Purification and Characterization of Phenolic Compounds from the Leaves of Cnestis ferruginea (De Candolle): Investigation of Antioxidant Property
A. Abass Khan,
A. Ur Rahman
The presence of yet to be explored radical scavengers in the leaf extracts of Cnestis ferruginea (De Candolle) have been reported. Thus, the present study is focused on isolating and characterizing these radical scavengers for possible therapeutic use as lead drug candidates against oxidatively induced pathological conditions. The defatted methanol extracts (MECF) of the leaves of Cnestis ferruginea was fractionated into Chloroform (CF), Ethylacetate (EF), Butanol (BF) and Water (WF) fractions. Total antioxidant property, superoxide anion, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities and inhibition of xanthine oxidase activity by the fractions were determined. The extent of inhibition of Fe2+/ascorbate stimulated lipid peroxidation in mitochondrial membranes was also evaluated. All fractions and crude extracts exhibited antioxidant properties at different degrees. The pattern of DPPH radical scavenging activity and inhibition of mitochondrial membrane lipid peroxidation by MECF and EF were similar. β-sitosterol, para-hydroxyphenol and six other sub-fractions were isolated from EF. The DPPH radical scavenging activities of the sub-fractions were not significantly (p>0.05) different from one another. Robustaside B or 6'-(3", 4"-dihydroxycinnamoyl) arbutin was also isolated from sub-fraction III from EF using sephadex LH 20. Structural elucidation of all compounds was determined by spectroscopy (IR, UV, 1D and 2D 1H and 13C NMR). The present study reported for the first time the isolation and radical scavenging activity of Robustaside B-an arbutin derivative and para-hydroxyphenol from leaves of Cnestis ferruginea. Leaf extracts of Cnestis ferruginea was demonstrated to be a rich source of antioxidants.
to cite this article:
R.A. Adisa, A. Abass Khan, I. Oladosu, A. Ajaz, M.I. Choudhary, O.O. Olorunsogo and A. Ur Rahman, 2011. Purification and Characterization of Phenolic Compounds from the Leaves of Cnestis ferruginea (De Candolle): Investigation of Antioxidant Property. Research Journal of Phytochemistry, 5: 177-189.
Received: May 05, 2011;
Accepted: September 28, 2011;
Published: October 29, 2011
Cnestis ferruginea (De Candolle) (Capparaceae) (Irvine,
1961) is a climber of genus Cnestis ferruginea DC (Connaracea). In
Nigeria, it is locally referred to as Gboyin-gboyin, Akara-aje, Omu-aje by the
Yorubas; Ukpe, Ibi-eka by Binis, Amunketa, Okpe-nketa, or Okpe-isi-uketa by
Igbos and Utinabua, Usiere-ebua by the Efiks. It has a wide distribution in
Africa and is specifically found in deciduous and secondary scrub such as Udi-Plateau.
The fruits are orange-red with velvety hairs on the follicle and have leaves
ten inches long with eight pairs of leaflets. All leaf parts being ferruginously
pubescent and the flowers are white with ferruginous sepals. The local uses
of the plant include as laxative, remedy for dysentery and gonorrhoea (Dalziel,
1937). In West-Africa the bitter fruit juice is widely used for the treatment
of tooth-ache, mouth and skin infections (Boakye-Yiadom
and Konning, 1975) while the bark is rubbed on the gums for pyorrhea (Okwu
and Iroabuchi, 2004).
The first reported chemistry of C. ferruginea was by Olugbade
et al. (1982) when phytochemistry revealed the presence of coumarin,
flavonoid, squalene, β-sitosterol and triacontal-1-ol in its roots. Five
years later, the petroleum ether fraction of C. ferruginea fruit was
demonstrated to contain octacosanyl stearate, 1-myristo-2-stearo-3-palmitin
(Ogbechie et al., 1987). At exactly one decade
after the first report, a novel isoflavone glycoside, afrormosin-7-O-beta-D-galactoside
with antimicrobial activity against Staphylococcus aureus and Escherichia
coli was isolated in the fruit testa (Parvez and Rahman,
1992). The compound also possess antifungal effects against Candida albicans
but was unable to inhibit the growth of Aspergillus niger. Other compounds
such as squalene, myricyl alcohol, β-sitosterol, cyanidin, delphinidin
and apigenidin (Ogbede et al., 1986) have also
been isolated from the plant. Extracts of roots, stems and leaves of Cnestis
ferruginea have been shown to possess antibacterial and anticonvulsant (Declume
et al., 1984) activities. Recently, Atere and
Ajao (2009) reported the adverse effects of the crude alkaloid fraction
of the roots on hepatic functions. The methanol extracts of leaves of C.
