COMPARATIVE STUDY OF ANTIOXIDANT ACTIVITY BETWEEN ETHANOLIC AND AQUEOUS EXTRACT OF CLEOME RUTIDOSPERMA

COMPARATIVE STUDY OF ANTIOXIDANT ACTIVITY BETWEEN ETHANOLIC AND AQUEOUS EXTRACT OF CLEOME RUTIDOSPERMA

Anup K. Chakraborty*, Manoj S. Charde ,Harekrishna Roy , Satyabrata Bhanja  and M. Behera

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jeypore College of Pharmacy , Rondapalli, Jeypore, Koraput, Odisha, India

Dean Academics and Head of PG Departments, NRI group of Institutions , Bhopal, MP, India

ABSTRACT

Cleome rutidosperma is traditionally used in the treatment of paralysis, epilepsy, convulsions, spasm, pain and skin disease. Extraction of the aerial parts of the plant Cleome rutidosperma using water and ethanol and evaluation of their antioxidant activity has been envisaged in this present research work. Both the extracts exhibits significant antioxidant activity in DPPH, Nitric oxide and hydroxyl radical induced In-vitro assay methods. The DPPH radical inhibition (%) was 53.13, 57.13 and 79.12 for CRA, CRE and ascorbic acid respectively. The Cleome rutidosperma extracts (CRA and CRE) showed significant free radical scavenging action against nitric oxide (NO) induced release of free radicals at the concentrations 250μg/ml, showing 29.22% and 63.32% of NO inhibition, respectively. The CRA and CRE extracts (25-400μg/ml) significantly scavenged the hydroxyl radical generated by the EDTA/H₂O₂ system, when compared to that of ascorbic acid.

INTRODUCTION: Cleome rutidosperma (Capparidaceae) is a low-growing herb, up to 70 cm tall, found in waste herb, grounds and grassy places with trifoliate leaves  and small, violet-blue flowers, which turn pink as  to West Africa, although it has become naturalized in various parts of tropical America as well as Southeast Asia ^{1, 2}. According to traditional use, the different parts of the plants of Cleome genus are used as stimulant, antiscorbutic, antihelmintic, vesicant, rubefacient and carminative ³. The anti-plasmodial, analgesic, locomotor, antimicrobial, diuretic, laxative ^{4, 5} activities of Cleome rutidosperma were reported earlier.

Cleome rutidosperma is traditionally used in the treatment of paralysis, epilepsy, convulsions, spasm, pain and skin disease. The popular use of the roots, however, refers mainly to its analgesic, anti-inflammatory and antihelmintic activity ⁶. However, there are no scientific reports on the antioxidant activity of this plant. Therefore, in the light its use in traditional medicine, the present study was undertaken to investigate free radical scavenging activity of the ethanolic and aqueous extract of Cleome rutidosperma using In-vitro assay methods.

MATERIALS AND METHODS:

Plant materials: The Plant material (whole plant) was collected from the forests of Ganjam district of Odisha, India during September 2008 and was authenticated at Botanical Survey of India, Shibpur, Howrah and West Bengal, India. The fresh aerial parts were washed under running tap water to remove adhered dirt, followed by rinsing with distilled water, shade dried and pulverized in a mechanical grinder to obtain coarse-powder.
Preparation of extracts: The aerial parts of the plant were powdered. 150g of powder was subjected to extraction using Soxhlet apparatus with various solvents like water and ethanol. The solvent was then removed under reduced pressure which will give a greenish-black colored sticky residue. The prepared extracts were then subjected to antioxidant activity studies.

Evaluation of Antioxidant activity of the Cleome rutidosperma:

Scavenging of DPPH radical ^{7, 8}: This assay is based on the measurement of the scavenging ability of the antioxidant test substances towards the stable radical. The free radical scavenging activity (Yokazawa et al., 1998) of the extracts (CRA and CRE) was examined in vitro using DPPH radical. The test extracts were treated with different concentrations from a maximum of 250μg/ml to minimum of 4μg/ml. The reaction mixture consisted of 1 ml of 0.1mM DPPH in ethanol, 0.95 ml of 0.05 M Tris-HCl buffer (pH 7.4), 1 ml of ethanol and 0.05 ml of the extract. The absorbance of the mixture was measured at 517 nm exactly 30 sec after adding the extract. The experiment was performed in triplicate and the % of scavenging activity was calculated using the formula;

100 - [100/ blank absorbance × sample absorbance] 

Scavenging of nitric oxide ^{9, 10}: Sodium nitroprusside (Sreejavan Rao, 1997) (5M) in standard phosphate buffer solution was incubated with different concentration of the test extracts dissolved in standard phosphate buffer (0.025M, pH 7.4) and the tubes were incubated at 25⁰ C for 5 hrs. After 5 h, 0.5 ml of incubated solution was removed and diluted with 0.5 ml Griess reagent (prepared by mixing equal volume of 1% sulphanilamide in 2% phosphoric acid and 0.1% naphthylethylene diamine dihydrochloride in water). The absorbance of the chromophore formed was measured at 546 nm. The control was also carried out in similar manner using distilled water instead of extracts. The experiment was performed in triplicate and % scavenging activity was calculated using the formula;

100 - [100/ blank absorbance × sample absorbance]

The activity was compared with ascorbic acid, which was considered as standard antioxidant.

