Phytochemical Analysis And In Vitro Antioxidant Activity .

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Suhas Manoharan et al /J. Pharm. Sci. & Res. Vol. 8(6), 2016, 512-516Phytochemical Analysis and In vitro AntioxidantActivity of Jojoba OilSuhas ManoharanI BDS Student,Saveetha dental college andhospitalsNo.162 , PH road,Chennai – 600077Vishnupriya.VAssociate professorDepartment of BiochemistrySaveetha dental college andhospitalsNo.162 , PH road,Chennai – 600077Gayathri.RAssistant professorDepartment of BiochemistrySaveetha dental college andhospitalsNo.162 , PH road,Chennai – 600077AbstractAimTo evaluate the phytochemical constituents and in vitro antioxidant activity of jojoba oil.ObjectiveJojoba oil is the liquid produced with the seed of the Simmondsia chinensis. Phytochemical constituents and in vitroantioxidant activity of jojoba oil is analysed.BackgroundThe antioxidant defence system protects the cells against the free radicals. The antioxidant defence mechanism is affected byage, diet and health condition of individual .When formation of free radicals overtakes the antioxidant defence system, the freeradicals start attacking the cell and resulting in several physiological disorders like Alzheimer’s disease, cancer, diabetes, livercirrhosis and rheumatism .Jojoba oil is found as an additive in many cosmetic products, especially those marketed as beingmade from natural .ReasonThis research is done to analyse the phytochemical constituents and antioxidant activity of jojoba oil.Result.Phytochemical tests and in vitro antioxidant assays were done and it was found out that jojoba oil contained antioxidantproperty.INTRODUCTIONSimmondsia chinensis or simply Jojoba belongs to thefamily Simmondsiaceae , is mostly a woody, evergreen,perennial shrub that produce small seeds, which containswaxy liquid very similar to spermaceti[1,2] . Jojoba oil andwax is produced from its seeds. Jojoba is used to encourageweight loss, improve liver functions, elevate bodyimmunity, also provides remedy for cancer and promoteshair growth[3]. Most of these uses were extensively studiedand the plant wax and extracts showed promising activityas skin emollient[4,5], anti-acne, anti psoriasis[6], antiinflammatory[7] and anti- hypercholesterolemia[8]. Jojobacontains 50% by weight oil which is more than soybeansand most oil seed crops . Jojoba oil is unique due to itsunusual properties that differ from other oil seeds. Thecomplete absence of glycerin makes it liquid wax and notfat. This jojoba liquid contains natural antioxidants and isused traditionally to treat sunburn , headache , chaffed skin, wounds and sore throat. In human studies , it wasdiscovered that sulphurized jojoba oil can be used in thetreatment of acne whereas unmodified jojoba wax can beused for treatment of psoriasis. The flavonoid profile ofjojoba plant fruits may place this family among otherfamilies which are rich in flavones methyl ethers andflavonoid content which make the pericarp a valuablesource for antioxidant and hepato-protective compounds [9].Phytochemicals are basically plant chemicals , that are inactive , non nutrient plant blend that are found in most plantfoods such as grains , vegetables and fruits .Phytochemicals prevent the substances we eat , drink andbreathe from becoming carcinogenic , stimulate damagedcells to commit suicide before they can reproduce , regulatehormones , prevent DNA damage and also slows growthrate of cancer . Thereby reducing the risk of cancer andother major chronic diseases.[10]Jojoba is powerful combination of antioxidant andmoisturising properties. The human body has a strongantioxidant defence mechanism . This mechanism protectsthe body from free radicals. When activity of free radicalsovertakes the antioxidant defence mechanism it results inmajor chronic diseases such as cancer , Alzhemeirs diseaseetc.MATERIALS AND METHODSJojoba oil used in the study was purchased from cyprusenterprises argumbakam ,Chennai , India. The reagentsrequired for the procedure was procured from Himedia.PHYTOCHEMICAL TESTSThe phytochemical tests were conducted according toKoleva et al [19]Test for carbohydratesTo 2ml of plant extract, 1ml of Molisch’s reagent and fewdrops of concentrated sulphuric acid were added. Presenceof purple or reddish colour indicates the presence ofcarbohydrates.512

