Abstract: This research aimed to investigate the antioxidant effect from rhizomes of K. rotunda for finding the active compounds by DPPH free radical scavenging activity assay. The chloroform-soluble extract of the rhizomes of K. rotunda showed significant scavenging effect on the on 1,1-diphenyl-2-picryl hydrazyl (DPPH) free radicals (IC50 = 180 μg mL-1). Two compounds of the chloroform-soluble extract were isolated and identified. Compound 1, 2`-hydroxy-4,4`,6`-trimethoxy-chalcone was found as the active constituent (IC50 = 142 μg mL-1). Compound 2, (+)-crotepoxide, was inactive (IC50= 1516 μg mL-1). The structures of compounds 1 and 2 were identified based on the basis of spectral evidence, Mass Spectrophotometry (MS) and 2D-NMR (2 dimension of Nuclear Magnetic Resonance) data including Heteromolecular Multiple Quantum Coherence (HMQC) and Heteromolecular Multiple Bond Correlation (HMBC) and comparison to published values.
INTRODUCTION
Kaempferia rotunda L. is a very closely related to Kaempferia angustifolia Rosc. (syn. K. roxburghiana Schult.; K. undulata Teysm. and Binnend.; K. gilbertii W.Bull.) (Zingiberaceae). In Java these two plants have same local name as Kunci Pepet and Kunir Putih (Riswan and Setyowati, 2000). This plant is indigenous to south-east Asia and cultivated in Indonesia for medicinal purposes (Woerdenbag et al., 2004). It is distributed from eastern Himalaya, Laos, Vietnam, Thailand and Java. K. rotunda is found in teak forest, low land rice and on calcerous marl up to 150 m altitude. In Java, this plant is cultivated and flowers from October to January. This small herb, with its small roots, tubers and rhizomes are fragrant and traditionally used as abdominal pain, dysentery, diarrhea, cold, obesity, astringent (cosmetic) and after childbirth (Ibrahim, 2003). Leaves and rhizomes are eaten fresh or cooked as vegetable, used in cosmetic powder and as a food flavoring spice. Recently, the dried powder of K. rotunda rhizomes is famous for traditional prevention and treatment for cancer diseases. The dried powder of the rhizomes is easily available and sold in Indonesian traditional medicine markets. This powder is eaten directly or made as a drink by adding sugar and hot water. It has been speculated that the active compounds are either its curcuminoids or polysaccharides. Formulation containing the ethanol soluble extracts of the rhizomes of K. rotunda in combination with the extracts of Boesenbergia pandurata, Allium tuberosum and Phyllanthus niruri possessed platelet activating factor. The combined extracts were used for the atopic dermatitis as skin external use agent for rough skin prevention which possessed improvement and preventive effect to various skin diseases of eczema (Maeda and Ota, 2001). A formulation for skin-lightening cosmetics comprised K. rotunda extracts containing melanin formation and tyrosinase inhibitors was reported to be safe and effective. Earlier phytochemical study from this plant and related species led to the isolation of (-)-pipoxide and the 2+-zeylenol-related substances, (-)-(1R,2S,3R,4S)-2-benzoyloxymethylcyclohex-5-ene-1,2,3,4-tetrol 1,4-dibenzoate and (1R,2S,3R,4S)-2-hydroxymethylcyclohex-5-ene-1,2,3,4-tetrol 1,4-dibenzoate, together with 2`-hydroxy-4,4`,6`-trimethoxychalcone, crotepoxide, boesenboxide and (+)-zeylenol (Pancharoen et al., 1996). In this study, we investigated the antioxidant effect from rhizomes of K. rotunda for finding the active compounds by DPPH free radical scavenging activity assay.
MATERIALS AND METHODS
This research was conducted at Natural Product and Pharmaceutical Laboratory, Research Centre for Chemistry, Indonesian Institute of Sciences (LIPI) on 2004-2005.
General experimental procedures: Melting points were measured on a Buchi B-540 apparatus and uncorrected. UV and IR spectra were measured on Hitachi U-2000 spectrometer in MeOH and on Perkin Elmer FT-IR spectrometer in KBr, respectively. 1H- and 13C-NMR spectra were measured on a Varian VXR-500 instrument at 500 MHz using TMS as internal standard. EIMS and HR-EIMS, CIMS (iso-butane for a gas) were measured on a Hitachi M-4100 instrument. TLC was performed using Silica gel 60 F254, 0.25 mm (Merck), with detection provided by UV light (254 nm) and by spraying 10% H2SO4 solution, followed by heating, or 5% FeCl3 reagent. Gravity column chromatography was performed using silica gel for column chromatography (Merck).
