Preliminary Phytochemical Analysis And In Vitro .

Shivanand Bhat et al /J. Pharm. Sci. & Res. Vol. 9(8), 2017, 1283-1287Preliminary Phytochemical Analysis and in vitro antioxidantPotential of Fruit Stalk ofCapsicum annuum var. glabriusculum (Dunal) Heiser &PickersgillShivanand Bhat1,2*, L. Rajanna3Department of Botany, Government Arts and Science College, Karwar, Karnataka 581301, India2Reaserch and Development Section, Bharathiar University, Coimbatore, Tamil Nadu 641046, India3Department of Botany, Bengaluru University, Bengaluru, Karnataka, 560056, India1AbstractThe aim of the present study was to assess and evaluate the therapeutic value of fruit stalk of Capsicum annuum var.glabriusculum (Dunal) Heiser & Pickersgill found across homegardens of Uttar Kannada. The study involved the screening ofphytochemical (qualitative) and quantitative estimation of phenolic content, flavonoid content and to assess their antioxidantpotential. Antioxidant activity was evaluated using different in vitro assays such as 2,2-Diphenyl-1-picrylhydrazyl ulfonic acid (ABTS), reducing power assay and total antioxidant capacity. The studyindicated presence of different phytochemicals and constituted fair amount of phenolics and flavonoids, and exhibitedantioxidant activity in a dose dependent manner. Thus, the present study presents a comprehensive knowledge on thetherapeutic value of C. annuum var. glabriusculum fruit stalk which is commonly utilized for medicinal purpose acrosshomegardens of Uttara Kannada.Keywords: Homegardens, Phenolic content, Phytochemicals, Antioxidant potential, Medicinal plants.INTRODUCTIONPlants are basic providers of food, fodder and fuel. Apartfrom these inherent properties, plants also act as reservoirof medicines that are found in the form of secondarymetabolites. Till date several potential phytochemicals havebeen isolated and the structure has been elucidated fromwide array of plants. Based on their bioactivity they areoften categorized as medicinal plants and non-medicinalplants. A great number of plants have been exploited insearch of phytochemicals. Extensive studies have beenreported in assessment of antimicrobial and antioxidantactivities [1]. However the search for novel phytochemicalsis still in progress for the increasing incidence of diseasesdue to ever increasing population. Phytochemicals havereceived a great deal of attention due to the very reason thatthey are non-toxic and often show any side effects [2].Phytochemicals as a whole is a vast group and generallycomprise phenolics, tannins, flavonoids, alkaloids, saponinsand carotenoids to name a few [3]. Among these classes ofcompounds phenolics is the most extensively studied groupwherein several bioactivities have been attributed to itincluding antimicrobial, antioxidant, anti-inflammatory,anti-diabetic, anti-hypertensive, anticancer and many more[4].Excessive free radicals or reactive oxygen species (ROS)generated in the body result in oxidative stress which isresponsible for multiple chronic diseases includingneurodegenerative diseases and cardiovascular diseases [5].Several studies have hypothesized that secondarymetabolites of plants are able to scavenge free radicalsnaturally [6]. Phenolics and non-phenolic compounds suchas vitamins play a vital role in inhibiting oxidative stress.Several potential phenolic compounds that are widelydistributed across plants act as antioxidants. Numerousantioxidants have been identified and isolated from variousparts of plant. Nevertheless some antioxidants arechemically synthesized such as Butylated hydroxytoluene(BHT), Butylated hydroxyanisole (BHA) Propyl gallate(PG) and Tert-butylhydroquinone (TBHQ) that are knownto cause several severe side effects. As a result the quest forsearch of potential natural antioxidants has been the maingoal of phytochemists.Homegardens serve to be primary source of medicine forwide array of diseases across Western Ghats. Traditionally,several plant species of homegardens are used for treatmentof health ailments [7, 8]. Locally, C. annuum var.glabriusculum (Dunal) Heiser & Pickersgill is used ascoolant and to cure ulcer. Flavonoids and antioxidantactivity of Capsicum annuum cultivars has been welldocumented [9, 10]. Peppers are found to comprise goodquantity of flavonoids especially Quercetin and luteolin.Though Capsicum annuum has been highly domesticated asa spice, Ethanopharmacological studies have shown thatCapsicum species are effective medicines against severalhealth ailments including arthritis, rheumatism, snake biteand wounds [11]. However, understanding its medicinalvalue is relatively essential as studies have proved thatgeographical variations/environmental factors have impact.Hence in the present study an attempt was made to evaluatethe therapeutic value of fruit stalk of C. annuum var.glabriusculum commonly grown across homegardens ofUttara Kannada.MATERIAL AND 3ethylbenzothiazoline-6-sulfonic acid) diammonium salt(ABTS), 2,2-diphenyl-1- picrylhydrazyl hydrate (DPPH)were purchased from Sigma Aldrich, Bangalore. All otherchemicals used were of analytical grade and purchasedfrom Merck, Bangalore.1283

