Indian Journal of Chemistry Vol. 398, February 2000, pp. 125- 131

Indole and flavanoid constituents of Wrightia tinctoria, W. tomentosa and W. coccinea

A V Muruganandam & S K Bhattacharya

Department of Pharmacology, Institute of Medical Sciences, Banaras Hindu University, Varanasi- 221 005, India

and

S Ghosal*

R&D Centre, Indian Herbs Ltd., Saharanpur 247 001, India

Received 30 December 1998; accepted 10 June 1999

Members of the genus Wrightia, viz. W. tinctoria, W. tomentosa and W. coccinea, have been investigated for their chemical constituents with a view to locating their bioactive principles. lndigotin 3, indirubin 6, tryptanthrin 8, isatin 5, an-thranillate 7 and rutin 9 have been isolated and identified as major constituents of W. tinctoria and W. 101nentosa. Anthra-nillate 7 and rutin 9 are the major constituents of W. coccinea. The identities of these compounds have been established by comprehensive chromatographic (HPTLC, HPLC) and spectroscopic ( UV-VIS, IR , El-MS) techniques, using markers and by synthesi s, where possible. While indigotin is found to be native in the living plants (in fresh leaves), indirubin was found to be an artifact formed only during drying process after harvesting of the leaves. This transformation is presumably caused by the intact hydrolytic enzyme system(s) and by autoxidation. Seasonal variation studies of the chemical constituents of leaves, using HPTLC and HPLC analyses, revealed similar variation patterns in the three species. The concentration of in-digotin-indirubin combination steadily increases from August to November. In contrast, concentration of isatin and anthra-nillate increases in the months of December and January, at the expense of indigotin-indirubin . !satin is produced by the autoxidation of indigotin. Tryptanthrin concentration also increases, periodically, in May (at the expense of isatin) and in January. Plausible pathways for the formation of these indole metabolites are appraised on the basis of circumstantial and synthetic evidence.

Members of the genus Wrightia (Family: Apocyana-ceae) are widely distributed in Asia, Africa and Aus-tralia ' . The seeds of W. tinctoria R.Br. are used in traditional medicines (Ayurveda and Siddha), in India as a carminative, astringent , aphrodisiac and as tonic . It is also recommended for the treatment of infections of chest (in asthma), colic and as diuretic2 . Extracts of leaves exhibit antimicrobial activity against Bacil-lus substilis, Staphyloccous aureus, Pseudomonas aeruginosa and Escherichia colP. Extracts of leaves and roots possess hypotensiv~ properties 1• An oil (777 oi l®), prepared from the leaves of W. tinctoria is currently used in the treatment of psoriasis, for which there is no treatment available in the conventional (synthetic) medicine4·5·6 . Despite, holding high me-dicinal potentiality, the species has been considered as an adulterant of Holarrhena antidysenterica1•

Earlier chemical investigations on W.tinctoria re-ported the occurrence of indigo 3 1, tryptanthrin 87 and several triterpenoids (lupeol, urosolic acid8 and wrightial9) and phyto-steroidal (s itbsterol and conge-

ners) constituents in the leaves and barks of this plant. However, practically nothing was known earlier about the sea~>onal variations, if any, of these con-stituents, and their attendand impact on the therapeu-tic property of the extracts·. It was the objective of the present investigation to bridge this gap. Also, the oc-currence of anthranilic acid 7 (and equivalents), indi-rubin 6, isatin 5, and the flavanoid glycoside rutin 9 is· reported for the first time in this species. Addition-ally, two other members of Wrightia , viz. W. tomen-tosa Roem & Shul t and W. coccinea Sims, included in the present study, were also partially investigated be-fore, in respect of only terpenoidal constituents' . We have studied in detail the chemical constituents of fresh and preserved leaves of these species at differ-ent periods of vegetation . The findings constitute the subject of this paper.

Results and Discussion lndigotin 3, indirubin 6, isatin 5, tryptanthrin 8,

indoxyl-yielding glycosides (1, R= glycosyl), anthra-

126 INDIAN J CHEM, SEC 8, FEBRUARY 2000

nillate and rutin 9 were isolated from W. tinctoria and W. tomentosa, in varying yields, depending upon the period of collection (Seasonal variations) (Figure 1 and 2). W. cocinea, on the other hand, afforded only anthranillate and rutin, and no indolic constituents. The identities of these compounds were established

by comprehensive chromatographic and spectroscopic analyses, by direct comparison with authentic mark-ers and by chemical synthesis, where possible.

