determination+of+biologically+active+constituents

8/13/2019 Determination+of+Biologically+Active+Constituents

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LS VI R Journal of Chromatography A, 742 (1996) 127-130

JOURN L OFCHROM TOGR PHY

e te r mi n at i on o f b i o l og i c a l l y ac t i ve c on s ti tu e n t s i n e n t e l l aa s i a t i c a i

EK . Inam dar , R .D. Yeo l e , A .B . G hogare , N .J . de SouzaResearc h centre Hoe chst India Ltd. Mu lund B omb ay In dia

Received 29 March 1995; revised 14 March 1996; accepted 15 March 1996

A b s t r a c t

A high-performance liquid chromatographic method is described, using an octadecylsilylated silica co lumn forsimultaneous quantitat ive determination of the bioactive terpene acids: asiatic acid, madecassic acid and their respectiveglycosides, asiaticoside and madecassoside, in the plant C e n t e l l a a s i a t i c a . The method is suitable for estimating thesecompounds in plant material. The method is also suitable for the routine assay of pharmaceutical preparations containing a C.a s i a t i c a extract.K e y w o r d s : C e n t e l l a a s i a t i c a ; Madecassoside; Asiaticoside; Madecassic acid; Asiatic acid; Terpenes

1 I n t r o d u c t i o n

C e n t e l l a a s i a t i c a (Linn) is an ethnomedical plantused in different contine nts by diverse ancient cul-tures and tribal groups. In India, it is usuallydescribed under the name of Mandukaparni in theAyurved ic system of medic ine [1]. Different uses areclaimed for the plant, the more co mmon b eing its useas a wound heali ng agent [2], constituent of braintonics for the mentally retarded [3,4]. These prop-erties have been ascribed to the active principles:asiatic acid (I), asiaticoside (II), madecassic acid(III) and madecassoside (IV). These are pentacyclictriterpenes, found to display chronic venous insuf-ficiency [5], varicose vein and wound healing prop-erties. They belong to the fl-amyrin ursolic acidgroup, as shown in Scheme 1.

Corresponding author.~This paper is dedicated to Dr. E. Baltin, Managing Director,Hoechst India Ltd., on the occasion of his 60th birthday.

Madecassol, a formulation based on C . a s i a t i c aplant extract, when applied locally on wounds in ratsprompted the proliferation of granulation and in-creased tensile strength [6]. It decreased the woundarea of the skin necrosis induced by burn [7,8].

HOR=H RI :H I II .R :O H R I :H

- o - o o

H O ~ O H O H H ~ _ ~ O H O HHO O H HO O H

Scheme 1. Structures of asiatic acid (I) , asiaticoside (II),madecassic acid (III) and madecassoside (IV).0021-9673/96/ 15.00 Copyright 1996Elsevier Science B.V. All rights reservedP I I S0021-9673 (96)00237-3


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128 P . K . I n a m d a r e t a l . / J . C h r o m a t o g r . A 7 4 2 1 9 9 6 ) 1 2 7 - 1 3 0

F o r t h e e s t i m a t i o n o f t h e t e r p e n e a c i d s ( I , I I I ) a n dt h e g l y c o s i d e s ( I I , I V ) o f C as ia t i ca t he t i t r im e t r i cm e t h o d o f a n a ly s i s i s r e p o r t e d [ 9 ]. T h e m e t h o dd e t e r m i n e s o n l y t o t a l t e r p e n e a c i d s c o n t e n t . F o r t h eg l y c o s i d e s d e t e r m i n a t i o n , t h e e x t r a c t m u s t f i r s t b eh y d r o l y s e d t o c o n v e r t t h e m t o a c i d s a n d t h e n t h et o t a l t e r p e n e a c i d s a r e c h e m i c a l l y e s t i m a t e d . H e n c et h i s m e t h o d i s n o n - s e l e c t i v e , n o n - s p e c i f i c a n d l a c k sp r e c i s i o n a n d a c c u r a c y .

A r e v e r s e d - p h a s e g r a d i e n t H P L C m e t h o d w h i c h i ss u p e r i o r t o t h e r e p o r t e d a n a l y t i c a l m e t h o d i s d e -v e l o p e d t o a s s a y t h e f o u r ( I - I V ) c o n s t i t u e n t s i n t h ec r u d e p l a n t e x t r a c t s . T h e m e t h o d i s f u r t h e r u s e d f o rg a l e n i c a l p r e p a r a t io n s a n d t h e m o n i t o r i n g o f t h ei s o l at i o n p r o c e s s o f t h e s e p r o d u c t s f r o m t h e p l a n t so b t a i n e d f r o m v a r i o u s l o c a t i o n s i n I n d i a . T h e n e wm e t h o d o f a n a l y s i s i s p r e c i s e , a c c u r a t e a n d r a p i d .