Suhas Manoharan et al /J. Pharm. Sci. & Res. Vol. 8(6), 2016, 512-516Test for tanninsTo 1ml of plant extract, 2ml of 5% ferric chloride wasadded. Formation of dark blue or greenish black indicatesthe presence of tannins.Test for saponinsTo 2ml of plant extract, 2ml of distilled water was addedand shaken in a graduated cylinder for 15minuteslengthwise. Formation of 1cm layer of foam indicates thepresence of saponins.Test for flavonoidsTo 2ml of plant extract, 1ml of 2N sodium hydroxide wasadded. Presence of yellow colour indicates the presence offlavonoids.Test for alkaloidsTo 2ml of plant extract, 2ml of concentrated hydrochloricacid was added. Then few drops of Mayer’s reagent wereadded. Presence of green colour or white precipitateindicates the presence of alkaloids.Test for quinonesTo 1ml of extract, 1ml of concentrated sulphuric acid wasadded. Formation of red colour indicates presence ofquinones.Test for glycosidesTo 2ml of plant extract, 3ml of chloroform and 10%ammonia solution was added. Formation of pink colourindicates presence of glycosides.Test for cardiac glycosidesTo 0.5ml of extract, 2ml of glacial acetic acid and fewdrops of 5% ferric chloride were added. This was underlayered with 1 ml of concentrated sulphuric acid.Formation of brown ring at the interface indicates presenceof cardiac glycosides.Test for TerpenoidsTo 0.5ml of extract, 2ml of chloroform was added andconcentrated sulphuric acid was added carefully. Formationof red brown colour at the interface indicates presence ofterpenoids.Test for phenolsTo 1ml of the extract, a few drops of Phenol Ciocalteaureagent was added followed by few drops of 15% Sodiumcarbonate solution. Formation of blue or green colourindicates presence of phenols.Test for coumarinsTo 1 ml of extract, 1ml of 10% NaOH was added.Formation of yellow colour indicates presence ofcoumarins.Steroids and phytosteroidsTo 1ml of plant extract equal volume of chloroform wasadded and subjected with few drops of concentratedsulphuric acid appearance of brown ring indicates thepresence of steroids and appearance of bluish brown ringindicates the presence of phytosteroids.PhlobatanninsTo 1ml of plant extract few drops of 2% HCL was addedappearance of red colour precipitate indicates the presenceof Phlobatannins.AnthraquinonesTo 1ml of plant extract few drops of 10% ammoniasolution was added, appearance pink colour precipitateindicates the presence of Anthraquinones.ANTIOXIDANT ASSAYNitric oxide radical inhibition assaySodium nitroprusside in an aqueous solution atphysiological pH spontaneously generates nitric oxide; itinteracts with oxygen to produce nitrite ions, which can beestimated by the use of GriessIllosvoy reaction (Garrat,1964). In the present investigation, GriessIllosvoy drochloride(0.1%w/v)instead of 1-naphthylamine (5%). The reaction mixture (3ml) containing sodium nitroprusside (10 mM, 2 ml),phosphate buffer saline (0.5 ml) and different concentrationof oils (200–600 µg) or standard solution (0.5 ml) wereincubated at 25 C for 150 min. After incubation, 0.5 ml ofthe reaction mixture containing nitrite was pipetted andmixed with 1 ml of sulphanilic acid reagent (0.33% in 20%glacial acetic acid) and allowed to stand for 5 min nediaminedihydrochloride (1%) was added,mixed and allowed to stand for 30 min. A pink colouredchromophore was formed in diffused light. The absorbanceof these solutions was measured at 540 nm against thecorresponding blank. Ascorbic acid was used as positivecontrol. The scavenging activity was calculated using theformula.% of Inhibition (A of control – A of Test)/A of control *100DPPH Assay:The antioxidant activity of the extracts was measured onthe basis of the scavenging activity of the stable 1, 1diphenyl 2-picrylhyorazyl (DPPH) free radical according tothe method described by Brand-Williams et al. with slightmodifications. 