Abstract: Phytochemical evaluation of Chenopodium murale Linn. whole plant,revealed the presence of two flavonoids along with two steroidal glycosides, which were identified as 3,7-Dihydroxy-3`-(4-hydroxy-3-methylbutyl)-5,6,4`-trimethoxyflavone (1), 5,7-Dihydroxy-3`-(2-hydroxy-3-methyl-3-butenyl)-3,6,4`-trimethoxyflavone (2), βSitosterol 3-O-βD-glucoside (3),Stigmasterol 3-O-βD-glucoside (4). Although these were known compounds, but isolated for the first time from this plant specie. The characterization of all these compounds was achieved by various spectroscopic methods and the results were compared with the literature.
Plant description: Chenopodium murale Linn. is a medicinal plant that grows in waste places, on rubbish, by road sides and also in crops, in U.S.A., Mexico, Brazil, Argentina Republic, Barbados, Porto Rico and India (Robert and Henry, 1991). This plant belongs to the family Chenopodiaceae also called as goosefoot family that contains about 102 genera and 1400 species of annual and perennial herbs and shrubs scattered throughout the world (Marie, 1965). The genus Chenopodium consists of 200 species (Boulos, 1983).
Chenopodium murale is an erect annual plant, up to 60 cm in height. It is slightly mealy and its leaves are 2-8 x 1-6 cm broad, ovate, angular sides, lobed, sharply toothed, base wedge shaped and stalked long or short. Flowers are in slender spikes, forming loose dense axillary clusters. Seeds are sharply keeled, dotted and horizontal (Bamber, 1916; Davis, 1967).
Uses in traditional medicine: The importance of Chenopodium species is due to their wide variety of medicinal properties. Plants belonging to this genus are reported to have wide applications in folk medicines; as an anthelmintic, stomachic, antispasmodic, diaphoretic, emmenagogue, for the pain of amenorrhea, as an abortifacient and for the relief of asthma, catarrh and migraine (Watt and Breyer, 1962; Vasishita, 1989). In Mexico, Chenopodium is used for medicinal purpose to treat different illnesses, conditions or discomfort, e.g., sterility, digestive problems, anxiety, depression, hair loss and cough etc (Vega and Iskander, 1997). Some flavonoids isolated from C. murale showed antihypertensive activity (Ahmad and Elmazar, 1997). Various Chenopodium species have been reported to have anthelmintic properties (Lozoya and Lozoya, 1982).
Previously isolated chemical constituents: Chemical studies of members of this genus have been concerned with essential oils (Rustenbekova et al., 1974; De-Paschual et al., 1983); a wide variety of flavonoids (Bahrman et al., 1985; Ahmad et al., 2000 and El-Sayed et al., 1999); sterols and steroidal oestrogens like substances (Van-Rompuy and Zeevaart, 1979; Bathory et al., 1982), alkaloids and coumarins (Rizk, 1986a and b).
Newly isolated chemical constituents: During the present work, we isolated four compounds for the first time from Chenopodium murale L. which were identified as 3,7-Dihydroxy-3`-(4-hydroxy-3-methylbutyl)-5,6,4`-trimethoxyflavone (1), 5,7-Dihydroxy-3`-(2-hydroxy-3-methyl-3-butenyl)-3,6,4`-trimethoxyflavone (2), β-Sitosterol 3-O-β-D-glucoside (3),Stigmasterol 3-O-β-D-glucoside (4).
MATERIALS AND METHODS
Instrumentation: All melting points were recorded in glass capillary tubes using Buchi 535 melting point apparatus.
Optical rotations were measured on JASCO DIP-360 (Japan Spectroscopic Co. Ltd., Tokyo, Japan) digital polarimeter.
The Ultraviolet (UV) spectra were recorded in methanol on Hitachi UV-3200 (Hitachi Corporation, Tokyo, Japan) spectrophotometer.
The Infrared (IR) spectra were measured on JASCO IRA-1 (Japan Spectroscopic Co. Ltd., Tokyo, Japan) and a Shimadzu IR-460 (Shimadzu Corporation, Tokyo, Japan) Infrared Spectrometers.
