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Two new isopimarane diterpenes from the feces of Trogopterus xanthipes
Fitoterapia 81 (2010) 381–384
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f i t o t e
Two new isopimarane diterpenes from the feces of Trogopterus xanthipes Nian-Yun Yang, Wei-Wei Tao, Min Zhu, Jin-Ao Duan ⁎, Jian-Guo Jiang Jiangsu Key Laboratory for TCM Formulae Research, Nanjing University of Traditional Chinese Medicine, Nanjing 210046, China
a r t i c l e
i n f o
Article history: Received 4 July 2009 Accepted in revised form 19 November 2009 Available online 30 November 2009 Keywords: Trogopterus Feces Wulingzhic acid A Wulingzhic acid B Antithrombin Antiplatelet aggregation
a b s t r a c t Chemical investigation of Trogopterus Feces has led to the isolation of two new isopimarane diterpenes, wulingzhic acid A (1) and wulingzhic acid B (2). Their structures were elucidated by chemical and extensive spectral analysis. Compounds 1 and 2 exhibited weak activity of antithrombin and moderate activity of antiplatelet aggregation in vitro. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Trogopterus Feces, called “Wulingzhi”, are the dry stool of Trogopterus xanthipes Milne-Edwards (Petauristidae). Trogopterus Feces have the function of invigorating blood and relieving pain, and are often used in treatment of amenorrhea, menses pain and postpartum abdominal pain in traditional Chinese medicine. Modern studies have indicated that Trogopterus Feces mainly consisted of the chemical constituents of triterpenoids, diterpenoids and phenolic acids, and possess the pharmacological action of antiplatelet aggregation and antiinflammatory [1–3]. In our present study different solvent extracts of Trogopterus Feces were screened, which showed that the ethyl acetate extract was the important active part. So the ethyl acetate extract was chemically investigated and two new isopimarane diterpenes were isolated. Wulingzhic acid was an isopimarane diterpene from Trogopterus Feces, which was reported to have pharmacological functions of antiplatelet aggregation and antibiosis [1]. In this study, we deal with the isolation and structural elucidation of two new isopimarane
⁎ Corresponding author. E-mail address: [email protected] (J.-A. Duan). 0367-326X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2009.11.006
diterpenes, wulingzhic acid A (1) and wulingzhic acid B (2) (Fig. 1), and report some of their anticoagulative activities. Their structures were elucidated by means of chemical and extensive spectroscopic analysis. 2. Experimental 2.1. General experimental procedures Optical rotations were measured on a JASCO P-1020 polarimeter. IR spectra were taken on a Nicolet IR-100 FT-IR spectrometer in KBr discs. NMR spectra were measured on a Bruker AV-500 MHz (500 MHz for 1H NMR and 125 MHz for 13C NMR) using TMS as internal standard and chemical shifts were recorded as δ values. ESI-MS and HR-ESI-MS spectra were obtained on a Micromass Q/TOF Mass Spectrometer. Anticoagulative assay was performed on coagulation analysis instrument LG-PABER-I. The blood sample was treated on Anke TDL40B centrifugal machine. Silica gel for column chromatography (CC) (200–300 mesh) and TLC plates (10–40 µm) were the products of Qingdao Marine Chemical Co., Ltd (Qingdao, China). Thrombin was purchased from Xisen Sanhe Co., Ltd (Leling, China). Adenosine diphosphate (ADP) was purchased from Beijing Zhongqin Scientific Instrument Co. Ltd (Beijing, China). Rabbit (3.8 kg) was supplied by Shanghai Sikelai Experimental Animal Co., Ltd (Shanghai, China).
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N.-Y. Yang et al. / Fitoterapia 81 (2010) 381–384
Fig. 1. Structures of wulingzhic acid and compounds 1–2.