ferruginea have been reported to inhibit haemoglobin glycosylation in
vitro (Adisa et al., 2004). More recently,
methanol and ethylacetate fraction of C. ferruginea were confirmed to
contain highly potent antihyperglycaemic principles capable of attenuating secondary
complications of diabetes such as atherosclerosis, liver and renal dysfunction
(Adisa et al., 2010).
Polyphenolic compounds including flavonoids are established to have antioxidant
properties and have been isolated from many natural sources (Nijveldt
et al., 2001; Heijnen et al., 2002;
Arya and Yadav, 2011; Butkhup and
Samappito, 2011). Several reports have shown that these substances are effective
against myriads of oxidative stress-induced disease conditions including cancer,
neurodegenerative disorders, ageing and diabetes (Ames et
al., 1993; Jain et al., 2011). Meanwhile,
there is renewed interest in the investigation of natural products for the discovery
of new bioactive substances with better pharmacological activities (antioxidant
and anticancer). These drug candidates could serve as substitutes to synthetic
drugs adverse effects (Kim et al., 2003).
Therefore, the present study was designed to investigate the phytochemistry
of leaves of C. ferruginea. Bioactive compounds with antioxidant property
would be isolated, purified and characterized using bioassay guided isolation.
MATERIALS AND METHODS
Spectroscopic measurements: One dimensional (1D) NMR (1H-NMR,
13C-NMR, DEPT 135 and DEPT 90) and two dimensional (2D) NMR spectra
were recorded in deuterated solvents (CDCl3 or MeOD or DMSO) on Bruker
AM-400, or 500 MHZ spectrometers. Chemical shifts were measured in ppm (δ)
and coupling constants (J) are given in Hz. Electron Impact Mass spectra (EIMS)
was recorded on Varian MAT 312 double focusing spectrometer or on a Finnigan
MAT 311 with MASS SPEC data system. Peak matching and Field Desorption (FD)
experiments were performed on Finnigan MAT 312X mass spectrometer. Fast atom
bombardment mass spectra (FAB-MS) were recorded on Jeol HX 600 mass spectrometer.
Exact molecular formulae were determined by High Resolution Mass Spectrometry
(HRMS). Glycerol or thioglycerol were used as reference compounds for FAB (+ve).
Melting points of compounds were determined on the Yanaco micromelting point
apparatus and were uncorrected. UV and IR spectra were recorded on Hitachi-UV-3200
and Jasco 320-A spectrophotometers, respectively. Column chromatography was
carried out using silica gel 60 Merck; 70-230 mesh; pore diameter (0.06-0.20
mm) and Sephadex LH 20 (Reverse Phase 18F254s (Merck KGa A) 64271
Darmstadt Germany). Thin layer chromatography (TLC) was done on silica gel 60
pre-coated plates, F-254 (Merck) and Reverse Phase 18F254s (Merck
KGa A) 64271 Darmstadt Germany) pre-coated plates.
Plant material: Fresh leaves of C. ferruginea were collected at the Forest Reserve Area of International Institute of Tropical Agriculture Ibadan in the month of October, 2004. The leaves were immediately rinsed of debris and shade-dried for one week on laboratory trays. The dried leaves were powdered and weighed. Leaves were authenticated and identified at the Herbarium, Forestry Research Institute of Nigeria (FRIN) Ibadan, Oyo State where specimens (Voucher No. 106524) were deposited. The laboratory experiments were continuously on-going up to year 2008.
Animals: Male wistar strain albino rats (180-200 g, 16 weeks old) used in this study were bred and housed at the animal house of HEJ Research Institute of Chemistry, University of Karachi, Karachi under a 12 h light/dark condition. The experimental design was approved by the Animal Care Ethics Committee of H.E.J Research Institute of Chemistry, University of Karachi, Karachi and the protocols conformed to the National Institute of Health (NIH) guidelines. Animals had free access to food (rat chow) and water throughout the experimental period.
