTPA ester of compound 2 [(R)MTPA-2] white powder, m.p. 193–196 ◦ C, 1 H NMR (CDCl3 , 500 MHz) ı 7.44 (6H, m, MTPA-ArH-3, 5), 7.36 (9H, m, MTPAArH-2, 4, 6), 3.89 (3H, m, H-10, 12, 15), 3.55 (9H, s, MTPA-OCH3 ), 1.68 (1H, m, H-11a), 1.65 (1H, m, H-11b), 1.55 (4H, m, H-13, 14), 1.45 (4H, m, H-9, 16), 1.23–1.30 (38 H, m, H-2–8 and H-17–28), 0.88 (6H, t, J = 6.6 Hz, H-1, 29). ESI-MS: m/z 1105 [M+H]+ .
2.4. Acetylation of compounds 1 and 2 Compounds 1 (2 mg) and 2 (2 mg) were dissolved in 3 mL of Ac2 O–pyridine (1:1) and kept at room temperature for 48 h to yield crude products, respectively. 3 mL of water was added into the reaction products, and the solution was extracted three times with EtOAc (3 × 3 mL), respectively. The resulting extractions were concentrated under reduced pressure to afford their corresponding diacetate derivatives (1a and 2a), which were analyzed by ESIMS–MS.
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Fig. 1. ESI-MS/MS fragmentation of compounds 1 and 2.
2.5. Preparation and purification of MTPA esters 1 mg nonacosanetriol was dissolved in dry CH2 Cl2 (0.5 mL) and sequentially added dry pyridine (0.4 mL), DMAP (0.5 mg), and (R)-MTPA (6 L). The reaction mixtures were stirred at room temperature for 24 h. The resulting mixture was dried over Na2 SO4 and concentrated under reduced pressure. The residue, diluted with CH2 Cl2 , was purified by microcolumn chromatography on silica gel (60–80 m) and eluted with 5 mL hexane–EtOAc (8:2). Finally, the eluent was dried in vacuo, thus yielding the corresponding MTPA ester derivative namely S-Mosher esters. The R-Mosher ester was prepared and purified by using (S)-MTPA following the same procedure as described above (Kobayashi et al., 2001; Freire et al., 2009; Chen et al., 2011). 2.6. Anticoagulative assay All samples were dissolved in ethanol. The anticoagulative activities of compounds 1–10, petroleum ether extracts, ethyl acetate extracts, n-butanol extracts, H2 O extracts, positive control (heparin sodium) and blank (ethanol) were evaluated by the TT method. A rabbit was sacrificed to obtain plasma (50 mL). The plasma was put in a plastic cup for 3 min at 37 ◦ C, together with 100 mL thrombin solution of 15 U mL−1 diluted by 0.1 mol mL−1 Tris–HCl buffer (pH 7.4) and 10 mL sample solution; meanwhile, the coagulation analysis instrument was started up to record the thrombin clotting time. Each analyte was tested for six times, and an average value was calculated. TT prolongation rate and lgTT prolongation rate were calculated to assess the anticoagulative activity of the samples. 2.7. Antiplatelet aggregation activity Evaluation of antiplatelet aggregation activities of the different solvent extracts, pure compounds 1–10, positive control (aspirin) and blank (ethanol) were performed using an antiplatelet aggregation method. A rabbit was sacrificed to obtain PRP and PPP (Yang et al., 2011). Platelet count was adjusted to 3 × 105 with PPP. Added to the PRP, each sample was incubated at 37 ◦ C with stirring for 1 min before the addition of ADP (10 M) as inducer of platelet
aggregation. Aggregation was measured by a turbidimetric method. Platelet aggregation was expressed as the percentage change with the difference of light transmittance between PRP and PPP as 100%. Each analyte was tested for six times, and an average value was applied. Antiplatelet activity was expressed as the percent inhibition of the control value.
3. Results and discussion The petroleum ether extracts of Gbs were subjected to repeated silica gel liquid chromatography to obtain two new fatty alcohols: (7S,8R,11S)-nonacosanetriol (1)and (10R,12R,15S)nonacosanetriol (2), eight known compounds: ginkgolic acid (3) (Yamagiwa et al., 1987), hydroginkgolic acid (4) (Fu et al., 1962), sciadopitysin (5) (Markham et al., 1987), ginkgetin (6) (Markham et al., 1987), isoginkgetin (7) (Krauze-Baranowska et al., 2005), ginkgolide A (8) (Llabres et al., 1989), ginkgolide B (9) (Llabres et al., 1989) and ginkgolide C (10) (Llabres et al., 1989). The structures of compounds 1 and 2 were elucidated as follows. Compound 1 was obtained as a white powder. Its molecular formula was established as C29 H60 O3 by HR-ESI-MS (m/z 479.7761 [M+Na]+ ). Its positive ESI-MS showed characteristic peaks at m/z 457 [M+H]+ , and 479 [M+Na]+ . The 1 H NMR (CD3 OD) of 1 showed proton signals of two terminal methyls at ı 0.89 (6H, t, J = 7.0 Hz), 20 methylenes at ı 1.24–1.39 (40H, br. m), and three oxygenated methine at ı 3.80 (1H, m), 3.58 (1H, m), and 3.40 (1H, m). The 13 C NMR (CD OD) spectrum of 1 revealed carbon signals for three 3 oxygenated methine at ı 76.4, 76.0, and 72.4. The above data indicated a long chain hydrocarbon with three hydroxyl functions in 1 (Gao et al., 2001; Li et al., 2009). As shown in Fig. 1(1), the ESI-MS–MS spectrum showed a set of characteristic peaks at m/z 203 → 185 → 167 → 149, 173 → 155 → 137, 283 → 265, and 253 formed by ␣-fission and loss of H2 O, which suggested the hydroxyl group was located at C-11. Other diagnostic fragment ions at m/z 85, 371 → 353 → 335 → 317, 115 → 97, 341 → 323 → 305, and 145 → 127 → 109, showed hydroxyl groups at C-7, C-8, and C-11. Compound 1 was subjected to acetylation (Zhang et al., 2007), and the final reaction product of 1a was analyzed by ESIMS and ESI-MS–MS. Its ESI-MS–MS gave characteristic peaks at
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Fig. 2. ESI-MS/MS fragmentation of acetylation products 1a and 2a.