2.2. Materials Trogopterus Feces were collected in June of 2008 from Hebei province of China, and identified as the dry stool of T. xanthipes by associate professor Nianyun Yang of Nanjing University of Traditional Chinese Medicine. A voucher specimen (GS-20080610) was kept in the Herbarium of Nanjing University of Traditional Chinese Medicine. 2.3. Extraction and isolation The air-dried and powdered Trogopterus Feces (3 kg) were extracted with 60% C2H5OH (2× 50 L) for 2 h under reflux, and the combined extracts were concentrated under reduced pressure. The resulting extract (370 g) was then suspended in H2O and extracted successively with ethyl acetate, n-butanol to give the respective extracts after solvent removal. The combined ethyl acetate layers were evaporated under reduced pressure to leave the residue (277 g), which was chromatographed on silica gel (2 kg) eluting with CHCl3–CH3OH, stepwise gradient (100:0 → 5:1) and 5 fractions were collected. Fr. 3 (10 g) was separated by silica gel [CH2Cl2–CH3OH (20:1)] to obtain compounds 1 (150 mg) and 2 (100 mg). Wulingzhic acid A (1): white powder, [α] D20 +49.0 (c 0.10, CH3OH); IR (KBr) λmax 3430, 2911, 1695, 1454, 1389 1258, 1034, 965, 720 cm− 1; 1H NMR (CD3OD, 500 MHz), see Table 1; 13 C NMR (CD3OD, 125 MHz), see Table 1; ESI-MS: m/z m/z 351 [M − H]−, 387 [M +Cl]−, 703 [2 M− H]− and 739 [2 M + Cl]−; HR-ESI-MS: m/z 351.2163 [M −H]− (C20H31O5, calc. 351.2171). 20 Wulingzhic acid B (2): white powder, [α]D +17.7 (c 0.11, CH3OH); IR (KBr) λmax 3430, 2909, 1693, 1461 1270, 1060, 722 cm− 1; 1H NMR (CD3OD, 500 MHz), see Table 1; 13C NMR (CD3OD, 125 MHz), see Table 1; ESI-MS: m/z 333 [M− H]− and 667[2 M − H] − ; HR-ESI-MS: m/z 333.2059 [M − H] − (C20H29O4, calc. 333.2066). 2.4. Clotting time of rabbit plasma thrombin Evaluation of anticoagulative activities of the different solvent extracts and compounds 1–2 was performed by using thrombin time (TT) method. All samples were dissolved in ethanol respectively. Rabbit common carotid artery was cut off to take a sample of blood, which was mixed with anticoagulant (3.8% sodium citrate) in the proportion of 9 to 1,
and the mixture was centrifuged at 2500 rpm for 15 min to collect the plasma. The plasma (50 μL) was put in a plastic cup for 3 min at 37 °C, and 100 μL thrombin solution of 15 U mL− 1 diluted by 0.1 mol mL− 1 pH 7.4 Tris–HCl buffer was also put in the plastic cup along with 10 μL sample solution, meanwhile, the coagulation analysis instrument was started up so that the thrombin clotting time was recorded. The same experiment was done for the positive control drug heparin sodium and the blank solvent ethanol. Each analyte was tested several times, and an average value was applied. TT prolongation rate was calculated to assess the anticoagulative activities of the samples.
Table 1 1 H (500 MHz) and 13C (125 MHz) NMR spectral data of compounds 1–2 in CD3OD (δ, ppm; J, Hz). 1
2
1
H (m, J Hz)
1α 1β 2 3α 3β 4 5 6α 6β 7α 7β 8 9 10 11α 11β 12α 12β 13 14 15 16a 16b 17 18 19 20
1.09 (t, 11.9) 2.02 (m) 3.80 (dddd, 11.6, 11.6, 4.3, 4.3 Hz) 1.67 (t, 11.6) 1.89 (m) 1.91 (d, 2.8) 1.27 (m) 1.34 (m) 2.15 (m) 2.27 (ddd, 14.5, 5.6, 2.9) 1.85 (m) 1.64 1.52 1.30 1.47
(m) (m) (m) (m)
5.40 3.24 3.61 3.43 0.98
(brs) (dd, 8.7, 2.6) (dd, 11.2, 2.6) (dd, 11.2, 8.7) (s)
1.21 (s) 0.87 (s)
13
C
1
H (m, J Hz)
48.5 1.08 (t, 11.9) 2.10 (m) 64.9 3.82 (dddd, 11.7, 11.7, 4.3, 4.3 Hz) 46.4 1.68 (t, 11.5) 1.89 (m) 44.7 50.4 2.05 (m) 25.0 1.76 (m) 1.94 (m) 36.2 5.37 (brd, 5.1)
13
C
48.5 64.8 46.5 46.2 45.9 25.8 122.6
137.3 52.8 2.04 (m) 39.7 19.6 1.78 (m) 1.43 (m) 31.5 3.52 (dd, 11.6, 5.6)
135.3 52.4 37.7 29.6
38.9 129.9 1.93 (m) 81.2 5.89 (dd, 17.5, 10.8) 64.0 5.02 (dd, 17.5, 1.4) 5.00 (dd, 10.8, 1.4) 22.7 0.84 (s) 181.4 18.5 1.27 (s) 16.2 0.98 (s)
43.1 45.9 148.3 112.4
75.8
15.1 181.3 18.8 16.5
N.-Y. Yang et al. / Fitoterapia 81 (2010) 381–384
383
Fig. 2. Key HMBC and NOESY correlations of compounds 1–2.
