Journal of Scientific & Industri al Research Vol. 63, Fehruary 2004, pp. 18 1- 186

Characterisation of sapindosides in Sapindus mukorosii saponin (reetha saponin) and quantitative determination of sapindoside B*

D Saxena, R Pal , A K Dwivedi t and S Singh Division uf Pharmaceutics, Central Drug Research Institute (CDRI),Luck now

Received: 23 June 2003; accepted: 03 December 2003

Hi gh performance thin layer chromatography (HPTLC) met hod as well as high performance liquid ch romatographic (HPLC) met hod combined with ES-MS are developed and va lidated fo r fingerprinti ng (profiling) Sapilldlls sa/JOllill and quantitati ve determination of Sapindoside B in bulk drug samples of Sapindlls sapoll ill and its fo rmul ati on Consap cream. The separation of saponins usi ng TLC is achi eved on precooted si li ca gel plates using chl oroform: methanol: water as mobile phase . Detecti on of the spots is done at the 630 nm . The identificat ion of sapon ins is done by LC-MS, using Electro Spray Ioni sation [ES lltec hniqu e. The HPLC met hod in vo lves chromatog raphy of the saponins on reverse phase (C- 18) column , using acetonitri le-water gradicnt as mobil e phase. The detcction is done by UV -visible detector at a wavelengt h of 2 1 5 nm. Calibrati on graphs are fou nd to be linear over the range 11-220 ~g in HPTLC and 30-200 ~g/mL in HPLC meth od. The methods deve loped are being used for the analysis of the bu lk drug samples and Consa p cream samples .

Keywords: Sapilldlls supollill , Sapindoside-B, Spermicidal, HPLC, HPTLC, LC-MS

Introduction The Reetha plant Sapindus l11ukorosii is found across

the Indian subcontinent . It has been used in the Indian househo ld since long, as a c lean sing agent. Its fruit was found to co nta in vari ous saponin s, which are mainly res ponsibl e fo r its c leansin g prope rti es. Sapindlls s(lponin l.~ , (ra ffia saponin ) is the n-buta no l so lubl e fraction, obta ined from the e thano lic ex tract of the fruit pericarp of the plant Sapindus lI1ukorosii . The saponin fracti on cons ists of several saponins and most of them are derived from hederagenin l . The spermicidal action of the

saponins was found to be assoc iated with the B-amyrin C-28 carboxy lic acid type of sapogenins linked to a particular sequence of sugar moie ti es·l . The mixture of saponins' ·6 isolated from Sapindus l11ukorosii were taken for furth er drug developme nt at CDRI, Lucknow. For thi s, it was incorporated in a c ream re ferred to as Con sap cream, and its phase III multi-centric c lini cal trial s are complete in seven State Med ica l Colleges and three Family Welfare Ce ntres. Th e c ream has been found to be safe and efficacious, and permission for marketing from the Drug Controlle r General of India is received. The process has been handed over to indu stry for manufacturing,

*CDRI Communi cat ion No. 63 16; Accepted in AAPS Annual Meeting and Expositi on, Toronto , November 2002. 'Correspo nding author. E-mai l: A .. _K __ DW IVED I@'yahon .c,lm, Tel: 91 -522-22 1 24 1 111~, exl.- 430 1.4302. Fax: 9 1-522-2223938

There are seve ral m e th o d s u sed for sapo nin determination in plant materials. It is observed that the biolog ical and spectrophotomeric methods which are being used for saponin dete rmination provide, to a large extent, valuable results on saponin concentrations in pl ant material but these dete rminati ons do not provide accurate data and have to be recognized as approximate, GLC, TIC on norma l and reversed-phases is be ing used for routine determination of saponins in plant material. HPLC is the most-wide ly used method fo r saponin de termin ati o n. However the lack of chromophores limits the cho ice of de tec tion method . The pre-column derivati sation7 with benzoyl chloride , coumarin or4-bromophenacy l bromide has been used in some cases, a ll ow ing UV detection. New hyphenated techniques, combining HPLC with MS and NMR a re d eve lo pin g rapidl y and allow o n- lin e identification of separated saponinsx among other methods being deve loped~ · ' (). A colourimetric assay method for the estimation of the total saponin conte nt of the spermicidal ree fh a saponin "was also repo rted. Th e present study desc rib es tw o diffe re nt m e th od s for analy s is of sapindosides within the saponin mi xture . The identity of six sapindosides, inc luding sapindos ide 8 , was estab li shed in the sapindus saponin mi xture by LC-MS technique. We have developed and used HPLC and HPTLC methods for the fingerprinting and estimation of sapindoside-8 , one of the major active constituents, in bulk drug samples of sapindus saponin and its formulation consap cream .

