Triterpenoids of Akebia quinata callus tissue

Phymc~try. Vol. 25, No. 7. pp. 1625-1628. Printed in Great Britain.

TRITERPENOIDS

0031~9422/86 S3.OO+O.W Pcrgamon Journals Ltd.

1986.

OF AKEBIA AKIRA IKUTA

and HIDEII ITOKAWA

Tokyo College of Pharmacy. 1432-l. (Rcreiwd Key Word

Index--Akcbia qutio:

QUUVATA CALLUS TISSUE

Horinouchi,

1 Nceunber

Hachioji

Tokyo,

Japan

1985)

Lardizabalaaaq callus tissue;

triterpate;

sterol.

Abstract-From tissue culture of Akebia quinata, three new triterpenes were isolated together with two known triterpenes, oleanolic acid and mesembryanthemoidgenic acid. The new triterpenes were characterized by spectroscopic means as 3~-hydroxyXt-norokan-12,20(29)dien-28-oic acid, 3cpi-~norolean-l220(29~ien-28-oic acid and 3fl-hydroxy-29(or 3O~aLolean-12-en-28-oic acid.

INTRODUCllON

RESULTS AND DISCUSSION

Callus tissue cultures were established from the stem of A. quinata (Akebi in Japanese) and they were cultured in the dark on Murashige and Skoog’s medium containing 2.4-D (0.1 and 1.0 ppm) with kinetin (0.1 ppm). The callus tissues were extracted suctxssively with methanol and ethyl acetate and both extracts combined. The material was chromatographed on a silica gel column to give oleanolic acid (l), the new triterpenes 2, 3 and 5, mesenbryanthemoidigenic acid (4) and a mixture of sitosterol (6) and stigmastcrol (7). The structure of 1 was established by spectroscopic means and identification with an authentic sample (see

Akebia quinata Decne. is a creeping, woody vine which is widely distributed in Japan and East Asia. Its air dried stems are called Mokutsu in Japan, and it is used as a traditional oriental medicine in Japan and China, for example as an antiphlogistic, diuretic and analgesic. The stems [l-3], seed [4,5] and pericarp [6,7] have been reported to contain a number of tritcrpenoid saponins which on acid hydrolysis yield hedcragenin. oleanolic acid and norarjunolic acid, respe&vely. as sapogenins. Also anthocyanins have been reported from the flowers [8]. We now report on the triterpenc components of callus tissue of this plant.

COOH

CHO

HO

4 8

R’ = Me,

RI =

R’=CHIOH,R’=

CHIOH

5

me 1625

1626

A.

IKIJ’TA

and H. ITOKAWA

Experimental), Compound 2, on treatment with diaxomethane yielded a methyl ester (Zb).The mass spectrum of compound 2b showed [M]’ at m/z 454 and it had the molecular formula C,,,H4e03 as determined by high resolution mass spectromttry. The mass spectrum of 2b exhibited the typical retro-DielsAlder fragmentation of the C-ring of the /Lamyrin A”- skeleton [9]. The strong peaks at m/z 246 (a) and 187 [246-COOMe] (59) were observed (Scheme 1). The mass number of the peak (a) is 16 mass units less than that of the peak due to the retroDiets-Alder type fragmentation of oleanolic acid methyl ester (1 b). The *H NMR spectrum of 2b show five tertiary methyl signals, two protons at 64.61 and 4.62 ascribable to the exe-methylene protons, and the H-18 proton at 2.79 (dd) [lo]. Therefore, compound 2bis presumed to be a 30norolean- 12cne derivative carrying an exe-methylene group in ring D or E. The 13CNh4R chemical shifts (Table 1) of 2b were found to be similar to the authentic sample oleanolic acid methyl ester (lb) but with predictable differences. The low-field carbon signals at 6148.0 (s) and 106.8 (t) were assigned to the exo-methylene C-20 and C-29, respectively. The hydroxyl group at C-3 must have a /&equatorial configuration according to the signal at 678.7 (d) [ 1I] and also from the *H NMR chemical shift and the coupling pattern of the proton at C-3. The other assignments were supported by comparison of the “C NMR spectrum of 2b with that of lh Thus the structure of compound 2 was established as 3/?-hydroxy-30-noroltn-12,20(29)dien28oic acid (akebonoic acid).

