Evaluation of Antioxidant, Antibacterial, Alpha Amylase Enzyme Inhibition

Journal of Pharmacognosy and Phytochemistry 2018; 7(6): 2326-2333 E-ISSN: 2278-4136 P-ISSN: 2349-8234 JPP 2018; 7(6): 2326-2333 Evaluation of antioxidant, antibacterial, alpha Received: 09-09-2018 Accepted: 13-10-2018 amylase enzyme inhibition activities and GC-MS analysis of leaves extract of Gymnema sylvestre L. Gayathri S Sathyabama Institute of Science and Technology, OMR Road, Gayathri S, Sivaraj C, Sangeetha ST, Arumugam P and A Manimaran Chennai, Tamil Nadu, India Sivaraj C Abstract Armats Biotek Training and Gymnema sylvestre is an important medicinal plant which belongs to the family Apocynaceae, commonly Research Institute, Guindy, used herb in Ayurveda. The common name is gurmar, meaning of “sugar destroying,” was given because Chennai, Tamil Nadu, India of the plant has antisacharogenic property (suppresses the taste of sugar). The aim of the present study was to evaluate the antioxidant, antibacterial activities and to identify the bioactive compounds by GC- Sangeetha ST MS analysis of methanol leaves extract of G. sylvestre. Different antioxidant assays were carried out for Sathyabama Institute of Science evaluating antioxidant activity. The maximum DPPH radical scavenging activity was 54.08±0.15% at and Technology, OMR Road, 120 µg/mL concentration with the IC50 of 104.66 µg/mL concentration. The maximum DPPH radical Chennai, Tamil Nadu, India scavenging activity was 54.08±0.15% at 120 µg/mL concentration with the IC50 of 104.66 µg/mL ·- concentration. The maximum superoxide radical (O2 ) scavenging activity was 62.48±0.20% at 120 Arumugam P 3+ Sathyabama Institute of Science µg/mL concentration with the IC50 of 72.93 µg/mL concentration. The maximum Fe reduction and 6+ and Technology, OMR Road, Mo reduction were 83.11±0.17% and 62.03±0.36% at 120 µg/mL concentration with the RC50 of 43.79 Chennai, Tamil Nadu, India µg/mL and 75.14 µg/mL concentration respectively. The maximum α-amylase enzyme inhibition was 70.86±0.94% at 60 µg/mL concentration with the IC50 of 75.40 µg/mL concentration. A Manimaran P.G and Research Department of Keywords: G. sylvestre, antibacterial, antioxidant activity, DPPH, GC-MS Advanced Zoology and Biotechnology, Government Arts 1. Introduction College for Men, Nandanam, G. sylvestre is a perennial, woody climber belonging to family Asclepiadaceae or the “milk Chennai, Tamil Nadu, India [1] weed” family . G. sylvestre commonly known as sakkarai kolli, has been broadly used in [2] indigenous systems of Indian medicine due to its numerous therapeutic properties . G. sylvestre is native to India, found in tropical and subtropical regions, but widely well distributed in the southern part of China, tropical Africa, Malaysia, and Sri Lanka [3]. The leaves are opposite, usually elliptic, elongate oval (1.25–2.0 inch × 0.5–1.25 inch) shaped and have soft hairs on the upper surface, inflorescence is lateral umbel in cymes; follicles are terete and lanceolate, up to 3 inches in height. Flowers are greenish white in colour. Corolla is pale yellow in colour, valvate, campanulate with single corona with 5 fleshy scales. The calyx- lobes are long, ovate, obtuse, and pubescent. Carpels-2, unilocular, ovules locules may be present, anther connective produced into a membranous tip [4, 5] . 1.1 Taxonomy Kingdom: Plantae Phylum: Tracheophyta Subphylum: Angiosperms Class: Eudicots Order: Gentianales Family: Apocynaceae Genus: Gymnema Species: sylvestre Binomial name: Gymnema sylvestre Correspondence A Manimaran P.G and Research Department of Advanced Zoology and Biotechnology, Government Arts College for Men, Nandanam, Chennai, Tamil Nadu, India Fig 1: Gymnema sylvestre ~ 2326 ~ Journal of Pharmacognosy and Phytochemistry 2. Materials and Methods that sequence. The reaction was started by illuminating the 2.1 Collection of leaves and preparation of extract reaction mixture for 15 min. After illumination, the The leaves of G. sylvestre were collected from the market at absorbance was measured at 590 nm in UV-Vis Mylapore, Chennai, Tamil Nadu, India. The leaves were Spectrophotometer. Ascorbic acid was used as standard washed, shade dried for 10 d and powdered in mechanical reference. The percentage of inhibition was calculated as: blender. About 10 g of leaves powder was soaked in methanol for 72 h. The greenish supernatant liquid was filtered by filter paper and condensed in a hot plate at 50°C, which yields gummy extract. 