Flavonoid Glycosides from Tribulus terrestris L. orientalis

1. Introduction

     The genus Tribulus L. (Zygophyllaceae) has 25 species in the world [1]. In the Flora Iranica four species have been reported for this genus which include T. terrestris L., T. pentandrus Forssk., T. ochroleucus Maire and T. longipetalus subsp. macropeterus. The former one is characterized by having four spines in mericarps of fruits in which two of them are shorter than two other ones. This species includes with three varieties: T. terrestris var. orientalis (Kerner) G. Beck, T. terrestris var. robustus Boiss., and T. terrestris var. bicornis (C. A. Mey) Hadidi [2].

      T. terrestris L. var. orientalis is a widespread and summer- tolerant plant which distributed in arid and semi-arid parts of the world include Africa, Middle East, East Asia, East, Center and South of Europe and Australia, Mediterranean region and SWAsia. The plant is distributed in most parts of Iran [2].

     T. terrestris L. is used in the traditional medicine of many countries for treatment of cardiac diseases, edema, eye trouble, skin disorders, urinary troubles and stones in the bladder and as a diuretic, aphrodisiac [3]. It has been shown to increase the free serum testosterone [4] and to be effective in the treatment of sexual and erectile dysfunction by conversion of its phytochemical derivative, protodioscine to dehydro-epi androsterone (DHEA) [5]. It has protective effect on genetic damage [6] and stimulates melanocyte proliferation in the treatment of vitiligo [7]. Moreover, a nematocidal activity has been reported [8]. Its action on motor activity, muscle tone and restorative tonic for vigor, and mainly used to improve performance in sports [9]. The plant is reported to contain steroidal saponins, alkaloides, and flavonoides [10-16]. T. terrestris is also known for having antimicrobial, antiacetylcholine and haemolytic activity [17-19]. For treatment of kidney troubles, it is either used alone or in combination with Zea mays [20]. The quantities and presence of important metabolites depend on the various parts of the plant used [16].

    There are no investigations on the flavonoides of it in Iran. In this article, we report the isolation and the structural charac-terization of two quercetin glycosides and one kaempferol glycoside.

Table 1. UV data of flavonoid glycosides from T. terrestris var. orientalis.

2. Materials and methods

2.1. Plant material

     Aerial parts of the plant were collected which grows as weed in farming lands as dominated plant after harvesting crops. A voucher specimen (Golestan Province, 9 km E Kalaleh, near to Ajansangarly village, ca. 200 m, 11.8.2006, Ajani 10050) is deposited in the Central Herbarium of Medicinal Plants (ACECR), Karaj, and Tehran Province, Iran.

2.2. General experimental procedures

     Melting points were taken on a Reichert-Jung apparatus (Vienna, Austria). Ultraviolet spectra were recorded on a Shimadzu 160A spectrometer (Kyoto, Japan). Electron Ionization Mass Spectra (EIMS) were determined on a Finnigan MAT SQ 70 (California, USA) at 70eV. H-NMR and C-NMR spectra were measured in CDC1₃ with tetramethylsilane (TMS) as an internal standard using a varian 400 Unity plus spectrometer. FTIR spectra were recorded on a Nicolet 550 spectrometer (Madison, WI, USA). Column chromatography (CC) was conducted with silica gel (kieselgel 60, 60-100 mesh ASTM; Merck, Darmstalt, Germany) and thin-layer chromatography (TLC) with Merck silica gel 60 F254 on glass plates.

2.3. Extraction procedures

     The shade dried and powdered aerial parts (50 g) of the plant were extracted with 80% MeOH (2 l) by perculation for 72 h. The solvent was evaporated by vacuum distillation at 45 ºC to produce a gummy residue and 50% of methanolic extract was re-dissolved in MeOH and the 50% MeOH soluble part was partitioned by ethyl acetate. The 50% MeOH phase was subjected to column chromatography over silica gel (60-120 mesh) and eluted with CHCl₃: MeOH mixtures with increasing concentrations methanol to give 4 fractions. Fraction 2 was re-chromatographed on a sephadex LH- 20 column using MeOH as eluent, to yield 1 and 2 (150, 100 mg). Fraction 4 was chromatographed on a silica gel CC and eluted with EtOAC: MeOH mixtures with increasing concentrations of methanol to give 3 fractions. Fraction 3 was rechromatographed on a sephadex LH- 20 column using MeOH as eluent to yield 3 (12 mg).

2.4. Identification

     The isolated flavonoides were identified by UV, ¹H and ¹³C NMR and mild acid hydrolysis. UV, ¹H and ¹³C NMR data of the isolated flavanones are shown in Tables 1 and 2. Acid hydrolysis: Compounds 1, 2 and 3 each in a mixture of HCl 8% and MeOH (2 ml) were separately refluxed for 2 h. The reaction mixtures were reduced in vacuum to dryness, dissolved in H ₂O (2 ml) and neutralized with Na₂CO₃ . The neutralized products were subjected to TLC analysis (eluent: EtOAc- MeOH- H₂O- HOAC, 6:2:1:1 and pc reluent: N-BuOH-HOAC-H₂O (4:5:7) and C₆H₆-N- BuOH-H₂O-Pyridin (1:5:3:3). The chromatograms were sprayed with aniline hydrogen phthalate followed by heating. The sugars were identified after comparison with authentic samples.