2.5. Platelet aggregation in rabbit platelet-rich plasma (PRP) Rabbit common carotid artery was cut off to take a sample of blood, which was mixed with an anticoagulant (3.8% sodium citrate) in the proportion of 9 to 1, then centrifuged at 1000 rpm for 10 min at room temperature to give PRP. Centrifugation of the remaining blood at 3000 rpm for 10 min yielded plateletpoor plasma (PPP). Platelet count was adjusted to 3 × 105 with PPP. The samples were added to the PRP (0.200 ml), then the mixture was incubated at 37 °C with stirring for 1 min before the addition of ADP (10 μM) as inducer of platelet aggregation. Aggregation was measured by a turbidimetric method. Changes in light transmission caused by platelet clotting were measured using an aggregometer. Platelet aggregation was expressed as the percentage change with the difference of light transmittance between PRP and PPP as 100%. The same experiment was done for the positive control drug Aspirin and the blank solvent ethanol. Each analyte was tested several times, and an average value was applied. Antiplatelet activity was expressed as the percent inhibition of the control value [4]. 3. Results and discussion Compound 1 was isolated as a white powder. The molecular formula of 1 was established as C20H32O5 by HR-ESI-MS (m/z 351.2163 [M− H]−). The negative ESI-MS showed a quasi molecular ion peak at m/z 351 [M− H]−, 387 [M+ Cl]−, 703 [2 M − H]− and 739 [2 M + Cl]−. The IR absorption at 3430 and 1695 cm− 1 suggested the presence of hydroxyl and carboxyl groups. 1 showed a resemblance with wulingzhic acid in their 1 H NMR and 13C NMR spectra (Table 1). The difference between the two diterpenes was in the positions of the hydroxyl group and olefinic bond. The 13C NMR spectrum of 1 indicated one carboxyl group [δ 181.4 (s)] and two olefinic carbons [δ 137.3 (s), 129.9 (d)]. Two oxygenated methine carbons and one oxygenated methene carbon resonating at δ 81.2 (d), 64.9 (d), and 64.0 (t) in the 13C NMR spectrum, along with their 1H NMR
data [δ 3.24 (1H, dd, J = 8.7, 2.6 Hz), 3.80 (1H, dddd, J = 11.6, 11.6, 4.3, 4.3 Hz), and 3.61 (1H, dd, J = 11.2, 2.6 Hz), 3.43 (1H, dd, J = 11.2, 8.7 Hz)], indicated that 1 had two oxygenated methine carbons and one oxygenated methene carbon. Its 1 1 H, H-COSY spectrum showed the mutual correlations among the protons at δ 3.24, 3.61 and 3.43, which were suggested to be assigned to H-15, H-16a and H-16b. In the HMBC experiment (Fig. 2), H-15 showed long-range correlation with an olefinic carbon (δ 129.9) assignable to C-14, so the olefinic carbon at δ 137.3 was assigned to C-8. The HMBC spectrum also revealed three methyl protons at δ 1.21 (3H, s) correlated with a carboxyl carbon (δ 181.4), which suggested that the methyl proton was H-19 and the carboxyl carbon was C-18. The multiplicity and coupling constants of the signal at δ 3.80 could only arise by interaction of an axial proton with two neighboring axial and two equatorial protons (Jax-ax = 11.6 Hz, Jax-eq = 4.3 Hz), hence the signal at δ 3.80 was assigned to H-2 which was axial and β-oriented. The splitting pattern of H-2 is quite similar to that of the C2β proton of amoenolide A isolated from Amphiachyris amoena [5]. The structure and relative configuration of 1 were analyzed by 1H,1H-NOESY experiment (Fig. 2). In its NOESY spectrum, H-2 correlated with H-19 and a methyl proton at δ 0.87 (3H, s) assignable to H-20, H-20 and H-17 (δ 0.98, 3H, s) both correlated with a proton at δ 1.52 (1H, m) assignable to H-11β, so H-19, H-20 and H-17 are Table 2 Antithrombin activities of the different solvent extracts and compounds 1–2 ( ¯x ± s, n = 6 ∼ 8). Sample 60% Ethanol extract Ethyl acetate extract n-Butanol extract Compound 1 Compound 2 Heparin sodium “–” stands for no effect.