2 E x p e r i m e n t a l2 1 R e a g e n t s

A c e t o n i t r i l e a n d m e t h a n o l ( H P L C g r a d e ) w e r ef r o m E . M e r c k ( I n d i a ) , a s i a t i c a c i d ( I ) a n da s i a t i c o s i d e ( I I ) w e r e o b t a i n e d f r o m C a r l R o t h( K a r l s r u h e , G e r m a n y ) . S p e c t r a l d a t a f o r m a d e c a s s i ca c i d ( I I I ) a n d m a d e c a s s o s i d e ( I V ) , i s o l a te d b y K i l o -s c a l e l a b o r a t o r y , H o e c h s t ( B o m b a y , I n d i a ) , w e r e i na g r e e m e n t w i t h r e p o r t e d v a l u e s . A s t o c k s o l u t i o n o fa ll th e c o m p o u n d s I - I V w a s p re p a re d ( 1 0 m g / m l o fe a c h ) i n m e t h a n o l - w a t e r ( 9 0 : 1 0 ) . T h e s t o c k s o l u t i o nw a s d i lu t e d to o b ta i n s o lu t io n s 0 . 5 - 7 . 5 m g / m l f o rd e t e r m i n a t i o n o f t h e li n e a r i t y r a n g e . D o u b l e d i s t i ll e dw a t e r w a s f i l t e r e d t h r o u g h a 0 . 4 5 - / z m m e m b r a n ef i l t e r . T h e p l a n t m a t e r i a l w a s c o l l e c t e d f r o m t h ef a c t o r y g a r d e n a n d i d e n t i f i e d a s C as ia t i ca ( H O E9 2 4 ) b y D r . V . S h a h o f H o e c h s t I n d i a . A v o u c h e rs p e c i m e n i s p r e s e r v e d a t t h e H e r b a r i u m o f th eR e s e a r c h C e n t r e , H o e c h s t I n d i a . C e n t e l l a s e t a b l e t sw e r e f r o m S c h a r p e r ( I t a l y ) a n d m a d e c a s s o l o i n t m e n tw a s f r o m L a b o r a t o r i e s S y n t e x ( F r a n c e) .2 2 S a m p l e p r e p a r a t i o n

Table IGradient conditions for HPLCTime Flow-rate Pump A Pump B Curve(min) (ml/m in) water( ) acetonitrile( )00 1.4 80 2030 1.4 45 55 635 1.4 45 5545 1.4 80 20 6

b r o w n e x t r a c t ( 0 . 1 5 g ) . T h e d r i e d c r u d e e x t r a c t w a sa c c u r a t e l y w e i g h e d ( ~ 1 0 0 m g ) , d i s s o l v e d i n m e t h a -n o l - w a t e r ( 9 0 : 1 0 ) a n d m a d e u p t o 1 0 m l . T h es o l u t i o n w a s f i l t e r e d t h r o u g h a 0 . 4 5 - m f i l t e r ( W a -t e r s M i l l i p o r e ) a n d t h e c l e a r f i l t r a t e w a s u s e d f o rH P L C a n a l y s i s .

F i v e t a b l et s o f c e n t e ll a s e , e a c h w e i g h i n g a p p r o x .7 0 m g , w e r e p o w d e r e d . A q u a n t i t y e q u i v a l e n t t o o n et a b l e t w a s a c c u r a t e l y w e i g h e d a n d w a s t r a n s f e r r e d t oa 2 5 - m l c e n t r i f u g e t u b e . A 1 0 - m l v o l u m e o f m e t h a -n o l - w a t e r ( 9 0 : 1 0 ) w a s a d d e d a n d v o r t ex e d f o r 5m i n . T h e s u s p e n s i o n w a s c e n t r i f u g e d f o r 1 0 m i n . T h ep r o c e d u r e w a s r e p e a t e d t h r e e t i m e s . T h e s u p e r n a t a n tw a s e v a p o r a t e d u n d e r v a c u u m . T h e r e s i d u e o b t a i n e dw a s d i s s o lv e d i n 1 0 m l m e t h a n o l - w a t e r ( 9 0 : 1 0 ) a n du s e d f o r t h e H P L C a s s ay . A 5 0 0 - m g s a m p l e o fo i n t m e n t w a s v o r t ex e d w i t h 1 0 m l m e t h a n o l - w a t e r( 9 0 : 1 0 ) f o r 2 0 m i n , c e n t r i f u g e d a n d f il te r ed t h r o u g h a0 . 4 5 - m f il te r ( W a t e r s M i l li p o r e ) a n d t h e c l e a rs o l u t io n w a s u s e d f o r t h e H P L C a s s a y .2 3 Ch r o m a t o g r a p h y

