Formulation And Evaluation Of Natural Antioxidant Cream .

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Available online at www.ijpcr.comInternational Journal of Pharmaceutical and Clinical Research 2016; 8(9): 1305-1309ISSN- 0975 1556Research ArticleFormulation and Evaluation of Natural Antioxidant Cream ComprisingMethanolic Peel Extract of Dimocarpus longanRavindran Muthukumarasamy*, Alifah Ilyana, Nur ‘Afini Fithriyaani, Nur Ain Najihah, NurAsyiqin, Mahendran SekarFaculty of Pharmacy and Health Sciences, University Kuala Lumpur – Royal College of Medicine Perak, Ipoh, Perak,Malaysia, 30450.Available Online:20th September, 2016ABSTRACTPhoto aging is a common problem that occurs in our community due to ongoing exposure to ultraviolet rays. The use ofantioxidants is an effective approach to prevent symptoms related to photo-induced aging of the skin. Thus, the presentstudy was to prepare and evaluate the antioxidant cream comprising the methanolic peel extracts of Dimocarpus longanfor their radical scavenging activity. Antioxidant activity of peels and seeds methanolic extract (by continuous hotpercolation-soxhletation) of D. longan was assessed by using stable 2,2 -Diphenyl-1-picryl hydrazyl (DPPH). The extractof the D. longan fruits contained three major polyphenolic compounds which are corilagin, gallic acid, and ellagic acidwhich are responsible for the antioxidant properties. Methanolic extracts of both peels and seeds of D. longan exhibitedhigh radical scavenging properties, the IC50 result revealed that peels extract were having higher antioxidant propertieswith 23.5 µg/ml compared to the seeds 32.13 µg/ml. Based on the higher scavenging activity the peels was chosen toprepare as formulation. Thus, the cream was formulated with 2.5% of peels extract by fusion method with incorporationof two different emulsifying agents for two formulations (F1 & F2). The evaluation of the formulations was done ondifferent parameters like pH, spreadability, rheological study, non-volatile matter at 105 C, physical stability of creamand microbial limit test. Both the formulations (F1 & F2) were showed good pH, homogeneity, appearance, ease to remove,good consistency, spreadability and no microbial growth. However, after four weeks of storage formulation of F1 showedcracking and phase separation. The evaluation parameters of the formulated cream F2 showed good results and are safe touse for skin. The present results indicates that the D. longan fruit peel extract has a good potential for cosmetic productdevelopment.Keywords: Dimocarpus longan, photoaging, antioxidant, evaluation, DPPH.INTRODUCTIONMalaysia’s climate is categorized as equatorial; being hotand humid throughout the year and Malaysian folks willexpose more to ultraviolet rays. Ultraviolet rays willfacilitate the aging process and will cause the decrease inskin elasticity. For this reason, the investigations of radicalscavenging activities are done to know the ability of theextract to combat or delay the photo aging process.D.longan is commonly known as dragon eye fruit orlongan, which is most prevailed fruits in Southeast Asia. Itcomes from the Sapindaceae family which is the samegroup as Litchichinensis L. (litchi) and Nepheliumlappaceum L. (rambutan). The extract of the D.longanfruits contained three major polyphenolic compoundswhich are corilagin, gallic acid, and ellagic acid which areresponsible for the antioxidant properties. Despite manyresearch is still ongoing for D.longan, however so far thelongan fruits extract have not been used as an antioxidantformulation for skin care. Thus, the present study focusedto develop a formulation that may scavenge free radicalsand protect skin against oxidative damage. The study fromNair et. al., concluded that it is possible to develop creams*Author for Correspondence:containing herbal extracts having antioxidant property andthey can be used as the alternative as a barrier to protectskin1. The litchi pericarps are proven to have highantioxidant cue to its high in phenolic compounds content2.The antioxidant activity of rambutan is due to the presenceof ellagic acid and gallic acid in the rambutan areresponsible for the antioxidant activity in the fruits 3. Thelongan seeds exhibited three major polyphenoliccompounds which are corilagin, gallic acid and ellagicacid. Longan seed water extract shows the scavengingactivity as good as white tea and dried longan pulp havethe least scavenging activity4. The study conducted toobserve the ability of natural products to suppress theproduction of oxidative stress and increasing enzymaticantioxidants in tissues. The result revealed that waterextract longan pericarp can inhibit production of oxidation,elevated antioxidant enzyme activities, and decreasedinflammatory response5. The longan seeds extracts havethe MMPIs activity which probably correlates with thehigh antioxidant properties in the D. longan6.MATERIAL AND METHODS

