p-Hydroxybenzoic Acid and Kaempferol from Desmodium triquetrum

Desmodium triquetrum (Fabaceae) is widely distributed in Indonesia and used as herbal medicines for diuretic, hemorrhoids, tonics, and anti-inflammatory. Two known compounds, p-hydroxybenzoic acid (1) and kaempferol (2), were isolated from the MeOH extract of the leaves of Desmodium triquetrum. The structures of isolated compounds were determined based on H and C NMR data. The MeOH extract showed very active with 6,5 μg/mL in addition, compound (1) and (2) showed weak cytotoxicity against murine cell leukemia P-388 with IC50 55,0 and 24,7 μg/mL, respectively. Compound (1) was the first reported in this species.


Introduction
Desmodium triquetrum or known as the synonym Tadehagi triquetrum, is a plant native to China, India, Sri Lanka, Australia and Southeast Asian countries such as Indonesia [1]. D. triquetrum produced significant anti-inflammatory and antioxidant activity, for example the leaves have potent hepatoprotective and antioxidant activities against CCl4-induced liver toxicity [2], besides the chloroform and alcoholic extracts of D. triquetrum (L.) was reported have their antibacterial properties [3]. The phytochemistry of the plants has revealed that this species produces diverse secondary metabolites, such as the leaves of this plant contains tannins, alkaloids, flavonoids and the fruits of this plants contains saponin and flavonoids, while the roots contain alkaloids, saponins, flavonoids, tannins [2,4,5].
Some of these compounds from D. triquetrum showed various bioactivities, such as flavonoid derivatives, baicalein, naringin and neohesperidin as antioxidant [1] or lignin derivative, adehaginosin reported to have hypoglycemic activity on Hep G2 cells [6]. As a continuation of phytochemical studies, in this paper, we report the isolation of secondary metabolites and preliminary cytotoxic evaluation of the compounds from the leaves of D. triquetrum.

General
1 H and 13 C NMR spectra were measured at JEOL ECA500 at 500 MHz ( 1 H) and 125 MHz ( 13 C). Chemical shifts are given on sigma (ppm) scale with acetone-d6 solvent. Vacuum liquid chromatography (VLC) and centrifugal planar chromatography (CPC) were carried out using Merck silica gel 60 GF254 art. 7731 and 7749, respectively. For column chromatography, were used silica Sephadex LH-20. Solvents such as MeOH, acetone, EtOAc, and n-hexane, for extraction, fractionation, and purification were of technical grades, which were distilled before used, while CHCl3 used in the purification was a proanalysis grade. For thin layer chromatography (TLC) analysis, precoated silica gel plates (Merck Kieselgel60 GF254 0.25 mm thickness) were used, and spots were detected by UV irradiation and spraying with cerium sulfate followed by heating.

Plant material dan isolation
The dried leaves powdered of D. triquetrum (850 g) (collected from Solo, central java in 2005) were macerated with methanol three times (each 24 h) at room temperature. The extract was then evaporated under vacuum to give acetone extract (81 g). The MeOH extract was applied to a silica gel vacuum liquid chromatography (VLC) and eluted with n-hexane-EtOAc (10:0-0:10, MeOH) to give five major fractions (Frs. A-E). Fraction B (0.7 g) was purified on radial chromatography eluted with CHCl3-MeOH = 39:1) to give compound (1) (12 mg). Fraction E (0.7 g) was purified on column chromatography by Sephadex LH-20 eluted methanol to give compound (2) (10 mg).

Cytotoxic assay
The cytotoxicity assay was conducted according to the method described previously. The IC50 value is the concentration required for 50% growth inhibition. Each assay and analysis were run in triplicate, averaged, and the results are shown in table 1. Compound (1) has a pair of doblet δH 7.90 and 6.91 ppm (2H) in 1 H NMR, suggest as psubstitution of aromatic. Also, in 13 C NMR the presence of 167.4 ppm as carboxylic acid and four aromatic part (122.6; 132.7; 115,9 and 162.5) showed a comparison of the NMR data with those reported confirmed the structure compound (1) as p-Hydroxybenzoic acid [7]. Compound (1) is a part of hydroxybenzoic acid derivatives (HBAs), which are phenolic compounds with a general structure C6-C1. Four HBAs, including vanillic acid (3-methoxy-4-hydroxy), syringic acid (3,5-dimethoxy-4-hydroxy), protocatechuic acid (3,4dihydroxy) and p-hydroxybenzoic acid, are constituents of lignin. In addition, the previous investigation reported the D. triquetrum contains a lot of lignin and some other extra complexes such as tannin [1]. Compound (1) can be found from other plant such as Daucus carota, Vitis vinifera, Xanthophyllum rubescens, Pterocarpus santalinus [8] and Camellia oleifera [7]. This compound showed a variety of activities such as antimicrobe [9] nematicidal, antiviral and antiinflammatory [10,11].
In preliminary cytotoxic evaluation against murine cell leukemia P-388, the MeOH extract showed very active with 6,5 μg/mL, after fractionation and purification resulted in compound (1) and (2), have been shown weak cytotoxicity.

Conclusion
Two compounds had been isolated from methanol extract of D. triquetrum, p-hydroxybenzoic acid (1) and kaempferol (2). Compound (1) was the first reported in this species. Even though both compounds exhibited weak cytotoxicity against murine cell leukemia P-388 in preliminary cytotoxic evaluation, but the MeOH extract showed very active with 6,5 μg/mL. This result suggested that D. triquetrum still has many potentials since this species reported contained a lot of flavonoid and alkaloids derivatives, which commonly possess a broad spectrum of biological activity.