Shivanand Bhat et al /J. Pharm. Sci. & Res. Vol. 9(8), 2017, 1283-1287Collection of plant material and identificationThe plant was collected between May to September acrosshomegardens of Uttara-Kannada, Karnataka, India. TheBotanical name was authenticated using multiple floras atthe Department of Botany, Government Arts and ScienceCollege Uttara Kannada, Karnataka, India. Theethnobotanical information of the plants considered for thepresent study is listed in Table 1.Solvent extractionThe plant sample was shade dried at room temperature(31 4 C) for 21 days, further they were homogenized intofine powder with the aid of electric grinder (TTK Prestige,India). The methanolic extract was prepared by macerating10 g of plant sample in 100 ml of methanol for duration of48 h with constant shaking using a rotary shaker (Remi,India). The crude extract was filtered through a Whatmannfilter paper No. 1. Further the excess solvent was removedusing a rota-evaporator (Ika, Bangalore, India). Finallyfiltrate was evaporated to dryness in a water bath at 60 C.A known concentration of 10mg/ml was prepared whichwas stored at -20 C for further analysis.Qualitative phytochemical analysisThe crude methanolic extract was subjected for qualitativephytochemical screening following the protocols asdescribed by Harborne [12].Quantification of total phenolic contentThe total phenolic content (TPC) in the methanolic plantextract was estimated by following Folin-Ciocalteau’s (FC)method as previously reported by Kumar et al [13].Different aliquots of plant extracts and standards (1mg/ml)were taken in series of test tubes and volume was made upto 3 ml by adding deionized water. FC reagent (0.5ml) wasadded to each tube which was incubated for 3 min thensodium carbonate (20%, 2ml) was added, vortexed andfurther these tubes were kept in boiling water bath for 1min. Absorbance was recorded at 650 nm using a doublebeam UV-Visible Spectrophotometer (U-2900, Hitachi,Japan) against reagent blank. Gallic acid was used as areference standard and calibration curve was plotted (20100 µg/ml) and TPC in plant extracts were expressed asmilligrams of gallic acid equivalents per gram dry weight(mg GAE/g dw).Quantification of total flavonoid contentTotal flavonoid content (TFC) of the methanol extracts wasdetermined following the protocol described by Helmja etal [14], with slight modifications. Briefly, Known aliquotsof extracts (500 μg/ml) and various concentrations ofstandards were pipetted out in a series of test tubes andvolume was made up to 5 ml with deionized water. Sodiumnitrite (5%; 0.3 ml) was added which was incubated for 5min to this aluminium chloride solution (10%; 0.6 ml) wasadded with further incubation for 5 min, and, sodiumhydroxide solution (1 M, 2 ml) was added and total volumewas made up to 1 ml with distilled water. Absorbance wasrecorded at 510 nm against blank using a double beam UVVisible spectrophotometer (U-2900, Hitachi, Japan). Astandard curve was plotted using various concentrations ofQuercetin (20-100 μg/ml). From the standard curve,concentration of flavonoids in the crude extracts weredetermined and expressed as milligrams of Quercetinequivalents per gram dry weight (mg QE/g dw).In-vitro antioxidant assaysDPPH radical scavenging activityDPPH free radical scavenging activity determinedspectrophotometrically following the protocol of Blois [15]and as reported by Kumar et al [16]. Briefly, 0.2mM ofDPPH solution was mixed to various concentrations ofsample (1-5 mg/ml), vortexed and incubated for 30 min at37 2 C in dark absorbance was read at using a doublebeam UV-Visible spectrophotometer (UV-1800, Shimadzu,Japan). Methanol with no added samples was taken ascontrol. Percentage radical scavenging activity (RSA) wascalculated using the formula:% RSA (Acontrol-Asample/ Acontrol) 100, where,A absorbance at 517 nm.ABTS assayABTS assay was performed as per the procedure describedby Re et al [17]. Briefly, 7.4 mM ABTS solution and 2.6mM potassium persulfate solution was prepared. Workingsolution was prepared by mixing these two stock solutionsin equal volume and reaction mixture was kept forincubation in dark for overnight at room temperature. 1 mlreacted ABTS solution was diluted with 60 ml of methanolto attain an absorbance of 1.170 0.02 units at 734 nm usinga UV-Visible Spectrophotometer (UV-1800, Shimadzu,Japan). A 300 µl of different concentrations (200-1000µg/ml) of crude methanolic extracts were mixed with 2700µl of the reacted ABTS solution, which was allowed toreact in dark. The absorbance was recorded at 734 nm.Total antioxidant capacityTotal antioxidant activity of the plant extracts wasdetermined following protocol of Prieto et al [18] withslight variations in reaction volume. 0.2 ml of crudeextracts of various concentrations (12.5, 25, 50, 100 µg/ml)were taken in a test tube to which 1.8 ml of reagent (0.6 Msulfuric acid, 28 mM sodium phosphate, and 4 mMammonium molybdate) was added. The tubes wereincubated at 95 C for 90 min in a water bath and allowed tocool. The absorbance was measured at 695 nm against ablank using double beam UV-Vis spectrophotometer (UV1800, Shimadzu, Japan). The values are represented as µgascorbic acid equivalents per gram dry weight (µgAAE/gdw).Reducing power activityReducing power activity of test samples was determinedfollowing Phosphomolybdenum method as described byOyaizu et al [19]. Briefly, Plant extracts of variousconcentrations 200-1000 µg/ml (made up to 1.0 ml withmethanol) was taken in a series of test tubes to which 2.5ml of phosphate buffer (0.2 M, pH 6.6) and 2.5 ml ofpotassium ferricyanide (1%) was added, and allowed tostand at 50 C for 20 min in a water bath. Further 2.5 ml of1284