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Antioxidant Activity of Clematis Brachiata Thunb. Leaf

M. Mostafa*
BCSIR Laboratories, Dhaka-1205, Bangladesh

S. Ahmed
BCSIR Laboratories, Dhaka-1205, Bangladesh

AJ Afolayan
Center for Phytomedicine Research, Department of Botany, University of Fort Hare, Alice 5700, South Africa

Abstract/ Executive Summary:

The antioxidant activity, total phenolic and flavonoid contents of different extracts of the Clematis brachiata Thunb leaves were determined. The antioxidant activity was evaluated using spectroscopic methods against 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) and 2,2′-azinobis [3-ethylbenzothiazoline-6-sulphonic acid] diammonium salt radical cation (ABTS). Folin Ciocalteu method was used to determine the total phenolics and Aluminium Chloride Colorimetric method was used to determine the flavonoids contents in these extracts. The results showed that the methanol extract of the leaf exhibited the highest antioxidant activity with the value of 180.45 ±2.4 µg mL^-1 in DPPH and 60 ±0.80 µg mL^-1 in ABTS assay among the extracts. The methanol extract contains more phenolic compounds (178±2.20 mg/g as garlic acid equivalent per g dry matter) and the acetone extract contains more flavonoids (135.11±1.20 mg/g as quercitin equivalent per g dry matter) among the extracts. This study provides the evidence that the leaves of the Clematis brachiata Thunb could be a good source of natural antioxidant.

Keywords: Clematis brachiata; Antioxidant activity; Phenolic; Flavonoid

Location: BCSIR Laboratories, Dhaka-1205, Bangladesh

Start Date:

End Date:

Program Area: Development of Host and Medicinal Plants

Commodity: Medicinal Plants

Objectives:

The present study is an attempt to evaluate the antioxidant activity, total phenolics and total avonoids contents of the different extracts of C. brachiata leaves.

Methodology:

Plant material Clematis brachiata was collected from a natural population within the premises of the University of Fort Hare, Alice, South Africa. The plant was identified by Prof DS Grierson of the Department of Botany, University of Fort Hare. A voucher specimen (M. Mostafa med. 2008/1) was prepared and deposited at the Giffen Herbarium of the University. Preparation of extract The dried leaves of the plant were pulverized and portions of 50 g each were separately extracted in hexane, acetone, methanol and water for 24 h on an orbital shaker (Stuart Scientific Orbital Shaker, UK). The extracts were filtered using a Buchner funnel and Whatman no. 1 filter paper. The acetone and methanol extracts were evaporated to dryness under reduced pressure at 40^oC using a vacuum rotary evaporator (Laborot 4000-efficient, Heldolph, Germany), while the water extract was freeze-dried with Savant Refrigerated Vapor Trap (RVT4104, USA). Antioxidant activity DPPH free radical scavenging assay The radical scavenging activities of the extracts on DPPH radical was determined by using the method described by Liyana-Pathirana and Shahidi (2005), with slightly modification. Briefly, one ml of DPPH solution (0.135 mM) in methanol was mixed with one ml of varying concentrations of the extracts. The reaction mixture was vortexed thoroughly and left in the dark at room temperature for 30 min. The absorbance of the mixture was read at 517 nm using butylated hydroxytoluene (BHT) as standards. A blank solution was prepared containing the same amount of methanol and DPPH. The ability to scavenge DPPH free radical was calculated from the expression: (%) DPPH radical scavenging activity = [(Abscontrol - Abssample)/ (Abscontrol)] × 100, where Abscontrol was the absorbance of DPPH radical + methanol and Abssample was the absorbance of DPPH radical + sample extract/ standard. The IC50 (the concentration of sample required to decrease the absorption at 515 nm by 50%) was calculated to quantify the antioxidant activity. ABTS radical scavenging assay The method described by Re et al. (1999) was adopted for the ABTS radical scavenging assay. The stock solution (equal volumes of 7 mM ABTS salt and 2.4 mM potassium persulfate) was allowed to stand in the dark for 14 hrs at room temperature. The resultant ABTS+ solution was diluted with methanol until the absorbance of 0.70 ± 0.01 at 734 nm was attained. Varying concentrations of the plant extracts (1 mL) was reacted with 1 ml of the ABTS+ solution and the absorbance read at 734 nm within 1-3 min using the spectrophotometer (Beckman DU-7000, USA) using butylated hydroxytoluene (BHT) as standards. The percentage inhibition was calculated from the expression: (%) ABTS radical scavenging activity = [(Abs_control - Abs_sample)/ (Abs_control)] × 100, where Abs_control was the absorbance of ABTS radical + methanol and Abssample was the absorbance of ABTS radical + sample extract/ standard. The IC50 (the concentration of sample required to decrease the absorption at 734 nm by 50%) was calculated to quantify the antioxidant activity (Table I). Determination of total phenolic The total phenolic content of the extracts was determined using the modi?ed Folin-Ciocaltu method (Wolfe et al., 2003). Brie?y, 1 mL extract (1mg/mL) was mixed with 5 ml Folin-Ciocalteu reagent (1:10 v/v distilled water) and 4 ml (75 g/L) of sodium carbonate. The mixture was vortexed for 15 s and allowed to stand for 30 min at 40^oC for colour development. The absorbance was read at 765 nm with a spectrophotometer (Beckman DU 700, USA). Total phenolic content was determined as mg of gallic acid equivalent per g of dry extract using the equation obtained from a standard gallic acid calibration curve: y = 6.993 x + 0.0379, R2 = 0.9995. where × is the absorbance and y is the gallic acid equivalent (GAE). All the tests were performed in triplicate. Determination of total flavonoid Aluminium chloride colourimetric assay was used to determine the total flavonoid contents in the extracts as previously reported method described by Ordonez et al. (2006). Briefly 0.5 mL of 2% AlCl3 was prepared in ethanol and was then added in 0.5 ml of the extracts. The mixture was allowed to stand for 60 min at room temperature and the absorbance was read at 420 nm with a spectrophotometer (Beckman DU 700, USA). The extracts were evaluated at a final concentration of 0.1 mg/mL. Total flavonoid content was calculated and expressed as mg of quercetin equivalent per g of dry extract using the equation obtained from a standard quercetin calibration curve: y = 43.862 x - 0.1757, R2 = 0.9931. where × is the absorbance and y is the quercetin equivalent (QE). All the tests were performed in triplicate.

Source of Information: Bangladesh J. Sci. Ind. Res. 53(3), 185-192, 2018

Article Link (Online Journal): DOI: http://dx.doi.org/10.3329/bjsir.v53i3.38264

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The high antioxidant activities of methanol and acetone extracts might be due to their flavonoid and phenolic contents. Therefore, the antioxidant activity of these extracts of C. brachiata may explain its use to treat inflammations and wounds. The current investigation also confirmed that leaves of Clematis brachiata can be considered as potential natural antioxidants that play a major role in human health as free radical scavenger.

Knowledge Management: Journal