Hydroxyl Radical Scavenging activity ¹¹: The hydroxyl radical scavenging activity was measured by studying the competition between deoxyribose and the extract for hydroxyl radicals generated from the Fe³⁺/ascorbate/EDTA/H₂O₂ system_. The reaction mixture contained deoxyribose (2-8mM), FeCl₃ (0.1mM), EDTA (0.1 mM), ascorbate (0.1 mM), H₂O₂ (1mM), KH₂PO₄-KOH buffer(20mM, pH 7.4) and various concentrations (25-400μg/ml of extracts and standard 10-80μg/ml) of standard drug in a final volume of 1 ml. The reaction mixture was incubated for 1 hr at 37°C, deoxyribose degradation was measured at 532 nm (Mary et al., 2002).

Statistical Analysis ¹²: The data on in-vitro studies were reported as mean ± Standard deviation (n=5). For determining the statistical significance, standard error mean and analysis of variance (ANOVA) at 5% level significance was employed. The P values<0.05 were considered as significant ¹⁴.

RESULTS:

DPPH Scavenging: The aqueous (CRA) and ethanolic extracts (CRE) of the Cleome rutidosperma showed promising free radical scavenging effect of DPPH in a concentration dependent manner upto a concentration of 250μg/ml. The CRE showed more scavenging activity than CRA. The reference standard ascorbic acid also shows a significant radical scavenging potential in the concentration of 1 μg/ml. The DPPH radical inhibition (%) was 53.13, 57.13 and 79.12 for CRA, CRE and ascorbic acid respectively in table 1.

Nitric oxide Scavenging: The Cleome rutidosperma extracts (CRA and CRE) showed significant free radical scavenging action against nitric oxide (NO) induced release of free radicals at the concentrations 250 μg/ml, showing 29.22% and 63.32% of NO inhibition, respectively. Ascorbic acid was used as reference standard. The % inhibition is shown in Table 2.

OH Radical Scavenging: The CRA and CRE extracts (25-400 μg/ml) significantly scavenged the hydroxyl radical generated by the EDTA/H₂O₂ system, when compared to that of ascorbic acid. The percentage scavenging of OH radicals by CRA and CRE was increased in a dose dependent manner. The standard ascorbic acid (10-80 μg/ml), also showed scavenging effect (Table 3).

TABLE 1: IN VITRO FREE RADICAL SCAVENGING ACTIVITY OF AQUEOUS AND ETHANOLIC EXTRACTS OF CLEOME RUTIDOSPERMA BY DPPH METHOD

CRA= Aqueous extract, CRE= Ethanolic extract, *P<0.05

TABLE 2: IN VITRO FREE RADICAL SCAVENGING ACTIVITY OF AQUEOUS AND ETHANOLIC EXTRACTS OF CLEOME RUTIDOSPERMA BY NITRIC OXIDE SCAVENGING METHOD

CRA= Aqueous extract, CRE= Ethanolic extract, *P<0.05

TABLE 2: IN VITRO FREE RADICAL SCAVENGING ACTIVITY OF AQUEOUS AND ETHANOLIC EXTRACTS OF CLEOME RUTIDOSPERMA BY HYDROXYL RADICAL SCAVENGING METHOD

CRA= Aqueous extract, CRE= Ethanolic extract

DISCUSSION: The ethanolic extract of the aerial parts of Cleome rutidosperma possesses significant anti inflammatory activity. Reactive oxygen species (ROS) generated endogenously or exogenously are associated with the various diseases such as atherosclerosis, diabetes, cancer, arthritis and aging process. ROS plays an important role in the pathogenesis of inflammatory diseases. Thus antioxidants which can improve these disorders, the free radical scavenging activity of the extracts were evaluated based on the ability to scavenge the DPPH. This assay is highly important to provide information about the reactivity of organic compounds with stable free radicals, because of the odd number of electrons. DPPH shows a strong absorption band at 517 nm in visible spectrum (deep violet color). As the electron became paired of in the presence of free radical scavenging, the absorption vanishes and the resulting discoloration stoichiometrically coincides with the number of electrons taken up. The bleaching of DPPH absorption is representative of the capacity of the test drugs to scavenge the free radicals independently. Hydroxyl radical is the principal contributor for tissue injury. The formation of hydroxyl radical from fenton reaction was quantified using 2, D-deoxyribose degradation. The extracts CRA and CRE inhibited hydroxyl scavenging activity.

Sodium nitroprusside serves as a chief source of free radicals. The absorbance of the chromophore formed during diazotization of nitrite with sulphanilamide and subsequent coupling with naphthylethylene diamine is used as a marker for NO scavenging activity (Mukherjee et al., 1989). The chromophore formation was not complete in the presence of extracts of Cleome rutidosperma (CRA and CRE), which scavenges the NO thus formed from the sodium nitroprusside and hence the absorbance decreases as the concentration of the extracts (CRA and CRE) increases in the dose dependent manner.

ACKNOWLEDGEMENT: Authors are thankful to the all the members of department of Pharmacognosy as well as Pharmaceutical Chemistry, Jeypore College of Pharmacy and also to NRI group of institutions for their co-operation during the whole research work. Authors are also thankful to the Botanical Survey of India, Shibpur, Howrah and West Bengal, India for authentication and identification of this plant.

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