Compound 3, on treatment with diaxomethane, yielded a methyl ester (3b), which was found to be similar to 2b but with predictable ditTerences. The low-field “CNMR signal at 676.2 (6) was assigned to C-3 with the aconfiguration of the hydroxyl group. Other signals at 633.0 (f), 25.2 (1).37.4 (s)and 49.0 (d) were due to C-l. C-2, C-4 and C-5, respectively [ 121 (Table 1). These values for the chemical shifts also agreed with the ar-configuration of the C-3 hydroxyl group, and this was also supported by the ‘H NMR chemical shift and the coupling pattern of the C-3 proton. The values of the chemical shifts for other carbons were similar to those of 2b. The mass spectrum of 3b exhibited the typical retro-Diets-Alder fragmentation and its fragments had the same m/z values to those of 2b (Scheme 1). Thus, the structure of compound 3 was established as 3-epi-3@norolean-l2,20(29)dien-28-oic acid (3-epi-akebonoic acid). The compounds 2 and 3 are reported for the first time from natural sources. A noroleanane type triterpene, norarjunolic acid [7], has been isolated from the pericarp of A. quinato and other eupteleogenins have been reported from Eupteleo polandra (Eupteleaceae) [ 131. The 30-noroleanane type compounds are rare as natural products. The mass spectrum of 4b exhibited significant peaks at m/z 278 (a) and 247 [278 -CH,OH], which could be assigned to fragments of the D and E rings derived by retro-Diels-Alder cleavage and m/z 207 (b) and I89 which could be unequivocally assigned to fragments of the A and B rings by a cleavage of the C ring and elimination of the hydroxyl groups (Scheme 1).

\ r-Iv “.&T I

I

34

CooMe

‘I

m/t 454

2b 3b

mfz 246

+

m/z 187 CH;

R = OH

CH;

\

R-OH HO@ R

&

(b) m/z 189

m/z 207

\

H

4b Sb

R(29)

= CHIOH

COOMe

\

m/z 486

R(29 or 30 ) = CHO m/z 484 (0) (from

4b)

m/z 278

(b)

m/z 207

Scheme

mfz 247

-

I. Fragmentation

m/z 189

of compounds

2-5.

m/z 187

1628

A.

IKUTA

aad

37.1 (5). 37.5 (I). 38.8 (s), 39.4 (s), 41.4 (a), 41.6 (s), 47.0 (s). 47.2 (I), 47.7 (d), 51.6 (q), 55.2 (a), 79.2 (4 123.2 (a), 142.3 (s), 177.5 (sh 106.8 (I), 148.0 (s) MS m/r (rd. int): 454.3485 (ak. for C,,H,O,, 454.3489) [M]’ (IO), 246 (45). 207 (23), 189 (ll), 187 (100). Merhy13-epi-30-ncwolean-12,20(29)dien-28-cure (3a akebnic acid methyl ester, 3b). Mp 200-202” (CHCI,-MeOHA colourless needles, 8.3 mg, [K]E+ 118.1” (~0.166; CHCI,); IR v=cbn-‘: 1705, 1635. ‘H NMR (CD&): 60.72 (s, 3H). 0.84) (s. 3H), 0.92 (s, 3H), 0.95 (s, 3H), 1.16 (s, 3I.Q 2.79 (&f, J=4.8.13.4Hz,IH),3.41(brs,lH),3.6O(s,3H)4.61.4.62(cachs, IH),5.35(r,J = 4 Hz. 1H). “CNMR(CDCl,):615.1 (q), 16.9(q), 18.3 (r), 22.3 (q), 23.3 (r), 23.4 (r), 26.2 (q), 25.2 (1).27.7 (r), 28.3 (qh 30.0 (r), 32.7 (r), 37.1 (s), 37.5 (I), 33.0 (r), 37.4 (s), 39.5 (s), 41.4 (d), 41.8 (s). 47.1 (s), 47.2 (I), 47.5 (d), 51.6 (q), 49.0 (d), 76.2 (d), 123.0 (d), 143.0 (s), 177.5 (s), 107.0 (r), 148.2 (s). MS m/z (rd. int.): 454.3483 (cak. for C,,H,O,. 454.3486) [M] + (17), 246 (55), 207 (16), 189 (40), 187 (100). Mesembryanrhewwidigenica&i merhyl ester (4)) Mp 200-203” (CHCI,-MeOH), colourless crystal, 2.1 mg, [a]g+ loo” (~0.022; CHCl,); IR~~~,urt-‘: 1710. ‘HNMR (CD&): 60.76 (s, 3H), 0.78 (s, 3H). 0.92 (s, 3H). 0.97 (s, 3HA 0.99 (s, 3H), 1.15(~,3H).2.90(dd,J=4.13Hz,1H),3.25(m,1H),3.30(s.2H), 5.32 (I. J = 4 Hz. 1H). “C NMR (CDCl,)zI 15.3 (q), 15.6 (q), 16.9 (q), 18.4 (r), 19.0 (q), 23.1 (1).23.5 (r), 26.0 (q), 27.2 (CA27.7 (1). 28.1 (q), 31.6 (I), 32.7 (I), 35.8 (s), 37.1 (s), 38.5 (1). 38.8 (s). 39.3 (s), 40.1 (I). 40.5 (d), 41.6 (s), 47.0 (s), 47.7 (4 55.3 (d), 51.6 (q), 74.4 (I), 79.1 (d). 122.8 (a), 143.2 (s), 178.1 (5). MS m/z (rcl. int.): 486 [Ml’ (5). 278 (13), 247 (100). 207 (19), 189 (6). 187 (13). Methyl 38-hydroxy-29(or 3O)-al-olea12-ew28-oare (Sbb Amorphous (0.6 mg) [K]E’ +212” (~0.066; CHCl,); lRv~a,cm-‘: 1720. 1700; ‘HNMR (CDCl,): 60.75 (s, lH), 0.77(s, lHA0.91 (s. lHA0.98 (s, 1HA 1.13 (s, lH), 1.16(s, lHA2.92 (dd, J = 4, 13 Hz, lH), 3.25 (m, 1H). 5.35 (t,J - 4m lH), 9.39 (s, I H). MS m/z (rel. int.): 484 [M]+ (4.1), 247 (50). 207 (33), 189 (36). 187 (100). AcknmvledgemenrtWe thank H. lzumi, The MaIi&al Plant garden of this college. for helpful dbcwsion about the plant