1ml of 0.1mM DPPH solution in methanolwas mixed with 1ml of essential oil solution of varyingconcentrations (200, 400 and 600µg). Corresponding blanksample were prepared and L-Ascorbic acid (1-100 µg/ml)was used as reference standard. Mixer of 1ml methanol and1ml DPPH solution was used as control. The decrease inabsorbance was measured at 517nm after 30 minutes indark using UV-Vis spectrophotometer. The inhibition %was calculated using the following formula.% of Inhibition (A of control – A of Test)/A of control *100ABTS Assay:ABTS radical scavenging activitywas performedaccording to the protocol by Re R, Pellegrini N et al [12].The stock solution was prepared by mixing equal volumesof 7 mM ABTS solution and 2.45 mM potassium persulfatesolution followed by incubation for 12 h at roomtemperature in the dark to yield a dark-colored solutioncontaining ABTS radicals. Free radical scavengingactivity was assessed by mixing Different concentration ofessential oil (200, 400 and 600 μg) with 3.0 ml of ABTSworking standard. The decrease in absorbance wasmeasured exactly 1 min after mixing the solution, the finalabsorbance was noted up to 6 min. Ascorbic acid was usedas positive controls. The scavenging activity was estimated513

Suhas Manoharan et al /J. Pharm. Sci. & Res. Vol. 8(6), 2016, 512-516based on the percentage of ABTS radicals scavenged by thefollowing formula:% scavenging [(A0 As)/A0] 100Where A0 is absorption of control, AS is absorption oftested extract solutionRESULTS AND DISCUSSIONThe following results were obtained after performing thephytochemical tests .;TABLE 1 :S.NoPhytochemical TestsJojoba oil1Carbohydrates testWeakly 2Tannins testWeakly 3Saponins test-4Flavonoids test-5Alkaloid test-6Quinones test 7Glycosides test-8Cardiac glycosides test S.NoPhytochemical Tests9Terpenoids test10Phenols test11Coumarins test12Steroids &PhytosteroidsJojoba oil Weakly Steroids13Phlobatannins test-14Anthraquinones test- Present- AbsentFrom the phytochemical tests done , Jojoba oil shows astrong presence of quinones , cardiac glycosides andterpenoids. Cardiac glycosides are a class of medicinesused to treat cardiac anomalies like irregular heartbeats.Carbohydrates , tannins and phenols also show a weakpresence. Quinones suggest anticancer potential of jojobaoil and phenols prove the strong antioxidant property ofjojoba oil[23]. Tannins are also know for theirantimutagenetic properties.[24]ANTIOXIDANT ASSAYTABLE 2 : NITRIC OXIDE SCAVENGING ASSAYConcentration (µg)ControlJojoba OilAscorbic acid% Jojoba Oil% of Ascorbic 1859.0561224584.94897959TABLE 3 : DPPH SCAVENGING ASSAYConcentration (µg)Control2004006001.0681.0681.068Jojoba Oil0.7520.5530.385% of Inhibition29.5880149848.2209737863.95131086GRAPH 1 : NITRIC OXIDE SCAVENGING ASSAY90100Jojoba OilJojoba Oil90Ascorbic acid70806070504030% of Inhibition50.3745318470.9737827787.82771536GRAPH 2 : DPPH SCAVENGING ASSAY% of Inhibition% of Inhibition80% of InhibitionAscorbic Acid0.530.310.13Ascorbic Acid605040302020101000200400Concentration (µg)600200400600Concentration (µg514

Suhas Manoharan et al /J. Pharm. Sci. & Res. Vol. 8(6), 2016, 512-516Concentration (µg)200400600TABLE 4 : ABTS ASSAYJojoba oilAscorbic 30.713% Jojoba Oil26.7882187946.0028050559.18653576% of Ascorbic acid43.1977559665.4978962183.45021038GRAPH 3 : ABTS ASSAY9080% of inhibition70Jojoba oilAscorbic acid6050403020100200400600Concentration (µg)Three tests were conducted to assess the in vitroantioxidant activity of jojoba oil. Nitric oxide scavengingassay clearly shows that there is a gradual increase in thepercentage of inhibition of jojoba oil as the concentrationincreased. In DPPH scavenging assay , for a concentrationof 200 µg , the percentage inhibition of jojoba oil wasfound to 29.58 . In ABTS radicle scavenging assay , thepercentage of inhibition showed a similar increase withincrease in concentration, 200 µg showed 26 %inhibition.All the test were done against ascorbic acid asstandard.The results shows that jojoba oil posses a strongin