Isolation and characterisation of flavonoids from the leaves of medicinal plant Orthosiphon stamineus

1.1 Orthosiphon stamineus

Benth, known locally as ‘Misai kucing’ belongs to the Lamiaceae family. Malaysia and Indonesia have a tropical climate with high temperature and rainfall all year, which have enabled the plant to flourish extensively.

The leaves of Orthosiphon stamineus, Benth are commonly used as herbal tea for diuresis, to treat rheumatism, diabetes, urinary lithiasis, oedema, eruptive fever, influenza, hepatitis, jaundice, biliary lithiasis, and hypertension (Sumaryono et al., 1991; Tezuka et al., 2000; Shibuya et al., 1999).

O. stamineus is also used in Southeast Asia, for the treatment of eruptive fever, epilepsy, gallstone, hepatitis, rheumatism, hypertension, syphilis and renal calculus. In South East Asia the tea prepared from leaves is taken as beverage to improve health and for treatment of kidney disease, bladder inflammation, gout and diabetes (Hegnauer, 1996; Wangner, 1982). O. stamineus (OS) contains several chemically active constituents such as terpenoids (diterpenes and triterpenes), polyphenols (lipophilic flavonoids and phenolic acids), and sterols (Tezuka et al., 2000). The therapeutics effects of OS have been ascribed mainly to its polyphenol, which has enzyme inhibition and antioxidant activity. Lipophilic flavonoids isolated from OS was reported to show radical-scavenging activity toward the diphenylpicryihydrazyl radical and inhibition of 15-lipoxygenase from soybeans used as model for mammalian 15-lipoxygenase (Lyckander and Malterud, 1996). Research indicates that the isolated flavones sinensetin and 3′-hydroxy-5,6,7,4′-tetramethoxyflavone from OS exhibited diuretic activity in rats after intravenous administration of 10 mg/kg body weight, therefore, the diuretic effect of OS extracts could be partially due to its lipophilic flavones content (Schut and Zwaving, 1993).

Living cells are constantly exposed to reactive oxygen species, some of which are capable of initiating lipid peroxidation by abstracting of allylic proton from polysaturated fatty acid. This process leads to lipid peroxidation products, which is responsible for cellular damage as a result of oxidative stress (Halliwell and Gutteridge, 1999). Xanthine oxidase is considered to be an important biological force of these superoxide radicals; hence, inhibition of XO may assist in preventing gout and kidney stones (Cos et al., 1998; Lavelli et al., 2000). The recent surge of interest in chemistry of this plant has led to the isolation of more than 50 components with different biological activities. In our recent research, methanol extract of the leaves were separated by a series of chromatographs to give six flavonoid compounds. On the basis of chemical and spectral analyses their structures were elucidated as eupatorin (Wollenweber and Mann, 1985) (1), sinensetin Bombardelli et al., 1972 (2), 5-hydroxy-6,7,3′,4′-tetramethoxyflavone (Schneider and Tan, 1973) (3), salvigenin Zhong and Wu, 1984 (4), 6-hydroxy-5,7,3′-trimethoxyflavone (Zhong and Wu, 1984) (5) and 5,6,7,3′-tetramethoxy-4′-hydroxy-8-C-prenylflavone (Hossain et al., 2001) (6).

2.1 General

IR spectra were recorded (KBr discs) on an FT-IR spectrometer (ν_max in cm⁻¹). ¹H NMR spectra were recorded on a Bruker R-32 (300 MHz) instrument in CDCl₃ with TMS as an internal standard (chemical shifts in δ, ppm). UV spectra were recorded on HATACHI, U-2000 spectrophotometer Ultrospeck in methanol (λ_max in nm). Mass spectra were recorded on a Varian 3800 mass spectrometer. TLC was performed with silica gel GF₂₅₄. All solvents were analytical reagent grade.

2.2 Plant material

O. stamineus Benth (Lamiaceae) leaves were collected from the Island of Penang. The plant was identified and a voucher specimen was deposited in the herbarium of the School of Biology, University Sains Malaysia.