H.

~OKAWA

rpecimm. We are very grateful to Miss T. Hasegawa and Y. Arai for their assistance in the experimental work. Thanks are due to The Raeucb Center, Tabho Phamwxutical Co., Ltd., for ‘HNMR spawaI mastamcnt and Mr. Y. shida and Miss Y. Kaneko. The Central A~Iytical Laboratory of this college. for meaaurcmcnt of ma8s spectra

ItEFepENczs 1. Fujita, M, Itokawa, H. and Kumckawa, Y. (1974) Yaku&u Zasshi 94, 189. 2. Fujita, M.. Itokawa, H. and Kumekawa, Y. (1974) Yakugaku Zauhf 94.194. 3. Kumckawa, Y., Itokawa, H. and Fujita. M. (1974) Chem. Pharm. Bull, 22 2294. 4. Higuchi, R., Miyahara, K. and Kawasaki. T. (1972) Chem. Pharm. Bull. 20, 1935. 5. Ii&hi, R. and Kawamaki,T. (1972) Chem. Phumt. Bull. 20, 2143. 6. Higuchi, R. and Kawasaki, T. (1976) Gem. Pham Bull. 24, 1021. 7. H&hi, R. and Kawasaki, T. (1976) Chem. Pharm. Bull. 24. 1314. 8. Ishikura, N. and Nagamino. N. (1976) Phyruchemisrry IS, 442. 9. Budzikicwicz,H,Wilson,J.M.andDjaas.si,C.(l%3)J.Am Gem. Sot. E&3688. 10. Cheung. H. T. and Yan. T. C. (1972) Ausr. J. Chem. 25,2003. 11. Tori, K., Sco, S.. Shimaoka, A. and Tomita, Y. (1974) Tktmhedroa~ Letters 4227. 12. Dclgado, M. C. C, Silva, M. S. Da and Bras Fo., R. (1984) Phyrochemisrry 23,2289. 13. Murata, T.. I& S., Itishi, M., Goto, M. and Morita. K. (lW5) Terrahedron Letters 3215. 14. Turach, B.. Leckrcq. J. and Cbiurdogloy G. (1965) Temhedron Letters 4161. 15. Tumch, B.. Savoir. R., OflInger, R. and Chiurdoglu, G. (1967) Terrahedron Letters 539.