Preparation of rat liver mitochondria: Mitochondrial fraction
were isolated from liver homogenate of male Wistar strain albino rats (180-200
g) as described by Johnson and Lardy (1967). The weighed
livers were washed in ice-cold 1.15% KCl solution and then chopped before homogenizing
in 0.25 M sucrose (ice-cold). The homogenate was centrifuged twice at 3000 rpm
for 5 min. The supernatant was further centrifuged at 10,000 rpm for 20 min
to pellet the mitochondria. The mitochondria were washed twice in 0.25 M sucrose
at 10,000 rpm, dispensed into pre-cooled eppendorf tubes and used fresh.
Protein estimation: The protein content was determined using the method
of Lowry et al. (1951).
Extraction procedures: The air-dried, powdered (1 kg) leaves of C.
ferruginea (D.C) were exhaustively extracted separately with n-hexane (100%)
and methanol (95% v/v) (Sigma Aldrich Chemical Co. St Louis USA), in a giant
size Soxhlet apparatus at 60°C continuously for 12 h in each case. The crude
extracts were collected and concentrated with a rotary evaporator at 40°C.
The methanol extract concentrate of C. ferruginea (MECF) obtained was
a greenish brown substance. MECF (234.9 g) was dissolved in distilled water
and partitioned with equal volume of chloroform, ethylacetate and butanol in
succession to yield the Chloroform (13%), (CF), Ethylacetate (EF) (18%), Butanol
(BF) (7.2%) and Water (WF) (7.2%) fractions. The water fraction was lyophilized
in a freeze dryer. The ethylacetate fraction (EF, 20 g) was subjected to column
chromatography on silica gel (Merck; 70-230 mesh; pore diameter 0.06-0.20 mm)
and eluted with gradients of dichloromethane-hexane (15:85, 2L), dichloromethane-methanol
(98:2, 2.5 L; 85:15, 1 L; 85:15, 0.75 L; 85:15, 0.5 L; 80: 20, 1 L; 75:25, 1.75
L; 75:25, 2.5 L) at a flow rate of 24 mL min-1. The fractions were
subjected to Thin Layer Chromatography (TLC) in solvent systems (40:60; H2O:
MeOH) on Reverse Phase 18F254s (Merck KGa A) 64271 Darmstadt Germany)
chromatographic plates. The fractions were combined, concentrated on a rotary
evaporator and weighed.
The subfractions obtained were further screened qualitatively on TLC plates by spraying with DPPH, AlCl3 and FeCl3.
Determination of antioxidant activities: Antioxidant activities of MECF, HF, EF, CF, BF and WF were determined using in vitro free radical generating systems such as horse radish peroxidase catalysed oxidation of 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), Fe2+/ascorbate induced-mitochondrial membrane lipid peroxidation, PMS-NADH-NBT reduction system and inhibition of xanthine oxidase activity assay.
Total antioxidant activity: The total antioxidant activity of extracts
of C. ferruginea was measured using horse radish peroxidase catalysed
oxidation of ABTS with a slight modification of the method described by Cano
et al. (2002). The reaction mixture (200 μL) contained 1 mm
ABTS, 30 μM H2O2 and 6 μM horse radish peroxidase
in acidified ethanol (phosphoric acid 0.7% w/v). The test sample, control or
standard (Trolox) was added to the reaction medium in a microtitre plate and
the decrease in absorbance at 730 nm was determined after 5 min at 27°C
in a spectramax 384 USA (Molecular devices).
Superoxide anion radical scavenging activity: The superoxide
anion radical scavenging activity of MECF, HF, EF, CF, BF and WF was assessed
by a slight modification of the method described by Nishikimi
et al. (1972) and De Gaulejac et al. (1999)
in the Phenazine Methosulphate (PMS)-NADH coupling reaction system that generates
superoxide anion radical which reduces nitroblue tetrazolium (NBT). The observed
decrease in absorbance at 560 nm in the presence of antioxidants indicates the
consumption of superoxide anion in the reaction mixture (Oktay et al.,
2003) at a temperature of 27°C. The continuous formation of the blue colour
of formazan dye was monitored for 5 min immediately after the addition of PMS.
The percent radical scavenging activity of samples was determined in comparison
with a DMSO treated control group and propyl gallate group (positive control).
