Phytochemistry. Vol. 35. No. 1, PP. 275-276. 1994 Printed in Great Britain.

FLAVONOID GLYCOSIDES FROM ZNDZGOFERA

003 I -9422/94 S6.00 + 0.00 Q 1993 Pergamon Pnxs Ltd

HEBEPETALA

AURANGZEB HASAN,* MOHAMMED FARMAN and IFITKHAR AHMED

Department of Chemistry, Quaid-i-Azam University, Islamabad, Pakistan

(Received in reuisedjorm 22 June 1993)

Key Word Index--Indi~ofera hebepetala; Leguminosae; flavonoids; flavonol glycosides; kaempferol 7-alloside and 3,7_diarabinoside.

Abstract-Two new flavonoid glycosides, kaempferol 7-alloside and 3,7diarabinoside have been characterized from leaves of Zndigoferu hebepetala, together with the known glycosides, kaempferol 3-arabinoside-7-rhamnoside and 3- rhamnoside-7-arabinoside.

INTRODUCTION

Indigofera hebepetala (Benth. ex Baker) is a deciduous shrub widely distributed in Pakistan. The genus Indi- gofira is rich in organic and fatty acids and some flavonoids. Chemical investigation of I. tinccoria has revealed rotenoids [ 11, coumarins and flavonoids as the major biologically active constituents [2, 33. De Moraese- Souze and her collaborators have reported the isolation of two new 2-aryl-3-methylbenzofurans from leaves of I. microcarpa [S] and kaempferol 3,5-digalactoside has been reported from the leaves of I. hirsuta [5]. Pro- tocatechuic acid, p-hydroxy benzoic acid, apigenin 7,4’- diglucoside, apigenin 7-rhamnoglucoside and kaempferol 3-neohesperidoside have been characterized from I. mys- orensis leaves [6], while a phytochemical investigation of I. heterantho has led to the characterization of some leaf flavonoid aglycones [7,8] and phenolic acids [9]. Also a nitropropanoyl glucopyranoside has been recorded from 1. su@ucticosa [lo]. In the present investigation of I. hebepetalu we now report two new flavonol glycosides (1 and 2) and two rare kaempferol diglycosides (3 and 4) from leaf tissue.

RESULTS AND DISCUSSION

The glycosides (l-4) were isolated by multiple 2D-PC from a methanolic leaf extract of I. hebepetala (see Experimental). Acid hydrolysis of each glycoside with 2 N hydrochloric acid, followed by the usual workup, gave kaempferol as the only aglycone, in each case. The sugar of the monoglycoside 1 was identified as allose by TLC [ll] in five solvent systems compared with standard sugar samples. The position of attachment of the sugar moiety was determined by UV spectral analysis with the usual shift reagents [12, 133. The glycosylation site was found to be at C-7 of the aglycone (no effect on Band II

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with sodium acetate). On alkaline hydrolysis, 1 remained unchanged and no organic acid was detected, showing it was not acetylated. Compound 1 was therefore identified as kaempferol 7-alloside.

The sugars obtained after acid hydrolysis of the digly- cosides were identified by co-chromatography as arabin- ose for 2 and rhamnose and arabinose for both 3 and 4. The position of attachment in each case was found to be at C-3 and C-7 of the aglycone, by UV spectral analysis ( l2- 17 nm hypsochromic shift of band I in methanol and no effect on band II by the addition of sodium acetate). Compounds 2-4 all remained unchanged after alkaline hydrolysis. Therefore, 2 was identified as kaempferol 3,7- diarabinoside and 3 and 4 were assigned the structures kaempferol 3-arabinoside 7-rhamnoside and kaempferol 3-rhamnoside 7-arabinoside, respectively, after mild acid hydrolysis with 0.1 N sulphuric acid to give the respective kaempferol 7-rhamnoside and 7arabinoside.

Although numerous glycosides of kaempferol are known, including 3-allosides [14, 15-J 1 and 2 are re- ported here for the first time, while 3 and 4 have been reported only once previously [ 163.

EXPERIMENTAL

Plant material. Indigofera hebepetala was collected in September 1991, at Kashmir Point, Mume Hills (Pakis- tan) and authenticated by Dr Mir Ajab Khan of the Biology Department, Quaid-i-Azam University, Islama- bad. A voucher specimen no. 63662 is deposited in the herbarium of the above cited department.

Extraction. Soluble compounds were exhaustively ex- tracted from 1 kg air-dried leaves of the plant with 85% MeOH. Extracts were coned to H,O under vacuum, defatted with CHCl,, and repeatedly extracted with EtOAc. The EtOAc extract was evapd to dryness and the dry residue (2.1 g) dissolved in MeOH. The methanolic soln was sepd by multiple SD-PC (Whatman No. 1) using BAW (n-BuOH-HOAc-H,O, 4: 1: 5 upper phase) and

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15% HOAc. Individual spots corresponding to com- pounds l-4 were eluted with MeOH and further purified by ZD-TLC (Polyamide 6F, Riedel-de-HBen) using C,H,-MeCOEt-MeOH (4:3:3) and 15% HOAc. Com- pounds l--4 were obtained as crystals from MeOH, and identified by standard techniques using R, values and UV spectrometry with diagnostic reagents. Sugars were iden- tified, after acid hydroysis with 2 N HCI, followed by co- chromatography with standard samples in 5 solvent systems: BAW. n-BuOH-HOAc-H,O (4: 1: 5, top layer); BEW, n-BuOH-EtOH-EtOH (4: 1:2.2); BTPW, n-BuOH-toluene-pyridine-H,O (5: 1: 3: 3) and phenol satd with H,O on No. 1 Whatman paper and BAEW, n-BuOH-HOAc-Et,O-H,O (9:6:3: 1) and pyridine- EtOAc-HOAc-H,O (5: 5: 1: 3) on TLC cellulose using aniline hydrogen phthalate as spray reagent. Mild acid hydrolysis with 0.1 N H$O* was used for partial hy- drolysis of the diglycosides.