Ravindran et al. / Formulation and Evaluation Figure 1: Fruits of Dimocarpus longan.Formulation 1Formulation 2Figure 2: Formulated antioxidant creamsChemicals2,2 -Diphenyl-1-picryl hydrazyl (DPPH) was obtainedfrom Sigma Aldrich Co, St Louis, USA. Ascorbic acid wasobtained from S.D. Fine Chem, Ltd., Biosar, India. Allother chemicals used were of analytical grade.Collection and Identification10 kg of D. longan fruits was purchased from the localmarket and identified. Care was taken to select healthyfruits, the selected fruits were washed carefully with waterto remove dust and foreign materials.ExtractionThe peels and seeds of D. longan fruits were separated anddried in the oven at 40 C for 48 hours and grinded tocoarse powder using blender. The dried powder of peels(250 g) and seeds (250 g) of the fruits were individuallyextracted with methanol as solvent using Soxhletextraction method. Both the extracts were concentrated todryness under reduced pressure and controlled temperatureusing rotary evaporator. The percentage yield of theextracts were calculated. The collected extracts werestored in air tight containers in refrigerator at 4 C untilfurther study.Qualitative Phytochemical Analysis6The stock solution was prepared from the crude extract andwas dissolved in 10 ml of its own mother solvent. Theobtained stock solution were subjected to preliminaryphytochemical screening.Test for alkaloids: Mayer’s reagent, Dragondroff’sreagent, Hager’s reagent and Wagner’s reagent.Test for carbohydrates: Molisch test, Fehling’s test andTable 1: Composition of antioxidant cream.ComponentsAmount (% w/w)Active IngredientFormulation 1 Formulation 2(F1)(F2)D.longan Peel extract 2.5 %2.5 %Oily PhaseStearic acid7.00 %7.00 %Cetyl alcohol2.00 %2.00 %Mineral oil20.00 %20.00 %Aqueous PhaseGlycerin10.00 %10.00 %Methyl paraben0.05%0.05%Tween 802.00 %Triethanolamine2.00 %(TEA)Deionised water q.s100 %100 %Benedict’s test.Test for proteins: Biuret test.Test for glycosides: Keller-Killiani test, Borntrager’s testand Legal test.Test for fixed oils: Spot test.Test for tannins and phenolic compounds: Lead acetate testand gelatin test.Test for flavonoids: Shinoda’s test, test with Sodiumhydroxide solution and test with sulphuric acid.Test for steroids: Libermann- Burchard test.In vitro Antioxidant activityThe in vitro method is based on the inhibition. Samples areadded to a free radical – generating system, inhibition ofthe free radical action is measured and this inhibition isrelated to antioxidant activity of the sample. Method varygreatly as to the generated radical, the reproducibility ofthe generation process, and the endpoint that is used fordetermination. Both the extracts were tested for in vitroantioxidant activity. The final concentration of the extractand standard solutions used were 1000, 500, 250, 125,62.5, 31.25, 15.625 and 7.812 µg/ml. the absorbance onding blank solution.The percentage inhibition was calculated by using thefollowing formula.% Inhibition OD control – OD Sample 100OD controlIC50, which is the concentration of the sample required toscavenge 50% of free radicals was calculated.DPPH AssayThe present study on estimation of free radical scavengingactivity of peels and seeds of D. longan on 2,2-diphenyl1-picrylhydrazyl (DPPH) free radical was determined8.Reagents2, 2-Diphenyl-1-picryl hydrazyl solution (DPPH, 100µM): Accurately weighed 22 mg of DPPH in 100 ml ofmethanol. From this stock solution, 18 ml was diluted to100 ml with methanol to obtain 100 µM DPPH solution.Preparation of Extract SolutionsAccurately weighed 21 mg of each extracts and dissolvedin 1 ml of freshly distilled DMSO to obtain solutions of 21mg/ml concentration. These solutions were serially dilutedseparately to obtain the lower concentrations.IJPCR, Volume 8, Issue 9: September 2016Page 1306

Ravindran et al. / Formulation and Evaluation Table 2: Nature, Percentage Yield of the Extracts.ExtractNaturePercentageYieldCrudemethanol Light Yellow 5.26extract (Seeds)semisolidCrudemethanol Dark brown 30.10extract (Peels)semisolidTable 3: Phytochemical analysis of the extracts.PhytoconstituentsSeed extractPeel desPPFixed oilsPPTannins and Phenolic PPcompoundsFlavonoidsPPSteroidsPPA Absent, P PresentPreparation of Standard SolutionsAccurately weighed 10 mg of ascorbic acid and dissolvedin 0.95 ml of freshly distilled DMSO to get 10.5 mg/mlconcentration. These solutions were serially dilutedseparately to obtain the lower concentrations.ProcedureTo 2 ml of DPPH solution, 100 µl of each of the extract orstandard solution was added seperately. The solution wereincubated at 37 C for 30 min and the absorbance of eachsolution was measured at 490 nm using UVspectrophotometer9.Preparation of FormulationD. longan methanolic peels extract is used to prepare theantioxidant cream. For testing the maximum stabilitybetween the formulations F1 and F2, two types