Inhibition of xanthine oxidase activity assay: The inhibition
of xanthine oxidase activity by MECF and fractions-HF, CF, EF, BF and WF of
C. ferruginea was evaluated according to the method described by Lee
et al. (1998).
Fe2+/ascorbate induced-mitochondrial membrane lipid peroxidation:
Mitochondrial membrane lipid peroxidation was stimulated in Fe2+/ascorbate
system spectrophotometrically using the Thiobarbituric Acid Reactive Substances
(TBARS) index as described by Varshney and Kale (1990).
The absorbance of the clear pink supernatant was measured against a reference
blank of distilled water at 532 nm using a Camspec 106 spectrophotometer.
DPPH radical scavenging activity: DPPH radical scavenging activity
of MECF and fractions-HF, CF, EF, BF and WF was evaluated by a slight modification
of the method described by Tomohiro et al. (1994).
Sub-fractions III-VII and pure compounds (1 mM) obtained from column chromatography
of EF on silica gel and sephadex LH 20 were also evaluated for DPPH radical
scavenging activity at varying concentrations (12.5-200 μg mL-1)
and IC50 values were determined using the EZ-Fit software (Parrella
Scientific Inc. USA). Propyl gallate was used as positive control.
Statistical analysis: All experiments were run in triplicates and the
data were expressed as Mean±SD for each group and the analysis of variance
was used to calculate the statistical significance. Values with p<0.05 were
Spectral information: Compound B: FAB-MS = 110.036, Calculated MS for C6H6O2 =110.03678; IR-C-H = 2925, O-H = 3258.5/3624, 1601.2, C = C-1514 and 1365.5, C-O = 1216.1/1096.4, aromatic = 827/758.8, UV = 189 nm, 388.8 nm. Synonyms: para-hydroxyphenol or quininol.
Compound C: Amorphous powder, 1H-NMR spectral data (CD3OD)
δ: 3.40 (1H, t, J = 7.18 Hz, H-4'), 3.43 (1H, t, J = 6.5 Hz, H-3'), 4.39
(1H, dd, J = 11.8, 6.5 Hz, H-6'), 4.51 (1H, dd, J =11.8, 1.8 Hz, H-6'), 4.71
(1H, d, J = 7.1 Hz, H-1'), 6.27 (1H, d, J = 15.9, Hz, H-8"), 6.66 (2H, d, J
= 8.1 Hz, H-3, H-5), 6.78 (2H, J = 8.1, H-5'), 6.94 (1H, d, J = 8.1 Hz, H-5'),
6.94 (1H, d, J = 8.1 Hz, H-6'), 7.04 (1H, J = 1.3, H-2'), 6.93 (2H, d, J = 8.1
Hz, H-2, H-6), 7.98 (1H, d, J = 15.9 Hz, H-7"). For 13C-NMR spectral data (CD3OD)
melting point = 143; FAB MS (positive ion mode) m/z: HR-FAB MS negative ion
mode) m/z: 433, calculated for C21H23O10: 435.1219.
Synonym (s): 6-O-Caffeoylarbutin, Robustaside B.
The Total Antioxidant (TA) activity of MECF presented in Fig. 1 is greatest (85.5%, 200 μg mL-1) compared to in CF (44.4%), WF (37.7%), HF (33.3%), BF (23.6%) and EF (5.3%), respectively. TA activity of MECF was not significantly different (p>0.05) from that of allopurinol (96.6%, 1 mM) (standard antioxidant).
As clearly presented in Fig. 1, MECF at 200 μg mL-1 significantly (p<0.05) scavenged superoxide anion radical by 80.4% while similar concentrations of BF, CF, WF, EF and HF elicited 78.6, 68, 57.5, 54 and 30% radical scavenging activities, respectively. There was no significant difference (p<0.05) between the superoxide anion radical scavenging activity of MECF and BF compared to that by allopurinol (85.7%), the standard antioxidant. Furthermore, radical scavenging activity of CF, EF and WF were not significantly different from one another.