Plant material: The fresh rhizomes of K. rotunda (5 kg) were collected in Purworejo Central Java, the plant was identified by Dr. S. Riswan and the voucher specimen was deposited at Herbarium Bogoriense, Bogor.
Extraction and isolation: The dried powdered rhizomes (1.2 kg) were defatted with n-hexane (1x2 L) to yield 18 g (IC50>1000 μg mL-1) of hexane extract. The defatted materials then was extracted with methanol (3x2.5 L) and evaporated to give the MeOH extract (85 g; IC50 = 398 μg mL-1). The methanol extract was partitioned in chloroform and water, 1 L each and evaporated to obtain chloroform soluble extract (35 g; IC50=180 μg mL-1) and water soluble part extract. The water-soluble part was partitioned further with ethyl acetate (1:1, 1 L each), to obtain ethyl acetate soluble extract (9 g; IC50=192 μg mL-1) and water part. The water part was partitioned with n-butanol (1 L), then both extract were evaporated to obtain n-butanol-soluble extract (8 g; IC50 = 251 μg mL-1) and water-soluble extract (32 g; IC50>1000 μg mL-1). The chloroform, ethyl acetate and n-butanol extracts were the active extracts. The ethyl acetate and n-butanol extracts were not studied further. The chloroform extract was subsequently passed through a silica gel column chromatography with chloroform as the solvent system to give 9 main fractions. Fraction 5 (IC50 = 160 μg mL-1) was found to be the active fraction. Further chromatographic elution of the Fraction 5 with a mixture of chloroform and methanol (99:1) then chloroform and methanol (97:3) as the solvent systems, compounds 1 and 2 were isolated.
Assay for DPPH free radical scavenger activity: The antioxidant
assay was performed on scavenging effect of stable free radicals 1,1-diphenyl-2-picryl-hydrazyl
(DPPH) (Sigma). An extract of 4 mg was dissolved in 4 mL Dimethyl Sulfoxide
(DMSO) to obtain 1000 μg mL-1 as mother solution of test
sample. This test samples were diluted with ethanol to concentrations
of 10, 40, 200 and 1000 μg mL-1 for extracts and 10, 20,
50, 100 and 200 μg mL-1, for pure compounds, respectively.
The test samples were mixed with the ethanol solution of 300 μM DPPH
in 90-well micro-titer plate and incubated at 37°C for 30 min. The
absorption was measured at 515 nm. Inhibition percentage of the test samples
was compared to that of control (DMSO), as shown in Table
1. The positive control test were use solutions of t-Butyl Hydroxy
Anisole (BHA), t-Butyl Hydroxy Toluene (BHT) and ascorbic acid (Vit C).
The IC50 value is the ability of the test sample to scavenge
50% of free radicals, DPPH (Yen and Chen, 1995).
| Table 1: | DPPH free radical scavenging effects from extracts, fractions and isolates of K. rotunda rhizomes |
| *Extracts or fractions were considered active when IC50 < 400 μg mL-1; Pure compounds were considered active when IC50 < 200 μg mL-1 | |
RESULTS AND DISCUSSION
DPPH free radical scavenging activity: Scavenging effect values of extracts and isolates were various. The active compounds were presumed in the chloroform, ethyl acetate- and n-butanol-soluble extracts. Table 1 showed the scavenging effects of n-hexane-, chloroform-, ethyl acetate-, n-butanol- and water-soluble extracts. This fact indicated that more than one compound were active. In the chloroform-soluble extract, Compound 1 and 2 were isolated. Only Compound 1 showed DPPH free radical scavenging effect activity. However, Compound 1 was less active to standard controls BHA, BHT and ascorbic acid, used in this experiment. This result at least gave indication that the traditional used as cancer prevention and anticancer traditional medicines of the rhizomes of K. rotunda having correlation to the active of Compound 1 (as antioxidant) and compound 2 that has been known as potential anticancer compound (Kupchan et al., 1968; Shing and Tam, 1998). The speculation that probably the active compounds were its curcuminoids or polysaccharides as claimed in traditional application has not been known.