2.3 Extraction

Dried leaves of the plant (1 kg) were milled into powder and then extracted with direct methanol (10 L) in a Soxhlet extractor for 36 h. The extract was evaporated in a rotatory evaporator and dried by vacuum pump. The methanolic extract (50 g) was suspended on water and extracted successively with n-hexane, chloroform, ethyl acetate, and n-butanol to yield n-hexane (5.5 g), chloroform (11.5 g), ethyl acetate (9.3 g) and n-BuOH-soluble (5.2 g) fractions, respectively. Chloroform soluble fraction (10 g) was subjected to chromatography on silica gel (60–120 mesh, Merck) eluted with ethyl acetate–n-hexane (7:3) solvent system. By repeated chromatography to give five major fractions (Fraction-1, 0.176 g; Fraction-2, 0.216 g; Fraction-3, 1.00 g; Fraction-4, 0.074 g; Fraction-5, 0.143 g; Fraction-6, .0103 g).

2.4 Fraction-1

Fraction-1 obtained from column chromatography was further purified by preparative TLC over silica gel GF₂₅₄ using ethyl acetate–hexane (3:2) as the developing solvent. It was a yellow crystal, m.p. 196–197 °C (lit. Wollenweber and Mann, 1985, m.p. 196 °C); MS: (M⁺, 344); UV: 340, 275, 210 nm; IR (KBr): 3470, 3250, 2840, 1650, 1605, 1599, 1478, 1365, 1050 cm⁻¹; ¹H NMR (δ values, DMSO-d₆): 3.72 (s, 3H, 6-OCH₃), 3.88 (s, 3H, 4′-OCH₃), 3.96 (s, 3H, 7-OCH₃), 6.76 (s, 1H, H-3), 6.88 (s, 1H, H-8), 7.10 (d, 1H, H-5′), 7.45 (s, 1H, H-2′), 7.68 (dd, 1H, H-6′), 9.48 (s, 1H, 3′-OH), 12.55(s, 1H, 5-OH). It was characterized as 5,3′-dihydroxy-6,7,4′-trimethoxyflavone (Wollenweber and Mann, 1985) (Eupatorin, 1).

2.5 Fraction-2

Isolated fraction was further purified by preparative TLC over silica gel GF₂₅₄ using ethyl acetate–hexane (3:2) as the developing solvent. It was crystallized from methanol as a white powder (2, 6 mg); m.p. 175–176 °C (lit. Wangner, 1982, m.p. 175–176 °C); MS: (M⁺ 373); UV: 327, 282, 213 nm; IR (KBr):2850, 1645, 1600, 1590, 1476, 1378, 1215, 1189, 870 cm⁻¹; ¹H NMR (δ values, DMSO-d₆): 3.76 (s, 3H, 5-OCH₃), 3.78 (s, 3H, 6-OCH₃), 3.84 (s, 3H, 4′-OCH₃), 3.88 (s, 3H, 3′-OCH₃), 3.96 (s, 3H, 7-OCH₃), 6.78 (s, 1H, H-3), 7.12 (d, 1H, H-5′), 7.18 (s, 1H, H-8), 7.52 (s, 1H, H-2′), 7.68 (d, 1H, H-6′). It was characterized as 5,6,7,3′,4′-pentamethoxyflavone (Bombardelli et al., 1972) (sinensetin, 2).

2.6 Fraction-3

The isolated mixture was worked-up in the usual way and purified by preparative TLC over silica gel GF₂₅₄ using benzene–acetone–ethyl acetate (7:5:1) as the developing solvent. It was crystallized from methanol as orange needles colour (9 mg); m.p. 102 °C; MS: (M⁺, 462); UV: 228, 254, 370 nm; IR: 3415, 1645, 1600, 1590, 1375, 1365 cm⁻¹; ¹H NMR: (δ values, DMSO-d₆): 3.41 (s, 12H, –CH₂OCH₃ × 4), 5.29 (s, 8H, –CH₂OCH₃ × 4), 6.13 (s, 1H, H-3), 6.48 (s, 1H, H-8), 6.97 (s, 1H, H-6), 7.10 (s, 1H, H-2′), 7.29 (d, 2H, J = 9 Hz, H-5′ and H-6′), 12.75 (s,1H, 5-OH). It was characterized as 5-hydroxy-6,7,3′,4′-tetramethoxyflavone (Schneider and Tan, 1973) (3).