2.2 Qualitative phytochemical analysis 2.5.2 ABTS˙+ radical cation scavenging assay The methanol leaves extract of G. syvestre was subjected to The antioxidant capacity was estimated in terms of the different classes of phytoconstituents, using specific reagents ABTS●+ radical cation scavenging activity [11]. ABTS●+ was and following standard methods [6]. obtained by reacting 7 mM ABTS solution in 5 mM of phosphate-buffered saline (pH 7.4) with 2.45 mM potassium 2.3 Estimation of total phenols persulfate and the mixture was left to stand in dark at room Folin-Ciocalteau reagent method was used to determine the temperature for 12-16 h before use. The ABTS solution total phenolic compounds [7] with slight modifications. One (stable for 2 days) was diluted with 5 mM phosphate-buffered hundred µL of methanol leaves extract (1mg/mL) was mixed saline (pH 7.4) till to reach an absorbance of 0.70±0.02 at 734 with 900 µL of methanol and 1 mL of Folin Ciocalteau nm. To the various concentrations (20-120µg/mL) of reagent (1:10 diluted with distilled water). After 5 min, 1 mL methanol leaves extract of G. sylvestre, 500 µL of diluted of 20% (w/v) of Na2CO3 solution was added. The mixture ABTS●+ solution was added. The absorbance was measured was then allowed to stand for 30 min incubation in dark at after 10 min incubation at 734 nm. Ascorbic acid was used as room temperature. The absorbance was measured at 765 nm the standard reference. The ABTS●+ radical cation scavenging in UV-Vis spectrophotometer. The total phenolic content was activity was expressed as: expressed in terms of gallic acid equivalent (µg/mg of extract), which is a common reference compound. 2.4 Estimation of total flavonoids The total flavonoid content was determined using aluminium chloride reagent method with slight modification [8]. Five 2.5.3 Ferric (Fe 3+) reducing power assay hundred µL of methanol leaves extract (1mg/mL) was mixed The reducing power of methanol leaves extract of G. sylvestre with 500 µL of methanol and 500 µL of 5% (w/v) sodium was determined by Fe 3+ reduction method with slight nitrite solution followed by 500 µL of 10% (w/v) aluminium modification [12]. One mL of leaves extract of different chloride solution was added and incubated for 5 min at room concentrations (20 - 120 µg/mL) was mixed with 1 mL of temperature. Then 1 mL of 1 M NaOH solution was added phosphate buffer (0.2 M, pH 6.6) and 1 mL of potassium and the total volume was made up to 5 mL with distilled ferricyanide [K3Fe (CN)6] (1 % w/v). The mixtures were then water. Absorbance was measured at 510 nm in UV-Vis incubated at 50°C in water bath for 30 min. One mL of spectrophotometer. The result was expressed as (µg/mg of trichloroacetic acid (10 % w/v) was added to each mixture. extract) quercetin equivalent. Then 1 mL of freshly prepared FeCl3 (0.1% w/v) solution was added and the absorbance was measured at 700 nm in UV-Vis 2.5 In vitro antioxidant assays spectrophotometer. Ascorbic acid was used as the standard 2.5.1 DPPH˙ radical scavenging assay reference. The percentage of reduction was calculated as: The antioxidant activity of methanol leaves extract of G. sylvestre was measured on the basis of stable DPPH free radical reduction method [9]. One mL of 0.1 mM DPPH solution in methanol was mixed with 1 mL of various concentrations (20-120 μg/mL) of leaves extract. The mixture was then allowed to stand for 30 min incubation in dark. One 2.5.4 Phosphomolybdenum reduction assay mL methanol and 1 mL DPPH solution was used as the The antioxidant capacity of methanol leaves extract of G. control. The decrease in absorbance was measured using UV- sylvestre was assessed by Mo6+ reduction method [13]. The Vis Spectrophotometer at 517 nm. Ascorbic acid was used as leaves extract with concentrations ranging from 20 to 120 the standard reference. The percentage of inhibition was μg/mL was combined with 1 mL of reagent solution calculated as: containing ammonium molybdate (4 mM), sodium phosphate (28 mM) and sulphuric acid (600 mM). The reaction mixture was incubated in water bath at 95oC for 90 min. The absorbance of the coloured complex was measured at 695 nm in UV-Vis spectrophotometer. Ascorbic acid was used as the standard reference. The percentage of reduction was ●- 2.5.2 Superoxide radical (O2 ) scavenging assay calculated as: Superoxide radical scavenging activity was carried out by the method of Ravishankara et al [10]. Different concentrations of leaves extract (20-120 μg/mL) of G. sylvestre was mixed with 50 mM of phosphate buffer (pH 7.8), 1.5 mM of riboflavin, 12 mM of EDTA and 50 mM of NBT solutions and added in ~ 2327 ~ Journal of Pharmacognosy and Phytochemistry 2.6 Antidiabetic activity desirable concentrations. Tetracycline was used as the 2.6.1 Alpha amylase enzyme inhibition assay standard with the concentration of 25 µg. All the plates α- amylase enzyme inhibition assay was carried out based on containing sample loaded wells were incubated for 24 h at the starch-iodine test [14]. The total assay mixture was 37°C. After the incubation period, zone of inhibition in each composed of various concentration (20-120 µg/mL) of leaves plate, for each concentration of extract and standard were extract of G.

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