Free radical reactions have been implicated in the etiology of several human diseases including atherosclerosis, ischemic heart disease, the ageing process, inflammatory lesions, diabetes mellitus, different immunosuppressive disorders, metabolic disorders, neuromuscular degenerative conditions etc (Berlett and Stadtman, 1997). Antioxidants may act by decreasing oxygen concentration, intercepting singlet oxygen, preventing first chain initiation by scavenging initial radicals, binding metal ion catalysts, decomposing primary products to non-radical compounds and chain-breaking to prevent continued hydrogen abstraction from substrates. There are two basic categories of antioxidant namely synthetic and natural ones. Restriction on the use of synthetic antioxidants is being imposed because of their carcinogenicity (Bronen, 1975; Ito et al., 1983). Thus the interest in natural antioxidants has been increased considerably. As resources of natural antioxidants much attention has been paid to plants (Couladis et al., 2003; Linn et al., 2003).
Galega purpurea (Papilionaceae) a plant popularly known as kolinji in Tamil and vempali in Telugu, which thrives in Southern parts of India. It grows on hard stony ground too difficult to be rooted. The various parts of the plant are widely used in the folk medicine for the treatment of cough, asthma, bilious febrile attacks, arthritis and rheumatism. Decoction of the root useful in the management of enlargement and obstruction of the liver, spleen and kidney. Also the root is useful in the treatment of dyspepsia, chronic diarrhoea and ulcers (Nadkarni, 1976). The objective of the present investigation is to evaluate the antioxidant and antimicrobial properties of methanol extract of Galega purpurea root.
MATERIALS AND METHODS
Plant material: The roots of the plant Galega purpurea (Family:
Papilionaceae) were collected from Erode district of Tamilnadu, India. The plant
material was taxonomically identified by Botanical Survey of India, Kolkata.
A voucher specimen (No. GMG 03/05) has been preserved in the laboratory for
future reference. The collected plant material was dried under shade and then
powdered with a mechanical grinder and stored in airtight container. The dried
powder material of the leaves was defatted with petroleum ether and the marc
thus obtained was then extracted with methanol in a soxhlet apparatus. The solvent
was completely removed under reduced pressure and a semisolid mass was obtained.
The dried MEGP was dissolved in water and used for the present study. All the
studies were conducted in Division of Pharmaceutical Chemistry and Pharmacology,
Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India
in the month of December 2006.
Chemicals: 1,1-diphenyl-2-picryl-hydrazyl (DPPH), α-tocopherol, ascorbic acid, thiobarbituric acid, dodecyl sulphate, ferrous ammonium sulphate were purchased from Sigma Chemical Co. Ltd., USA. All other unlabeled chemicals and reagents were analytical grade.
Phytochemical screening: The extract was screened for the presence of various constituents employing standard screening test (Trease and Evans, 1983). Conventional protocol for detecting the presence of steroids, alkaloids, tannins, flavonoids, glycosides, etc., was used.
Lipid peroxidation assay: The peroxide formation was measured by the method of Ohkawa et al. (1979) by measuring the color of thiobarbituric acid reactive substances (TBARS) formed at the end of the reaction. Malondialdehyde (MDA), which is formed as the end product in lipid peroxidation will react with thiobarbituric acid (TBA) to give TBARS, which is pink in color measured at 530 nm. The reaction mixture contained rat liver homogenate (0.1 mL, 25% w/v) in Tris-HCl buffer (20 mM, pH 7.0), KCl (150 mM), ferrous ammonium sulphate (0.8 mM), ascorbic acid (0.3 mM) and various concentrations of MEGP (10-100 μg mL-1) in a final volume of 0.5 mL was incubated for 1 h at 37°C (Bishayees and Balasubramanian, 1971).
The incubated reaction mixture (0.4 mL) was treated with 0.2 mL of 8% sodium dodecyl sulphate (SDS), thiobarbituric acid (1.5 mL, 8%) and acetic acid (1.5 mL, 20%, pH 3.5). the total volume was then made up to 4 mL by adding distilled water and kept in a water bath at 100°C for 1 h. After cooling, 1 mL of distilled water and 5 mL of a mixture of n-butanol-pyridine (15:1, v/v) were added and shaken vigorously. The absorbance of the organic layer was measured at 560 nm after centrifugation. The percentage inhibition of lipid peroxide formation was determined by comparing the results of the extract-treated and untreated samples. Αlpha tocopherol was used as a reference compound.