2.7 Fraction-4

Isolated fraction from the column was further purified by preparative TLC over silica gel GF₂₅₄ using ethyl acetate–n-hexane (7:3.5) as the developing solvent. It was crystallized from petroleum spirit as yellow needles colour (5 mg); m.p. 144 °C (lit. Lyckander and Malterud, 1996, m.p. 145 °C); MS: (M⁺, 346); UV: 228, 254, 370 nm; IR: 3450, 1645, 1600, 1590, 1375, 1365 cm⁻¹; ¹H NMR: (δ values, DMSO-d₆): 3.91, 3.94 and 3.99 (3s, 9H, OCH₃ × 3), 6.67 (s, 1H, H-3), 7.13 (d, 1H, J = 9.5 Hz, H-2′), 7.74 (d, 2H, J = 9.5 Hz, H-5′ and H-6′), 12.45 (s, 1H, 5-OH). It was characterized as salvigenin (Zhong and Wu, 1984) (4).

2.8 Fraction-5

It was purified by preparative TLC over silica gel GF₂₅₄ using ethyl acetate–hexane (7:3.5) as developing solvent to give compound 5 (6 mg) and compound 6 (4 mg). Compound 5: It was crystallized from methanol-water as yellow coloured needles (4 mg); m.p. 172 °C (lit. Zhong and Wu, 1984. m.p. 172–174 °C); MS: (M⁺, 426); UV: 232, 258, 365 nm; IR: 3470, 1642, 1595, 1590, 1375, 1365 cm⁻¹; ¹H NMR (δ values, DMSO-d₆): 3.91, 3.94 and 3.97 (3s, 9H, OCH₃ × 3) 6.45 (s, 1H, H-3), 6. 65 (s, 1H, H-8), 7.05 9d, 1H, J = 9 Hz, H-2′), 7.45 (d, 2H, J = 9 Hz, H-5′ and H-6′), 12.75 (s, 1H, 6-OH). It was characterized as 6-hydroxy-5,7,3′-trimethoxyflavone (Zhong and Wu, 1984) (5). Compound 6: It was crystallized from methanol an orange coloured needles; m.p. 134 °C (lit. Hossain et al., 2001, m.p. 134 °C); MS: (M⁺, 426). UV: 318, 288, 213 nm. IR (KBr): 3435, 2964, 2936, 2832, 2361, 1649, 1610, 1595, 1452, 1376, 1364, 1271, 1200, 1128, 1046, 1030, 879, 832, 800 cm⁻¹. ¹H NMR (δ values, CDCl₃): 1.60 (s, 6H, C(CH₃)₂, 3.51 (d, 2H, J = 7 Hz, –CH₂–CH⚌), 3.90, 3.94, 3.99 and 4.00 (4s, 12H, –OCH₃ × 4), 5.55 (t, 1H, J = 7 Hz, –CH₂–CH⚌), 6.96 (s, 1H, H-3), 6.99 (d, 1H, J = 9 Hz, H-2′), 7.44 (m, 2H, H-5′ and H-6′), 12.76 (s, 1H, 4′-OH); ¹³C NMR (δ values, CDCl₃): 153.47 (C-2), 126.27 (C-3), 183.07 (C-4), 159.16 (C-5), 90.97 (C-6), 159.16 (C-7), 104.95 (C-8), 153.47 (C-9), 111.10 (C-10), 119.50 (C-1′), 126.27 (C-2′), 112.75 (C-3′), 146.45 (C-4′), 112.75 (C-5′), 119.50 (C-6′), 77.80 (C-1″), 77.58 (C-2″), 77.38 (C-3″), 76.95 (C-4″), 77.38 (C-5″), 61.23 (5-OCH₃), 56.69 (7-OCH₃), 56.54 (6-OCH₃), 55.64 (3′-OCH₃). It was characterized as 5,6,7,3′-tetramethoxy-4′-hydroxy-8-C-prenylflavone (Hossain et al., 2001) (6).