Isolation of the antioxidant: The methanol extract of the K.
rotunda rhizomesshowed weak activity as DPPH scavenger. However,
when this extract was partitioned between chloroform and water, ethyl
acetate and water then n-butanol and water, the chloroform-soluble extract
showed significant activity. The fraction of silica gel cut column showed
that the significant active fraction was fraction 5. Compound 1 and 2 were isolated from this fraction.
| Compound 1, 2: | `-hydroxy-4, 4`, 6`-trimethoxy-chalcone |
| Compound 2: | (+)-crotepoxide |
Compound 1: Yellow amorphous; mp 151-153°C; UV (MeOH): λmax(log ε) = 258 (4.20), 368 (3.50); IR (KBr): vmax = 3432 (OH), 1710 (CO), 1686 (ArH), 1615 cm-1 (Ar); 1H-NMR (500 MHz, CDCl3): δ 14.37 (1H, 2`-OH), 7.81 (2H, s, Hα and Hβ), 7.57 (2H, d, J = 10 Hz, H-2 and H-6), 7.94 (2H, d, J = 10 Hz, H-3 and H-5), 6.12 (1H, d, J = 2.8 Hz, H-3`), 5.98 (1H, d = 2.8 Hz, H-5`), 3.93 (3H, s, 6`-OCH3), 3.87 (3H, s, 4-OCH3) and 3.85 (3H, s, 4`-OCH3), 13C-NMR (125 Hz, CDCl3): δ 192.7 (C = O), 168.4 (C-2`), 166.1 (C-4`), 162.5 (6`), 161.4 (C-4), 142.5 (C-β), 130.1 (C-2 and C-6), 128.4 (C-1), 125.2 (C-α), 114.4 (C-3 and C-5), 106.5 (C-1`), 93.9 (C-3`), 91.3 (C-5`), 55.6 (4`-OCH3), 55.5 (4-OCH3) and 55.4 (2`-OCH3); HR-EIMS: m/z (rel. int.) = 315.1057 [M+H]+ (20), 314.1127 [M]+ (100), 313.0906 [M-H]+(65), 297.1076 [M-OH]+(10), 286.0604 [M-C2H4]+ (15), 207.0656 [M- C2H4-C5H3O]+ (35), 180.0381 [M-C9H10O]+ (34), 161.0576 [M- C2H4-C6H5O3]+ (9), 152.0436 [M- C9H10O-CO]+ (10), 137.0110 [M- C9H10O-CO-CH3]+ (9), 134.0658 [M-C9H8O4]+ (38), 121.0678 [M-C9H8O4 -CH]+ (38). HR-EIMS: m/z = 314.1127 [M]+ (Calcd for C18H18O5, 314.1154).
Compound 1 was identified as 2`-hydroxy-4,4`,6`-trimethoxy chalcone, based on the physical and spectroscopic data of UV, IR, low and high resolution mass, 1H- and 13C-NMR (gHSQC and gHMBC) and comparison to published values (Pancharoen et al., 1989). This compound was recently isolated from K. angustifolia and K. rotunda rhizomes (Pancharoen et al., 1989; Sirat et al., 2001). The stereochemistry of the double bond at C-α and C-β was identified as a trans, judging from the fact tat the α, β-protons of 2`-hydroxy-4,4`,6`-trimethoxy chalcone did not split to a doublet (Kurosawa et al., 1978; Song-San et al., 1989; Lien et al., 2000). The gSQC spectrum showed that the 1H-resonance at δ 7.81 (s) is crossed related to δ 145.5 (C-β) and 125.2 (C-α) supported that the H-α and H-β did not split. The gHMBC spectrum showed that the singlet peak at δ 7.81 were crossed related to 13C-NMR at δ 192.7, 130.1 and 128.4. The 1H-NMR resonance at δ 7.57 (H-2 and H-6) showed crossed related peaks to 13C-NMR at δ 114.4, 128.4, 142.5 and 161.4. The 1H-NMR resonance at δ 7.94 (H-3 and H-5) showed crossed related peaks to 13C-NMR at δ 128.4 (C-1), 130.1 (C-2 and C-6) and 161.4. The 1H-NMR resonance at δ 6.12 (H-3`) showed crossed related peaks to 13C-NMR at δ 168.4 (C-2`), 166.1 (C-4`), 106.5 (C-1`) and 91.3 (C-5`). The 1H-NMR resonance at δ 5.98 (H-5`) showed crossed peaks to 13C-NMR at δ 166.1 (C-4`), 162.5 (C-6`), 106.5 (C-1`) and 93.9 (C-3`). The methoxy peaks at δ 3.93, 3.87 and 3.85 showed crossed peaks at δ 162.5 (C-6`), 161.4 (C-4) and 166.1 (C-4`), respectively. The hydroxy peak (δ 14.37, -OH), showed crossed related peak to δ 168.4 (C-2`).
