In – vitro antioxidant activity of aqueous and ethanolic extract of Coscinium fenestratum root and Embelia ribes flower

 

Basavaraj H ¹   , Purnima Ashok. ²

¹Government College of Pharmacy, Bangalore.

²KLE University’s College of Pharmacy, Bangalore.

*Corresponding Author E-mail: basavarajhulkoti@gmail.com

 

ABSTRACT:

The present study envisaged to evaluate in-vitro anti oxidant activity of ethanolic and aqueous extracts of roots of Coscinium fenestratum and extracts of Embelia ribes flower, by estimating nitric oxide scavenging activity, ferric thiocynate and total reduction capacity and carrying out DPPH assay of increasing concentration of extracts and compared with ascorbic acid, in view of investigations of pharmacological activities reported, extensive chemistry and being a part of folklore medicine. Review of literature revealed paucity of reports of in- vitro study of parts and extract chosen for the study. Results revealed concentration dependent increase in nitric oxide, DPPH free radical, ferric thiocyanate inhibition / scavenging activity, and similar total reduction capacity, indicated by increase in absorbance by ethanolic and aqueous extracts of roots of Coscinium fenestratum and of Embelia ribes flower. Ethanolic extracts possess relatively higher scavenging activity than aqueous extracts.  The results identifies for the first time, the ethanolic and aqueous extracts of roots of Coscinium fenestratum and extracts of Embelia ribes flower, possess in-vitro antioxidant activity.

 

 

INTRODUCTION:

There are some speculations that the generation of free radicals inside the body in some physiological conditions is resulting in the cellular changes and development of several disorders including cancer and this could be neutralized by the antioxidants from different medicinal plants. Several studies have shown that plant derived antioxidant nutraceuticals scavenge free radicals and modulate oxidative stress-related degenerative effects.^{1, 2} Free radicals have been implicated in many diseases such as cancer, atherosclerosis, diabetes, neurodegenerative disorders and aging.^{3, 4}

 

Coscinium fenestratum is a critically endangered medicinal liana found in Western Ghats, India ⁵ , known as false Columba or tree turmeric in English.⁶  Previous studies have reported that the stem extracts possesses hepatoprotective,⁷ antioxidant,⁸ antibacterial,⁹ antidiabetic,¹⁰ and anti-malarial activities.¹¹  

 

The root and stem from Indonesia was reported to contain berberine and jatrorrhizine as the major alkaloids, besides berberrubine, N, N–dimethyllinilind carpine, palmatine and thalifendine, sitosterol and stigmasterol.⁶ Embelia ribes Burm – a common shrub, mostly climbing, rarely small trees, distributed in tropical and sub-tropical regions of the world.⁶  It has been employed in India, since ancient times by the local tribes as anthelmentic and is administered as powder, usually with milk followed by purgative. Aqueous extracts of the fruit is reported to possess antibacterial⁵, antitubercular¹³,  antioxidant ¹⁴,  cardioprotective ¹⁵ ,  antifungal ¹⁶  ,  antidiabetic ¹⁷ ,  and  antitumor ¹⁸ ,  properties in experimental animal and also reported to be rich source  chemicals and those isolated include embelin, quercitol, christembine, a volatile oil ⁵  and valangin.¹⁹

 

Literature review revealed that ethanolic and aqueous extracts of roots of Coscinium fenestratum (CFRAE, CFREE) and flowers of Embelia ribes (ERFAE, ERFEE) have not been investigated, despite reported multiple biological properties of other parts of these chosen herbs. In this perspective, current studies envisaged evaluating in vitro antioxidant activity and identify the potential choice as next nutraceuticals for disease prevention and health promoting qualities. A similar concentration of ascorbic acid (AA) was used as reference drug for comparison purposes. 

MATERIALS AND METHODS:

Collection, authentication and extraction: 

Roots of Coscinium fenestratum  roots and Embelia ribes Burm flowers were collected from Udupi, Karnataka and was identified and authenticated   by Dr. Siddamallayya from Regional Research Institute (Ay.) Bangalore, Karnataka state and herbarium retained (RI/BNG/SMP/Drug Authentication/2007-08/699/700) for future reference.  The shade dried roots and flowers (500 g)   reduced to coarse powder, defatted with petroleum ether (60–80^OC) and  then extracted with 5 liters of 90% ethanol using soxhlet apparatus.  After drying the mark to remove traces of solvent, was soaked in distilled water for 48 hours and both the extracts were concentrated under vacuum.  Extracts, so obtained was preserved in air tight container, in cool and dry place.

 

Assessment of in-vitro antioxidant activity:

Nitric oxide scavenging activity:

Nitric oxide scavenging activity was carried out according to method described.²⁰ Nitric oxide radicals were generated from sodium nitroprusside solution in phosphate buffer saline at physiological pH (7.4). Sodium Nitroprusside solution (10 mM) was mixed with test samples / standard antioxidant solution (1 ml each) in different concentration ranges (25, 50 and 75 µg/mL).The mixture was incubated at 25°C for 150 mins. To this incubated solution(1ml), 1 ml of Griess reagent (Equal portion of 1 % Sulphanilamide, 2 % O–Phosphoric acid and 0.1 % Naphthyl ethylene diamine dihydrochloride) was added.  Absorbance of formed chromophore was measured at 546 nm (Jasco V – 530 UV).

 

DPPH free radical scavenging activity: The free radical scavenging activity of extracts was measured by 1, 1-diphenyl-2-picryl-hydrazil (DPPH) ²¹  Briefly 0.1 mM solution of DPPH in ethanol was prepared (1 ml) and was added to 3 ml of test solution in water at different concentrations (25-75 µg/mL). The mixture was allowed to stand at room temperature for 30 min. Then the absorbance was measured at 517 nm.