Dosage (µg mL− 1) 4
1.0 × 10 1.0 × 104 1.0 × 104 100 100 50
TT prolongation rate (%) 23.62 ± 1.34 39.32 ± 1.89 – 1.52 ± 0.09 2.34 ± 0.10 73.25 ± 3.14
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N.-Y. Yang et al. / Fitoterapia 81 (2010) 381–384
Table 3 Antiplatelet aggregation activity of the different solvent extracts and compounds 1–2 ( ¯x ±s, n = 6 ∼ 8). Sample
Dosage (µg mL− 1)
Inhibition ratio (%)
60% Ethanol extract Ethyl acetate extract n-Butanol extract Compound 1 Compound 2 Aspirin
1.0 × 104 1.0 × 104 1.0 × 104 100 100 27
35.94 ± 2.14 82.23 ± 5.49 38.21 ± 2.97 28.10 ± 2.59 32.52 ± 2.80 47.10 ± 3.60
all β-oriented. The C-15 epimers of isopimaran-15,16-diols were found to show different chemical shifts; the C-15R isomer at δ 78.5/78.2 and the C-15 S isomer at δ 75.5/73.0 [6,7]. In case of compound 1 the C-15 had chemical shift of δ 81.2. On this basis an (R)-configuration may be assigned for this centre. 2D NMR experiments, including 1H,1H-COSY, NOESY, HSQC and HMBC, allowed us to make an unambiguous and complete assignment of the 1H and 13C NMR spectra of 1. The optical direction of compound 1 was the same as that of wulingzhic acid and hallol [8], which possessed similar isopimarene structures. Consequently, compound 1 was identified as 2α,15R,16-trihydroxy-8(14)-isopimarene-18-oic-acid, named wulingzhic acid A. Compound 2 was isolated as a white powder. The molecular formula of 2 was established as C20H30O4 by HR-ESI-MS (m/z 333.2059 [M− H]−). The negative ESI-MS showed a quasi molecular ion peak at m/z 333 [M− H]− and 667 [2 M − H]−. The IR absorption at 3430 and 1693 cm− 1 suggested the presence of hydroxyl and carboxyl groups. By comparison of the 1H and 13C NMR data of 2 with 1 (Table 1), their spectral data were similar, except for that 2 showed a group of terminal olefinic protons [δ 5.89 (1H, dd, J = 17.5, 10.8 Hz), 5.02 (1H, dd, J = 17.5, 1.4 Hz), 5.00 (1H, dd, J = 10.8, 1.4 Hz)] and two terminal olefinic carbons [δ 148.3 (d), 112.4(t)] in its NMR spectra. Its 1H,1H-COSY spectrum showed the mutual correlations among the protons at δ 5.89, 5.02 and 5.00, which suggested an olefinic bond at C-15 (δ 148.3). 2 also showed an additional oxygenated methine carbon [δ 75.8 (d)] and its corresponding proton [δ 3.52 (1H, dd, J = 11.6, 5.6 Hz)]. In the HMBC experiment (Fig. 2), the proton (δ 3.52) showed longrange correlation with C-15 and a methyl carbon [δ 15.1 (q)] assignable to C-17, so the proton at δ 3.52 was assigned to H-12. The HMBC spectrum also revealed a proton [δ 2.05 (1H, m)] correlated with two methyl carbons at δ 18.8 (q, C-19) and 17.0 (q, C-20) and an olefinic carbon at δ 122.6 (d), which suggested that the proton (δ 2.05) was assigned to H-5 and the olefinic
carbon (δ 122.6) was assigned to C-7. H-2 [δ 3.82 (1H, dddd, J = 11.7, 11.7, 4.3, 4.3 Hz)] was axial and β-oriented. H-19, H-20 and H-17 were all β-oriented, which was deduced from the 1H,1H-NOESY spectrum (Fig. 2) of 2. The NOESY correlation between H-12 and H-15 suggested that H-12 was axial and α-oriented. 2D NMR experiments, including 1H,1HCOSY, NOESY, HSQC and HMBC, allowed us to make an unambiguous and complete assignment of the 1H and 13C NMR spectra of 2. The optical direction of compound 2 was also the same as that of wulingzhic acid and 2α-hydroxy7,15-isopimardiene-18-oic-acid [8]. Thus, compound 2 was identified as 2α,12β-dihydroxy-7,15-isopimardiene-18-oicacid, named wulingzhic acid B. Anticoagulative bioassay called optimized thrombin time and antiplatelet aggregation methods was successfully developed for determining antithrombin activities of a series of Chinese blood-activating medicines and some active components in our lab [9]. Trogopterus Feces are the dry stool of T. xanthipes which feed on leaf twigs of Biota orientalis, and the isolated isopimarane diterpenes 1–2 might be metabolic products of T. xanthipes. In vitro anticoagulative activities of the different solvent extracts and compounds 1–2 were tested using the thrombin time and antiplatelet aggregation methods in this study. As can be appreciated by the data summarized in Tables 2 and 3, ethyl acetate extract was the important active part, and compounds 1 and 2 could slightly prolong thrombin time and moderately inhibit platelet aggregation. The results suggested that these isopimarane diterpenes are the potential anticoagulative constituents of Trogopterus Feces. Acknowledgment This research work was supported by the major program of the National Natural Science Foundation of the Higher Education Institutions of Jiangsu Province (06KJA36022). References [1] [2] [3] [4] [5] [6] [7] [8] [9]
Yang DM, Su SW, Li X. Acta Pharmacol Sin 1987;22:756. Numaya A, Yang PM, Takahashi C. Chem Pharm Bull 1989;37:648. Numaya A, Takahashi C, Miyamoto T. Chem Pharm Bull 1990;38:942. Iyobe A, Uchida M, Kamata K, Hotei Y, Kusama H, Harada H. Chem Pharm Bull 2001;49:822. Mathuna DO, Doskotch RW. J Nat Prod 1995;58:1407. Wenkert E, Ceccherelli P, Raju MS, Polonsky J, Tingoli M. J Org Chem 1979;44:146. Subrahmanyam C, Rao BV, Ward RS, Hursthouse MB, Hibbs DE. Phytochemistry 1999;51:83. Cambie RC, Burfitt IR, Goodwin TE, Wenker E. J Org Chem 1975;40:3789. Liu L, Ma HY, Duan JA, Tang YP, Su SL, Guo JM. Chin J Experiment Tradit Med Formulae 2009;15:68.