T h e W a t e r s H P L C s y s t e m c o n s i s te d o f a h ig h -p r e s s u r e c o n s t a n t f l o w p u m p ( M o d e l 5 1 0 ) , a u t oi n j e c to r ( W I S P 7 1 2 ) , a U V s p e c t r o p h o t o m e t r i c d e t e c-t o r ( 4 9 0 m u l t i w a v e l e n g t h d e t e c t o r ) a n d a d a t a s t a t i o n( 8 4 0 w i t h D i g i t a l 3 5 0 c o m p u t e r ) . C h r o m a t o g r a p h i cs e p a r a t io n w a s p e r f o r m e d w i t h a / ~ B o n d a p a k , C j s 1 0/ z m , 3 0 x 0 . 3 9 c m , S . S . c o l u m n ( W a t e r s ) w i t h aw a t e r - a c e t o n i t r i l e m o b i l e p h a s e , w i t h U V d e te c t i o na t 2 2 0 n m a n d a t t e n u a ti o n o f 0 .1 A U F S . G r a d i e n tc o n d i t i o n s a r e d e s c r i b e d i n T a b l e 1 . A 2 0 - / z l v o l u m eo f s a m p l e w a s i n j e c t e d o n t o t h e c o l u m n .

F o r e x t r a c t i o n , 1 g d r ie d , f i n e l y p o w d e r e d m a t e r i a lw a s s t i r r e d w i t h m e t h a n o l - w a t e r ( 9 : 1 ) ( 2 1 0 m l )f o r 5 h a t a m b i e n t t e m p e r a t u r e a n d f i l t e r e d . T h ef i l t r a t e w a s e v a p o r a t e d t o d r y n e s s t o o b t a i n a d a r k

3 R e s u l t s a n d d i s c u s s i o nT o a c c o m p l i s h o p t i m a l s e p a r a t i o n o f th e C .

as ia t i ca e x t r a c t , d i f f e r e n t e x p e r i m e n t a l v a r i a b l e s l i k e


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P.K. lnamdar et al. / J. Chromatogr. A 742 1996) 127 -13 0 129

Table 2Features of the developed HPLC methodCompound LOD L t R

/zg) mg/ml) rain)Madecassoside 3 0.5-3. 5 10.60Asiaticosside 3 0.5-3. 5 12.70Madecassic acid 2.5 0.5-2.5 22.23Asiatic acid 2.5 0.5 -4. 0 26.87

eluent strength, composition and flow-rate wereattempted. Isocratic HPLC was first used to achievea separation of the four compounds by using differ-ent variants. However, due to the large difference in

tmln 15 20 25 30 ~5

Fig. I. Spect rum index plot of madecassos ide IV), asiaticasideIlL madecassic acid III ) and asiatic acid I).

polarity of the triterpene acids and their glycosides,either the two acids (I and III) (with high con-centration of acetonitrile or methanol), or the twoglycosides (II and IV) (with low concentration ofacetonitrile or methanol) were separated. Hence, itwas decided to use a linear gradient, so that all thefour compounds (I-IV) could be separated in asingle run. The optimum separation was obtained byusing a linear gradient of acetonitrile in water from20 to 55 in 30 min. The retention times (t~) of thecompounds I-IV are presented in Table 2 along withthe limits of detection (LOD) and linearity range (L).The LOD was calculated as the ratio of peak heightto three-times the baseline noise. The linearity rangewas determined by injecting the solutions of theconcentrations ranging from 0.5 to 7.5 mg/ml each.The standard curves exhibited excellent linearity forthe, admittedly rather short, concentration rangegiven in Table 2 and regression analysis showedcorrelation coefficient greater than 0.99 with inter-cept values that did not deviate significantly fromorigin for all the four constituents.

The peak purity of the individual compounds I- IVwas examined between 200 and 350 nm using aphotodiode array detector (Waters 991 detector). Fig.1 represents the spectrum index plot. None of thepeaks showed absorbance beyond 230 nm.

Fig. 2 represents a chromatogram of the plantextract which demonstrates the optimum separationand ideal quantitative evaluation of individual con-stituents in the plant extract. In some of the plantextracts, an unidentified peak eluting just beforeasiatic acid is observed, but it does not interfere withthe analysis of asiatic acid.

Extraction efficiency was studied by addingknown amounts of pure compounds to the previously

ZJU3 37

1153 9~

4 51

IIIv

Fig. 2. HPLC analysis of the crude extract of C. asiatica.


