C031-9422/92 $5.tX1+0.00 Pergamon Press plc

Phytochem~stry,Vol. 31, No. 3, pp. 1049 1050, 1992 Printed in Great Britain.

TRITERPENOIDSAPONINSFROM SHEN-RU

CLINOPODIUMPOLYCEPHALUM

XUE, JIN-QI LIU and GANG WANG

Department of Pharmacy, Anhui College of Traditional Chinese Medicine, Hefei, Anhui 230038, China (Received in revisedform 11 August 1991) Key Word Index --Clmopodium polycephalum; Labiatae; triterpenoid saponin; clinopodiside A, X-ray diffraction.

Abstract-A new triterpenoid saponin, clinopodiside A, has been isolated from Clinopodium polycephalum. Its structure was established by spectroscopic methods and X-ray diffraction analysis as 3-0-/I-D-glucopyranosyl( 1+6)[~-D-glUCOpy~aUOSyl(1-+4)]-j?-D-glucopyranosyl-olean-l 1,13(18)-diene-3~,16fi,23,28-tetrol.

INTRODUCTION

Clinopodium polycephalum (Vant) C. T. Wu et Hsuan which is called ‘Duanxueliu’ in Chinese is an annual herb native to the Anhui province. The whole plant is used in folk medicine for the treatment of haemorrhagia disease. We describe the isolation and structure elucidation of a new triterpenoid saponin, clinopodiside A.

and at 673.3 in genin A. This indicated that the three sugar residues are linked at C-3 of the aglycone. Genin A crystallized in the monoclinic, space group P2,, a=12.952 (4), b=6.674 (5), c=15.084 (6) A, /3 =101.94 (3), V=1276 (1) A, Z=2, D,=1.23 gcme3; p (MoKJ = 0.74 cm- ‘, F (000) = 520. The structure (Fig. 1) was solved by direct methods and refined to a final R =0.056 using 1468 observed MoK, reflections with I > 3oji).

RESULTS AND DISCUSSION Clinopodiside A, C4sH7sO19 (FAB-MS m/z 981 [M +Na]+), exhibited strong IR bands at 3480 and 1639 cm-’ due to hydroxy groups and unsaturated olefin,

respectively. Its UV absorption at 242, 250 and 259 nm indicated the presence of a heteroannular diene [l]. The ‘HNMR spectrum showed the presence of six tertiary methyl singlets in the range 60.73-1.08 (18H, s). The signals at 6 5.55 and 6.42 (2H, d, J = 11 Hz) were assigned to H-11 and H-12 olefinic protons. The H-3 signal was observed at 6 3.37 (lH, m). The signals at 6 3.73 and 4.24, and 63.57 and 4.15 (each 2H, ABq, J= 11 Hz) were assigned to an axial -CH,Oand to an equatorial -CH,O- group at C-4 and C-17. Three anomeric proton signals were observed at 64.31,4.47 and 4.65 (each lH, d, J = 7.3 Hz) and indicated /I-configuration for C-l of each glucose residue. The presence of three sugars linked with one another was confirmed by the 13CNMR spectral data (Table 1) in which three anomeric signals were observed at 6103.7, 105.2 and 105.5. The C’-4 and C’-6 signals of the glucosyl residue were shifted downfield to 680.6 and 69.3 in comparison with saikosaponin C [2]. thus indicating that the two terminal glucoses were linked at the C’-4 and C’-6 position of glucose. This was indicated by the FAB mass spectrum: m/z 797 [M-Glc +H]+,635[M-2xGlc+H]+,487[3xGlc+H]+,325 [2 xGlc+H]’ and 163 [Glc+H]+. The EI mass spectrum of full acetylated clinopodiside A exhibited fragment ions at m/z 842 [M -2 x terminal tetraacetylglucoseoxonium]+, 598 [clinopodigenin A triacetate]+ and 331 [terminal tetraacetylglucose - oxonium] +. Clinopodiside A, on mild acid hydrolysis, yielded genin A and only D-ghCOSC. The spectral data of genin A were in agreement with those of saikogenin A [2]. In addition, in the 13C NMR spectra of clinopodiside A and genin A the C-3 signal was observed at 688.7 in clinopodiside A 1049

Table 1. 13CNMR spectral data of the clinomopodiside A and genin A (DMS&&, ppm values) C

Saponin A

Genin A

C

Glc

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

38.8 26.0 88.7 43.0 55.2 18.3 32.2 39.9 53.6 31.6 126.4 125.0 135.3 42.6 34.4 76.7 43.3 132.7 39.0 31.9 35.6 29.0 65.5 12.6 18.1 17.3 22.2 64.7 25.2 32.9

38.7 28.0 73.3 43.1 48.3 18.2 32.3 39.8 53.6 37.6 126.3 124.9 135.2 42.1 34.4 76.3 43.4 132.7 39.2 31.7 35.9 29.0 67.2 12.2 18.2 17.7 21.6 64.3 24.5 32.0

1’ 2 3’ 4 5 6 1” 2” 3” 4” 5” 6” 1”’ 2” 3”’ 4” 5” 6”’

105.5 75.1 76.7 80.6 76.8 69.3 105.2 74.8 76.3 71.3 76.7 62.3 103.7 74.9 76.3 71.6 76.7 62.4

Short Reports

1050

C-29 Fig. 1. The molecule configuration of genin A (C3,,H4s04).