Proton magnetic resonance (1H NMR) spectra were run in CDCl3, CD3OD and (CD3)2CO using TMS as an internal standard at 300 MHZ, 400 MHZ or 500 MHZ on Bruker AC-300, AM-300, AM-400 or AMX-500 nuclear magnetic resonance spectrometers with aspect 3000 data systems at a digital resolution of 32 K. The 13C NMR spectra were recorded in CDCl3, CD3OD and (CD3)2CO at 75, 100 or 125 MHZ on the same instruments.
Low-resolution electron impact mass spectra were recorded on a Finnigan MAT 311 with MASPEC Data System. Peak matching, field desorption (FD) and field ionization (FI) was performed on the finnigan MAT 312 mass spectrometer. High-resolution mass measurements and fast atom bombardment (FAB) mass measurement were carried out on (Jeol) JMS HX 110 mass spectrometer. FAB source using glycerol or thioglycerol as the matric and cesium iodide (CsI) as internal standard was used for accurate mass measurements.
Chromatography: Column chromatography was carried out on silica gel 60 (E.Merck), (70-230 mesh). Precoated silica gel GF-254 preparative plates (20x20, 0.5 mm thick) (E. Merck) were used for preparative thick layer chromatography. Purity of the samples was also checked on the same precoated plates.
Spraying reagents: Ceric sulphate [Ce(SO4)2] reagent was used for the visualization of compounds. Ceric sulphate (0.1g) and trichloroacetic acid (0.1g) were dissolved in 4 ml distilled water. The solution was boiled and concentrated Sulphuric acid was added drop wise until the disappearance of turbidity.
Plant material: The plant was collected from Matta, District Swat, NWFP, Pakistan during the month of July 2001. The plant was identified by Mehboob-ur-Rehman, plant taxonomist Government College Matta, District Swat, NWFP, Pakistan and verified by Dr.Abdur-Rashid, Chairman Department of Botany, University of Peshawar, NWFP, Pakistan, where voucher specimens were deposited in their Herbarium.
Extraction: The shade dried plant material was chopped into small pieces and finally pulverized into fine powder. The powdered plant material (10 Kg) was soaked in methanol (3 x 10 L), with occasional shaking, at room temperature. After 15 days, the methanol soluble materials were filtered off. The filtrate was concentrated under vacuum at low temperature (40°C) using rotary evaporator. A dark greenish crude extract (253 g) was obtained.
Fractionation: The crude methanolic extract (253 g) was suspended in distilled water (500 ml) and partitioned with n-hexane (3 x 500 ml), chloroform (3 x 500 ml), ethyl acetate (3 x 500 ml) and n-butanol (3 x 500 ml) to yield the n-hexane (24 g), chloroform (61 g), ethyl acetate (30 g), n-butanol (35 g) and aqueous (73 g) fractions, respectively.
Isolation: The chloroform soluble fraction (61g) of the crude extract of C. murale was subjected to gravity eluted column chromatography over silica gel. Elution was carried out with n-hexane (100%), n-hexane : CHCl3, CHCl3 (100%), CHCl3 : MeOH and MeOH (100%) in increasing order of polarity to obtain six fractions i.e. n-hexane (100%) (Fraction A), n-hexane : CHCl3 (7:3) (Fraction B), n-hexane : CHCl3 (1:1) (Fraction C), n-hexane : CHCl3 (2:8) (Fraction D), pure CHCl3 (Fraction E) and CHCl3: MeOH (9:1) (Fraction F), on pooling of the similar fractions on TLC profile.
The Fraction C eluted with n-hexane : CHCl3 (1:1) was resubjected to column chromatography over silica gel using n-hexane: EtOAc in increasing order of polarity to obtain three major fractions i.e. n-hexane : EtOAc (9.5:0.5) (C-1), (9.0:1.0) (C-2) and (8.5:1.5) (C-3), respectively. The fractions (C-1), eluted with n-hexane : EtOAc (9.5 : 0.5) were subjected to PTLC using the same solvent system as column which yielded 3,7-Dihydroxy-3`-(4-hydroxy-3-methylbutyl)-5,6,4`-trimethoxyflavone (1) (20 mg).
The Fraction E eluted from the main column with CHCl3 (100%) was subjected to repeat column chromatography over silica gel using solvent system n-hexane : acetone in increasing order of polarity to obtain four major fractions i.e n-hexane : acetone (8.5:1.5) (E-1), (8.0:2.0) (E-2), (6.5:3.5) (E-3) and (6.0:4.0) (E-4), respectively.