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f i t o t e
Two new isopimarane diterpenes from the feces of Trogopterus xanthipes Nian-Yun Yang, Wei-Wei Tao, Min Zhu, Jin-Ao Duan ⁎, Jian-Guo Jiang Jiangsu Key Laboratory for TCM Formulae Research, Nanjing University of Traditional Chinese Medicine, Nanjing 210046, China
a r t i c l e
i n f o
Article history: Received 4 July 2009 Accepted in revised form 19 November 2009 Available online 30 November 2009 Keywords: Trogopterus Feces Wulingzhic acid A Wulingzhic acid B Antithrombin Antiplatelet aggregation
a b s t r a c t Chemical investigation of Trogopterus Feces has led to the isolation of two new isopimarane diterpenes, wulingzhic acid A (1) and wulingzhic acid B (2). Their structures were elucidated by chemical and extensive spectral analysis. Compounds 1 and 2 exhibited weak activity of antithrombin and moderate activity of antiplatelet aggregation in vitro. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Trogopterus Feces, called “Wulingzhi”, are the dry stool of Trogopterus xanthipes Milne-Edwards (Petauristidae). Trogopterus Feces have the function of invigorating blood and relieving pain, and are often used in treatment of amenorrhea, menses pain and postpartum abdominal pain in traditional Chinese medicine. Modern studies have indicated that Trogopterus Feces mainly consisted of the chemical constituents of triterpenoids, diterpenoids and phenolic acids, and possess the pharmacological action of antiplatelet aggregation and antiinflammatory [1–3]. In our present study different solvent extracts of Trogopterus Feces were screened, which showed that the ethyl acetate extract was the important active part. So the ethyl acetate extract was chemically investigated and two new isopimarane diterpenes were isolated. Wulingzhic acid was an isopimarane diterpene from Trogopterus Feces, which was reported to have pharmacological functions of antiplatelet aggregation and antibiosis [1]. In this study, we deal with the isolation and structural elucidation of two new isopimarane
⁎ Corresponding author. E-mail address: [email protected] (J.-A. Duan). 0367-326X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2009.11.006
diterpenes, wulingzhic acid A (1) and wulingzhic acid B (2) (Fig. 1), and report some of their anticoagulative activities. Their structures were elucidated by means of chemical and extensive spectroscopic analysis. 2. Experimental 2.1. General experimental procedures Optical rotations were measured on a JASCO P-1020 polarimeter. IR spectra were taken on a Nicolet IR-100 FT-IR spectrometer in KBr discs. NMR spectra were measured on a Bruker AV-500 MHz (500 MHz for 1H NMR and 125 MHz for 13C NMR) using TMS as internal standard and chemical shifts were recorded as δ values. ESI-MS and HR-ESI-MS spectra were obtained on a Micromass Q/TOF Mass Spectrometer. Anticoagulative assay was performed on coagulation analysis instrument LG-PABER-I. The blood sample was treated on Anke TDL40B centrifugal machine. Silica gel for column chromatography (CC) (200–300 mesh) and TLC plates (10–40 µm) were the products of Qingdao Marine Chemical Co., Ltd (Qingdao, China). Thrombin was purchased from Xisen Sanhe Co., Ltd (Leling, China). Adenosine diphosphate (ADP) was purchased from Beijing Zhongqin Scientific Instrument Co. Ltd (Beijing, China). Rabbit (3.8 kg) was supplied by Shanghai Sikelai Experimental Animal Co., Ltd (Shanghai, China).
382
N.-Y. Yang et al. / Fitoterapia 81 (2010) 381–384
Fig. 1. Structures of wulingzhic acid and compounds 1–2.