“CH, OH

On the basis of these data, the structure of clinopodiside A was elucidated as 3-O-/hD-ghCOpyranOSyl( l-+6)[B-D-gh.ICOpyranOSyl(

1-*4)]~-DglUCOpyranOSyl-Olean-

11,13(18)-diene-3b,16/3,23,28-tetrol. EXPERIMENTAL Mps: uncorr; ‘H NMR (400 MHz) and r3C NMR (100 MHz): DMSO-d,, TMS as int. standard. Plant material. The whole plant of Clinopodium polycephalum C. T. Wu et Hsuan was collected in June 1989 at Anhui province. A voucher specimen is deposited in the Herbarium of Anhui College of Chinese Traditional Medicine. Extraction and isolation. The dried and powdered whole plant (20 kg) was completely extracted with 80% EtOH. The coned aq. extract was partitioned between Et,0 and HzO. The aq. layer was extracted with EtOAc and then extracted with n-BuOH saturated with HzO. The n-BuOH fraction (50 g) was subjected to MPLC on a silica gel column eluted with CHClsMeOH-Hz0 (70: 30: 3) in different ratios giving 40 frs. Frs 15 21 (3 g) were rechromatographed on a silica gel column eluted with CHCl,-EtOAc-MeOH (2: 1: 2) to afford clinopodiside A (0.9 g), B (0.1 g) and C (0.06 g). Clinopodiside A. Powder, 7OOmg, mp 249-251” (MeOH), [a];$ + 10.7 (EtOH), (Found: C 60.10; H 8.19%; C,,H,,O,, requires: C 60.01; H 8.20%). UV A:$” nm: (logs) 242 (4.1l), 250 (4.17), 259 (4.00); IR vhy cm -‘: 3480,2930,1630,901; ‘H NMR: S0.73,0.84,0.86,0.90,0.94, 1.08 (3H each, s, Me x 6), 3.37 (lH, m, H-3), 3.83 (lH, m, H-16), 3.57 and 4.15 (1H each, ABq, J= 11 Hz, Hz-28), 3.73 and 4.24 (1H each, ABq, .J = 11 Hz, Hz-23), 6.42 (lH, d,J=ll Hz,H-l2),5.55(1H,dd,J=ll,4Hz,H-ll),4.31(lH,d, J=7.3 Hz, H-l’), 4.47 (lH, d, J=7.3 Hz, H-l”), 4.65 (lH, d, J =7.3 Hz, H-l”‘); FAB-MS: m/z 981 [M+Na]+, 797, 635, 487, 325, 163. Acetylation ofclinopodiside A. Acetylation of clinopodiside A

gave the fully acetylated clinopodiside A which crystallized from MeOH as a microcrystalline powder, mp 152-153.3”. (Found: C 59.03; H 6.96%, C74H,0403z, requires: C 58.73; H 7.11%.) IR v$,f’ cm-‘: 2940, 1750, 1367, 1233, 1063, 1036; EI-MS: m/z 842, 810, 768, 598, 561, 521, 331, 239,229, 187, 169. Mild hydrolysis of clinopodiside A. Clinopodiside A was hydrolysed with 5% HCI-MeOH at 80” for 30 mm to afford genm A and o-glucose. Genin A crystallized from MeOH as needles, mp 289-290”. (Found: C 76.18; H 10.40%; C 30H 480 4 requires: C 76.22; H 10.24%.) UV n&y” nm (log): 242 (4.22) 250 (4.32), 260 (4.11); IR v:; cm-‘: 3460, 1630; ‘HNMR: 60.74,0.80,0.83, 0.93,0.96,1.10(3Heach,s,Mex6),3.80(1H,m,H-3),3.34(1H,m, H-16), 3.55 and 4.14 (1H each, ABq, J= 11 Hz, 2H-28),3.71 and 4.25 (1H each, ABq, J= 10 Hz, 28-23) 6.38 (lH, d, J = 11 Hz, H12), 5.65 (lH, dd, J= 11,4 Hz, H-11); EI-MS: m/z 473 [M + l]‘, 472 [Ml’, 454,441,436,424, 388, 239, 187. 107, 69. Acetylation ofgenin A. Genin A (50 mg) was acetylated with AczO and pyridine by the usual method to give crystals (40 mg), which were recrystallized from MeOH to afford the tetraacetate genin A, mp 92-94” (Found: C 71.22; H 8.81, C 38H 560 s requires: C 71.30; H 8.61). IR v:;; cm -I: 1700; ‘HNMR: 60.71,0.81,0.96, 0.98,1.01, l.lO(3H each, s, Me x 6), 1.98,2.02,2.03,2.04(3H each, s, Ac x 4), 3.78 (2H, dd, J = 11, 11 Hz, CCHz-OAc), 4.13 (2H, dd, J= 12, 12 Hz, C-CHz-OAc), 4.74-5.10 (2H, m, CH-OAc); EIMS: m/z 640 [Ml’, 580,567,520,507,447,387,253,239,187,69. Acknowledgements-The authors are grateful to Prof. Shi-Hua Huang for identification of the plant materials and Ye-Shi Li for her assistance in this work. REFERENCES

1. Kubota, T. and Tonami, F. (1967) Tetrahedron 23, 3333. M. (1980) Chem. Pharm. Bull.

2. I&ii, H. and Nakamura, 28, 2367.