 

Ferric thiocynate method: The antioxidant activity of extracts was determined according to the ferric thiocyanate method in linoleic acid. ²²   For stock solutions, 10 mg of different extract dissolve in 10 ml ethanol. Then, 25, 50 and 75 (µg/mL) of test or standard sample in 2.5 ml of potassium phosphate buffer (0.4 M, pH 7.0) were added to 2.5 ml linoleic acid emulsion in potassium phosphate buffer (0.4 M, pH7.0). 5ml linoleic acid emulsion consists of 17.5 µg Tween -20, 15.5 µl linoleic acid and 0.04 M potassium phosphate buffer (pH 7.0). Tween-20 was used as an emulsifier. On the other hand, 5.0 ml control consists of 2.5 ml linoleic acid emulsion and potassium phosphate buffer (0.4 M, pH7.0). The mixed solution was incubated at 37⁰C in a glass flask and in dark. After the mixture was stirred for 3 minutes the peroxide value was determined by reading the absorbance at 500 nm by using spectrophotometer. The inhibition of lipid peroxidation in percentage was calculated by following equation. Inhibition of lipid peroxidation (%) = 100 − [(A₁/A₀) × 100] where A₀ is the absorbance of the control reaction and A₁ is the absorbance in the presence of the test sample or standard compound.

 

Total Reduction capability: The total reduction capability was estimated using the method of Oyaizu. ²³   The different concentrations of extract from stock solution (25, 50 and 75µg/mL) in distilled water was mixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide [K₃Fe- (CN)₆] (2.5 ml 1%). The mixture was incubated at 50 ⁰ C for 20 min. A portion (2.5ml) of trichloroacetic acid (10%) was added to the mixture, centrifuged for 10 min at 100 × g. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl₃ (0.5 ml, 0.1 %), and the absorbance was measured at 700 nm.

 

RESULT AND DISCUSSION:

Result of nitric oxide scavenging activity, DPPH free radical scavenging activity, ferric thiocynate method and total reduction capability are graphically represented as shown in Figure 1, 2, 3 and 4.

 

 

 

Concentration dependent increase in percentage inhibition / scavenging activity was observed in ethanolic and aqueous extract. Ethanolic extract of roots Coscinium fenestratum demonstrated relatively higher in-vitro antioxidant activity than aqueous extract in all methods employed in the assessment of antioxidant activity. Antioxidant activity of Embelia ribes flowers was also of similar nature, but, of lesser magnitude. In nitric oxide scavenging activity, the IC ₅₀ (the inhibitory concentration at which there is 50% reduction of free radical) of ethanolic extract of Coscinium fenestratum roots and Embelia ribes flower were found to be 32 μg/ml and 28 μg/ml, respectively.  As mentioned earlier, secondary metabolites reported may be responsible for observed scavenging activity of the extracts. ^{8, 14}

 

Oxidative stress, in which large quantities of reactive oxygen species (ROS) like hydrogen peroxide, superoxide (^*O ^-₂), hydrogen radical (OH ^-), singlet oxygen, and nitrogen species are generated, is one of the earliest responses to stress. These ROS have a role in disease and aging in animals. ²⁴   The anti oxidative system protects the organism against ROS-induced oxidative damage. There are restrictions on the use of synthetic antioxidants, such as BHT, as they are suspected to be carcinogenic. ²⁵  Natural antioxidants, therefore, have gained importance.  

 

Nitric oxide was generated from sodium nitroprusside and measured by the Greiss reduction. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrate ions that can be estimated by use of Greiss reagent. Scavengers of nitric oxide compete with the oxygen, leading to reduced production of nitric oxide.²⁶

 

DPPH is a stable free radical at room temperature and accepts an electron or hydrogen radical to become a stable diamagnetic molecule. The reduction capability of DPPH radicals was determined by the decrease in its absorbance at 517 nm, which is induced by antioxidants.²⁰    The significant decrease in the concentration of the DPPH radical may be due to the scavenging ability of extracts of Coscinium fenestratum and Embelia ribes.

 

Metal chelating capacity of extracts was significant, since it reduced the concentration of the catalysing transition metal in lipid peroxidation. It was reported that chelating agents are effective as secondary antioxidants due to they reduce the redox potential, thereby stabilizing the oxidized form of the metal ion. The data obtained from above study reveal extracts demonstrate a marked capacity for iron binding, suggesting that their action as peroxidation protector may be related to its iron binding capacity.

 

As mentioned earlier, secondary metabolites reported may be responsible for observed scavenging activity of the extracts. ^{8, 14}   The reducing capacity of the plant extract components may serve as a significant indicator of its potential antioxidant activity. A higher absorbance indicates a higher ferric reducing power. Previous studies have reported that the electron donation capacity of bioactive compounds is associated with antioxidant activity. ²⁷

 

CONCLUSION: 

Ethanolic and aqueous extract of Coscinium fenestratum roots and Embelia ribes flower possess in-vitro   free radical scavenging property, therefore, are potential antioxidants.

 

ACKNOWLEDGEMENT:

Authors acknowledge Dr. T.N. Shivanand, Senior Scientist, IIHR, Bangalore for help extended to identify and collection of samples used in this investigations and Prof (Dr.) Desai BG, Principal, KLE University’s College of Pharmacy, Bangalore for extending infrastructural support to carry out this investigations.  

 

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Received on 09.04.2012          Modified on 12.04.2012

Accepted on 20.04.2012         © RJPT All right reserved