During preservation and ai r drying of fresh leaves of W. tinctoria and W. tomentosa, chemical trans-formation of indole metabolites into artifacts oc-

Figure 1 - Seasonal variations of chemical constituents of Wrightia tinctoria

0 .8 --- INDIGOTIN(1)

- - INDIRUBIN(2)

- -)IC- · TRYPTANTHRIN(3) 0.7 --o-ISATIN(4)

-Jir - ANTH RANILLATE(5 ) -----RUTIN~(6~) ____ ~

0 .6

' 0 .5

~ 0 .4

3: '$. 0 .3

0 .2

0 .1

0 J N FEB MAR APR MAY JUN JUL AUG SEP OCT N O V D c

-0 .1

Months

Figure 2 - Seasonal variations of chemical constituents of Wrightia tomentosa

MURUGANANDAM eta/.: INDOLE & FLA VAN OlD CONSTITUENTS OF WRIGHT/A TINCTORIA 127

OH HO

9

curred. This was established by qualitative and quan-titative HPTLC and HPLC analyses.

Although both indigotin and indirubin were re-ported to co-occur in indigo - yielding plants

10, we

observed that fresh leaves of W. tinctoria and W. to-mentosa were completely devoid of indi rubin (HPTLCIHPLC, using markers) and contained only indigotin. Additionally, fresh leaves contained in-doxyl-yielding-0-glycoside(s) 1, isatin 5, tryptanthrin 8 and anthranillate, among other nitrogenous prod-ucts, and rutin 9. Dried leaves of these plants, on the other hand, contained indirubin along with other con-stituents of fresh leaves. Thus, absence of indirubin in fresh leaves and its presence in the dried leaves sug-gested that indirubin was not a native compound in W. tinctoria and W. tomentosa. It was an artifact pro-duced during the drying process of leaves by the in-tact enzymatic (hydrolytic) transformation and autoxidation of indoxyl glycoside(s) . Hence, the changes in the contents of indigotin and indirubin, observed during the seasonal variation studies , are complementary and should be considered on the ba-sis of indigotin - indirubin combined contents (Fig-ures 1 and 2).

The concentration of indigotin-indirubin combined (relative abundance 0.436-0.780%) was found to steadily increase from August to November. By con-trast, the concentration of isatin (rei. abundance 0.018-0.38 1% ), anthranillate (rei. abundance 0.054-0.123%) were found to increase in the months of De-cember and January when the concentrations of the indigotin - indirubin were considerably declined. Furthermore, the concentration of tryptanthrin 8, was fountl to increase periodically, such as in May (sharp increase) (rei. abundance 0.690-0.793% (Figures 1 and 2) (in lieu of the decreased concentration of isa-tin), and in January (increased steadily , rei. abun-dance 0.426-0.583% ). Tryptanthrin was found to be a native constituent of Wrightia. The seasonal cold and heat stresses in the producer plants had, conceivably, resulted in the metabolism of indigotin into isatin, enroute to tryptanthrin 8 (Scheme III). Al so, accu-mulation of anthranillate, which is another building

block of tryptanthrin (Scheme Ill), was observed during this period (January).

Although W. tinctoria and W. tomentosa are replete in indole metabolites, tryptophan per se would not seem to be a precursor of the indole metabolites 1-8, since tryptophan was conspicuously absent (HPTLC) in different parts of these species. Also, complete ab-sence of any tryptophan - derived indole alkaloid in these Apocynaceous species lends credence to this conjecture. In fact, rigorous and careful testing of the Wrightia extracts, in this study, has ruled out the oc-currence of any true alkaloid. It is also interesting to note that the biosynthetic sequence in W. coccinea is arrested at the anthranillate stage (no indoxyl derived metabolites was detected).