The Fraction E-4 was a binary mixture which was further CC using n-hexane : ethylacetate (4:6) as eluent and finally purified by preparative TLC using solvent system n-hexane: acetone (7.0:3.0) and n- hexane: ethylacetate: methanol (6.0:3.5:0.5) that afforded 5,7-Dihydroxy-3`-(2-hydroxy-3-methyl-3-butenyl)-3,6,4`-trimethoxyflavone (2) (16 mg).
The Fraction F obtained from the main column using CHCl3: MeOH (9:1) gave a binary mixture, which was further chromatographed over silica gel using chloroform and increasing the polarity with methanol. The fractions eluted with CHCl3: MeOH (9:1) yielded β-Sitosterol 3-O-β-D-glucoside (3) (10 mg) and with CHCl3: MeOH (8:2) afforded Stigmasterol 3-O-β-D-glucoside (4) (15 mg).
RESULTS AND DISCUSSION
The isolation and purification of the chemical constituents were undertaken from the Chloroform soluble fraction of the crude extract during which compound 1-4 were isolated by utilizing repeat column chromatography and preparative TLC. The structure elucidation of these isolated chemical entities was carried out through standard chemical and physical methods including UV, IR, MS, 1H and 13C-NMR (Mabry, Markham and Thomas, 1970; Markham and Mabry, 1975) and comparative studies from the literature.
Table 1 shows 1H-NMR and Table 2 shows 13C-NMR spectral data of compound 1, 2, 3 and 4 respectively.
Compound (1) was assigned the molecular formula C23H26O8 by HR-EIMS showing [M]+ peak at m/z 430.1629 (calcd for C23H26O8, 430.1627). It gave positive Shinoda and negative Quastel test for flavonoids. The IR spectrum showed the absorption at 3380 cm-1 indicating the presence of hydroxyl groups, 2905 and 1190 cm-1 for methoxyl groups and a, β-unsaturated C=O at 1650 and 1590 cm-1. The UV spectrum showed absorption at 271 and 340 nm suggesting it to be flavonoid (Voirin, 1983).
The 1H NMR spectrum included signals of ABX splitting pattern of phenyl ring B (δ 7.98, 7.96 7.12), the signals for the side chain (δ 3.46, 2.67, 1.65, 1.40 and 0.98) and the signals for three methoxyl groups (δ 3.84, 3.81 and 3.69) attached at C-5, C-4' and C-6 positions respectively. The aromatic proton of ring A also showed the singlet at δ 6.56 indicating a 5,6,7-oxygenated substitution pattern. Acetylation provided a triacetate, confirming the presence of three hydroxyl groups.
The 13C NMR spectrum (BB and DEPT) revealed the presence of four methyl, three methylene, five methines and eleven quaternary carbon atoms. Comparison of 13C NMR data with those of literature (Itrat et al., 2001) confirmed the same substitution pattern in rings A/B and the side chain.
Mass spectroscopic techniques exhibited peaks at m/z 430 [M]+, 358 [M-C4H8O]+, 357 [M-C4H9O]+, 343 [M-C4H8O-CH3]+, 221 [M-C10H9O5]+, 152 [M-C15H18O5]+. The fragmentation pattern showed that two methoxyls and one hydroxyl group were in ring A, one methoxyl group and the side chain was in ring B and the remaining hydroxyl was on C-3.
The position of side chain at C-3´ was determined by HMBC experiment in which methylene protons of side chain at δ 2.67 showed 2J correlation to C-3' (δ130.8) and 3J interactions with C-2' (δ 129.5), C-4' (δ156.6) and C-2" (δ 33.0) confirming its position at C-3'. The compound (1) is having a methoxyl group in ring B. Its position at C-4´ was confirmed by HMBC correlations. The protons of methoxyl at C-4' (δ 3.81) showed 3J interactions with C -4' (δ 156.6). The aromatic protons of ring B showed HMBC correlations; the proton at δ 7.12 (H-5') with C-4' (δ 156.6), C-3' (δ 130.8) and C-1' (δ 122.2), the proton at δ 7.84 (H-2') showed cross peaks (2J and 3J) with C-4' (δ 156.6), C-2 (δ 152.0), C-1" (δ 27.4), C-6' (δ 127.9) and the proton at δ 7.78 (H-6') also correlated with C-4' (δ 156.6), C-2 (δ 152.0) and C-2' (δ 129.5) confirming the substitution pattern of ring B.