Similarly, 200 μg mL-1 of MECF and EF significantly (p<0.05) inhibited the activity of xanthine oxidase by 80 and 87.1% compared to control (Fig. 1). There was no significant difference (p<0.05) in the inhibitions by MECF, EF and allopurinol-the standard antioxidant. Inhibitions by HF, CF, BF and WF of 43.3, 44.2, 39.5 and 48.5%, respectively were also not significantly different from one another (p<0.05) but significantly different from MECF, EF and allopurinol (Fig. 1).
MECF and EF demonstrated similar pattern of inhibition of mitochondrial lipid peroxidation in Fe2+/ascorbate system (Fig. 1). Maximum inhibition of mitochondrial lipid peroxidation (93%) was obtained at 0.2 mg mL-1 of EF and MECF, respectively compared to inhibitions by other fractions. Increasing the concentrations to over 0.3 mg mL-1, showed no significant difference (p<0.05) in the inhibitions by EF, MECF and CF.
||Comparison of antioxidant properties of 200 μg mL-1
of methanolic crude extracts and purified fractions of Cnestis ferruginea.
The 200 μg mL -1 each of HF-hexane fraction; CF-chloroform
fraction, EF-ethylacetate fraction, BF-Butanol fraction, WF-water fraction
were tested for TA-Total antioxidant (ABTS), radical (DPPH, superoxide anion)
scavenging activities (RSA), inhibition of Fe2+/ascorbate-induced
lipid peroxidation (LPO) and xanthine oxidase activity (IXO) spectrophotometrically
at different wavelengths. Values are Mean±SD of three independent experiments.
One-way ANOVA was used for comparisons of multiple groups
||DPPH radical scavenging activities of varying concentrations
of subfractions of ethylacetate and methanol fractions of Cnestis ferruginea
Similarly, MECF and EF (200 μg mL-1) showed very strong DPPH radical scavenging activity (RSA) of 88.8 and 87%, respectively (Fig. 1). But BF showed moderate DPPH RSA of 68.2% while CF and WF exhibited 40 and 48.5% activity, respectively. HF did not show any significant (p>0.05) RSA against DPPH radical at this concentration. Increasing concentrations (12.5-200 μg mL-1) of MECF and EF, scavenged DPPH radicals in a concentration-dependent manner (Fig. 2) and the IC50 values obtained for MECF, EF and BF thereafter, were 26.9, 51.9 and 54.4 μg mL-1, respectively (Table 1).
EF was further purified on silica gel column chromatography because it exhibited similar RSA and anti-lipid peroxidation activity with MECF. Six Sub-fractions (III-VIII) and two pure compounds (A and B) were obtained from this separation process. All these fractions also exhibited very strong DPPH radical scavenging activity of not less than 90% at 200 μg mL-1 which were not significantly different (p<0.05) from each other. Increasing concentrations of these sub-fractions indicated an IC50 value in the order VII> V>VI>III>VIII>IV (Table 1). Although, sub-fraction III showed similar radical scavenging activity (96%) with others but the yield was highest (2.4 g, 0.12%). The chromatograms of these fractions were separately sprayed with DPPH radical solution (1 mg mL-1), FeCl3 and AlCl3. A particular band in the fractions was observed to change the purple colour of DPPH to yellow, FeCl3 to blue and AlCl3 to yellow. Fraction III appeared to contain the highest quantity of the constituents of this band compared to other fractions. Based on this, fraction III was purified further on sephadex LH 20 column and compound C was isolated. The isolated compounds were screened quantitatively for DPPH radical scavenging activity. Compounds B and C at 1 mM exhibited very strong DPPH radical scavenging activities of 86.4 and 85.3%, respectively compared to 92% radical scavenging activity of propylgallate (Fig. 3).
The structure of each compound A, B, C was elucidated by UV, IR, MS. 1H- and
13C-NMR and 2D-NMR methods as β-sitosterol, para-hydroxyphenol and Robustaside
B (6'-3", 4"-dihydroxycinnamoyl arbutin. These structures were also confirmed
by comparing spectral with previously reported spectral data.
|| DPPH free radical scavenging activity of varying concentrations
of extract and fractions from Cnestis ferruginea
||Each compound-Robustaside B, p-hydroxyphenol and propylgallate
(1 mM) were incubated with DPPH radical solution (300 μM) for 30 min.