m/z 491 ([M+Cl+3×H2 O−3×AcOH]− , 100), 581 ([M−H]− , 14), 521 ([M−H–AcOH]− , 10), 461 ([M–H−3×AcOH]− , 6), 497 (C29 H53 O6 , 3), 425 (C26 H49 O4 , 10), 353 (C23 H45 O2 , 5), 329 (C17 H29 O6 , 3), 325 (C21 H41 O2 , 18), 257 (C14 H25 O4 , 3), 253 (C18 H37 , 4), and 85 (C6 H13 , 3) in Fig. 2(1a). Assignments of all protons and carbons in 1 were also made on the basis of 1 H–1 H–COSY, HSQC, and HMBC spectra. In the 1 H–1 H–COSY spectrum of 1, the diagnostic correlations between H-7 at ı 3.40 (m) and H-8 at ı 3.58 (m), H-8 and H-9a at ı 1.73 (t), H-8 and H-9b at ı 1.53 (m), H-10a at ı 1.76 (t) and H-11 at ı 3.80 (m), H-10b and H-11 were observed in 1. These correlations strongly confirmed that 1 contained the partial structure CH2 CH(OH) CH(OH) CH2 CH2 CH(OH) . Meanwhile, the corresponding correlations were observed in HSQC and HMBC spectra. From these data, compound 1 was identified as 7,8,11nonacosanetriol. The absolute configuration of 1 was elucidated on the basis of spectroscopic evidence and the spectroscopic analyses of the (R)- and (S)-MTPA esters. The chemical shift of three chiral centers (7, 8, 11 positions) of 1 was identified by HSQC techniques. The sign of ıH [=(ıS − ıR )] was positive for H (6), H (7), and H (12), and negative for H (8), H (9), and H (10). The detailed ıH of the synthetic MTPA-1 which had the unambiguous absolute stereochemistry (Rieser et al., 1992; Gao et al., 2004; Ciavatta et al., 2010; Wenzel and Chisholm, 2011) is listed in Table 1. Based on the above evidences, the structure of compound 1 was established as (7S, 8R, 11S)-nonacosanetriol. Compound 2 was obtained as a white powder. Its molecular formula was established as C29 H60 O3 by positive ion HR-ESI-MS m/z 479.7706 [M+Na]+ . Its positive ESI-MS showed characteristic peaks at m/z 457 [M+H]+ , and 479 [M+Na]+ . The 1 H NMR (CDCl3 ) of 2 showed proton signals of two terminal methyls at ı 0.88 (6H, t, J = 6.6 Hz), methylenes at ı 1.26–1.29 (38 H, br. m),
and three oxygenated CH groups at ı 3.88 (3H, m). The 13 C NMR (CDCl3 ) spectrum of 2 revealed carbon signals for three oxygenated methine at ı 75.4, 74.5, and 72.8. These data inferred the existence of a straight-chain compound with three hydroxyl functions in 2 (Gao et al., 2001). As shown in Fig. 1(2), a set of characteristic peaks at m/z 197, 259 → 241 → 223 → 205, 229 → 211 → 193, and 227 → 209, were formed by ␣-fission and loss of H2 O, which suggested the hydroxyl group was located at C-15. Other diagnostic fragment ions at m/z 127, 329 → 311 → 293 → 275, 171 → 153, 285 → 267 → 249, 157 → 139, 299 → 281 → 263, 201 → 183 → 165, and 255 → 237, were derived from ␣-fission of the hydroxyl group (Manini et al., 2008), which suggested three OH groups present at C-10, C-12, and C-15. Compound 2 was also subjected to acetylation and the final reaction product (2a) was analyzed by ESI-MS and ESI-MS–MS, and its ESI-MS–MS gave characteristic peaks at m/z 479 ([M+Na+3×H2 O−3×AcOH]+ , 100), 582 ([M]+ , 8), 545 ([M+Na–AcOH]+ , 3), 487 ([M+Na−2×AcO]+ , 22), 428 ([M+Na−3×AcO]+ , 11), 455 (C26 H47 O6 , 3), 385 (C21 H37 O6 , 8), 383 (C23 H43 O4 , 3), 369 (C22 H41 O4 , 4), 313 (C18 H33 O4 , 6), 297 (C19 H37 O2 , 5), 285 (C16 H29 O4 , 6), 269 (C17 H33 O2 , 2), 213 (C13 H25 O2 , 2), 199 (C12 H23 O2 , 2), 197 (C14 H29 , 2), and 127 (C9 H19 , 2) in Fig. 2(2a). These fragment ions were derived from loss of HOAc or ␣-fission of the acetoxyl group, which revealed three OAc groups present at C-10, C-12, and C-15. Thus, compound 2 was elucidated as 10,12,15-nonacosanetriol. 1 H–1 H–COSY, HSQC, and HMBC experiments were done to further prove the structure of 2. The absolute stereochemistry of 2 was interpreted on the basis of spectroscopic evidence and the spectroscopic analyses of the Mosher’s esters. The sign of ıH [=(ıS − ıR )] was positive for H (11), zero for H (9), and H (16), and negative for H (13), and H (14). The detailed ıH of synthetic MTPA-2 which had the absolute stereochemistry (Rieser
Table 1 1 H NMR data for H (6)–H (16) from the (S)- and (R)-MTPA ester of compounds 1 and 2. Position
(S)-MTPA-1
(R)-MTPA-1
ıH (ıS − ıR )
Position
(S)-MTPA-2
(R)-MTPA-2
ıH (ıS − ıR )
6, 12 7 8 9 10
1.38–1.43 3.42 3.60 1.75 1.52
1.35–1.40 3.40 3.64 1.76 1.55
+0.03 +0.02 −0.04 −0.01 −0.03
9, 16 11 13, 14
1.45 1.73 1.52
1.45 1.68 1.55
0 +0.05 −0.03
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Table 2 Antithrombin activity of the different solvent extracts and compounds 1–10 (x ± s, n = 6). Samples
Dosage (g mL−1 )
TT prolongation (%)
(7S,8R,11S)-Nonacosanetriol (10R,12R,15S)-Nonacosanetriol Ginkgolic acid Hydroginkgolic acid Sciadopitysin Ginkgetin Isoginkgetin Ginkgolide A Ginkgolide B Ginkgolide C Petroleum ether extract Ethyl acetate extract n-Butanol extract H2 O extract Heparin sodium
100 100 100 100 100 100 100 100 100 100 1.0 × 104 1.0 × 104 1.0 × 104 1.0 × 104 50
10.02 11.42 5.61 6.21 12.22 9.82 32.86 35.27 48.32 29.25 45.47 40.51 25.34 22.00 77.99
et al., 1992; Gao et al., 2004; Ciavatta et al., 2010; Wenzel and Chisholm, 2011) is listed in Table 1. Based on the above evidences, the structure of compound 2 was established as (10R,12R,15S) – nonacosanetriol. The TT and antiplatelet aggregation methods were successfully developed in our laboratory for determining the anticoagulative activities of a series of Chinese blood-activating medicines and their components (Yang et al., 2009b). In vitro anticoagulative activities of the different solvent extracts and compounds 1–10 were tested by using the TT and antiplatelet aggregation methods in this study. The results are summarized in Tables 2 and 3. Petroleum ether extracts, and ethyl acetate extracts were the important active fractions, and ginkgolides (8–10) could remarkably prolong thrombin time and inhibit platelet aggregation, especially ginkgolide B (compound 9). Two new fatty alcohols (1 and 2) could also slightly prolong thrombin time and moderately inhibit platelet aggregation. Platelet activation could be inhibited by inhibiting thromboxane A2 pathway, ADP pathway, thrombin and phosphodiesterase (Sikka and Bindra, 2010). In this study platelet aggregation was induced by ADP in vitro, while compounds 1 and 2 could moderately reversed the platelet aggregation induced by ADP. So, compounds 1 and 2 might exhibit the antiplatelet activity by playing a role on the ADP pathway. Thrombin was a serine protease that played a key role in thrombosis. Thrombin was the most potent physiological agonist of platelet activation and aggregation which was generated on the platelet surface (Sakurai et al., 2004). Compounds 1 and 2 might act directly on thrombin to attain the antithrombin effect which is another way to exhibit the antiplatelet activity. Fatty alcohols, fatty acids, and their esters were important lipid compounds and had many biological and
Table 3 Antiplatelet aggregation activity of the different solvent extracts and compounds 1–10 (x ± s, n = 6). Samples
Dosage (g mL−1 )
Inhibition ratio (%)
(7S,8R,11S)-Nonacosanetriol (10R,12R,15S)-Nonacosanetriol Ginkgolic acid Hydroginkgolic acid Sciadopitysin Ginkgetin Isoginkgetin Ginkgolide A Ginkgolide B Ginkgolide C Petroleum ether extract Ethyl acetate extract n-Butanol extract H2 O extract Aspirin
100 100 100 100 100 100 100 100 100 100 1.0 × 104 1.0 × 104 1.0 × 104 1.0 × 104 100
25.70 27.20 3.78 4.05 17.16 6.78 29.14 21.43 36.75 21.21 42.74 46.58 29.71 22.03 70.20
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.20 1.73 0.58 0.74 2.21 2.09 3.65 1.90 0.82 1.49 4.23 3.38 2.40 2.33 3.11
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.92 1.76 1.09 1.40 1.07 1.05 0.95 1.22 1.07 1.94 1.65 1.69 1.62 1.74 2.60
