In Vitro Evaluation of Antioxidant Potentiality and Estimation of Total
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The Pharma Innovation Journal 2021; 10(3): 01-08 ISSN (E): 2277- 7695 ISSN (P): 2349-8242 NAAS Rating: 5.23 In vitro evaluation of antioxidant potentiality and TPI 2021; 10(3): 01-08 © 2021 TPI estimation of total phenolic and flavonoid content of the www.thepharmajournal.com Received: 02-01-2020 whole stem of Coffea benghalensis B. Heyne ex Schult Accepted: 05-02-2021 Shoriful Islam Sagor Shoriful Islam Sagor, Golam Mostofa Sajol, AKM Khurshid Alam and Department of Pharmacy, Faculty of Science, University of Adeeba Anjum Rajshahi, Rajshahi-6205. Bangladesh Abstract This study was carried out for the first time to evaluate the antioxidant properties and total phenolic and Golam Mostofa Sajol flavonoid content of the methanolic extract along with its organic and aqueous soluble fractions of the Department of Pharmacy, whole stem of Coffea benghalensis growing in Bangladesh. For evaluation of antioxidant properties Faculty of Science, University of Rajshahi, Rajshahi-6205. DPPH scavenging assay, ferric reducing capacity, hydroxyl radical scavenging assay and phospho Bangladesh molybdenum assay was used. In the DPPH free radical scavenging assay, the ethyl acetate soluble fraction of the crude methanol extract revealed the highest free radical and hydroxyl radical scavenging AKM Khurshid Alam activity with IC50 value of 3.22 ± 0.17 μg/ml and 12.88 ± 0.305 μg/ml, respectively while the standard Department of Pharmacy, BHT was 4.39 ± 0.02 μg/ml and 9.38 ± 0.075 μg/ml, respectively. All the test samples and the standard Faculty of Science, University of BHT exhibited good linear relationship in the ferric reducing capacity and phospho molybdenum assay. Rajshahi, Rajshahi-6205. Also, the test samples showed significant activities in the ferric reducing capacity and phospho Bangladesh molybdenum assay compared to the reference standard in a dose dependent manner. It was observed that the test samples contained considerable amount of bioactive compounds including total phenolic (ethyl Dr. Adeeba Anjum acetate soluble fraction giving the highest 22.631 ± 0.085 GAE/gm of dried sample) and flavonoid (pet Associate Professor, Department ether soluble fraction giving the highest 54.513 ± 1.500 CE/gm of dried sample) content. It can be of Pharmacy, University of concluded from the results that the crude methanolic extract along with it’s four soluble fractions of the Rajshahi, Rajshahi-6205, whole stem of Coffea benghalensis growing in Bangladesh are a good source of natural antioxidants. Bangladesh Keywords: Coffea benghalensis, antioxidant, free radical scavenging, hydroxyl radical scavenging, phospho molybdenum assay 1. Introduction Antioxidants are vital substances which possess the ability to protect the body from damage [1] caused by free radical induced oxidative stress . Over production of various forms of activated oxygen species, such as oxygen radicals and non-free radical species is considered to be the main contributor to oxidative stress, which has been linked to several diseases like atherosclerosis, cancer, and tissue damage in rheumatoid arthritis [2, 3]. Besides, hydroxyl radical is one of the most reactive oxygen species in the biological system. It reacts with cell membrane phospholipids polyunsaturated fatty acid moieties and causes damage to cell [4]. Synthetic antioxidants, such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), which are effective in their role as antioxidants, are commercially available and currently used in industrial processes. However, since suspected actions as promoters of carcinogenesis and other side effects have been reported, their use in food, cosmetic and pharmaceutical products has been decreasing [5-9]. Hence, there has been an upsurge of interest in naturally-occurring antioxidants from vegetables, fruits, flowers, leaves, oil-seeds, cereal crops, tree barks, roots, spices and herbs [10- 13]. Numerous crude extracts and pure natural compounds from fruits were reported to have antioxidant and radical-scavenging activities. Within the antioxidant compounds, flavonoids and phenolics, with a large distribution in nature, have been studied [14]. Phenolics or polyphenols, including flavonoids have received considerable attention because of their physiological functions such as antioxidant, antimutagenic and antitumor activities [15]. Corresponding Author: Medicinal plant is any plant where one or more of its organs contains substances which can be Dr. Adeeba Anjum used as therapeutic agents and which are precursors for synthesis and development of novel Associate Professor, Department therapeutic agents. The tendency on the use of medicinal plants had yet been placed on the of Pharmacy, University of [16] Rajshahi, Rajshahi-6205, treatment rather than prevention of diseases . Plants have been used for several years as a Bangladesh source of traditional medicine to treat various diseases and conditions [17]. ~ 1 ~ The Pharma Innovation Journal http://www.thepharmajournal.com Coffea benghalensis B. Heyne ex Schult. is a deciduous cotton plug and finally with filter paper (Whatman No. 1). shrub, commonly known as bonnyo koffee belonging to The filtrate was then concentrated to dryness, in vacuum by Rubiaceae family. In Nepal the flowers are used for excessive using rotary evaporator at 40 °C temperature to render the bleeding during menstruation [18]. Also, the fruit part of C. crude methanolic extract (21 gm). benghalensis showed antibacterial activities against Proteus The modified Kupchan partitioning protocol [23] was used for vulgaris, Escherichia coli, Vibrio cholerae, Salmonella typhi the partitioning of an aliquot of the concentrated methanol and Streptococcus aureus [19]. The native distribution of this extract (CME). The consequent partitionates were dried off to plant has been documented in Bangladesh (Sylhet to yield pet-ether (PESF), chloroform (CSF), ethyl acetate Chittagong), India (Arunachal Pradesh, Assam, Bengal, (EASF),and aqueous (AQSF) soluble materials (Table 1). The Meghalaya, Orissa, Rajasthan and Sikkim), East Himalaya, residues were then stored in a cool dry place until further use. Bhutan, Myanmar, Nepal, Thailand, Vietnam [20]. The leaf, pericarp and seed part of the plant exhibited nine Table 1: Modified Kupchan partitionates of C. benghalensis whole polyphenolic compounds of which caffeic acid, chlorogenic stem acid, ferulic acid, p-coumaric acid and sinapic acid are Crude extract/ Soluble fractions Weight (gm) phenolic acids and quercetin, isoquercitrin, rutin and CME 10 kaempferol are flavonols [19]. Also from the leaves a cafestol PESF 1.47 (bengalensol) was identified [21]. The fruit part of C. CSF 4.21 benghalensis showed antibacterial activities against Proteus EASF 0.14 vulgaris, Escherichia coli, Vibrio cholerae, Salmonella typhi AQSF 4.15 and Streptococcus aureus [19]. Literature survey showed that there are no phytochemical, biochemical or pharmacological 2.4 Antioxidant Activity information concerning the whole stem of Coffea 2.4.1 DPPH free radical scavenging assay benghalensis B. Heyne ex Schult. So, as part of our Following the method developed by Braca et al., 2001 [24] the continuing studies on medicinal plants of Bangladesh [22] antioxidant activity of the test samples was assessed by especially Coffea benghalensis B. Heyne ex Schult. growing evaluating the scavenging activities of the stable 1, 1- in Bangladesh we investigated the antioxidant potential in diphenyl-2-picrylhydrazyl (DPPH) free radical by using forms of free radical scavenging activity, ferric reducing synthetic antioxidant, butylated hydroxytoluene (BHT)as capacity, hydroxyl radical scavenging activity and total reference standards.according to this method absorbance was antioxidant capacity (Phosphomolybdenum assay), also total taken at 517 nm because the DPPH radical contains an odd phenolic and flavonoid content of the whole stem of C. electron, which is responsible for the absorbance and also for benghalensis was done for the first time, and we, herein, the visible deep purple color. When DPPH accepts an electron report the results of our preliminary studies. donated by an antioxidant compound, the DPPH is decolorized, which can be quantitatively measured from the 2. Materials and Methods change in absorbance and % of scavenging activity is 2.1 Chemicals calculated. According to this method 0.1 ml of each sample Most of the chemicals and reagents like Folin-ciocalteu (CME, PESF, CHSF, EASF and AQSF) was added to 3 ml of reagent, sodium phosphate, ammonium molybdate, ferric a 0.004% methanol solution of DPPH and five different chloride, methanol, DPPH, BHT, EDTA, FeSO4.7H2O, concentrations (25, 50, 100, 200 and 400 μg/ml) were made. aluminium chloride were purchased from Sigma chemical Absorbance was taken at 517 nm after 30 min and the company, USA. Potassium ferricyanide, trichloro acetic acid, percentage inhibition activity was calculated by using the sulfuric acid, sodium carbonate and hydrogen peroxide were following equation- purchased from Merck, Germany. But phosphate buffer, 2- deoxy-D-ribose, thiobarbituric acid were bought from Sigma- (I %) = (1 – Asample /Ablank) x 100 Aldrich, Japan. Gallic acid and catechin were obtained from Wako pure chemicals Ltd., Japan. All the chemicals and where, Ablank is the absorbance of the control (containing all solvents used were of analytical reagent grade and all are reagents except the test material and Asample is the absorbance commercially available. of the sample extractive. Extract concentration providing