Compound 2: White needle crystals; mp 148-150 °C; [α]D = +72o (c 0.5, CHCl3), UV (MeOH): λmax(log ε) = 259 (4.12), 296 (3.50), 312 (3.86); IR (KBr): vmax = 1756 (CO), 1720 (CO), 1686 (ArH), 1276, 1068 cm-1; 1H-NMR (500 MHz, CDCl3): δ 8.02 (2H, dd, J = 7.5; 1.5 Hz, H-2`and 6`), 7.58 (1H, dd, J = 7.5; 1.5 Hz, H-4`), 7.45 (2H, dd, J = 7.5; 7.5 Hz, H-3`and 5`), 5.69(1H, d, J = 9 Hz, H-4), 4.99 (1H, dd, J = 1.5, 9 Hz, H-3), 4.57 (1H, d, J = 12 Hz, H- 6), 4.23 (1H, d, J = 12 Hz., H-6), 3.65 (1H, d, J = 2.7 Hz, H-7), 3.44 (1H, dd, J = 2.7, 3.9 Hz, H-1), 3.09 (1H, dd, J = 1.6, 3.9 Hz, H-2), 2.11 (3H, s, CH3CO at C-4) and 2.02 (3H, s, CH3CO at C-3). Its 13C-NMR spectrum showed resonance at δ 170.21 (C = O acetyl at C-4), 169.89 (C = O acetyl at C-3), 165.98 (C = O benzyl), 133.72 (C-4`), 130.01 (C-2`and 6`), 129.40 (C-1`), 128.76 (C-3`and 5`), 70.61 (C-4), 69.72 (C-3), 62.69 (C-6), 59.60 (C-5), 54.01 (C-7), 52.80 (C-2), 48.25 (C-1), 20.83 (CH3- acetyl at C-4) and 20.79 (CH3-acetyl at C-3); EIMS: m/z (rel. int.) = 364 [M]+ (0.1), 363 [M-H]+ (0.5), 304 [M-acetyl]+ (0.1), 303 [M-H-acetyl]+(0.4), 260 [M-benzyl]+(0.4), 231 [M-CH-O-benzyl]+ (3.4), 227 [M- benzyl-COCH3]+ (13.5), 104 [M-CH2 O-benzyl-2 acetyl]+ (100); CI-MS (positive mode, isobutane) : 364 [M]+. HR-EIMS: m/z = 364.1118 [M]+ (Calcd for C18H20O8, 364.1158).
Compound 2 was identified as (+)-crotepoxide based on the physical (mp, αD) and spectroscopic data of UV, IR, low and high resolution mass, 1H- and 13C-NMR (COSY, DEPT, gHSQC and gHMBC) and to published values (Shing and Tam, 1998). This compound was first discovered from the fruits of Croton macrostachys (Kupchan et al., 1968), Piper futokadzura (Takahashi, 1969) and has been shown to display significant tumor-inhibitory activity against Lewis lung carcinoma in mice and Walker intramuscular carcinosarcoma in rats. Recently this compound was also isolated from the rhizomes of K. angustifolia and K. rotunda (Pai et al., 1970; Pancharoen et al., 1996; Sirat et al., 2001) and was synthesized (Shing and Tam, 1998). In HMQC spectrum, proton peaks at δ 8.02, 7.58, 7.45 had crossed related peaks with 13C- resonances at δ 130.01, 133.72 and 128.76, respectively and were assigned for 1H and 13C-of aromatic carbons, of 2`and 6`, 4`and 3`and 5`. Other proton peaks δ 5.69, 4.99, 3.65, 3.44 and 3.09 had crossed related peaks with 13C-resonances δ 70.61, 69.72, 54.01, 48.25 and 52.80, were assigned for 1H- and 13C-resonances for C-4, 3, 7, 1 and 2, respectively. The 13C-resonance peak at δ 62.63 had crossed peaks with 1H-resonance at δ 4.57 and 4.23 was assigned as C-6 and H-6, respectively. In HMBC spectrum aromatic proton of H-2`and 6` (δ 7.58) had crossed peaks with δ 165.98 and 133.72, assigned for C = O benzyl and C-4`. The H-3` and 5` (δ 7. 45) has crossed peaks with 129.40 and 133.72 assigned as C-1`and 4`. The resonance proton of H-6 (δ 4.23 and 4.57) had crossed peaks with δ 165.98, 70.61 (C-4) and 54.01 (C-7). The H-4 (δ 5.69) had crossed peaks with 13C-NMR at δ 54.01 (C-7), 52.80 (C-2) and 170.21 (C = O, acetyl. The H-3 (δ 4.98) had crossed peaks with 13C-NMR at δ_59.60 (C-5), 48.25 (C-1) and 169.89 (C = O, acetyl). The H-2 (δ 3.09) had crossed peaks with 13C-NMR at δ 70.61 (C-4) and 54.01 (C-7).
ACKNOWLEDGMENT
The authors wish to thank to Dr. Soedarsono Riswan, Herbarium Bogoriense, Research Center for Biology, Indonesian Institute of Sciences, for plant identification.