lgTT prolongation rate (%) 1.00 1.06 0.75 0.79 1.09 0.99 1.52 1.55 1.68 1.47 1.65 1.60 1.40 1.34 1.89
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.28 0.24 0.04 0.15 0.03 0.02 0.29 0.09 0.03 0.29 0.22 0.23 0.21 0.24 0.41
pharmacological functions including inhibiting cholesterol synthesis, increasing low-density lipoprotein processing, anti-thrombosis and anti-platelet aggregation activity (Yang et al., 2009b). This was the first report regarding the anticoagulative activities of biflavones in G. biloba, and isoginkgetin (7) showed significant antithrombin and antiplatelet aggregation activity. Acknowledgments This work was supported by A Project Funded by the National Science and Technology Support Program of China (2011BAI04B03), the Priority Academic Program Development of Jiangsu Higher Education Institutions (ysxk-2010), 2009 Program for New Century Excellent Talents by the Ministry of Education (NCET-09-0163), 2009 Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education, and Construction Project for Jiangsu Engineering Center of Innovative Drug from Blood-conditioning TCM Formulae. We are grateful to Mr. Wei Jiang (Taixing, Jiangsu province, China) for collecting samples. We are also pleased to thank Mr. Dongjun Chen (China Pharmaceutical University) for the help in NMR tests. References Carrier, D.J., van Beek, T.A., van der, Heijden, R., Verpoorte, R., 1998. Distribution of ginkgolides and terpenoid biosynthetic activity in Ginkgo biloba. Phytochemistry 48, 89–92. Chen, S.X., Wu, L., Yang, X.M., Jiang, X.G., Li, L.G., Zhang, R.X., Xia, L., Shao, S.H., 2007. Comparative molluscicidal action of extract of Ginko biloba sarcotesta, arecoline and mclosamide on snail hosts of Schistosoma japonicum. Pesticide Biochemistry and Physiology 89, 237–241. Chen, Y., Chen, J.W., Li, X., 2011. Cytotoxic bistetrahydrofuran annonaceous acetogenins from the seeds of Annona squamosa. Journal of Natural Products 74, 2477–2481. Choi, J.G., Jeong, S.I., Ku, C.S., Sathishkumar, M., Lee, J.J., Mun, S.P., Kim, S.M., 2009. Antibacterial activity of hydroxyalkenyl salicylic acids from sarcotesta of Ginkgo biloba against vancomycin-resistant enterococcus. Fitoterapia 80, 18–20. Choukchou-Braham, N., Asakawa, Y., Lepoittevin, J.P., 1994. Lepoittevin, isolation, structure determination and synthesis of new dihydroisocoumarins from Ginkgo biloba L. Tetrahedron Letters 35, 3949–3952. Ciavatta, M.L., Manzo, E., Nuzzo, G., Villani, G., Varcamonti, M., Gavagnin, M., 2010. Crucigasterins A-E, antimicrobial amino alcohols from the Mediterranean colonial ascidian Pseudodistoma crucigaster. Tetrahedron 66, 7533–7538. Deng, Q.C., Wang, L., Wei, F., Xie, B.J., Huang, F.H., Huang, W., Shi, J., Huang, Q.D., Tian, B.Q., Xue, S., 2011. Toxicity of 4 -O-methylpyridoxine-5 -glucoside in Ginkgo biloba seeds. Food Chemistry 124, 1458–1465. ˇ Freire, F., Lallana, E., Quinoá, E., Riguera, R., 2009. The stereochemistry of 1,2,3-triols revealed by 1 H NMR spectroscopy: principles and applications. Chemistry – A European Journal 15, 11963–11975. Fu, F.Y., Yu, D.Q., Sung, W.L., 1962. The chemical study of hydroginkgolinic acid. Acta Chimica Sinica 28, 52–56. Gao, J.M., Wang, C.Y., Zhang, A.L., Liu, J.K., 2001. A new trihydroxy acid from the ascomycete Chinese truffle Tuber indicum. Lipids 36, 1365–1366. Gao, J.M., Zhang, A.L., Chen, H., Liu, J.K., 2004. Molecular species of ceramides from the ascomycete truffle Tuber indicum. Chemistry and Physics of Lipids 131, 205–213.
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G. Zhou et al. / Chemistry and Physics of Lipids 165 (2012) 731–736
Jin, T.C., Chen, Y.W., Howard, A., Zhang, Y.Z., 2008. Purification, crystallization and initial crystallographic characterization of the Ginkgo biloba 11S seed globulin ginnacin. Acta Crystallographica 64, 641–644. Kobayashi, Y., Hayashi, N., Tan, C.H., Kishi, Y., 2001. Toward the creation of NMR databases in chiral solvents for assignments of relative and absolute stereochemistry: proof of concept. Organic Letters 14, 2246–2248. Krauze-Baranowska, M., Poblocka, L., El Hela, A.A., 2005. Biflavones from Chamaecyparis obtusa. Zeitschrift fur Naturforschung C 60, 679–685. Li, Z., Tran, V.H., Duke, R.K., Ng, M.C.H., Yang, D., Duke, C.C., 2009. Synthesis and biological activity of hydroxylated derivatives of linoleic acid and conjugated linoleic acids. Chemistry and Physics of Lipids 158, 39–45. Llabres, G., Baiwir, M., Sbit, M., Dupont, L., 1989. Ginkgolides A, B and C: a 1D and 2D NMR study. Spectrochimica Acta Part A 45, 1037–1045. Major, R.T., 1967. The ginkgo, the most ancient living tree. Science 157, 1270–1273. Manini, P., Camera, E., Picardo, M., Napolitano, A., d’Ischia, M., 2008. Biomimetic nitration of the linoleic acid metabolite 13-hydroxyoctadecadienoic acid: isolation and spectral characterization of novel chain-rearranged epoxy nitro derivatives. Chemistry and Physics of Lipids 151, 51–61. Markham, K.R., Sheppard, C., Geiger, H., 1987. 13 C NMR study of some naturally occurring amentoflavone and hinokiflavone biflavonoids. Phytochemistry 26, 3335–3337. Pan, W.G., Luo, P., Fu, R.B., Gao, P., Long, Z.F., Xu, F.Y., Xiao, H.B., Liu, S.G., 2006. Acaricidal activity against Panonychus citri of a ginkgolic acid from the external seed coat of Ginkgo biloba. Pest Management Science 62, 283–287. Rieser, M.J., Hui, Y.H., Rupprecht, J.K., Kozlowski, J.F., Wood, K.V., McLaughlin, J.L., Hanson, P.R., Zhuang, Z.P., Hoye, T.R., 1992. Determination of absolute configuration of stereogenic carbinol centers in annonaceous acetogenins by 1 H- and 19 F-NMR analysis of Mosher ester derivatives. Journal of the American Chemical Society 114, 10203–10213.
Sakurai, Y., Shima, M., Giddings, J., Takeyama, M., Kasuda, S., Nogami, K., Nishiya, K., Yoshioka, A., 2004. A critical role for thrombin in platelet aggregation under high shear stress. Thrombosis Research 113, 311–318. Sikka, P., Bindra, V.K., 2010. Newer antithrombotic drugs. Indian Journal of Critical Care Medicine 14, 188–195. Singh, B., Kaur, P., Gopichand, Singh, R.D., Ahuja, P.S., 2008. Biology and chemistry of Ginkgo biloba. Fitoterapia 79, 401–418. Tao, W.W., Yang, N.Y., Duan, J.A., Wu, D.K., Shang, E.X., Qian, D.W., Tang, Y.P., 2010. Two new nonacosanetriols from the pollen of Typha angustifolia. Chinese Chemical Letters 21, 209–212. van Beek, T.A., 2002. Chemical analysis of Ginkgo biloba leaves and extracts. Journal of Chromatography A 967, 21–55. Wenzel, T.J., Chisholm, C.D., 2011. Using NMR spectroscopic methods to determine enantiomeric purity and assign absolute stereochemistry. Progress in Nuclear Magnetic Resonance Spectroscopy 59, 1–63. Wu, X.Y., Mao, G.H., Zhao, T., Zhao, J.L., Li, F., Liang, L.H., Yang, L.Q., 2011. Isolation, purification and in vitro anti-tumor activity of polysaccharide from Ginkgo biloba sarcotesta. Carbohydrate Polymers 86, 1073–1076. Yamagiwa, Y., Ohashi, K., Sakamota, Y., Hirakawa, S., Kamikawa, T., 1987. Syntheses of anacardic acids and ginkgoic acid. Tetrahedron 43, 3387–3394. Yang, N.Y., Tao, W.W., Duan, J.A., 2009a. Four new long-chain aliphatics from the feces of Trogopterus xanthipes. Journal of Asian Natural Products Research 11, 1032–1039. Yang, N.Y., Tao, W.W., Duan, J.A., Guo, J.M., Cao, L.L., 2009b. Four new fatty acid esters from the feces of Trogopterus xanthipes. Lipids 44, 849–853. Yang, N.Y., Zhou, G.S., Tang, Y.P., Yan, H., Guo, S., Liu, P., Duan, J.A., Song, B.S., He, Z.Q., 2011. Two new ␣-pinene derivatives from Angelica sinensis and their anticoagulative activities. Fitoterapia 82, 692–695. Zhang, W.K., Xu, J.K., Zhang, X.Q., Yao, X.S., Ye, W.C., 2007. Sphingolipids with neuritogenic activity from Euphorbia sororia. Chemistry and Physics of Lipids 148, 77–83.