2.2. Materials Trogopterus Feces were collected in June of 2008 from Hebei province of China, and identified as the dry stool of T. xanthipes by associate professor Nianyun Yang of Nanjing University of Traditional Chinese Medicine. A voucher specimen (GS-20080610) was kept in the Herbarium of Nanjing University of Traditional Chinese Medicine. 2.3. Extraction and isolation The air-dried and powdered Trogopterus Feces (3 kg) were extracted with 60% C2H5OH (2× 50 L) for 2 h under reflux, and the combined extracts were concentrated under reduced pressure. The resulting extract (370 g) was then suspended in H2O and extracted successively with ethyl acetate, n-butanol to give the respective extracts after solvent removal. The combined ethyl acetate layers were evaporated under reduced pressure to leave the residue (277 g), which was chromatographed on silica gel (2 kg) eluting with CHCl3–CH3OH, stepwise gradient (100:0 → 5:1) and 5 fractions were collected. Fr. 3 (10 g) was separated by silica gel [CH2Cl2–CH3OH (20:1)] to obtain compounds 1 (150 mg) and 2 (100 mg). Wulingzhic acid A (1): white powder, [α] D20 +49.0 (c 0.10, CH3OH); IR (KBr) λmax 3430, 2911, 1695, 1454, 1389 1258, 1034, 965, 720 cm− 1; 1H NMR (CD3OD, 500 MHz), see Table 1; 13 C NMR (CD3OD, 125 MHz), see Table 1; ESI-MS: m/z m/z 351 [M − H]−, 387 [M +Cl]−, 703 [2 M− H]− and 739 [2 M + Cl]−; HR-ESI-MS: m/z 351.2163 [M −H]− (C20H31O5, calc. 351.2171). 20 Wulingzhic acid B (2): white powder, [α]D +17.7 (c 0.11, CH3OH); IR (KBr) λmax 3430, 2909, 1693, 1461 1270, 1060, 722 cm− 1; 1H NMR (CD3OD, 500 MHz), see Table 1; 13C NMR (CD3OD, 125 MHz), see Table 1; ESI-MS: m/z 333 [M− H]− and 667[2 M − H] − ; HR-ESI-MS: m/z 333.2059 [M − H] − (C20H29O4, calc. 333.2066). 2.4. Clotting time of rabbit plasma thrombin Evaluation of anticoagulative activities of the different solvent extracts and compounds 1–2 was performed by using thrombin time (TT) method. All samples were dissolved in ethanol respectively. Rabbit common carotid artery was cut off to take a sample of blood, which was mixed with anticoagulant (3.8% sodium citrate) in the proportion of 9 to 1,
and the mixture was centrifuged at 2500 rpm for 15 min to collect the plasma. The plasma (50 μL) was put in a plastic cup for 3 min at 37 °C, and 100 μL thrombin solution of 15 U mL− 1 diluted by 0.1 mol mL− 1 pH 7.4 Tris–HCl buffer was also put in the plastic cup along with 10 μL sample solution, meanwhile, the coagulation analysis instrument was started up so that the thrombin clotting time was recorded. The same experiment was done for the positive control drug heparin sodium and the blank solvent ethanol. Each analyte was tested several times, and an average value was applied. TT prolongation rate was calculated to assess the anticoagulative activities of the samples.
Table 1 1 H (500 MHz) and 13C (125 MHz) NMR spectral data of compounds 1–2 in CD3OD (δ, ppm; J, Hz). 1
2
1
H (m, J Hz)
1α 1β 2 3α 3β 4 5 6α 6β 7α 7β 8 9 10 11α 11β 12α 12β 13 14 15 16a 16b 17 18 19 20
1.09 (t, 11.9) 2.02 (m) 3.80 (dddd, 11.6, 11.6, 4.3, 4.3 Hz) 1.67 (t, 11.6) 1.89 (m) 1.91 (d, 2.8) 1.27 (m) 1.34 (m) 2.15 (m) 2.27 (ddd, 14.5, 5.6, 2.9) 1.85 (m) 1.64 1.52 1.30 1.47
(m) (m) (m) (m)
5.40 3.24 3.61 3.43 0.98
(brs) (dd, 8.7, 2.6) (dd, 11.2, 2.6) (dd, 11.2, 8.7) (s)
1.21 (s) 0.87 (s)
13
C
1
H (m, J Hz)
48.5 1.08 (t, 11.9) 2.10 (m) 64.9 3.82 (dddd, 11.7, 11.7, 4.3, 4.3 Hz) 46.4 1.68 (t, 11.5) 1.89 (m) 44.7 50.4 2.05 (m) 25.0 1.76 (m) 1.94 (m) 36.2 5.37 (brd, 5.1)
13
C
48.5 64.8 46.5 46.2 45.9 25.8 122.6
137.3 52.8 2.04 (m) 39.7 19.6 1.78 (m) 1.43 (m) 31.5 3.52 (dd, 11.6, 5.6)
135.3 52.4 37.7 29.6
38.9 129.9 1.93 (m) 81.2 5.89 (dd, 17.5, 10.8) 64.0 5.02 (dd, 17.5, 1.4) 5.00 (dd, 10.8, 1.4) 22.7 0.84 (s) 181.4 18.5 1.27 (s) 16.2 0.98 (s)
43.1 45.9 148.3 112.4
75.8
15.1 181.3 18.8 16.5
N.-Y. Yang et al. / Fitoterapia 81 (2010) 381–384
383
Fig. 2. Key HMBC and NOESY correlations of compounds 1–2.