1 ~2 J SCI IND RES VOL 63 FEBR UARY 2004

MlVOOUI 1 CO.fllUl <.;n (c-.;l.IttI.W . ... ~::im 1_. bIII.no); sa. (1.40.UU I 100

771.4

Figure 1- ES-MS spectra of sapindos ide B

2 Experimental P,-ocedure 2.1 Reagcnts and Standards

Stand a rd Sopine/Lls .\'{/pollin , was pro vid e d by

C hemical Techno logy Divi sion, o f thi s Institute. HPLC

grade acetonitrile, methanol and chloroform were. obtained from Mis E Me rc k (India) Limited . Triple di still ed wate r,

from an all g lass apparatu s, was L1 sed as so lvent. Other

reagents like, naphth orescorc ino l, ethano l and H ~SO., (96

per cent) used were of analyti cal grade.

2.2 Isolation and Purification of Sapindosidc 13

Pure sa pind os id e B wa s o bt a in e d b y co lumn c hromatography o f the Sopilldus saponin and the identity o f sapindoside B was established by ES-MS. To iso late

pure sapindos ide B, fro m the saponin mi xture, enri ched frac ti o n o f sapind os ide B was pre pared by co lumn c hro matog raph y, us in g s ili ca co lumn and mixture o f c hl oroform, methano l and wate r as e luent . Thi s enri ched frac ti on was again loaded on a silica column. The fractions were e luted with chl oroform: methanol: wate r (89.5 : 10:0.5 - 84.5: 15:0.5) and those corresponding to sapindoside B we re poo led, co ncentra ted to dry ness und e r reduced pressure. F in a l purificati on of sapindos ide B was done by prcp-TLC using so lvent system o f chloroform: methan o l: wa te r (50 : 14: I ). The MS o f thi s purified sapindoside B

(Figure T) uSlIlg ES MS sh owed a ma jo r peak at 905 (M+Na).

2.3 Apparatus and Chromatographic Condi tions

HPTLC sys te m was equippe d Wi th aut o -TLC

sample r-3 with air pressure injec tor (CA MAG ). sy ringe injector with 50 ilL loop, twin chambe r TLC devel-oper

and Camag Scanne r ATS-3 with tungs te n, mercury and deute rium lamps. HPTLC separati on was achi eved on a

silica gel pre-coated pl ate (20x 20c m, S i ge l 60 Fl,) from

Merck , for detecti on o f the spots CAMA G TLC scanner was used . Data was acquired and processed , using a Camag

HPTLC software CATS - 4. HPLC workstation used was from Sh imadzu, Japan:

equipped with SCL-I OA VP syste m cont ro ll er, LC- I OAT VP twin pump, SPD- IOA VP U V- VI S de tec tor. and

Rheod yne inj ec to r w ith 20 Il L inj ec tio n loo p . T he separation was achi eved on a 511 Lic hros phere RP C- 18 (25 x 0.4 cm) Li chrocart reverse phase anal yti ca l co lumn (Merck). The data acquisiti on and process in g was el one

by us ing Shimadzu HPLC soft ware c ia". -VP (V5 .03). LC-MS ex periment was performed o n a Mi crom<lss

Quattro II triple quadrupo le mass spectrometer. T he ES r capi Ilary was set at 3.5 k V and the cone \ o ll age was 40V.

The spectra was co ll ected in 6s scans .tIlei the fin a l prin h we re the averaged spec tra o f 6-8 sca n:-. .

SAXENA el al. : CHARACTER ISATION OF SAPINDOSIDES IN SA PIND US MUKOIWSII

2.3.1 Preparation of Stock and Working Standard Solution

Stock standard so luti on containin g I mg/mL of standard soluti on was prepared by disso lving 10 mg of sap ind os id e B in 10 mL of methanol. The working standards were prepared by serial diluti on from the stock soluti on of sapindos ide B accord ing ly in the range of 30 to 200 pg/mL in methano l.

2.3.2 Prepa ration of Sample Solution

The samples of the supine/us saponin (25 mg) each from different batches we re dissolved in 10 mL methanol and analysed.