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Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip
Two new nonacosanetriols from Ginkgo biloba sarcotesta Guisheng Zhou a , Xin Yao a , Yuping Tang a,∗ , Nianyun Yang a , Hanqing Pang a , Xuan Mo a , Shaoqing Zhu a , Shulan Su a , Dawei Qian a , Chun Jin b , Yong Qin b , Jin-ao Duan a,∗ a b
Jiangsu Key Laboratory for High Technology of TCM Formulae Research, Nanjing University of Chinese Medicine, Nanjing 210046, China Jiangsu Shenlong Pharmaceutical Co. Ltd., Yancheng 224200, China
a r t i c l e
i n f o
Article history: Received 9 June 2012 Received in revised form 29 August 2012 Accepted 31 August 2012 Available online 6 September 2012 Keywords: Ginkgo biloba Sarcotesta Fatty alcohol Anticoagulative activity Stereochemistry
a b s t r a c t Two new fatty alcohols named as (7S,8R,11S)-nonacosanetriol (1) and (10R,12R,15S)-nonacosanetriol (2), along with eight known compounds including ginkgolic acid (3), hydroginkgolic acid (4), sciadopitysin (5), ginkgetin (6), isoginkgetin (7), ginkgolide A (8), ginkgolide B (9) and ginkgolide C (10) have been isolated from the petroleum ether extract of Ginkgo biloba sarcotesta. Their structures were elucidated by means of chemical and extensive spectroscopic analysis. The absolute stereochemistry of compounds 1 and 2 was elucidated on the spectroscopic analysis of the R- and S-MTPA esters. Compounds 1 and 2 exhibited slight activity of antithrombin and moderate activity of antiplatelet aggregation in vitro. This was the first report regarding the anticoagulative activities of biflavonoids in G. biloba, and isoginkgetin (7) showed significant antithrombin and antiplatelet aggregation activity. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Ginkgo biloba is a deciduous and dioecious gymnosperm species (Major, 1967) originated in China and is the sole survivor of the ancient family of Ginkgoaceae (Carrier et al., 1998). Although G. biloba has been above 200 million years old, its true value has induced a range of attentions all around the world till the recent two decades. It contains a large number of active compounds, the most important of which are flavanol glycosides and terpene lactones. Its amazing vitality has attracted an increasing exploration into potential application in health, foods and supplements (van Beek, 2002; Singh et al., 2008). Male and female flowers are born on different plants and female plants bear a yellowish-green plumlike “fruit” (aril) (Choukchou-Braham et al., 1994) which is indeed the seed of ginkgo in gymnosperms. The outer malodorous fleshy layer is called sarcotesta. It surrounds an ovoid nut, which is called “Baiguo” in China and “Gin-nan” in Japan (Deng et al., 2011).
Abbreviations: ADP, adenosine diphosphate; COSY, correlation spectroscopy; DMAP, 4-dimethylaminopyridine; ESI, electrospray ionization; Gbs, Ginkgo biloba sarcotesta; HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear singular quantum correlation; HR, high resolution; MS, mass spectrometry; NMR, nuclear magnetic resonance; PPP, platelet poor plasma; PRP, platelet rich plasma; RMTPA, R-(−)-␣-methoxy-␣-(trifluoromethyl)phenylacetyl chloride; S-MTPA, S-(+)␣-methoxy-␣-trifluoromethylphenylacetyl chloride; TMS, tetramethylsilane; Tris, trishydroxymethylaminomethane; TT, thrombin time. ∗ Corresponding authors. Tel.: +86 25 85811916; fax: +86 25 85811916. E-mail addresses: [email protected] (Y. Tang), [email protected] (J.-a. Duan). 0009-3084/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemphyslip.2012.08.003
Ginkgo seeds have been used in traditional Chinese medicine (TCM) and as a foodstuff for centuries throughout Asia (Jin et al., 2008). However, Gbs, the epicarp of mature seeds which is peeled from G. biloba seeds, was treated as waste and discarded abundantly in soil and water, thus it polluted soil and poisoned fish in rivers and lakes (Pan et al., 2006). It has become an urgent problem to the local enterprises and governments of the planting area for its poison to the environment (Wu et al., 2011). Modern studies indicated that Gbs consists of ginkgolic acid and polysaccharide, which were reported to have many biological and pharmacological functions, including antitumor, anti-inflammatory, pesticidal and antibacterial properties (Chen et al., 2007). In Xingan (Guangxi province, China) the fruit juice produced during the peeling process was diluted with water and used as an insecticide by local peasants. Satisfactory results were achieved in the control of many kinds of insects, indicating the presence of some insecticidal constituents (Choi et al., 2009). In our preliminary experiment, 95% EtOH extract of Gbs showed anticoagulative activity. And then, in our present study, different solvent extracts of Gbs were tested, and the results showed that the petroleum ether extract had strong anticoagulative activity. Therefore, the petroleum ether extract of Gbs was chemically investigated, which resulted in the isolation of two new fatty alcohols, named as (7S,8R,11S)-nonacosanetriol (1) and (10R,12R,15S)-nonacosanetriol (2), together with eight known compounds including ginkgolic acid (3), hydroginkgolic acid (4), sciadopitysin (5), ginkgetin (6), isoginkgetin (7), ginkgolide A (8), ginkgolide B (9) and ginkgolide C (10). Some nonacosanetriols or similar compounds were isolated from nature, such as 7,8,10-nonacosanetriol and 7,9,10-nonacosanetriol from Typha
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angustifolia (Tao et al., 2010), 8,15-nonacosanediol and 6,13nonacosanediol from Trogopterus xanthipes (Yang et al., 2009a), and 9,10,11-trihydroxy-(12Z)-12-octadecenoic acid from Tuber indicum (Gao et al., 2001). The structures of the two new compounds were elucidated by means of chemical and extensive spectroscopic analysis. Furthermore, their anticoagulative activities were also evaluated. 2. Experimental 2.1. Materials and chemicals Gbs was collected on November 2010 from Taizhou, Jiangsu Province, China, and identified as the sarcotesta of Ginkgo biloba L. by Dr. Hui Yan (Department of Pharmacognosy, Nanjing University of Chinese Medicine, China). A voucher specimen (BG-20101120) was deposited at the Herbarium in Jiangsu Key Laboratory for TCM Formulae Research. Silica gel for column chromatography (CC) (60–80 m) and thin-layer chromatography plates (10–40 m) was purchased from Qingdao Marine Chemical (Qingdao, China). All solvents used were of analytical grade (Nanjing Chemical Plant, Nanjing, China). RMTPA and S-MTPA were obtained from Sigma Chemical Co. (St. Louis, MO). Thrombin was purchased from Xisen Sanhe (Leling, China). Tris was the product of Shanghai Jingxi Chemical Industry (Shanghai, China). Sodium citrate was purchased from Shanghai Lingfeng Chemical Reagent (Shanghai, China). Heparin sodium (biotech grade, 150 U mg−1 ) was purchased from Amresco (Solon, USA). ADP was purchased from Beijing Zhongqin Scientific Instrument Co. Ltd. (Beijing, China). A rabbit (3.8 kg) was supplied by Shanghai Sikelai Experimental Animal (Shanghai, China). 2.2. Apparatus Melting points were determined with a WRS-lB melting point apparatus (Shanghai Precision and Scientific Instrument, Shanghai, PR China) which was uncorrected. Optical rotation was measured on a Perkin-Elmer 341 polarimeter. NMR spectra were measured on a Bruker AV-500 MHz (500 MHz for 1 H NMR and 125 MHz for 13 C NMR), using TMS as internal standard; chemical shifts were recorded as ı (ppm) values. ESI-MS and HR-ESI-MS spectra were obtained on a Micromass Q/TOF mass spectrometer. Anticoagulative assay was performed on an LG-PABER-I coagulation-analysis instrument. The blood sample was treated on an Anke TDL-40B centrifuge. 2.3. Extraction and isolation Air-dried and powdered Gbs (25 kg) were extracted two times with 95% EtOH (2 × 250 L) under reflux for 2 h at 70–80 ◦ C each time. The extracts were concentrated under reduced pressure, and then they were suspended in H2 O and extracted successively with petroleum ether, ethyl acetate and n-butanol to give the respective extracts after solvent removal. The combined petroleum ether extracts were evaporated under reduced pressure to leave a residue (250 g) which was chromatographed on silica gel (2.5 kg) eluted with a petroleum ether–EtOAc stepwise gradient (100:0 → 1:1) and 5 fractions were collected. Fr. 4 (30 g) was separated by silica gel (petroleum ether–EtOAc, 5:1) to obtain compounds 1 (50 mg) and 2 (30 mg); Fr. 1 (60 g) was separated by silica gel (petroleum ether–EtOAc, 20:1) to obtain compounds 3 (100 mg) and 4 (60 mg); Fr. 3 (40 g) was separated by silica gel (petroleum ether–EtOAc, 8:1) to obtain compounds 5 (50 mg), 6 (10 mg) and 7 (10 mg), and Fr. 5 (40 g) was separated by silica gel (petroleum ether–EtOAc 3:1) to obtain compounds 8 (20 mg), 9 (30 mg) and 10 (10 mg).