Absorbances were read on microplate reader. Values are Mean±SD of three
independent experiments. One-way ANOVA was used for comparisons of multiple
groups. * Significantly different (p<0.05) from control
|| 13C-NMR and 1H-1H spectral
data (CD3OD, 400 MHZ) for Compound B
|| 13C-NMR and 1H-1H spectral
data (500 MHZ) for compound C
The spectral data for para-hydroxyphenol and Robustaside B or 6'-(3", 4"-dihydroxycinnamoyl)
arbutin are as shown on Table 2 and 3.
The measurement of antioxidant activity involves an assessment of the capacity
of the substance to inhibit peroxidation of membranes such as erythrocytes,
tissue homogenates, mitochondria, microsomes or liposomes (Halliwell,
1990). The phospholipids bilayers of cellular and subcellular membranes
are undoubtedly major targets for free radicals. Extracts or compounds inhibit
membrane phospholipids peroxidation probably by exerting a pharmacological effect
in the prevention of radical-induced oxidative pathological processes (Halliwell,
1991). The peroxidation of PUFAs on the cardiolipin of mitochondrial inner
membranes has been suggested to contribute to age-related decline of mitochondrial
function. Therefore, mitochondria are among cell-free systems usually chosen
to evaluate antioxidant effects on lipid peroxidation (Hsiao
et al., 1996). Exposure of mitochondria to Fe2+ and ascorbate
elicits lipid peroxidation by a mechanism whose induction step may primarily
involve site-bound iron-mediated decomposition of lipid hydroperoxides to yield
alkoxy or peroxyl radicals, leading to the chain reaction of lipid peroxidation
(Hsiao et al., 1996). DPPH radical is known to
abstract labile hydrogen (Constantin et al., 1990)
and therefore, the DPPH radical scavenging activity is not unrelated to the
inhibition of lipid peroxidation (Ratty et al., 1988;
Rekka and Kourounakis, 1991).
In the present study, MECF and its purified fractions scavenged the radicals
generated in in vitro systems to different extents. Among the six samples/extracts
tested, MECF showed the highest radical scavenging activity, total antioxidant
activity, inhibition of xanthine oxidase activity and mitochondrial lipid peroxidation.
Thus, it is a rich source of compounds possessing strong antioxidant properties
useful for chemotherapy of oxidant-induced pathological conditions in consonance
with the reports of Verghese et al. (2008). However,
the variation in the radical scavenging activity observed in other fractions
supports the claim that different extracts elicit their antioxidant activity
by different indices. This finding correlates with previous reports that a single
plant extract may not possess the activity of all the different measurements
of antioxidant activity (Cakir et al., 2003;
Annegowda et al., 2010). Fraction EF and MECF
showed similar and strong DPPH radical scavenging activity at 200 μg mL-1
probably by reducing DPPH radicals (violet) to DPPH-H (yellow). They also quenched
the radical chain reactions usually propagated by the OH. generated
by the Fe2+/ascorbate system to initiate lipid peroxidation to the
same extent. These data suggest that the same type of polyhydroxyl phenolic
containing compounds may be the major constituents of MECF and EF. This finding
confirms and proves that MECF and EF act as direct free radical scavenger. Thus,
explaining their strong radical scavenging activity and the low levels of TBARS
observed in the present study.
On this basis, the Ethylacetate Fraction (EF) was subjected to further purification on silica gel column using chromatographic techniques. Phytochemical screening of chromatograms of the sub-fractions with DPPH radical solution indicated that Fraction III contained high concentration of a band which markedly reduced DPPH radicals. Again, this band changed FeCl3 to blue and AlCl3 to yellow confirming the presence of trihydroxyl phenols and flavones, respectively. These observations were substantiated by a quantitative DPPH radical scavenging activity assay which indicated the presence of the radical scavenger in all the sub-fractions (Table 1). However, their DPPH RSA is concentration dependent and the strength varies at similar concentrations (Fig. 2). The IC50 values of these sub-fractions revealed their DPPH radical scavenging potential to be in the order VII>V>VI>III>VIII>IV (Fig. 2, Table 1). Sub-fraction III was further purified on sephadex LH 20 because of its high yield and the presence of large quantity of trihydroxylphenol and flavones compared to others. Compound C with RF value 0.43 was obtained from this separation.