2.3.2.1 Extraction of Saponin from Consap Cream

It was done, as described by Dwivedi et oJ! I.

2.3.3 HPTLC Method

Various spots of standard sap indoside B in the range of 5 .0-220 ~t g and three spots of I OO).1L each of the sample were loaded on the si I ica plate. T he plates were developed in the sol vent system - Ch loroform: Methanol: Water :: 50: 14: I . TLC was performed at 27 ± 3° C. The piates were re moved, dried and the spots were visua li zed by pos t c hromatography derivatization with naphthorescorcino l reagent l1 {naphthorescorcino\ 20 mg in 10 mL e thanol + 0.4 mL H

2S0

4 (96 per cent) and heating the plate at 100°

C for 10 min . The blue co loured spots were scanned at 630 flm .

2.3.4 HPLC method

The separat ion of different saponins was achi eved on C-18 (25x 0.4 cm, 5 /-1m) reverse phase column (Merck) and also on C-1 8 (30x 0.39 cm, 6).1) reverse phase co lumn (Waters). The so lve nt system was Acetonitrile:Water gradient starting with 90 to 20 water in 30 min. The e luents were detected at 2 15 nm , and chromatography was performed at 27 ±3() C.

LC-MS of Sapindosides in Sapilldll.l' sapollin

Using the above described method of HPLC, and ).1-bo ndapack C- 18 co lumn (Waters), the LC-MS was performed for enriched fractions of saponin s, obtained from co lumn chromatography. The identity of sapon in s was es tab li shed by Selected Ion Monitoring (SIM ) of the Total Io n C urre nt (TIC) at th e known mass numbers (Figure 2).

2.4 Accuracy and Precision

The accuracy of the method was calcul ated on the basis of the difference in the mean calcul ated and added co nce ntrat ions a nd th e p rec is io n was obt·a-i ne d by

ca lc ulating the inte r- and intra-day relative standard deviat ions (per centage R S O) LW.

2.5 Recovery

Known amount of sapindoside B was added to the mi xed contents of the sampl es/cream and sapinclos icle B in the sample/cream was determi ned by interpo lation on the corresponding calibration graphs.

3 Results and Discussion 3.1 Chromatography

Sap indus saponin is a mi xture of more than ten compo ne nt s. They are st ru c tu ra ll y re lated 4 and o nl y number and type of sugar moi eties attached constitute the differe nt saponins. Therefore proper reso luti on of th e individu a l components for qu a ntita ti o n as we ll as estimati on, among the saponins beco mes difficult task.

Initia l experiments, us ing s ili ca ge l TLC plates and severa l so lvent systems w ith diffe re nt ratio of e thyl acetate, methanol, chl oroform including wate r were tried. No proper separat ion of the individual components was o btained . Ch loroform : Methanol: Water (50: 14: I) gave the best separati on of the spot correspond ing to sap indoside B from ten other prominent spots (Figure 3).

The identity of purified fraction of sapindoside B fro m co lumn chromatography was con firm ed by LC-MS. Further the ac id hyd ro lys is of this purifi ed fract ion revealed the presence of three sugars xy lose, rhamnose, and arab inose . For HPLC, a C- 18 reverse phase ana lyt ica l co lumn and the acetonitrile-water g radient system gave proper separation of the Sapindoside B from the other sap indos ides (Figure 4).

Characterisaton of Sapindus sarilnins

For LC-MS, HPLC method, using C- 18 colu mn from Waters and MS were used . Authors were able to recognise six saponins (Figure I): sapindos ide A (M +Na=773) at 12.56 min , sapindos ide B (M+Na=90S) at 11 .6 min , sap indos ide C (M+Na= I 067) at 5.79min , sapindos ide D (M+Na= 1375) at 10 .9 Imin , muk o roz isaponin E I (M+ I=924) and mukuroz isaponin YI (M+I =1207) at 4 . 13min (Table I).

3.2 Selectivity and Specificity

The retention factor on HPTLC of sap indoside B was 0.48, and other sapindoside was fo und to be, 0 . 19, 0.26, 0.41, 0.59, 0.67, 0 .75 , 0 .83, 0.90, 0.94, respectively (Figure 3). Based on the no ise to s ignal ratio of 3, the

I H4 J SCI [ND RES VOL 63 FEBRUARY 2004

(a) mulcurozisaponin-Y 1 100 4.13

%

100 lib) sapindoside-C

%

27.18 13.g& O~ __ ~~~ __ ~-r~~~~~~~~-T~~~ ____ ~17;.~e~3 __ ~2~O=.30~~~~~~~~~~

100

%

'100

100

4 .15

LCO:lW(.;

100

%

12 ."