(7S,8R,11S)-nonacosanetriol (1) white powder, [␣]D 20 + 3.0◦ (c = 0.045, MeOH), m.p. 160–162 ◦ C, 1 H NMR (CD3 OD, 500 MHz) ı 3.80 (1H, m, H-11), 3.58 (1H, m, H-8), 3.40 (1H, m, H7), 1.76 (1H, t, J = 3.5 Hz, H-10a), 1.73 (1H, t, J = 3.5 Hz, H-9a), 1.53 (1H, m, H-9b), 1.51 (1H, m, H-10b), 1.45–1.50 (4H, br. m, H-6, 12), 1.24–1.39 (40H, br. m, H-2–5, 13–28), 0.89 (6H, t, J = 7.0 Hz, H-1, 29); 13 C NMR (CD3 OD, 125 MHz) ı 76.4 (C7), 76.0 (C-8), 72.4 (C-11), 40.4 (C-9, 10), 39.1 (C-6, 12), 34.1 (C-3, 27), 30.9–31.3 (C-4, 14–26), 27.4 (C-5), 26.9 (C-13), 24.1 (C-2, 28), 14.8 (C-1, 29). ESI-MS: m/z 455 [M−H]− , 457 [M+H]+ , 479 [M+Na]+ ; HR-ESI-MS: m/z 479.7761 [M+Na]+ (C29 H60 O3 Na, calc. 479.7747). C(7),C(8),C(11)-tris-(S)-MTPA ester of compound 1 [(S)-MTPA1] white powder, m.p. 175–180 ◦ C, 1 H NMR (CD3 OD, 500 MHz) ı 7.58 (6H, m, MTPA-ArH-3, 5), 7.41 (9H, m, MTPA-ArH-2, 4, 6), 3.83 (1H, m, H-11), 3.60 (1H, m, H-8), 3.56 (9H, s, MTPA-OCH3 ), 3.42 (1H, m, H-7), 1.75 (1H, m, H-9a), 1.70 (1H, m, H-10a), 1.56 (1H, m, H-9b), 1.52 (1H, m, H-10b), 1.38–1.43 (4H, m, H-6, 12), 1.27–1.38 (40H, m, H-2–5 and H-13–28), 0.90 (6H, t, J = 7.0 Hz, H-1, 29). ESI-MS: m/z 1105 [M+H]+ . C(7),C(8),C(11)-tris-(R)-MTPA ester of compound 1 [(R)-MTPA1] white powder, m.p. 169–173 ◦ C, 1 H NMR (CD3 OD, 500 MHz) ı 7.44 (6H, m, MTPA-ArH-3, 5), 7.36 (9H, m, MTPA-ArH-2, 4, 6), 3.81 (1H, m, H-11), 3.64 (1H, m, H-8), 3.55 (9H, s, MTPA-OCH3 ), 3.40 (1H, m, H-7), 1.76 (1H, m, H-9a), 1.73 (1H, m, H-10a), 1.57 (1H, m, H-9b), 1.55 (1H, m, H-10b), 1.35–1.40 (4H, br. m, H-6, 12), 1.30–1.35 (40H, br. m, H-2–5, 13–28), 0.89 (6H, t, J = 7.0 Hz, H-1, 29). ESI-MS: m/z 1105 [M+H]+ . (10R,12R,15S)-nonacosanetriol (2) white powder, [␣]D 20 − 8.0◦ (c = 0.015, MeOH), m.p. 185–187 ◦ C, 1 H NMR (CDCl3 , 300 MHz) ı 3.88 (3H, m, H-10, 12, 15), 1.61 (1H, m, H-11a), 1.58 (1H, m, H-11b), 1.42–1.48 (8H, br. m, H-9, 13, 14, 16), 1.26–1.29 (38 H, br. m, H-2–8, 17–28), 0.88 (6H, t, J = 6.6 Hz, H-1, 29); 13 C NMR (CDCl3 , 75 MHz) ı 75.4 (C-12), 74.5 (C-15), 72.8 (C-10), 38.6 (C-11), 38.0 (C-13, 14), 36.8 (C-9, 16), 32.0 (C-3, 27), 29.4–29.7 (C-4–7, 18–26), 26.0 (C-8), 25.4 (C-17), 22.6 (C-2, 28), 14.0 (C-1, 29). ESI-MS: m/z 457 [M+H]+ , 479 [M+Na]+ ; HR-ESI-MS: m/z 479.7706 [M+Na]+ (C29 H60 O3 Na, calc. 479.7747). C(10),C(12),C(15)-tris-(S)-MTPA ester of compound 2 [(S)MTPA-2] white powder, m.p. 209–213 ◦ C, 1 H NMR (CDCl3 , 500 MHz) ı 7.58 (6H, m, MTPA-ArH-3, 5), 7.42 (9H, m, MTPA-ArH2, 4, 6), ı 3.90 (3H, m, H-10, 12, 15), 3.56 (9H, s, MTPA-OCH3 ), 1.73 (1H, m, H-11a), 1.70 (1H, m, H-11b), 1.52 (4H, m, H-13, 14), 1.45 (4H, m, H-9, 16), 1.25–1.31 (38 H, m, H-2–8 and H-17–28), 0.88 (6H, t, J = 6.6 Hz, H-1, 29). ESI-MS: m/z 1105 [M+H]+ . C(10),C(12),C(15)-tris-(R)-MTPA ester of compound 2 [(R)MTPA-2] white powder, m.p. 193–196 ◦ C, 1 H NMR (CDCl3 , 500 MHz) ı 7.44 (6H, m, MTPA-ArH-3, 5), 7.36 (9H, m, MTPAArH-2, 4, 6), 3.89 (3H, m, H-10, 12, 15), 3.55 (9H, s, MTPA-OCH3 ), 1.68 (1H, m, H-11a), 1.65 (1H, m, H-11b), 1.55 (4H, m, H-13, 14), 1.45 (4H, m, H-9, 16), 1.23–1.30 (38 H, m, H-2–8 and H-17–28), 0.88 (6H, t, J = 6.6 Hz, H-1, 29). ESI-MS: m/z 1105 [M+H]+ .
