27-08-2012, 05:00 PM
Antioxidant activities and total phenolic content of Indian Orthosiphon
Antioxidant activities.doc (Size: 596 KB / Downloads: 48)
ABSTRACT
The crude methanolic leaf extract of Orthosiphon aristatus was analysed for the total phenolic content, and antioxidant activities. The possible radical scavenging and antioxidant activities of the methanolic extracts was investigated using different invitro methodologies:1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging, scavenging of superoxide anion radical-generated non-enzymatic system, nitric oxide scavenging, ferric thiocyanate method, reducing power, hydrogen peroxide scavenging ,metal chelating activities and FRAP assay. An HPLC method was developed for determination of the total phenolic content in the plant leaves and rosmarinic acid, a caffeic acid derivative, was found to be the major phenolic component. The results of the present study indicated that the antioxidant activity of the plant might be ascribable to its total phenolic content.
INTRODUCTION
Orthosiphon aristatus a member of the Lamiaceae family ,is a popular medicinal herb in south-east Asia. In Malaysia the tea prepared from leaves of Orthosiphon aristatus is taken as beverage to improve health and for treatment of kidney, bladder inflammation, gout, and diabetes ( Hegnauer, 1996; Wagner, 1982 ). Orthosiphon aristatus have been proven to exert anti-diabetic and lipid lowering effect in diabetic rats ( Sriplang et al 2007). In Malaysia, Orthosiphon aristatus is traditionally used to promote urination and to alleviate bladder and kidney discomfort ( Indubala 2000). The therapeutic effects of Orthosiphon aristatus have been ascribed mainly to its polyphenols, which have enzyme inhibition and antioxidant activity (Lyckander and Malterud, 1996). The aim of this work is to evaluate the total phenolic content and antioxidant properties of the methanolic extracts of Orthosiphon aristatus leaves. A rapid HPLC finger printing of the sample was achieved by reverse phase isocratic elution with acetonitrile and potassium phosphate buffer as mobile phase, with UV detection, based on the fact they are present in the samples.
Materials And Methods
BHA, BHT, nitroblue tetrazolium (NBT), ferrozine, ammonium molybdate,and linoleic acid from SISCO Reasearch Laboratories Pvt. Ltd. (Bombay), DPPH, Quercitin, and rosmarinic acid were purchased from Sigma Chemicals. All other chemicals and reagents used were of analytical reagent quality.
Plant Materials
The leaves of the plant, used for the study was collected from different parts of south India and were grown in the Botanical Garden, Department Of Botany, University Of Kerala. Voucher specimens are deposited in herbarium of the Department of Botany. The collected leaves were washed, chopped into pieces and air dried. Fifty gram of the powdered plant portion was filled in a soxhlet extractor and extracted with 300ml methanol (Boiling point – 64 to 65c) for ten hours. The extract was transferred to a conical flask, concentrated and evaporated to dryness in a vaccum rotary evaporator under reduced pressure (Harborne, 1973).
Isolation and estimation of total phenols [Mayr et al., 1995]
1gm of fresh tissue was finely chopped and put in boiling 80% methanol and refluxed for 10 min. The extract was cooled and the tissue was homogenized with a pestle for a few minutes. The homogenate was filtered and centrifuged for 10 min at 10000 g. The supernatant was taken for the estimation of phenols.
An aliquot of the sample was pipetted out and made up to 3 ml with 80 % methanol. 0.5 ml Folin-ciocalteau reagent was added and kept for 3 min. 2ml of 20% Na2CO3 was added to this and kept in a boiling water bath for 1 min. The white precipitate formed was centrifuged and the absorbance of the clear light blue solution was recorded at 650 nm against the reagent blank containing 3 ml 80% methanol, 0.5 ml Folin’s reagent and 2 ml of 20% Na2CO3. The reaction between phenols and an oxidizing agent, phosphomolybdate, in Folin-ciocalteau is responsible for the formation of the light blue solution. A standard graph of phenols was drawn with pyrocatechol by taking absorbance against the concentration and it was compared against the total phenolic content of the plant extract.
Statistical analysis
All data on total antioxidant activities are the average of duplicate analyses. The data were reported as mean ±standard deviation and analyzed by SPSS . one way analysis of variance was performed by ANOVA procedures. Significant differences between means were determined by Duncans’s multiple range tests. Values of p< 0.05 were regarded as significant.
Results And Discussion
Oxidative stress can be reduced with the provision of additional antioxidants. Antioxidants are closely related with the prevention of degenerative illness, such as cardiovascular, neurological diseases, cancer and oxidative stress dysfunction ( Diplock 1995). Many studies have shown that natural antioxidants in plants are closely related to their biofunctionalities such as the reduction of chronic diseases and inhibition of pathogenic bacteria growth, which