A distinct difference was observed in the sequence of formation of indigotin and indirubin. Synthetic evidence, obtained in this study, suggests that while indigotin was produced from the autoxidation of indi-can (indoxyl -0-P-o-L-glucoside), indirubin was formed from the condensation of free indoxyl and isatin (Schemes I and II) (See also Experimental).

Tryptanthrin was found to be a native compound in Wrightia. It was synthesized from the condensation of anthranillate and isatin (Scheme III). The steep in-crease in the concentration of tryptanthrin during ex-treme summer and winter seasons seem to indicate it as a stress metabolite of the producer plants. This product being a strong antimicrobial agent 11 fends off predators from the Wrightia species, when they are highly vulnerable to extraneous stresses.

!satin, a component of tribulin , an endogenous marker of stress, and a proven MAO inhibitor12 was detected for the first time in fresh and dried leaves of W. tinctoria and W. tomentosa. Earlier, we have re-ported the formation of isatin from tryptophan by in vitro oxidation studies using free radicals 13 . However, the absence of free tryptophan in Wrightia leaves in-dicated that, atleast in Wrightia, an alternative path-way exists for isatin formation. Oxidation of indigo-tin, as also of indoxyl glycoside 1 could be an alter-native route of its formation in Wrightia species. In consonance with this postulate, indigotin on TLC chromatogram was found to quickly transform into isatin after exposing it to sun light and air. Free in-doxy! produced during the acid hydrolysis of indoxyl-yielding glycoside(s) also resulted in isatin .

The known biological profiles of indole metabo-lites10-12 and our own findings would seem to suggest that the antimicrobial acti vity of Wrightia may be

128 INDIAN 1 CHEM, SEC B, FEBRUARY 2000

1 (R = !l-0-glucosyi)

-~RR~ [HOH] / ~-(0~ ~ [0)~-..---:;

H H H H

2 3

Scheme I -Plausible sequence of fo rmation ofindigotin from indican .

[HOH] '--7 ~OH _j _O] -? ~~0 0 H'~atr~

H H 4 5

4+5 -

ti

Cherne U -Plausible sequence of formation of indirubin from indoxyl and isatin .

0 r-- 0

((

11 011 lo;t_~'N •s ·-+ /'" 1 I --

0-. G-----,. /: H2 • O

MURUGANANDAM eta!.: INDOLE & FLA VANOID CONSTITUE TS OF WRIGHT/A T!NCTORIA 129

fonnic acid-acetic acid-water (100: 11:11:27, Solvent-3) were used as developers.

HPLC. Waters Associates HPLC assembly, with RP-18 reverse phase column, equipped with PDA and RI detectors was used. Methanol-water (80:20, Solvent-4) was used as el·:ent.

IR. (Perkin- Elmer), both KBr and nujol were used for preparing the samples.

El-MS. Mass spectrometra were obtained on a Hitachi M-4000 instrument, operating at an ionization potential of 70 eV.

Marker samples. Indigotin, indoxyl -~-glucoside,

isatin, anthranillic acid, tryptophan and rutin were procured from SIGMA Chemical Company, USA. Indirubin and tryptanthrin were synthesized and puri-fied by preparative. TLC using solvent-] as devel-oper.

Isolation of chemical constituents of Wrightia leaves. Fresh and dried leaves were separately proc-essed for the isolation of chemical constituents (Scheme IV).

Test for the presence of alkaloids. Powdered W. tinctoria leaves were defatted with light petrol (60-800C), using Soxhlet followed by continuous extrac-

Dried and powdered leaves of W. tinctoria (Ca I 0 g)

extracted with chlorofonn (Soxhlet, 10 h); solvent evapd.

. I Restdue from chlorofonn Marc ext. (1.025 g)

cc extracted. with methanol (Soxhlet, 10 h); solvent eva pd.

Fraction subjected to HPTLC, HPLC, prep. TLC Indigotin (3) (Yield 0.774% w/w} Indirubin (6) (Yield 0.248% w/w) }satin (5) (Yield 0.021% w/w)

Residue from methanol ext. (1.602 g)

cc

Fractions subjected to HPTLC, HPLC, prep.TLC

I Indirubin (6) (Yield 0.236% w/w) Tryptanthrin (8) (Yield 0.208 % w/w) Rutin (9) (Yield 0.190% w/w)

Marc (rejected)

Similar scheme was followed for the isolation of chemical constituents from W. tomentosa ~d W. coccinea.