2.4. Acetylation of compounds 1 and 2 Compounds 1 (2 mg) and 2 (2 mg) were dissolved in 3 mL of Ac2 O–pyridine (1:1) and kept at room temperature for 48 h to yield crude products, respectively. 3 mL of water was added into the reaction products, and the solution was extracted three times with EtOAc (3 × 3 mL), respectively. The resulting extractions were concentrated under reduced pressure to afford their corresponding diacetate derivatives (1a and 2a), which were analyzed by ESIMS–MS.
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Fig. 1. ESI-MS/MS fragmentation of compounds 1 and 2.
2.5. Preparation and purification of MTPA esters 1 mg nonacosanetriol was dissolved in dry CH2 Cl2 (0.5 mL) and sequentially added dry pyridine (0.4 mL), DMAP (0.5 mg), and (R)-MTPA (6 L). The reaction mixtures were stirred at room temperature for 24 h. The resulting mixture was dried over Na2 SO4 and concentrated under reduced pressure. The residue, diluted with CH2 Cl2 , was purified by microcolumn chromatography on silica gel (60–80 m) and eluted with 5 mL hexane–EtOAc (8:2). Finally, the eluent was dried in vacuo, thus yielding the corresponding MTPA ester derivative namely S-Mosher esters. The R-Mosher ester was prepared and purified by using (S)-MTPA following the same procedure as described above (Kobayashi et al., 2001; Freire et al., 2009; Chen et al., 2011). 2.6. Anticoagulative assay All samples were dissolved in ethanol. The anticoagulative activities of compounds 1–10, petroleum ether extracts, ethyl acetate extracts, n-butanol extracts, H2 O extracts, positive control (heparin sodium) and blank (ethanol) were evaluated by the TT method. A rabbit was sacrificed to obtain plasma (50 mL). The plasma was put in a plastic cup for 3 min at 37 ◦ C, together with 100 mL thrombin solution of 15 U mL−1 diluted by 0.1 mol mL−1 Tris–HCl buffer (pH 7.4) and 10 mL sample solution; meanwhile, the coagulation analysis instrument was started up to record the thrombin clotting time. Each analyte was tested for six times, and an average value was calculated. TT prolongation rate and lgTT prolongation rate were calculated to assess the anticoagulative activity of the samples. 2.7. Antiplatelet aggregation activity Evaluation of antiplatelet aggregation activities of the different solvent extracts, pure compounds 1–10, positive control (aspirin) and blank (ethanol) were performed using an antiplatelet aggregation method. A rabbit was sacrificed to obtain PRP and PPP (Yang et al., 2011). Platelet count was adjusted to 3 × 105 with PPP. Added to the PRP, each sample was incubated at 37 ◦ C with stirring for 1 min before the addition of ADP (10 M) as inducer of platelet
aggregation. Aggregation was measured by a turbidimetric method. Platelet aggregation was expressed as the percentage change with the difference of light transmittance between PRP and PPP as 100%. Each analyte was tested for six times, and an average value was applied. Antiplatelet activity was expressed as the percent inhibition of the control value.
3. Results and discussion The petroleum ether extracts of Gbs were subjected to repeated silica gel liquid chromatography to obtain two new fatty alcohols: (7S,8R,11S)-nonacosanetriol (1)and (10R,12R,15S)nonacosanetriol (2), eight known compounds: ginkgolic acid (3) (Yamagiwa et al., 1987), hydroginkgolic acid (4) (Fu et al., 1962), sciadopitysin (5) (Markham et al., 1987), ginkgetin (6) (Markham et al., 1987), isoginkgetin (7) (Krauze-Baranowska et al., 2005), ginkgolide A (8) (Llabres et al., 1989), ginkgolide B (9) (Llabres et al., 1989) and ginkgolide C (10) (Llabres et al., 1989). The structures of compounds 1 and 2 were elucidated as follows. Compound 1 was obtained as a white powder. Its molecular formula was established as C29 H60 O3 by HR-ESI-MS (m/z 479.7761 [M+Na]+ ). Its positive ESI-MS showed characteristic peaks at m/z 457 [M+H]+ , and 479 [M+Na]+ . The 1 H NMR (CD3 OD) of 1 showed proton signals of two terminal methyls at ı 0.89 (6H, t, J = 7.0 Hz), 20 methylenes at ı 1.24–1.39 (40H, br. m), and three oxygenated methine at ı 3.80 (1H, m), 3.58 (1H, m), and 3.40 (1H, m). The 13 C NMR (CD OD) spectrum of 1 revealed carbon signals for three 3 oxygenated methine at ı 76.4, 76.0, and 72.4. The above data indicated a long chain hydrocarbon with three hydroxyl functions in 1 (Gao et al., 2001; Li et al., 2009). As shown in Fig. 1(1), the ESI-MS–MS spectrum showed a set of characteristic peaks at m/z 203 → 185 → 167 → 149, 173 → 155 → 137, 283 → 265, and 253 formed by ␣-fission and loss of H2 O, which suggested the hydroxyl group was located at C-11. Other diagnostic fragment ions at m/z 85, 371 → 353 → 335 → 317, 115 → 97, 341 → 323 → 305, and 145 → 127 → 109, showed hydroxyl groups at C-7, C-8, and C-11. Compound 1 was subjected to acetylation (Zhang et al., 2007), and the final reaction product of 1a was analyzed by ESIMS and ESI-MS–MS. Its ESI-MS–MS gave characteristic peaks at
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Fig. 2. ESI-MS/MS fragmentation of acetylation products 1a and 2a.
m/z 491 ([M+Cl+3×H2 O−3×AcOH]− , 100), 581 ([M−H]− , 14), 521 ([M−H–AcOH]− , 10), 461 ([M–H−3×AcOH]− , 6), 497 (C29 H53 O6 , 3), 425 (C26 H49 O4 , 10), 353 (C23 H45 O2 , 5), 329 (C17 H29 O6 , 3), 325 (C21 H41 O2 , 18), 257 (C14 H25 O4 , 3), 253 (C18 H37 , 4), and 85 (C6 H13 , 3) in Fig. 2(1a). Assignments of all protons and carbons in 1 were also made on the basis of 1 H–1 H–COSY, HSQC, and HMBC spectra. In the 1 H–1 H–COSY spectrum of 1, the diagnostic correlations between H-7 at ı 3.40 (m) and H-8 at ı 3.58 (m), H-8 and H-9a at ı 1.73 (t), H-8 and H-9b at ı 1.53 (m), H-10a at ı 1.76 (t) and H-11 at ı 3.80 (m), H-10b and H-11 were observed in 1. These correlations strongly confirmed that 1 contained the partial structure CH2 CH(OH) CH(OH) CH2 CH2 CH(OH) . Meanwhile, the corresponding correlations were observed in HSQC and HMBC spectra. From these data, compound 1 was identified as 7,8,11nonacosanetriol. The absolute configuration of 1 was elucidated on the basis of spectroscopic evidence and the spectroscopic analyses of the (R)- and (S)-MTPA esters. The chemical shift of three chiral centers (7, 8, 11 positions) of 1 was identified by HSQC techniques. The sign of ıH [=(ıS − ıR )] was positive for H (6), H (7), and H (12), and negative for H (8), H (9), and H (10). The detailed ıH of the synthetic MTPA-1 which had the unambiguous absolute stereochemistry (Rieser et al., 1992; Gao et al., 2004; Ciavatta et al., 2010; Wenzel and Chisholm, 2011) is listed in Table 1. Based on the above evidences, the structure of compound 1 was established as (7S, 8R, 11S)-nonacosanetriol. Compound 2 was obtained as a white powder. Its molecular formula was established as C29 H60 O3 by positive ion HR-ESI-MS m/z 479.7706 [M+Na]+ . Its positive ESI-MS showed characteristic peaks at m/z 457 [M+H]+ , and 479 [M+Na]+ . The 1 H NMR (CDCl3 ) of 2 showed proton signals of two terminal methyls at ı 0.88 (6H, t, J = 6.6 Hz), methylenes at ı 1.26–1.29 (38 H, br. m),