DPPH scavenging assay: The free radical scavenging activity of MEGP
was measured by 1,1-diphenyl-2-picryl-hydrazil (DPPH) using the method of Blois
(1958). Used as a reagent, DPPH evidently offers a convenient and accurate method
for titrating the oxidizable groups of natural or synthetic antioxidants (Cao
et al., 1997). 0.1 mM solution of DPPH in methanol was prepared
and 1 mL of this solution was added to 3 mL of MEGP dissolved in water at different
concentrations (10, 25, 50, 75 and 100 μg). After 30 min, absorbance was
measured at 517 nm. Alpha tocopherol was used as a reference material. Lower
absorbance of the reaction mixture indicated higher free radical scavenging
activity. All the tests were performed in triplicate and the results averaged.
The percentage inhibition was calculated by comparing the absorbance values
of control and samples.
Microorganisms and media: Bacillus subtilis (ATCC 6633 Gram positive),
Staphylococcus aureus (ATCC 6538 Gram positive), Micrococcus luteus
(ATCC 10240 Gram positive), Escherichia coli (ATCC 9837 Gram negative),
Pseudomonas aureginosa (ATCC 9027 Gram negative), Salmonella typhimurium
(ATCC 43579 Gram negative) were used to determine antibacterial activity.
Fungal organisms such as Aspergillus niger (ATCC 16404), Candida albicans
(ATCC 10231) strains were employed for the determination of antifungal activity.
Bacteria and fungi were obtained from the stock cultures of the Central Drugs Laboratory (CDL), Kolkata and Indian Institute of Chemical Biology (IICB), Kolkata. The bacterial and fungal stock cultures were maintained on Muller Hinton agar and Sabouraud-dextrose agar slants respectively, which were stored at 4°C. Eight microorganisms maintained on nutrient agar base were used to assess the antimicrobial activity of the plant extract. The fungi were maintained on Sabouraud-dextrose agar, which is often used with antibiotics for the isolation of pathogenic fungi.
Antimicrobial screening: Agar cultures of the test microorganisms were
prepared as described by Mackeen et al. (1997). Three to five similar
colonies were selected and transferred to 5 mL broth with a loop and the broth
cultures were incubated for 24 h at 37°C. The MEGP was dissolved in dimethyl
sulfoxide with a magnetic stirrer. For screening, sterile 6 mm diameter filter
paper discs were impregnated with 100-1000 μg of the MEGP and placed in
Muller Hinton agar medium. The inoculums for each organism were prepared from
broth cultures. The concentration of cultures was to 1x105 colony
forming units mL-1. The results were recorder by measuring the zones
of growth inhibition surrounding the disc. Clear inhibition zones around the
discs indicate the presence of antimicrobial activity. All data regarding antimicrobial
activity are the average of triplicate analyses. The antibacterial amikacin
(10 μg mL-1) and antifungal griseofulvin (20 μg mL-1)
were used as reference standards as recommended by the National Committee for
clinical laboratory standards.
Statistical analysis: The experiments were done in triplicate. The results are given as the mean±SD. Student t-test was used for comparison between two means.
Phytochemical screening: The extraction of Galega purpurea root with petroleum ether ad methanol gave yields of 3 and 7.3%, respectively. The results of preliminary phytochemical screening of the methanol extract revealed the presence of steroids, flavonoids, tannins, alkaloids and glycosides (Table 1). Further separation of the specific phytochemical is in progress.
Antioxidant activity: Two in vitro tests, the DPPH test for direct free radical scavenging activity and the lipid peroxidation assay for antioxidant activity were used to assess the antioxidant properties of methanol extract of Galega purpurea.
In lipid peroxidation assay α-tocopherol was used as a standard drug. MEGP at all the tested concentrations inhibited the lipid peroxides formed from rat liver homogenate and the results were comparable to that of the standard drug (Table 2). In the DPPH test the ability of a compound to act as donor for hydrogen atom or electrons was measured spectrophotometrically. MEGP was able to reduce the stable radical DPPH to the yellow colored diphenyl hydrazine and the activity increased dose-dependently at the tested concentrations ranging from 10-100 μg mL-1. The strongest effect was measured for MEGP 100 μg mL-1 with an IC50 of 51.50±0.02 μg mL-1 (Table 3).