Scheme IV- Isolation of chemical constituents of Wrightia tinctoria

130 INDIAN J CHEM, SEC B, FEBRUARY 2000

tion with ethyl alcohol. The ethanolic extract was evaporated to dryness under reduced pressure. It was redissolved in 5% aqueous acetic acid and then ex-tracted with diethyl ether and chlroform. Thereafter, the aqueous acetic acid solution was basified (pH-8) with sodium bicarbonate and the liberated bases were extracted with ethyl acetate and n-butanol in succes-sion. The organic solvent extractives were washed with water, dried (anhydrous Na2S04) and evapo-rated. The residue on testing with modified Dragen-dorffs reagent (for alkaloids) showed the absence of any alkaloid. HPTLC reflectance spectra, however, showed the presence of several i'ndolic constituents which did not respond to Dragendorff reagent for al-kaloids.

Test for tryptophan. Methanol and ethyl alcohol extractives of W. tinctoria leaves were subjected to HPTLC analysis, using tryptophan for detecting pur-pose, by staining the TLC chromatogram with ninhy-drin reagent. The extractives were found to be devoid of tryptophan. HPLC analysis also ruled out the pres-ence of free tryptophan in the extracts. The absence of any tryptophan conjugate (eg. ester) in the extracts of Wrightia was established by the following hydro-lytic experiment, followed by HPTLC analysis as be-fore.

Hydrolysis of methanol extracts of Wrightia leaves. Methanol extracts of the three Wrightia spe-cies (1 g. each) were separately hydrolyzed with 0.5 N HCl (I mL). The mixture was kept at room tem-perature overnight. Thereafter, the extracts were evaporated to dryness . The residues were redissolved in methanol. The methanol extracts were subjected to HPTLC (Solvents I and 2) using authentic indigotin, indirubin, isatin, anthranillic acid and tryptanthrin as markers. While the presence of indigotin, indirubin, isatin, anthranillic acid and quercetin was detected in W. tinctoria and W. tomentosa, and anthranillic acid and quercetin in W. coccinea, tryptophan was consis-tantly absent in the hydrolyzed extractives. Further-more, the indirubin concentration in W. tinctoria and W. tomentosa extracts was found to increase after the hydrolysis. This observation indicates that indirubin was produced from the hydrolysis of indoxyl glyco-side (or equivalent) (via indoxyl~isatin) (Scheme II). In a separate experiment, similar hydrolysis of indican (=indoxyl-[3-o-glucoside) afforded initially the indoxyl, followed by isatin and indirubin in suc-cessiOn.

Indican (=indoxyl-[3-o-glucosiae) stored in cold

conditions, in the presence of air, resulted in the for-mation of indigotin in minor amount (HPTLC and HPLC). Indican, on the other hand, when stored un-der nitrogen atmosphere, did not produce any indigo-tin. This observation indicates a free radical mediated formation of indigotin from indican (Scheme 1). _

Synthesis of indirubin. The synthesis was at-tempted by us ing the following three sets of reactants: (A), indoxyl-[3-o-glucoside and isatin; (B), indoxyl-13-o-glucoside and indole; (C), indole and isatin.

(A)-Indoxyl-[3-o-glucoside (14.5 mg), in metha-nol (5 mL), was acidified to pH-3 (HCI) to yield free indoxyl in situ; isatin (7.5 mg) added to the freshly liberated indoxyl (HPTLC) and the mixture rcfluxed on a steam-bath for 6 hr. The product was worked-up as described later.

(B)-Indole (5.85 mg) was added to free indoxyl (indoxyl-[3-o-glucoside, 14.5 mg). The mixture was refluxed and processed as before.

(C)-To indole (5.85 mg) in acidic methanol (5 mL, pH-3), isatin (7.5 mg) was added and the mix-ture refluxed and processed as before.

Samples from the above reaction mixtures were withdrawn by capi llary at every 30 min . interval and subjected to HPTLC analysis (Solvent-!) . Indirubin, produced in about 34% yield from the reactants-A, was purified by preparative TLC (Solvent-!) and subjected to El-MS analysis for confirmation of iden-tity (m/z 262 (M+)). Reactants-B also yielded indiru-bin but in trace quantities (presumably via partial

conversion of indoxyl into isatin by aerial oxidation during hydrolysis). Reactants-C failed to produce any indirubin.