and three oxygenated CH groups at ı 3.88 (3H, m). The 13 C NMR (CDCl3 ) spectrum of 2 revealed carbon signals for three oxygenated methine at ı 75.4, 74.5, and 72.8. These data inferred the existence of a straight-chain compound with three hydroxyl functions in 2 (Gao et al., 2001). As shown in Fig. 1(2), a set of characteristic peaks at m/z 197, 259 → 241 → 223 → 205, 229 → 211 → 193, and 227 → 209, were formed by ␣-fission and loss of H2 O, which suggested the hydroxyl group was located at C-15. Other diagnostic fragment ions at m/z 127, 329 → 311 → 293 → 275, 171 → 153, 285 → 267 → 249, 157 → 139, 299 → 281 → 263, 201 → 183 → 165, and 255 → 237, were derived from ␣-fission of the hydroxyl group (Manini et al., 2008), which suggested three OH groups present at C-10, C-12, and C-15. Compound 2 was also subjected to acetylation and the final reaction product (2a) was analyzed by ESI-MS and ESI-MS–MS, and its ESI-MS–MS gave characteristic peaks at m/z 479 ([M+Na+3×H2 O−3×AcOH]+ , 100), 582 ([M]+ , 8), 545 ([M+Na–AcOH]+ , 3), 487 ([M+Na−2×AcO]+ , 22), 428 ([M+Na−3×AcO]+ , 11), 455 (C26 H47 O6 , 3), 385 (C21 H37 O6 , 8), 383 (C23 H43 O4 , 3), 369 (C22 H41 O4 , 4), 313 (C18 H33 O4 , 6), 297 (C19 H37 O2 , 5), 285 (C16 H29 O4 , 6), 269 (C17 H33 O2 , 2), 213 (C13 H25 O2 , 2), 199 (C12 H23 O2 , 2), 197 (C14 H29 , 2), and 127 (C9 H19 , 2) in Fig. 2(2a). These fragment ions were derived from loss of HOAc or ␣-fission of the acetoxyl group, which revealed three OAc groups present at C-10, C-12, and C-15. Thus, compound 2 was elucidated as 10,12,15-nonacosanetriol. 1 H–1 H–COSY, HSQC, and HMBC experiments were done to further prove the structure of 2. The absolute stereochemistry of 2 was interpreted on the basis of spectroscopic evidence and the spectroscopic analyses of the Mosher’s esters. The sign of ıH [=(ıS − ıR )] was positive for H (11), zero for H (9), and H (16), and negative for H (13), and H (14). The detailed ıH of synthetic MTPA-2 which had the absolute stereochemistry (Rieser
Table 1 1 H NMR data for H (6)–H (16) from the (S)- and (R)-MTPA ester of compounds 1 and 2. Position
(S)-MTPA-1
(R)-MTPA-1
ıH (ıS − ıR )
Position
(S)-MTPA-2
(R)-MTPA-2
ıH (ıS − ıR )
6, 12 7 8 9 10
1.38–1.43 3.42 3.60 1.75 1.52
1.35–1.40 3.40 3.64 1.76 1.55
+0.03 +0.02 −0.04 −0.01 −0.03
9, 16 11 13, 14
1.45 1.73 1.52
1.45 1.68 1.55
0 +0.05 −0.03
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Table 2 Antithrombin activity of the different solvent extracts and compounds 1–10 (x ± s, n = 6). Samples
Dosage (g mL−1 )
TT prolongation (%)
(7S,8R,11S)-Nonacosanetriol (10R,12R,15S)-Nonacosanetriol Ginkgolic acid Hydroginkgolic acid Sciadopitysin Ginkgetin Isoginkgetin Ginkgolide A Ginkgolide B Ginkgolide C Petroleum ether extract Ethyl acetate extract n-Butanol extract H2 O extract Heparin sodium
100 100 100 100 100 100 100 100 100 100 1.0 × 104 1.0 × 104 1.0 × 104 1.0 × 104 50
10.02 11.42 5.61 6.21 12.22 9.82 32.86 35.27 48.32 29.25 45.47 40.51 25.34 22.00 77.99
et al., 1992; Gao et al., 2004; Ciavatta et al., 2010; Wenzel and Chisholm, 2011) is listed in Table 1. Based on the above evidences, the structure of compound 2 was established as (10R,12R,15S) – nonacosanetriol. The TT and antiplatelet aggregation methods were successfully developed in our laboratory for determining the anticoagulative activities of a series of Chinese blood-activating medicines and their components (Yang et al., 2009b). In vitro anticoagulative activities of the different solvent extracts and compounds 1–10 were tested by using the TT and antiplatelet aggregation methods in this study. The results are summarized in Tables 2 and 3. Petroleum ether extracts, and ethyl acetate extracts were the important active fractions, and ginkgolides (8–10) could remarkably prolong thrombin time and inhibit platelet aggregation, especially ginkgolide B (compound 9). Two new fatty alcohols (1 and 2) could also slightly prolong thrombin time and moderately inhibit platelet aggregation. Platelet activation could be inhibited by inhibiting thromboxane A2 pathway, ADP pathway, thrombin and phosphodiesterase (Sikka and Bindra, 2010). In this study platelet aggregation was induced by ADP in vitro, while compounds 1 and 2 could moderately reversed the platelet aggregation induced by ADP. So, compounds 1 and 2 might exhibit the antiplatelet activity by playing a role on the ADP pathway. Thrombin was a serine protease that played a key role in thrombosis. Thrombin was the most potent physiological agonist of platelet activation and aggregation which was generated on the platelet surface (Sakurai et al., 2004). Compounds 1 and 2 might act directly on thrombin to attain the antithrombin effect which is another way to exhibit the antiplatelet activity. Fatty alcohols, fatty acids, and their esters were important lipid compounds and had many biological and
Table 3 Antiplatelet aggregation activity of the different solvent extracts and compounds 1–10 (x ± s, n = 6). Samples
Dosage (g mL−1 )
Inhibition ratio (%)
(7S,8R,11S)-Nonacosanetriol (10R,12R,15S)-Nonacosanetriol Ginkgolic acid Hydroginkgolic acid Sciadopitysin Ginkgetin Isoginkgetin Ginkgolide A Ginkgolide B Ginkgolide C Petroleum ether extract Ethyl acetate extract n-Butanol extract H2 O extract Aspirin
100 100 100 100 100 100 100 100 100 100 1.0 × 104 1.0 × 104 1.0 × 104 1.0 × 104 100
25.70 27.20 3.78 4.05 17.16 6.78 29.14 21.43 36.75 21.21 42.74 46.58 29.71 22.03 70.20
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.20 1.73 0.58 0.74 2.21 2.09 3.65 1.90 0.82 1.49 4.23 3.38 2.40 2.33 3.11
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1.92 1.76 1.09 1.40 1.07 1.05 0.95 1.22 1.07 1.94 1.65 1.69 1.62 1.74 2.60
lgTT prolongation rate (%) 1.00 1.06 0.75 0.79 1.09 0.99 1.52 1.55 1.68 1.47 1.65 1.60 1.40 1.34 1.89
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.28 0.24 0.04 0.15 0.03 0.02 0.29 0.09 0.03 0.29 0.22 0.23 0.21 0.24 0.41