10.Ql 11 .01

11.3g

10.e4 11 .5S

(c) mulcurozisaponin-EQ

(d) sapindoside-B

(e) sapindoside-B

(I) sapindoside-D

\)C8n cO)"

1,75 1.0008 2.1gee

o.LU~~~~~~~~~~~~~~~~~~~~ LC020QG Scan ES+

100

11051 l.lJ~

Figure 2- Selected ion monitoring by LC-MS of sapindoside: (a) Y I ; (b) C: (e) E I : (d) B; (e) A; (I) D

SAXENA et al. : CHARACTER ISATION OF SPINDOSIDES I SA PIND US MUKOROSII IRS

Tabl e 1- Finger-printing of sapindosides in sapilu/us

saponin by LC-MS

SI. Sapindos ides Mass M +sodium Retention time

Nu . ion (min )

Sapinuosiue- A 750 773 12.56

2 Sapi ndoside-B ~OQ 905 11.6

3 Sapindoside-C 1044 1067 5.79

4 Sapindoside-D 1352 1375 10.9 1

5 Mukurozisaponin-E I 924 947 13 .52

6 Mukurozisaponin-Y I 1206 1207 (M+I ) 4. 13

Tahl e 2- Estimation of sapindoside-B in sapil/{/us sapon in bu lk drug samples

Batch N(). Year of manufacture Per cent sapi ndoside B found

5- 1997 1997 5.4

4-1998 1998 7.2

2-2002 2002 7 .8

Tab le 3- Estimation o f sapindoside- B in con sap cream formul ation a ft e r storage

Year o f preparati on Label c laim (per cent w/w) Saponin extracti ve found (per cent w/w)

Sapindoside-R per cent estimated in extracted saponin

2.5 (J .97 2.22

95 2.5 1.38 6.33

97 2.5 2.39 8. 16

OulllltlA

-3r' .. .. Mil tMtI .. 1.u 0.4 RItI TIme

t 0.2 ~ <pO

0.0 ij <pO

<pO

0.0 2.1 1.0 7.' 10.0 121

detection limit in HPTLC was 5 ~g, however the limit of

quantitation was set at I 0 ~g.

The re tenti on time on HPLC of I (Figure 3) wa~

found to be about 14 min. U nde r the chromatographic

conditi ons menti oned above, peaks correspondi ng to othe r constituents did not inte rfe re with the peak o f ! (Figure 3) . The detecti on limit in thi s case was 10 f.l g and the limit of quantitation was set at 30 f.l g.

3.3 Linearity and Reproducibility

In HPTLC and HPLC both , extern a l standard ization

by peak area was used for the q uantitati ve determinati on

o f I (Figure 3) . In HPTLC the ca librati on curves were li near in the range of II to 220 ~g . with r=0 .99949.

Unknown conc. = A + (B* peak area), w here A = -12.62364 and B = 0 .0008, whi le in HPLC the ca librat ion

curve was linear in the range of 30 to 2 10 f.l g with r= 0 .9998.

~ ~ <pO

Figure 3-The HPTLC separation of sapindosidc B and uther co nstituent s: (a) Spots ( 1-6 ) and ( 13- 1 X) of sap indoside B

concentrati on range 5-220fl g. (h)S pots (7-8) o f sample I (c) Spots (9-10) of sample 2 (d) Spots ( 11 - 12 l of sample 3

0.4

~ ~

II)

~ ~ ... ~ ~

0.2

0 I I I I I ... <pO

0.0

11.0 17.1 20.0 22.1 21.0 27.' JO.O MInII'N

Figure 4- The II PLC separation of sap inu()s ide B and o ther constitucnt s

IX6 J SCI IND RES VOL 63 FEBRUARY 2004

Unknown cone. = A + (8 * peak area), where A = -4.7392 1 and 8 = 0.00049.

The reproducibility and accuracy of both the methods were determined by intra- and inter-day assay va riation . Re producibility and accuracy of the method were within the acceptable limit13

· ".

3.4 Recovery

The recovery from the con sap cream was checked by ex traction of saponi ns from the cream and estimation by the described method. It was found that the amount of saponins recovered from the cream was more than 90 per cent.