| Table 1: | 1H-NMR spectral data of compound 1, 2, 3 and 4 |
| Table 2: | 13C-NMR spectral data of compound 1, 2, 3 and 4 |
The position of other two methoxyls group was also confirmed by HMBC interactions; the methoxyl protons at C-5 position at δ 3.84 showed cross peak with C-5 (δ 159.6) and methoxyl protons at C-6 (δ 3.69) coupled with C-6 (δ 131.3). By comparing all these assignments with the literature (Itrat et al., 2001) the structure of (1) was confirmed as 3,7-Dihydroxy-3`-(4-hydroxy-3-methylbutyl)-5,6,4`-trimethoxyflavone.
Compound (2) was assigned the molecular formula C23H24O8 by HR-EIMS showing [M]+ peak at m/z 428.1475 (calcd for C23H24O8, 428.1471). It gave a red colour in the Shinoda test, typical for flavonoids. Negative results in the Quastel test indicated the absence of an ortho dihydroxyl moiety. The UV spectrum with λmax 272 and 338 nm also suggested it to be a flavonoid. The IR spectrum revealed the presence of the α,β-unsaturated carbonyl group (1680 and 1600 cm-1), a methoxyl group (2927 and 1193 cm-1) and hydroxyl groups (3430 cm-1).
The 1H NMR spectrum provided signals of functional groups including a chelated hydroxyl group at δ 12.94 (1H, s), methoxyl groups [δ 4.01 (3H, s, MeO-6), 3.91 (3H, s, MeO-4'), 3.82 (3H, s, MeO-3)] and an isoprenyl group comprising of a C5-unit which was revealed to be 2-hydroxy-3-methyl-3-butenyl, two one-proton double doublet at δ 3.02 (J = 13.7, 4.3 Hz) and 2.84 (J = 13.7, 8.3 Hz) assignable to 1" protons, a one-proton double doublet at δ 4.33 (J = 8.3, 4.3 Hz) assignable to a proton at the 2"-position bearing a hydroxyl group, a three-proton singlet at δ 1.82 (CH3 at 5") and two one-proton singlets at δ 4.90 and 4.83 assignable to an exo-methylene (CH2 at 4"). It also exhibited an ABX system (B ring) [δ 7.99 (1H, dd, J = 8.7, 2.3 Hz), 7.90 (1H, d, J = 2.3 Hz) and 6.98 (1H, d, J = 8.7 Hz)] and one aromatic proton (A ring) [δ 6.54 (1H, s, H-8)].
The 13C NMR spectrum (BB and DEPT) of (2) corroborated the presence of four methyl, two methylene, five methines and twelve quaternary carbons. The signal at δ 93.2 was typical of C-8 carbon of 5,6,7-oxygenated flavonoids.
The EIMS gave distinct peaks for flavanones at m/z 428 ([M]+), 358 ([M-70]+), 357 ([M-71]+), 343 ([M-70-CH3]+) and two typical daughter ions at m/z 183 ([A1+H]+) and 219 ([B2]+). This confirmed the presence of two hydroxyl groups and one methoxyl group in ring A and one methoxyl and the side chain in ring B and the remaining methoxyl group at C-3. The benzyl cleavage explained the loss of 71. a.m.u., while the loss of 70 a.m.u. was due to the β-cleavage of the side chain with H-transfer to the aromatic nucleus via a 1,6 rearrangement (Markham and Mabry, 1975).