vitro antioxidant property .CONCLUSIONThe phytochemical screening indicated that the testedjojoba oil extract contains secondary metabolites likesaponins, tannins, alkaloids, steroids and glycosides. Also ,jojoba extracts exhibited antioxidant activity. The resultsfrom the phytochemical screening suggests that theseextracts can have potential use as natural preservatives infood against the well-known agents of food-borne diseasesand food spoilage . Antioxidant-based formulations can beused for prevention and treatment of many illnesses such asatherosclerosis, stroke, diabetesThe potential to use jojobaoil’s antioxidant property for treatment of major chronicdiseases will open new horizons in the pharmaceuticalsindustry and will be dominant area of future ubitzki, K. and C. Bayer, Flowering plants, Dicotyledons :Malvales, Capparales, and non- betalain Caryophyllales. TheFamilies and genera of vascular plants. 2003, Berlin ; New York:Springer.Van Wyk, B.-E. and M. Wink, Medicinal plants of the world : anillustrated scientific guide to important medicinal plants and theiruses. 1st ed. 2004, Portland, Or.: Timber Press.14.15.Yaron, A., Metabolism and physiological effects of Jojoba oil, inThe chemistry and technology of jojoba oil, J. Wisniak, Editor. 1987,American Oil Chemists' Society: Champaign, Ill. p. 251-265.Taguchi, M., Test results on safety of Jobacohol (Jojoba alcohol) forcosmetic uses, in Proceedings of the 6th international conference onJojoba oil and its uses, J. Wisniak and J. Zabicky, Editors. 1985, BenGurion University: The Negev, Beer Sheva, Israel. p. 371-391.Brown, J., J. Arqette, and J. Reinhardt, Jojoba esters; A new familyof cosmetic emollients, in Proceedings of 9th internationalconference on Jojoba oil and its uses and of the 3rd internationalconference on new crops and products, L.H. Princen and C. Rossi,Miwa, T.K., Structural determination and uses of jojoba oil. J AmOil Chem Soc, 1984. 61(2): 407-410.Habashy, R.R., Abdel-Naim, A. B., Khalifa, A. E. and Al-Azizi, M.M., Anti-inflammatory effects of jojoba liquid wax in experimentalmodels. Pharmacol Res, 2005. 51(2): 95-105.Farag, R.S., M.M. Farag, and R.F.M. Ali, Use of sunflower oilmixed with jojoba and paraffin oils in deep-fat frying process. Int JFood Sci Technol, 2008. 43(7): 1306-1315.A.M. A. El- Halawany, “Pharmacognostical study of SimmondsiaChinenisis (Link) Schneider family Buxaceae (Simmondsiaceae)cultivated in Egypt. M.Sc, Thesis Pharmaceutical Science(Pharmacognosy), Pharmacognosy Department, Faculty ofPharmacy, Cairo University, Egypt, 2002.Rui Hai Liu2, Potential Synergy of Phytochemicals in CancerPrevention: Mechanism of Action1, J. Nutr. December 1, 2004, vol.134 no. 12 3479S-3485S.Garrat DC. (1964) The Quantitative analysisof Drugs. Chapman and Hall Ltd., Japan, 3: 456- 458.Brand-williams W, Cuvelier ME and Berset C.Use of free radicalmethod to evaluate antioxidant activity .Lebensmittel Wissenschaftand Technologie 1995; 28(1): 25-30.Re R, Pellegrini N, Proteggente A, Pannala A, Yong M, Rice-EvasC: Antioxidant activity applying an improved ABTS radical cationdecoloursation assay. Free Rad Biol Med 1999, 26:1231-1237.Sofowora A. Medicinal Plants and Traditional Medicinal inAfrica. 2nd Ed. Sunshine House, Ibadan, Nigeria: Spectrum BooksLtd; 1993. Screening Plants for Bioactive Agents; pp. 134–156.Harborne JB (1973). Phytochemical Methods: A guide to moderntechniques of plant analysis. Chapman and Hall, New York, pp. 279,3rd Edition.Smolenski SJ, Silinis H, Farnswoth NR (1974). Alkaloids screening.V. Lloydia, 37: 506-536.515

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Phytochemical Analysis and In vitro Antioxidant Activity of Jojoba Oil Suhas Manoharan I BDS Student, Saveetha dental college and hospitals No.162 , PH road, Chennai – 600077 Vishnupriya.V Associate professor Department of Biochemistry Saveetha dental college and hospitals No.162 , PH road, Chennai – 600077 Gayathri.R Assistant professor Department of Biochemistry Saveetha dental college .

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