2.5. Platelet aggregation in rabbit platelet-rich plasma (PRP) Rabbit common carotid artery was cut off to take a sample of blood, which was mixed with an anticoagulant (3.8% sodium citrate) in the proportion of 9 to 1, then centrifuged at 1000 rpm for 10 min at room temperature to give PRP. Centrifugation of the remaining blood at 3000 rpm for 10 min yielded plateletpoor plasma (PPP). Platelet count was adjusted to 3 × 105 with PPP. The samples were added to the PRP (0.200 ml), then the mixture was incubated at 37 °C with stirring for 1 min before the addition of ADP (10 μM) as inducer of platelet aggregation. Aggregation was measured by a turbidimetric method. Changes in light transmission caused by platelet clotting were measured using an aggregometer. Platelet aggregation was expressed as the percentage change with the difference of light transmittance between PRP and PPP as 100%. The same experiment was done for the positive control drug Aspirin and the blank solvent ethanol. Each analyte was tested several times, and an average value was applied. Antiplatelet activity was expressed as the percent inhibition of the control value [4]. 3. Results and discussion Compound 1 was isolated as a white powder. The molecular formula of 1 was established as C20H32O5 by HR-ESI-MS (m/z 351.2163 [M− H]−). The negative ESI-MS showed a quasi molecular ion peak at m/z 351 [M− H]−, 387 [M+ Cl]−, 703 [2 M − H]− and 739 [2 M + Cl]−. The IR absorption at 3430 and 1695 cm− 1 suggested the presence of hydroxyl and carboxyl groups. 1 showed a resemblance with wulingzhic acid in their 1 H NMR and 13C NMR spectra (Table 1). The difference between the two diterpenes was in the positions of the hydroxyl group and olefinic bond. The 13C NMR spectrum of 1 indicated one carboxyl group [δ 181.4 (s)] and two olefinic carbons [δ 137.3 (s), 129.9 (d)]. Two oxygenated methine carbons and one oxygenated methene carbon resonating at δ 81.2 (d), 64.9 (d), and 64.0 (t) in the 13C NMR spectrum, along with their 1H NMR
data [δ 3.24 (1H, dd, J = 8.7, 2.6 Hz), 3.80 (1H, dddd, J = 11.6, 11.6, 4.3, 4.3 Hz), and 3.61 (1H, dd, J = 11.2, 2.6 Hz), 3.43 (1H, dd, J = 11.2, 8.7 Hz)], indicated that 1 had two oxygenated methine carbons and one oxygenated methene carbon. Its 1 1 H, H-COSY spectrum showed the mutual correlations among the protons at δ 3.24, 3.61 and 3.43, which were suggested to be assigned to H-15, H-16a and H-16b. In the HMBC experiment (Fig. 2), H-15 showed long-range correlation with an olefinic carbon (δ 129.9) assignable to C-14, so the olefinic carbon at δ 137.3 was assigned to C-8. The HMBC spectrum also revealed three methyl protons at δ 1.21 (3H, s) correlated with a carboxyl carbon (δ 181.4), which suggested that the methyl proton was H-19 and the carboxyl carbon was C-18. The multiplicity and coupling constants of the signal at δ 3.80 could only arise by interaction of an axial proton with two neighboring axial and two equatorial protons (Jax-ax = 11.6 Hz, Jax-eq = 4.3 Hz), hence the signal at δ 3.80 was assigned to H-2 which was axial and β-oriented. The splitting pattern of H-2 is quite similar to that of the C2β proton of amoenolide A isolated from Amphiachyris amoena [5]. The structure and relative configuration of 1 were analyzed by 1H,1H-NOESY experiment (Fig. 2). In its NOESY spectrum, H-2 correlated with H-19 and a methyl proton at δ 0.87 (3H, s) assignable to H-20, H-20 and H-17 (δ 0.98, 3H, s) both correlated with a proton at δ 1.52 (1H, m) assignable to H-11β, so H-19, H-20 and H-17 are Table 2 Antithrombin activities of the different solvent extracts and compounds 1–2 ( ¯x ± s, n = 6 ∼ 8). Sample 60% Ethanol extract Ethyl acetate extract n-Butanol extract Compound 1 Compound 2 Heparin sodium “–” stands for no effect.
