Planta (Berl.) 92, 282--284 (1970)

Short

Communication

Abscisic Acid in the Leaf of Vernonia anthetmintica T. SANYAL,S. N. GANGCLu P. K . S m c A ~ a n d S. M. S m c A ~ Bose Institute, Calcutta Received March 3, 1970

Summary. A gTowth inhibitor was isolated in crystalline form from the leaf of Vernonia anthelmintica and was found to be identical with abseisic acid by biological activity and physical measurements namely, melting point, mixed melting point, UV-absorption, mass spectra and paper and thin-layer chromatography.

I s o l a t i o n of a n e w t u m o u r i n h i b i t o r , t h e s e s q u i t e r p e n o i d d i l a c t o n e , v c r n o l e p i n f r o m t h e l e a v e s of Vernonia hymenolepis (Compositae) has b e e n r e p o r t e d b y K u p c h a n et al. (1968). P r e s e n c e of an i n h i b i t o r of germAnation has b e e n r e p o r t e d for seeds of t h e local species Vernonia anthelmintica W i l l d . T h e seeds do n o t g e r m i n a t e in n o r m a l c o n d i t i o n s unless t h e y are l e a c h e d w i t h a b u n d a n t w a t e r ( C h a k r a v a r t y , 1968).This has led us t o e x a m i n e t h e l e a v e s of t h i s p l a n t , t h e l e a v e s a n d seeds of w h i c h h a v e b e e n k n o w n to h a v e v a r i o u s t h e r a p e u t i c uses f r o m t h e a n c i e n t v e d i c l i t e r a t u r e , a n d it was f o u n d t h a t t h e l e a v e s c o n t a i n abseisic a c i d (ABA). 5 kg fresh young leaves of V. anthelmintica were air dried at 60--70 ~ defatted with petroleum ether (B.P. 60--80 ~ and subsequently extracted with methanol. The methanolic extract was concentrated in a water bath and the residue was taken up with a large volume of ether. This ethereal solution was extracted with 3 % NaOH, the alkaline extract thus obtained was acidified with HC1 to pH 3 in an ice bath and further extracted with ether. This second ethereal extract was washed with a saturated aqueous solution of NaC1 until free from acid and dried over anhydrous Na2SO~. The ether was distilled off and the dark-green residue taken up with chloroform and chromatographed on a column of silica gel, eluted successively with petroleum ether (B.P. 60--80~ benzene, different mixtures of benzene:chloroform (3:1, 1 : l, 2:3, 1:2), and finally with 100% chloroform. The activity of each fraction was tested by a wheat-coleoptile extension bioassay (Fig. 1). The oil obtained in the benzene:chloroform, 2:3, fraction had considerable inhibitory activity. This oil was reclu'omatographed on a silica-gel column using petroleum ether (B.P. 60--80~ benzene, benzene chloroform mixtures (1:2, 1 : 1, 1:2) and 100% chloroform as successive eluents. The active fraction, again an oil, was this time obtained in the chloroform eluate, and was subjected to preparative thin-layer chromatography using silica gel G as adsorbent and ethyl acetate:methanol (75:25) as developer. The active fraction obtained in this procedure (l~f-0.71) was crystallised from benzene with a yield of 0.4 mg per kg of leaves. The melting point was determined to be 161 ~.

Abscisic Acid in Vernonia anthelmintica

283

120-a 1

0

0

~

8C 6C L ~12C b o

E~_100

o

~ o

8C

6E

12Q-c 100

8C 60 I

I

I

~

0.2 0.4. 0.6 0.8 Rf

I

Fig. 1. Wheat coleoptile bioassay with chromatograms of the leaf extract. Solvent, isopropanol: ammonia: water (10:1 : 1). The broken lines indicate significance at the 5% level, a = l g , b - - 5 g , c = 1 0 g Table. Comparison o/the physical properties o/the crystalline material/rom

Vernonia anthelmintica with those o/authentic A B A Properties

Crystalline material

Authentic ABA

U. V. absorption spectrum in ethanol, maximum Major peaks in mass spectrum

251 nm

251 nm

264 (M+), 246, 208, 205, 190 (base peak) 162,134, 111, 91, 69, 57, 43, 41 161 ~ 160--161 o

264 (M+), 246, 208, 205, 190 (base peak) 162, 134, 111, 91, 69, 57, 43, 41 ~ 161 ~ 160--161 ~

Melting point Mixed melting point Rf values: Thin-layer chromatography 0.71 (ethyl acetate: methanol, 75:25) Paper chromatography 0.73 (isopropanol : ammonia : water, 10:1:1) a Cornforth et al. (1966).

0.71 0.71

284

T. Sanyal et al. : Abscisic Acid in Vernonia anthr

1 mg of t h e crystalline m a t e r i a l o b t a i n e d was dissolved in 10 ml ethanol. A small p o r t i o n of this was t e s t e d for its b i o a c t i v i t y with a wheatcoleoptile extension bioassay. The results show strong inhibition, similar to t h a t b y a u t h e n t i c abscisie acid (ABA). The inhibition was to some e x t e n t overcome b y s i m u l t a n e o u s a d d i t i o n of gibberellie acid (GAa). C o m b i n a t i o n of a u t h e n t i c A B A with GAa gave similar results. The p h y s i c a l p r o p e r t i e s of the c o m p o u n d were f o u n d to be i d e n t i c a l with those of a u t h e n t i c A B A (Table). Due to t h e p a u c i t y of t h e material, o p t i c a l r o t a t i o n has n o t been d e t e r m i n e d . F r o m t h e results of t h e bioassays a n d p h y s i c a l properties, it m a y however be concluded t h a t t h e i n h i b i t o r y crystalline m a t e r i a l from the leaves of V. anthelmintica is identical w i t h abseisie acid. We express thanks to Dr. J. W. Cornforth, Shell Research Ltd., Mflstead Laboratory, Kent, U.K. for a gift of authentic abscisic acid. This research project was financed by a grant made by the United States Department of Agriculture under U.S.P.L. 480.

References Chakravarty, M. M. : Ann. Rep. on the study of enzymes of Vernonia anthelmintica Willd. U.S.P.L. 480 Project No. FG-In-314 (1968--1969). Cornforth, J. W., Milborrow, B. V., Ryback, G., Wareing, P. F. : Isolation of sycamore dormin and its identity with abscisin, II. Tetrahedron, Suppl. 8, pt. 2, 603--610 (1966). Kupchan, S.M., Hemingway, R. J., Werne, D., Karim, A., McPhail, A. T., Sire, G. A. : Verno]epin, a novel elemanolide dilactone tumour inhibitor from Vernonia hymenolepis. J. Amer. chem. Soc. 90, 3596 (1968). Dr. S. M. Sircar Bose Institute 93/1, Acharya Prafulla Chandra Road Calcutta-9, India