Identification of glycosides. Kaempferol 7-alloside (1). UV A.::” nm: 264,323, 365; + NaOMe 245,267, 335sh, 429 (inc.); + AlCI, 275, 306, 350, 397; + AICI,/HCl 276, 305, 348, 397; +NaOAc 265, 325, 375; + H,BOS 261, 323sh, 367. TLC (Polyamide 6F) R, (x 100) 59 (C,H,-MeCOEt-MeOH, 4:3:3), 13 (H,O-MeOH- n-BuOH-HOAc, 75: 15: 10:2), 21 (H,O-EtOH- MeCOEt-acetyl Me&O, 13:3:3: 1); dull yellow under UV changing to yellow with NH,.

Kaem&erol3,7-diarabinoside (2). UV lizH nm: 244sh, 266, 315sh, 347; +NaOMe 246, 274, 30lsh, 35Osh, 387 (inc.); +AICl, 275, 308, 352, 400, +AICI,/HCI 274, 304, 348, 399; +NaOAc 266, 307, 365; H,BO, 266, 3OOsh, 349. TLC (Polyamide 6F) R, (x 100) 61 (&H,- MeCOEt-MeOH, 4:3:3), 80 (H,O-MeOH-n-BuOH- HOAc, 75: 15: 10:2), 66 (H,O-EtOH-MeCOEt-acet- yl Me,CO, 13 : 3 : 3 : 1); dark purple under UV changing to yellow with NH,.

Kaempferol 3-arabinoside 7-rhamnoside (3). UV 1::” nm: 243sh, 266, 316sh, 345; + NaOMe 243, 274, 302sh, 351sh. 382 (inc.); +AICI, 276, 309, 352, 397; +AlCl,/HCl 275, 307, 350, 397; + NaOAc 265, 303, 370; H,BO, 266, 304sh, 345. TLC (Polyamide 6F) R, (x 100) 89 (C,H,-MeCOEt-MeOH, 4:3:3), 71 (H,O- MeOH-n-BuOH -HOAc, 75: 15: 10:2), 56 (H,O-EtOH- MeCOEt-acetyl Me,CO, 13: 3 : 3: 1); dark purple under UV changing to yellow with NH,.

Kaempferol 3-rhamnoside 7-arabinoside (4). UV 1%:” nm: 246sh, 266, 316sh, 345; + NaOMe 243, 274, 302sh, 35lsh, 382 (inc.); tAlCI, 276, 309, 352, 397;

Acknowledgement-The authors are thankful to the Na- tional Scientific Research Development Board (NSRDB) for financing this research work.

REFERENCES

1.

2. 3.

Kamal, R. and Mangla, M. (1984) Biol. Plant, 263, 202.

4.

Kamal. R. and Mangla, M. (1990) Herba Pol36, 3. Dzhuraed, Kh. Sh., Anikina, N. B. and Kazokova, N. M. (1986) Izv. Akad. Nauk Tadzh, SSR, Otd. Biol. Nauk. 3, 62. De Moraes-e-Souza, M. A., Lothar, W., Chiappeta, M., Alda, A., Maciel, G. M., De Mello, J. F., Delle Monache, F. and Messama I. (1988) Phytochemistry 27, 1817.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

Rao, J. U. M., Hanumiah, T., Rao, B. K. and Rao, K. V. J. (1984) Indian J. Chem. Sect. B. 23(B), 19. Adinarayana, D. and Sarada, M. (1987) J. Indian Chem. Sot. 64,648. Anila, T., Nancy, R. and Ahmed, S. R. (1982) J. Indian Chem. Sot. 59, 1007. Hasan, A., Waterman, P. and Iftikhar, N. (1989) J. Chromatogr. 466, 399. Hasan, A., Khan, M. A. and Iftikhar, N. (1991) Fitoterapia 52, 183. Garcez, W. S., Gaecez, F. B., Honda, H. K. and De Silva Antonio, J. B. (1989) Phytochemistry #), 1251. Harbome, J. B. (1984) Phytochemical Methods, p. 225. Chapman & Hall, London. Markham. K. R. (1982) Techniques of Flavonoid Identification. Academic Press, New York. Mabry, T. J., Markham, K. R. and Thomas, M. B. (1970) The Systematic Identification of Flavonoids. Springer, New York. Okuyuma, T., Hosoyama, K., Hirga, Y., Kurono, G. and Takemoto, T. (1978) Chem. Pharm. Bull. X,3071. Tsukasa, I. and Shunji, 0. (1990) Phytochemistry 29, 3639. Imperato, F. (1979) Experientia 35, 1134.

+AlCl,/HCl 275, 307, 350, 397; + NaOAc 265, 303, 370; HjB03 266, 304sh, 345. TLC (Polyamide 6F) R, (x 100) 85 (C,H,-MeCOEt-MeOH, 4:3:3), 76 (H,O- MeOH--n-BuOH-HOAc, 75: 15: 10:2), 65 (H,O-EtOH- MeCOEt-acetyl Me&O, 13:3:3: 1); dark purple under UV changing to yellow with NH,.