ofstabilizers were chosen such as tween 80 andtriethanolamine. The composition of the cream wereshown in Table 1. The formulation F1 and F2 adopts thesame method to prepare the cream. The oily phase andaqueous phase components were heated separately up to70 C and were mixed using homogenizer by addition ofmethyl paraben, extract and perfume. Care was taken forconstant and even mixing, the remaining deionised wateris added with continuous stirring until the mixture coolsand formed as cream. Base cream is prepared in the samemethod as formulation without extract. The formulatedcreams were shown in Figure 2.Evaluation of Antioxidant CreamThe following parameters were used to evaluate theantioxidant cream. The standard procedure was followedto evaluate all the parameters10.Physical PropertiesThe cream was observed for colour, odour and appearance.Determination of pHThe pH meter was calibrated using standard buffersolution. About 0.5 g of the cream was weighed anddissolved in 50 ml of distilled water and its pH wasmeasured.Determination of Emulsion Type (Dye test)The emulsion type was determined by using dye test. Thescarlet red dye is mixed with the cream. Placed a drop ofcream on a microscopic slide covers it with a cover slipand examined it under a microscope. If the disperseglobules appears colourless the ground is red, the cream isoil in water type. The reverse condition occurs in water inoil type cream. i.e. the disperse globules appear red in thecolourless ground.HomogenecityThe formulations were tested for the homogeneity byvisual appearance and by touch.After Feel EffectEmolliency, slipperiness and amount of residue left afterthe application of fixed amount of cream was checked.Loss on Drying1 g of cream was taken in china dish and kept in an ovenat 105 C for 2 hours.Rheological StudiesThe formulated cream was found to be non-newtonian.Take a fixed quantity 10 g of cream in a 10 ml beaker.Keep it impact for 1 hr. The beaker was inclined to oneside see whether consistency has changed or not. Thebeaker was again tilted and checked for pourability of thecream.Stability StudiesTo assess the formulation stability, stability studies werecarried out as per ICH guidelines. The cream filled bottlewas kept in humidity chamber maintained 30 2 C with65 5 % RH for two months. At the end of the studies,samples were analysed for the physical properties.Test for Microbial Growth in Formulated CreamsThe formulated creams were inoculated on the plates ofMuller Hilton agar media by streak plate method. Theplates were placed in the incubator and are incubated at37 C for 24 hours. After the incubation period, plates weretaken out and check the microbial growth by comparing itwith the control.RESULTS AND DISCUSSIONExtraction and Qualitative Phytochemical studiesThe percentage yield and nature of the extracts were givenin Table 2. The quantitative phytochemical analysis ofseeds and peels extracts showed the presence ofglycosides, flavonoids, tannins and phenolic compounds,carbohydrates, proteins, fixed oils and steroids as shown inTable 3.DPPH Radical Scavenging ActivityThe antioxidant activity of methanolic extracts of peels andseeds from D. longan were assessed using DPPH radicalscavenging activity. The results were shown in the Table 4and 5. The highest radical scavenging activity wasrecorded in the peels extract. Peels extract had the lowestconcentration to exhibit 50 % of the percentage inhibitionwhen compared to that of seeds extract. Moreover, whencompared to ascorbic acid, the ascorbic acid had higherantioxidant properties with 11.50 µg/ml compared to peels23.50 µg/ml and 32.13 µg/ml of seeds as shown in Table6. However, the extracts were found to be less activecompared to the standard ascorbic acid. Thus the result canconclude that the present study proposed that the D. longanIJPCR, Volume 8, Issue 9: September 2016Page 1307

Ravindran et al. / Formulation and Evaluation Table 4: DPPH radical scavenging activity of seeds extract.Concentration(µg/ml)S.D.Mean of % .28592.0510000.16294.98% inhibition SEM37.31 0.16239.87 1.61348.77 0.91174.05 0.43379.26 0.49190.97 0.58392.05 0.28594.98 0.162Table 5: DPPH radical scavenging activity of peels extract.Concentration (µg/ml)S.D.Mean of % 0596.5910000.41292.61% inihibition SEM40.91 1.5846.88 0.78353.41 0.48373.58 0.63994.6 0.62497.16 1.20696.59 0.50592.61 0.412Percentage of Inhibition (%)DPPH Scavenging Activity1009080706050403020100SeedsPeelsLinear (Seeds)Linear (Peels)0200400600800Concentration (µg/ml)10001200Figure 3: DPPH scavenging activity of peels and seeds.Table 6: IC50 value of standard vs sample extracts.ExtractsIC50 (µg/ml)Ascorbic acid11.50D. longan seeds extract32.13D. longan peels extract23.50peels methanolic extracts possess high potential ofantioxidant properties. The observed antioxidant effectscan be attributed majorly to the presence of polyphenoliccompounds in the D. longan. A