3.5 Application of Method in Pharmaceutical Analysis

Estimation of sapindoside B in the bulk samples for formulation de ve lopment was also done (Tab le 2) . Estimation of Sapindus saponin and sapindoside B in consap cream for vag inal irritation and efficacy stud ies after storage was also checked, using this method (Table 3).

4 Conclusions The HPTLC method so developed is simple and can

be eas ily used. Fu rther, due to post-chromatographic d e ri vatisa tion , u s in g naphthoresco rcin o l reagent si gnificant improvement in the sensitivity and limit of quantita ti on of sap ind os ide 8 was achieved . 8y thi s method proper resoluti on between Sapindus saponins was

also obtained. HPLC me thod also provides good resolution

between the Sap indus saponins. The method developed is sensitive and can be used for the analysis of bulk samples of Sapindus saponins and Con sap cream. Further, usi ng LC-MS , fingerprintin g of sapindus saponin can be done . Acknowledgement

The authors thank Director, CDRI, Lucknow for providing fellowship to 0 Saxena ..

References I

2

Garg H S , Setty B S. Kamboj V P & Khanna N M. A /lItlcess(or produOio//. ofspennicidal sClponins(mlll/lllIIi/s; 'll diall Pal No. 14 1240 (accepted 1974).

Garg H S, Selly B S, Kamboj V P & Khan lla r\ M, ProCl'ss/fJl'

productiOlI of.lllenllicidal saIIOllins/i¥}II I.!i·l lil.,' rIm/lilllllls s/Iecies, cOllllllOn.!), known as soap nut ; Intliall Pal No. ISO:DS (accepted 1975).

3 Selly B S, Kamboj V P; Garg H S , & Khann a N M. Spermicidal

pot e nti a l o f saponins iso la ted from Indi an medicina l plants: COlllraceptiun , 14 (5) ( 1976) 571-5n.

4 Dicliunwy ufnafllral prollucts ; Vol. 2 , J" ed (C hapman & Ha ll.

C hem ica l Data Base, Scient i fic Data D i \' is io n , London) 1994,1743-44.

5 Takagi K, Park E H & Kato H , Antiinllam matory activities of

Hedragenin and c rude saponin isolated from SJpindus mukurossi Gaertn , Chem Pharlll /Jull, 28(4) ( 1980) II x3-(;.

6 Nakayama K, Fujino H, Kasai R, Mitoma Y. Yata N & Tanaka 0, Solubilisation properties o f sapon ins from Sapindus mukuross i Gaertn , Chem Pharlll /Jull , 34(8) ( 1988) 327')-113.

7 Oleszek W A ,Chromatographi c dete rminati on u f pl anl saponins:, J ClrmlllalOgr-A, 967( 1) (2002) 147- 162.

8 Renukappa T, Roos G, Kl a iber I, Vogler B & Kraus W, Appli cation

of HPLC coupl ed to Nuclear magne tic resona nce spectromet ry, mass spectrometry and bioassay for the de te rmination of ac ti ve saponins from Bacopa monni era Wellst, J Clr m ll [{[ lrjgr A , X47( J-2) ( 1999) 109-1 16.

9 TLC- densitometric determination ofSaika sapollins in Buplellrllm root , Yaowu Fellx i Zazlri ,7(2) (1987) 104- 106: Cirelli Ahslr, 107 ( 1987) 13007k.

10 Ireland P A & Dziedzic S Z, Analysis o f soyhean s<lpogen ins by HPLC , J Chrollla/ogr-A, 325 ( 1985) 275-28 1.

II Dwi vedi A K, C haudhry M & Sarin J P S , Standardisation of a ne w spennicidal agenl Sapindus saponin and its estimation in its fo rmulation, Intliall J Pharlll Sci , 52(3) ( 191)0) 165· 167.

12 Ljubomir Krau s , COll c is e pra c tica l h flo" of Ihill lave r c/1J1)lIlalograpity (DESAGA Gmbh, He ide lherg).

13 Karnes H T & March C, Prec ision, accuracy and dat;l acceptance c rite ria in biopharmaceu tica l analys is, Phal'//[{[ 1\('.1' , JO( I 0) ( 1993) 1420-26.

14 IC I-I Guid e lin es o n va lidati o n of ana lyt ic a l procedures: methodology, Fed Regisl, 60( 1995) 11 200- 11 262.

15 ICH Guidelines on validation of analytical p('()l;etlures: definition and te rminology, Fed Rl'f.: isl , 62( 1997) 27464-27467.