The structure of compound B was elucidated by subjecting it to spectroscopic analyses. The HR-FAB MS spectrum of compound B (MZ-111) M++H+ was used to establish the molecular formula C6H6O2. The UV spectrum (λmax-388.3 and 189.2 nm) implied the presence of conjugated chromophore. The IR spectra indicated the presence of conjugated system C = C, signals at 2925 cm-1 represents C-H stretching vibrations. The characteristic signals of OH, C-O and aromatic system are revealed at 3258, 1216.1 and 827/758.8 cm-1, respectively. The standard 13C-NMR, DEPT as well as the polarization transfer experiment of compound B depicted 6 carbon atoms. The DEPT experiment showed resonance for two quarternary carbon, four methines, absence of methylene and methyl groups giving an attached proton formula of C6H4. This molecular formula possesses a double bond equivalent of 4 which was consistent with the exact mass measurement.
|| The structure of compound B (para-hydroxyphenol) and C (Robustaside
The clustering of signals between 3.24 and 3.28 is an indication of the presence of hydroxyl group moiety buttressing the IR signals at 3258 cm-1. The structure of compound B (Fig. 4) was proposed to be para-Hydroxyphenol based on comparison with reported spectral data (Table 2).
Also, the structure of the compound C (Fig. 4) was elucidated
by subjecting it to spectroscopic analyses. The HR-FAB MS spectrum of compound
C (m/z 453) M+H+ established the molecular formula C21H22O10.
The UV spectrum (λmax 328 and 199.2 nm) implied the presence
of conjugated chromophore. The 1H-NMR spectrum of compound C and
1H-1H COSY, showed signals for an ABC-system [a broad
singlet at δ 7.04 (δc 115.1 C-2") and a multiplet at δ6.94
(δc 117.9, 123.1, C-5" and C-6"). The carbon signals are 149.6
and 146.8 at C-3" and C-4", respectively. The 13C-NMR (Table
3) and HMQC spectra of this compound showed 17 signals assigned for 21 carbon
atoms in the molecule. These signals also included signals for a sugar moiety,
12 aromatic olefinic carbons and an ester carbonyl. The sugar moiety was identified
as glucose by comparing 13C-NMR spectral data with those reported
for methyl O-glucosides and by considering the glycosidation effect (Agrawal
et al., 1985). These spectral data (Table 3) suggested
an arbutin residue acylated at C-6" of the sugar moiety (δ4.39 and 4.51,
64.7) with a p-hydroxycinnamate (Occolowitz, 1964).
Fragments ions at m/z 324.3 and 162.2 (cinnamoyl) supports this partial structure.
The coupling constant of H-1' (J = 7.1 Hz) indicated a β configuration
of the sugar moiety. Compound C was named 6'-(3", 4"-dihydroxycinnamoyl) arbutin
or Robustaside B based on comparison with previously reported spectral data
and authentic standards (Dommisse et al., 1986).
This compound has an existing chemical structure previously isolated in Grevillea
robusta by Ahmed et al. (2000) along with
its several similar analogues. However, the present study was the first chemical
investigation of C. ferruginea leaves which led to the isolation of Robustaside
B and para-Hydroxyphenol from this source. Robustaside B belongs to the derivatives
originating from Arbutin (Ahmed et al., 2000;
He et al., 2006). Arbutins are known for their
diuretic and urinary anti-infective activities for centuries either as a plant
extract or in purified form (Robertson and Howard, 1987).
Furthermore, arbutin is known to be an inhibitor of melanin biosynthesis (Akiu
et al., 1988) and is used as a skin-whitening agent in cosmetics.
In addition, the present study revealed the radical scavenging activity of arbutin-derivative-Robustaside
B for the first time.
In conclusion, the present study isolated Robustaside B and para-Hydroxyphenol
which are phenolic compounds from the leaves of C. ferruginea for the
first time. Robustaside B and para-Hydroxyphenol were established to be potent
free radical scavengers. C. ferruginea was also identified from this
study to be a rich source of potent antioxidant principles which are direct
free radical scavengers. These bioactive agents would find therapeutic uses
in militating against age related disorders; cancer, diabetes, gout, liver and
kidney dysfunction; and other radical-induced pathological conditions.
This study was supported by the Third World Organization for Women in Science through their Postgraduate Training Fellowship received by Dr. Rahmat Adetutu Adisa in 2004 and utilized at H.E.J Research Institute of Chemistry, University of Karachi, Karachi.
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