pharmacological functions including inhibiting cholesterol synthesis, increasing low-density lipoprotein processing, anti-thrombosis and anti-platelet aggregation activity (Yang et al., 2009b). This was the first report regarding the anticoagulative activities of biflavones in G. biloba, and isoginkgetin (7) showed significant antithrombin and antiplatelet aggregation activity. Acknowledgments This work was supported by A Project Funded by the National Science and Technology Support Program of China (2011BAI04B03), the Priority Academic Program Development of Jiangsu Higher Education Institutions (ysxk-2010), 2009 Program for New Century Excellent Talents by the Ministry of Education (NCET-09-0163), 2009 Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education, and Construction Project for Jiangsu Engineering Center of Innovative Drug from Blood-conditioning TCM Formulae. We are grateful to Mr. Wei Jiang (Taixing, Jiangsu province, China) for collecting samples. We are also pleased to thank Mr. Dongjun Chen (China Pharmaceutical University) for the help in NMR tests. References Carrier, D.J., van Beek, T.A., van der, Heijden, R., Verpoorte, R., 1998. Distribution of ginkgolides and terpenoid biosynthetic activity in Ginkgo biloba. Phytochemistry 48, 89–92. Chen, S.X., Wu, L., Yang, X.M., Jiang, X.G., Li, L.G., Zhang, R.X., Xia, L., Shao, S.H., 2007. Comparative molluscicidal action of extract of Ginko biloba sarcotesta, arecoline and mclosamide on snail hosts of Schistosoma japonicum. Pesticide Biochemistry and Physiology 89, 237–241. Chen, Y., Chen, J.W., Li, X., 2011. Cytotoxic bistetrahydrofuran annonaceous acetogenins from the seeds of Annona squamosa. Journal of Natural Products 74, 2477–2481. Choi, J.G., Jeong, S.I., Ku, C.S., Sathishkumar, M., Lee, J.J., Mun, S.P., Kim, S.M., 2009. Antibacterial activity of hydroxyalkenyl salicylic acids from sarcotesta of Ginkgo biloba against vancomycin-resistant enterococcus. Fitoterapia 80, 18–20. Choukchou-Braham, N., Asakawa, Y., Lepoittevin, J.P., 1994. Lepoittevin, isolation, structure determination and synthesis of new dihydroisocoumarins from Ginkgo biloba L. Tetrahedron Letters 35, 3949–3952. Ciavatta, M.L., Manzo, E., Nuzzo, G., Villani, G., Varcamonti, M., Gavagnin, M., 2010. Crucigasterins A-E, antimicrobial amino alcohols from the Mediterranean colonial ascidian Pseudodistoma crucigaster. Tetrahedron 66, 7533–7538. Deng, Q.C., Wang, L., Wei, F., Xie, B.J., Huang, F.H., Huang, W., Shi, J., Huang, Q.D., Tian, B.Q., Xue, S., 2011. Toxicity of 4 -O-methylpyridoxine-5 -glucoside in Ginkgo biloba seeds. Food Chemistry 124, 1458–1465. ˇ Freire, F., Lallana, E., Quinoá, E., Riguera, R., 2009. The stereochemistry of 1,2,3-triols revealed by 1 H NMR spectroscopy: principles and applications. Chemistry – A European Journal 15, 11963–11975. Fu, F.Y., Yu, D.Q., Sung, W.L., 1962. The chemical study of hydroginkgolinic acid. Acta Chimica Sinica 28, 52–56. Gao, J.M., Wang, C.Y., Zhang, A.L., Liu, J.K., 2001. A new trihydroxy acid from the ascomycete Chinese truffle Tuber indicum. Lipids 36, 1365–1366. Gao, J.M., Zhang, A.L., Chen, H., Liu, J.K., 2004. Molecular species of ceramides from the ascomycete truffle Tuber indicum. Chemistry and Physics of Lipids 131, 205–213.
736
G. Zhou et al. / Chemistry and Physics of Lipids 165 (2012) 731–736
Jin, T.C., Chen, Y.W., Howard, A., Zhang, Y.Z., 2008. Purification, crystallization and initial crystallographic characterization of the Ginkgo biloba 11S seed globulin ginnacin. Acta Crystallographica 64, 641–644. Kobayashi, Y., Hayashi, N., Tan, C.H., Kishi, Y., 2001. Toward the creation of NMR databases in chiral solvents for assignments of relative and absolute stereochemistry: proof of concept. Organic Letters 14, 2246–2248. Krauze-Baranowska, M., Poblocka, L., El Hela, A.A., 2005. Biflavones from Chamaecyparis obtusa. Zeitschrift fur Naturforschung C 60, 679–685. Li, Z., Tran, V.H., Duke, R.K., Ng, M.C.H., Yang, D., Duke, C.C., 2009. Synthesis and biological activity of hydroxylated derivatives of linoleic acid and conjugated linoleic acids. Chemistry and Physics of Lipids 158, 39–45. Llabres, G., Baiwir, M., Sbit, M., Dupont, L., 1989. Ginkgolides A, B and C: a 1D and 2D NMR study. Spectrochimica Acta Part A 45, 1037–1045. Major, R.T., 1967. The ginkgo, the most ancient living tree. Science 157, 1270–1273. Manini, P., Camera, E., Picardo, M., Napolitano, A., d’Ischia, M., 2008. Biomimetic nitration of the linoleic acid metabolite 13-hydroxyoctadecadienoic acid: isolation and spectral characterization of novel chain-rearranged epoxy nitro derivatives. Chemistry and Physics of Lipids 151, 51–61. Markham, K.R., Sheppard, C., Geiger, H., 1987. 13 C NMR study of some naturally occurring amentoflavone and hinokiflavone biflavonoids. Phytochemistry 26, 3335–3337. Pan, W.G., Luo, P., Fu, R.B., Gao, P., Long, Z.F., Xu, F.Y., Xiao, H.B., Liu, S.G., 2006. Acaricidal activity against Panonychus citri of a ginkgolic acid from the external seed coat of Ginkgo biloba. Pest Management Science 62, 283–287. Rieser, M.J., Hui, Y.H., Rupprecht, J.K., Kozlowski, J.F., Wood, K.V., McLaughlin, J.L., Hanson, P.R., Zhuang, Z.P., Hoye, T.R., 1992. Determination of absolute configuration of stereogenic carbinol centers in annonaceous acetogenins by 1 H- and 19 F-NMR analysis of Mosher ester derivatives. Journal of the American Chemical Society 114, 10203–10213.
Sakurai, Y., Shima, M., Giddings, J., Takeyama, M., Kasuda, S., Nogami, K., Nishiya, K., Yoshioka, A., 2004. A critical role for thrombin in platelet aggregation under high shear stress. Thrombosis Research 113, 311–318. Sikka, P., Bindra, V.K., 2010. Newer antithrombotic drugs. Indian Journal of Critical Care Medicine 14, 188–195. Singh, B., Kaur, P., Gopichand, Singh, R.D., Ahuja, P.S., 2008. Biology and chemistry of Ginkgo biloba. Fitoterapia 79, 401–418. Tao, W.W., Yang, N.Y., Duan, J.A., Wu, D.K., Shang, E.X., Qian, D.W., Tang, Y.P., 2010. Two new nonacosanetriols from the pollen of Typha angustifolia. Chinese Chemical Letters 21, 209–212. van Beek, T.A., 2002. Chemical analysis of Ginkgo biloba leaves and extracts. Journal of Chromatography A 967, 21–55. Wenzel, T.J., Chisholm, C.D., 2011. Using NMR spectroscopic methods to determine enantiomeric purity and assign absolute stereochemistry. Progress in Nuclear Magnetic Resonance Spectroscopy 59, 1–63. Wu, X.Y., Mao, G.H., Zhao, T., Zhao, J.L., Li, F., Liang, L.H., Yang, L.Q., 2011. Isolation, purification and in vitro anti-tumor activity of polysaccharide from Ginkgo biloba sarcotesta. Carbohydrate Polymers 86, 1073–1076. Yamagiwa, Y., Ohashi, K., Sakamota, Y., Hirakawa, S., Kamikawa, T., 1987. Syntheses of anacardic acids and ginkgoic acid. Tetrahedron 43, 3387–3394. Yang, N.Y., Tao, W.W., Duan, J.A., 2009a. Four new long-chain aliphatics from the feces of Trogopterus xanthipes. Journal of Asian Natural Products Research 11, 1032–1039. Yang, N.Y., Tao, W.W., Duan, J.A., Guo, J.M., Cao, L.L., 2009b. Four new fatty acid esters from the feces of Trogopterus xanthipes. Lipids 44, 849–853. Yang, N.Y., Zhou, G.S., Tang, Y.P., Yan, H., Guo, S., Liu, P., Duan, J.A., Song, B.S., He, Z.Q., 2011. Two new ␣-pinene derivatives from Angelica sinensis and their anticoagulative activities. Fitoterapia 82, 692–695. Zhang, W.K., Xu, J.K., Zhang, X.Q., Yao, X.S., Ye, W.C., 2007. Sphingolipids with neuritogenic activity from Euphorbia sororia. Chemistry and Physics of Lipids 148, 77–83.