Antimicrobial activity: The initial screening of antibacterial activities
of methanol extract was assessed by the agar disc diffusion method (Table
4). The extract inhibited the growth of Gram-positive bacteria at a concentration
of 100 μg mL-1 which was the lowest concentration used in this
study. The diameters inhibitory zones were in the range of 7.46 to 16.88 mm.
However, 100 μg mL-1 concentration of the extract did not inhibit
the growth of Escherichia coli and Pseudomonas aureginosa. It
was also observed that the methanol extract exhibited antifungal activity against
Aspergillus niger and Candida albicans in a dose dependent manner(Table
||Preliminary phytochemical screening of extracts of Galega
|(-): Absent; (+): Present; (++): Abundant
||Effect of methanol extract of Galega purpurea on lipid
||Effect of methanol extract of Galega purpurea on DPPH
radical scavenging activity
||Antibacterial activity of methanol extract of Galega purpurea
and standard antibiotics
|- No Inhibition zone, BS-Bacillus subtilis, SA-Staphylococcus
aureus, ML-Micrococcus luteus, EC-Escherichia coli, PA-Pseudomonas
aureginosa, ST-Salmonella typhimurium; Values are mean±SD
(mm) of five separate experiments; Statistical value *p<0.05 when compared
||Antifungal activity of methanol extract of Galega purpurea
and standard antibiotics
|AN-Aspergillus niger, CA-Candida albicans. Values
are mean±SD (mm) of five separate experiments
When compared to standard commercial antifungal drug the methanol extract with
concentration 1000 μg mL-1 exhibit the zone of inhibition similar
to that of griseofulvin against Candida albicans (21.35±1.1 mm)
at the dose of 1000 μg mL-1.
About 5% or more of the inhaled oxygen (O2) is converted to Reactive Oxygen Species (ROS) such as O2¯, H2O2 and OH by univalent reduction of O2 (Maxwell, 1995). Antioxidant can act by scavenging reactive oxygen species by inhibiting their formation (e.g., by blocking activation of phagocytes), by binding transition metal ions and preventing formation of OH and/or decomposition of lipid hydroperoxides by repairing damage (e.g., α-tocopherol repairing peroxyl radicals and so terminating the chain reaction of lipid peroxidation) or by any combination of the above (Niwa et al., 1994).
In this study the antioxidative activity of the root extract of Galega purpurea was measured using lipid peroxidation assay and DPPH radical scavenging assay method. This good antioxidant activity of MEGP might be attributed to the presence of phytochemicals, such as flavonoids and biflavones (Wang and Wixon, 1995). The DPPH radical is considered to be a model for a lipophilic radical. A chain reaction in lipophilic radicals was initiated by the lipid autoxidation. The radical scavenging activity of the crude plant extract was determined from the reduction in the optical absorbance at 517 nm due to scavenging of stable DPPH free radicals. Positive DPPH test suggests that the samples are free radicals scavengers.
The result obtained from antimicrobial study demonstrated that methanol extract of root of Galega purpurea produced in vitro antibacterial and antifungal activity against some medically important microorganisms. The bacteria inhibited in this study, have been associated with infections which the local tribes treat with Galega purpurea extract. Also the fact that the extract inhibited Staphylococcus aureus which is associated with respiratory tract infections and chest complaints, provides some scientific rationale for the use of the extracts for bronchitis, chronic asthma etc. The Gram-positive bacteria appeared to be more susceptible than the Gram-negative bacteria (Khunt et al., 1994). The weak activity shown by the methanol extract against the Gram-negative bacteria could be due to the presence of compounds in the extract possessing lipophilic characteristics (Werner et al., 1979).
Thus, the lipid peroxidation assay, DPPH radical scavenging activity and antimicrobial activity strongly suggest that MEGP has antioxidant and antimicrobial properties. Further studies are in progress in our laboratory to evaluate the in vivo antioxidant potential of this extract in various animal models and phytochemical studies are required to establish the types of compounds responsible for the bioactivity of this medicinal plant and to determine the value of the ethnobotanical approach for the screening of plants as potential source of bioactive substances.