On the basis of the analysis of its 2D NMR spectra (HMQC and HMBC), (2) was deduced to be a flavanone with isoprenylated group. The two double doublets of methylene protons of the isoprenylated moiety at δ 3.02 and 2.84 correlated with C-3' (δ 127.4), C-2" (δ 75.3), C-2' (δ 131.3), C-3" (δ 147.0) and C-4' (δ 159.6), the proton germinal to the secondary hydroxyl group at δ 4.33 correlated with C-4" (δ 110.9) and the exo methylene protons at δ 4.90 and 4.83 coupled with C-2" (δ 75.3) and C-5" (δ 18.1). The protons of methyl group at δ 1.82 showed cross-peaks (J2 and J3) with C-3" (δ 147.0), C-4" (δ 110.9) and C-2" (δ 75.3) confirming the position of hydroxyl and olefinic groups in the prenyl moiety which was itself assigned to C-3'. The methoxyl protons at δ 3.91 also showed correlation with C-4' (δ 159.6) providing evidence for its location at C-4' position. The other methoxyl groups at δ 3.82 and 4.01 were also confirmed by HMBC interactions showing correlations with C-3 (δ 138.4) and C-6 (δ 130.0), respectively.
The ring A proton at δ 6.54 showed cross-peaks with C-7 (δ 155.0), C-9 (δ 152.2), C-6 (δ 130.0) and C-10 (δ 106.2), on the basis of which it could be assigned to C-8. The proton of the chelated hydroxyl group at δ 12.94 also showed HMBC interactions with C-5 (δ 152.8), C-6 (δ 130.0) and C-10 (δ 106.2).
The other aromatic protons of ring B also showed HMBC interactions; the proton at δ 6.98 (H-5') coupled with C-4' (δ 159.6), C-3' (δ 127.4) and C-1' (δ 122.6), the proton at δ 7.90 (H-2') coupled with C-4' (δ 159.6), C-2 (δ 156.1), C-1" (δ 37.0), C-6' (δ 128.7) and the proton at δ 7.99 (H-6') correlated with C-4' (δ 159.6), C-2 (δ 156.1) and C-2' (δ131.3) to confirm the substitution pattern of ring B. By comparing all these data with the published values (Itrat et al., 2002); the structure of compound (2)was, therefore, assigned as 5,7-Dihydroxy-3`-(2-hydroxy-3-methyl-3-butenyl)-3,6,4`- trimethoxyflavone.
β-Sitosterol 3-O-β-D-glucoside (3) was obtained as colorless crystals from chloroform soluble portion of the methanolic extract of C. murale on elution with CHCl3: MeOH (9:1). The HR-EIMS gave the molecular ion peak at m/z 576.4386 consistent with the molecular formula C35H60O6 (calcd for C35H60O6 576.4389). The fragmentation pattern in the mass spectrum of compound (3) was characteristic for sterol with double bond at C-5 (Wyllie et al., 1977).
The IR spectrum showed absorptions bands for hydroxyl group (3452 cm-1) and trisubstituted double bond (3044, 1646 and 814 cm-1).
The 1H-NMR data were similar to β-Sitosterol (Ian et al., 1976) except additional resonances at δ 5.33 (1H, d, J = 7.2 Hz) for the anomeric proton and between δ 3.82-4.42 (sugar protons geminal to hydroxyl groups).
On the basis of above evidence, comparison of 13C NMR data with the published values (Herbert et al., 1978; Adolfo and Pomilio, 1983), co-TLC and mixed m.p. with an authentic sample, the structure of (3) was established as β–Sitosterol 3-O-β-D-glucoside.
Stigmasterol 3-O-β-D-glucoside (4) crystallized as colorless crystals from chloroform soluble fraction of the methanolic extract of C. murale.
The molecular formula was established as C35H58O6 by HR-FAB-MS, which showed molecular ion peak at m/z 574.4231 (calcd. for C35H58O6 574.4233). The mass spectrum showed characteristic fragmentation pattern for sterols (Wyllie et al., 1977).
The 1H-NMR of (4) completely corresponded to the data for stigmasterol (Ian et al., 1976) except additional resonances at δ 5.23 (1H, d, J = 5.4 Hz) and between 3.84-4.44 corresponding to the sugar moiety. The 13C NMR spectrum was also in agreement with the published data for Stigmasterol (Herbert et al., 1978) except additional peaks for sugar moiety.
On the basis of above evidence as well as co-TLC and mixed m.p with an authentic sample, the structure of compound (4) was established as Stigmasterol 3-O-β-D-glucoside.
ACKNOWLEDGMENT
We are grateful to Dr. Farzana Shaheen Assistant Professor of Organic Chemistry and Dr. Khalid M. Khan Assistant Professor of organic chemistry, HEJ Research Institute of Chemistry, University of Karachi, Pakistan, for their help in measuring the MS and NMR spectra.