Dosage (µg mL− 1) 4
1.0 × 10 1.0 × 104 1.0 × 104 100 100 50
TT prolongation rate (%) 23.62 ± 1.34 39.32 ± 1.89 – 1.52 ± 0.09 2.34 ± 0.10 73.25 ± 3.14
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N.-Y. Yang et al. / Fitoterapia 81 (2010) 381–384
Table 3 Antiplatelet aggregation activity of the different solvent extracts and compounds 1–2 ( ¯x ±s, n = 6 ∼ 8). Sample
Dosage (µg mL− 1)
Inhibition ratio (%)
60% Ethanol extract Ethyl acetate extract n-Butanol extract Compound 1 Compound 2 Aspirin
1.0 × 104 1.0 × 104 1.0 × 104 100 100 27
35.94 ± 2.14 82.23 ± 5.49 38.21 ± 2.97 28.10 ± 2.59 32.52 ± 2.80 47.10 ± 3.60
all β-oriented. The C-15 epimers of isopimaran-15,16-diols were found to show different chemical shifts; the C-15R isomer at δ 78.5/78.2 and the C-15 S isomer at δ 75.5/73.0 [6,7]. In case of compound 1 the C-15 had chemical shift of δ 81.2. On this basis an (R)-configuration may be assigned for this centre. 2D NMR experiments, including 1H,1H-COSY, NOESY, HSQC and HMBC, allowed us to make an unambiguous and complete assignment of the 1H and 13C NMR spectra of 1. The optical direction of compound 1 was the same as that of wulingzhic acid and hallol [8], which possessed similar isopimarene structures. Consequently, compound 1 was identified as 2α,15R,16-trihydroxy-8(14)-isopimarene-18-oic-acid, named wulingzhic acid A. Compound 2 was isolated as a white powder. The molecular formula of 2 was established as C20H30O4 by HR-ESI-MS (m/z 333.2059 [M− H]−). The negative ESI-MS showed a quasi molecular ion peak at m/z 333 [M− H]− and 667 [2 M − H]−. The IR absorption at 3430 and 1693 cm− 1 suggested the presence of hydroxyl and carboxyl groups. By comparison of the 1H and 13C NMR data of 2 with 1 (Table 1), their spectral data were similar, except for that 2 showed a group of terminal olefinic protons [δ 5.89 (1H, dd, J = 17.5, 10.8 Hz), 5.02 (1H, dd, J = 17.5, 1.4 Hz), 5.00 (1H, dd, J = 10.8, 1.4 Hz)] and two terminal olefinic carbons [δ 148.3 (d), 112.4(t)] in its NMR spectra. Its 1H,1H-COSY spectrum showed the mutual correlations among the protons at δ 5.89, 5.02 and 5.00, which suggested an olefinic bond at C-15 (δ 148.3). 2 also showed an additional oxygenated methine carbon [δ 75.8 (d)] and its corresponding proton [δ 3.52 (1H, dd, J = 11.6, 5.6 Hz)]. In the HMBC experiment (Fig. 2), the proton (δ 3.52) showed longrange correlation with C-15 and a methyl carbon [δ 15.1 (q)] assignable to C-17, so the proton at δ 3.52 was assigned to H-12. The HMBC spectrum also revealed a proton [δ 2.05 (1H, m)] correlated with two methyl carbons at δ 18.8 (q, C-19) and 17.0 (q, C-20) and an olefinic carbon at δ 122.6 (d), which suggested that the proton (δ 2.05) was assigned to H-5 and the olefinic
carbon (δ 122.6) was assigned to C-7. H-2 [δ 3.82 (1H, dddd, J = 11.7, 11.7, 4.3, 4.3 Hz)] was axial and β-oriented. H-19, H-20 and H-17 were all β-oriented, which was deduced from the 1H,1H-NOESY spectrum (Fig. 2) of 2. The NOESY correlation between H-12 and H-15 suggested that H-12 was axial and α-oriented. 2D NMR experiments, including 1H,1HCOSY, NOESY, HSQC and HMBC, allowed us to make an unambiguous and complete assignment of the 1H and 13C NMR spectra of 2. The optical direction of compound 2 was also the same as that of wulingzhic acid and 2α-hydroxy7,15-isopimardiene-18-oic-acid [8]. Thus, compound 2 was identified as 2α,12β-dihydroxy-7,15-isopimardiene-18-oicacid, named wulingzhic acid B. Anticoagulative bioassay called optimized thrombin time and antiplatelet aggregation methods was successfully developed for determining antithrombin activities of a series of Chinese blood-activating medicines and some active components in our lab [9]. Trogopterus Feces are the dry stool of T. xanthipes which feed on leaf twigs of Biota orientalis, and the isolated isopimarane diterpenes 1–2 might be metabolic products of T. xanthipes. In vitro anticoagulative activities of the different solvent extracts and compounds 1–2 were tested using the thrombin time and antiplatelet aggregation methods in this study. As can be appreciated by the data summarized in Tables 2 and 3, ethyl acetate extract was the important active part, and compounds 1 and 2 could slightly prolong thrombin time and moderately inhibit platelet aggregation. The results suggested that these isopimarane diterpenes are the potential anticoagulative constituents of Trogopterus Feces. Acknowledgment This research work was supported by the major program of the National Natural Science Foundation of the Higher Education Institutions of Jiangsu Province (06KJA36022). References [1] [2] [3] [4] [5] [6] [7] [8] [9]
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