Synthesis olf tryptanthrin. The synthesis was at-tempted using the following two sets of reactants : (A), anthranillic acid and isatin ; (B), anthranillic acid and indoxyl-[3-o-glucoside.

(A). To anthranillate (6.5 mg) in acidic methanol (5 mL, pH-3), isatin (7.5 mg) was added and the mix-ture refluxed on a steam-bath for 6 hr. Thereafter, the reaction mixture was evaporated to dryness in vacuo. The residue was redissolved in methanol and sub-jected to HPTLC analysis.

(B)-Jndoxyi-[3-D-glucoside ( 14.5 mg) in metha-nol, was acidified to pH-3 (HCl) to yield free in-doxy!; anthranillic acid (6.5 mg) was added to freshly liberated indoxyl and the mixture refluxed and proc-essed as before.

It was observed that reactants-A yielded tryptan-

MURUGANANDAM eta!.: INDOLE & FLA VANOID CONSTITUENTS OF WRIGHT! A TJNCTORJA 131

thrin in appreciable amounts ( rei. abundance 48.7 %, HPTLC) . Reactants-B yielded tryptanthrin only in trace quantities (via partial conversion of indoxyl into isatin).

Anthranillate: Pale yellow amorphous powder, isolated from all the three species of Wrightia leaves; HPTLC: Rr 0.38 (Solvent-1), reflectance spectral de-tails, Amax (o.d.) 228 (0.66), 312 (0.94); HPLC: tR 3.41 min (Solvent-4); on acidic hydrolysis the compound yielded anthranillic acid (HPTLC).

lsatir.: Yellow colored powder, isolated from the fresh and dried leaves of W. tinctoria and W. tomen-tosa. HPTLC: Rr 0.42 (Solvent-2), reflectance spec-tral details, Arnax (o.d.) 231 (0.94), 306 (0.91); HPLC: tR 1.53 min (Solvent-4); El-MS : rnlz 147 (M+), 119,77.

Indigotin. Blue colored compound, isolated from the fresh and dried leaves of W. tinctoria and W. to-mentosa. HPTLC: Rr 0.59 (Solvent-2), reflectance spectral details, Amax (o.d .) 251 (0.67), 289 (0.93), 337 (0.58), 600 (0.46); HPLC: tR 4.04 min (Solvent-4) .

Indirubin. Pink colored compound, isolated only from the dried leaves of W. tinctoria and W. tomen-tosa. HPTLC: Rr 0.51 (Solvent-2), reflectance spec-tral details, Anmx (o.d.) 237 (0.46) , 292 (0.95), 369 (0.38), 540 (0.58); HPLC: tR 4.72 min (Solvent-4); El-MS : rnlz 262 (M+), 234,205, 179, ,158, 131, 119, 103.

Tryptanthrin: Pale yellow colored powder, iso-lated from the fresh and dried leaves of W. tinctoria and W. tomentosa . HPTLC: Rr 0.68 (Solvent-2), re-flectance spectral details, Arnax (o.d.) 250 (0 .98), 277 (0.42), 3 I I (0.51 ), 328 (0.27); HPLC: tR 2.66 min (Solvent-4); IR : (KBr) Vmax I 722, 1684 cm- 1 (C = 0 ), I 596 (C =C) ; El-MS : rnlz 248 (M+).

Rutin: Pale yellow colored crystal, isolated from all the three species of Wrightia leaves. Its identity was established by direct comparison with an authen-

tic sample of rutin . HPTLC: Rr 0.44 (Solvent-3), re-flectance spectral details, Amax (o.d.) 255(0.71), 378 (0.89); HPLC: tR 2.81 min (Solvent-4); on acidic hy-drolysis rutin afforded quercetin (HPTLC); El-MS rnlz 302 (M+).

Acknowledgment We are indebted to Messers Indian Herbs Ltd., Sa-

haranpur for technical and financial assistance. A VM thanks the CSIR, New Delhi for the award of a senior reaserch fellowship .

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