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130Table 3Results of centellase tablet assay

P.K. lnamdar et al. / J. Chromatogr. A 742 1996) 1 27 -130

Tablet Asiaticoside Mad ecassic acid Asiatic acidn = ( 4 3 )Mean amount (mg) 13.2 10.1 4.03S.D. 0.1 0.1 0.04Am ount found 44.1 33.8 13.5

Amount labelled 40.0 60.00 (madecassic acid+a siatic acid)

1 5 2 . 4 0

- 2 . 1 9 . ~ i5 I 0 1 5 2 O 2 ~

M b m l c s

I11 0 4 3 . 7 4

7 |' T

M ~ BFig. 3. HPLC analysis of ointment (A) and tablet (B) formulationscontaining C. asiatica extract.

a n a l y s e d p l a n t e x t r a c t . T h e e x t r a c t i o n e f f i c i e n c y ( n =4 ) f o r m a d e c a s s o s i d e , a s s ia t i co s i d e , m a d e c a s s i c a c i da n d a s i a t ic a c i d w a s f o u n d t o b e 9 8 . 1 ( R . S . D .0 . 4 ) , 9 7 . 3 ( R . S . D . 0 . 5 ) , 9 5 . 7 ( R . S . D . 0 . 8 )a n d 9 6 . 4 ( R . S . D . 0 . 6 ) r e s p e c t i v e ly .

T h e m e t h o d w a s f u r t h e r e m p l o y e d t o a s s es s t h ef o u r a c t i v e p r o d u c t s i n t h e b u l k i s o l a t i o n a n d p u r i f i -c a t i o n p r o c e s s o f a c t i v e c o n s t i t u e n t s f r o m p l a n tm a t e r i a l .

T h e m e t h o d w a s a l s o u s e d t o a s s ay t h e a c t iv ec o m p o n e n t s I - I V i n t h e c o m m e r c i a l f o r m u l a t i o n sw h i c h a r e a v a i l a b l e i n t h e E u r o p e a n m a r k e t . F i g . 3s h o w s t h e t y p i c a l a n a l y s e s o f M a d e c a s s o l , a n o i n t -m e n t , a n d C e n t e l l a s e , a t a b l e t f o r m u l a t i o n c o n t a i n i n gC. asiatica e x t ra c t . N o n e o f t h e f o r m u l a t i o n s s h o w e dt h e p r es e n c e o f m a d e c a s s o s i d e ( I V ) . T h e r e s u lt s o ft h e C e n t e l l a s e t a b l e t a s s a y a r e g i v e n i n T a b l e 3 . T h e

r e s u l ts a r e b a s e d o n e x t e r n a l s t a n d a r d q u a n t i t a t i o nm e t h o d .

T h e d e v e l o p e d m e t h o d i s a l so u s e d f o r t he e s t im a -t i o n o f t h e f o u r a c t i v e c o n s t i t u e n t s i n p h a r m a c e u t i c a lp r e p a r a t i o n s c o n t a i n i n g C. asiatica p l a n t e x t r a c t s ,v i z . t a b l e t s a n d o i n t m e n t s ( p r o d u c t s u n d e r d e v e l o p -m e n t ) f o r q u a l i t y c o n t r o l a n d t h e i r s t ab i l i t y s t u d ie s[ l O ] .

c k n o w l e d g m e n t sT h e a u t h o r s t h a n k D r . B . S . K a l a k o t i f o r s u p p l y i n g

t h e p l an t e x t r a c t s a n d M s . A r a t i P r a b h u f o r t e c h n i c a la s s i s t a n c e .

R e f e r e n c e s[1] K.M. Nadkami, Indian Materia Medica, Popular PrakashanBom bay, Rep., 1986, p. 662.[2] R.N. Chopra, I.C. Chopra, K.L. Handa, L.D. Kapur, Indigen-ous Drugs o f India, U.N. Dhur and Sons, Calcutta, 1985, p.351.[3] T. Kartnig, in L.E. Cracker and J.E. Simon, (Editors) Herbs,Spices and Medicinal Plants,Vol. 3, Oryx Press, AZ, 1988, p.

145.[4] M N.R . Appa Rao, K . Srinivasan and T. Koteshwara R ao,Indian J. Psychiatry, 19 (1977) 54.[5] J.E P ointel, H. Boccalon, M . Clorec, C. Ledvehat, M .Joubert, Angiology, 38 (1987) 46.[6] H.G . Vogel, N.J. De Souza, A.D 'Sa. Acta Therapeutica, 16(1990) 285.[7] Tsurumi, Kaito, Hiramatsu Yasuzo, Hayashi Motonide andFujimura H ajime, Oyo Yakuri, 7 (1973) 833.[8] Velasco Manuel, Romero Eduardo, Curr. Ther. Res Clin.Exp., 19 (1976) 121.[9] C. Castellani, A. Marai and E Vacchi, Boll. Chim. Farm ., 120(1981) 570.[10] P.K. lnamdar, R.D . Yeole, M.M. Srivastava and N.J . de-Souza, Drug Development and Industrial Pharmacy, 22(3)(1996) 211.

determination+of+biologically+active+constituents

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