comparison graph ofDPPH radical scavenging activity of peels and seeds ispresented in the Figure 3.Evaluation of CreamThe methanolic extract of D. longan peels were chosen toformulate the cream because of its higher antioxidantactivities when compared to seeds. The dye test confirmsthat the formulated creams were o/w type of emulsioncream. The pH of the formulated creams was found to be4.6 to 6.2. The formulated antioxidant cream wereevaluated for several physicochemical tests and the resultswere shown in Table 7. The formulated F1 and F2 creamsshowed pleasant odour and yellowish and light brownishcoloured creams respectively. The formulated cream wasnot greasy after application to the skin. The formulatedcreams were easily removable by washing with tap water.The cream showed homogenous distribution of extract inthe cream which was confirmed by visual examination.There was no change in colour of formulated cream uponkeeping for long time. After feel test showed that theformulated cream were emollient and slipperiness. Theloss on drying of the formulated cream was found to bewithin the limit to standard procedure. All thephysicochemical parameters were well maintained duringthe period of accelerated stability studies at temperatures80 C 0.1 C in refrigerator and at 25 C 1 C, 40 C 1 CIJPCR, Volume 8, Issue 9: September 2016Page 1308

Ravindran et al. / Formulation and Evaluation Table 7: Physicochemical Evaluation of the formulated llowish semisolid creamOdourGoodLoss on drying0.25 %SpreadabilityGoodAfter feelEmollients and slipperinessRemovalEasily removedStabilityUnstableMicrobial limit test 100 coloniesF1 Formulation 1, F2 Formulation 2in incubator for 8 weeks for both the formulations.However, the formulation 1 (F1) showed cracking and theglobules of the disperse phase become coalesced andshowed separation between oil and water phase. Theformulation 2 (F2) showed good stability in colour andconsistency until the end of accelerated study period.CONCLUSIONThe D. longan is commonly known as longan or dragoneye fruit in Malaysia and widely used for its medicinalvalue in the traditional system of medicine. The extract ofthe longan fruits contained three major polyphenoliccompounds which are corilagin, gallic acid, and ellagicacid which are responsible for the antioxidant properties4.The antioxidant creams are widely used today as it appearsto be an interesting way to safeguard the skin againstoxidative stress caused by various extrinsic sources. Tomaintain the effectiveness of antioxidants against freeradicals, it is important to stabilise the final formulation onits antioxidant properties. As a part of synergistic effectsthe current practice moves towards in the formulation ofdifferent combinations of antioxidants instead of singleantioxidant products. The present study revealed that thepeels were having higher radical scavenging activitycompared to that of seeds extract in DPPH method. Theevaluation test reveals that the formulated cream frompeels extract showed that it is safe to be used in the skin toprotect from extrinsic oxidation sources. Moreover, ourstudy presented that formulation of F2 is more stableduring the shelf storage. The research work suggests that,to ensure the quality and purity of the cream it must havethe consistency and uniformity in the ingredients of theherbal antioxidant cream. The trend of using herbal skincream is becoming in demand since it is proven that topicalapplication of anti-oxidant cream will be effective againstUV radiation and protect the skin from major consequenceof UV damage. In conclusion, the topical application of theformulated cream from D. longan extract will help inreducing oxidative damage and give the antioxidant effectto our skin due to its high antioxidant values. Inconclusion, the topical application of the formulated creamfrom D. longan extract will help in reducing oxidativedamage and give the antioxidant effect to our skin due toits high antioxidant values.F2HomogenousLight brown semisolid creamGood0.11 %GoodEmollients and slipperinessEasily removedStable for two months 100 coloniesREFERENCES1. Nair SS, Mathew M, & Sreena K. Formulation andevaluation of herbal cream containing Curcuma longa.International Journal of Pharmaceutical and ChemicalSciences, 2012, 4, 1362 - 68.2. Li W, Liang H, Wei Zhang M, Fen Zhang R, YuanDeng Y, Cheng Wei Z, Zhang Y, Jun Tang X. Phenolicprofiles and antioxidant activity of litchi (LitchiChinensis Sonn.) fruit pericarp from differentcommercially available cultivars. Molecules. 2012, 17,14954-967.3. Chiaw Mei WS, Ismail A, Esa NM, Akowuah GA, WaiHC, Seng YH. The Effectiveness of Rambutan(Nephelium lappaceum L.) Extract in Stabilization ofSunflower Oil under Accelerated Conditions.Antioxidant. 2014, 3, 371-86.4. Rangkadilok N, Worasuttayangkurn L, Bennett RN,Satayavivad. Identification and Quantification ofPolyphenolic Compounds in Longan (Euphorialongana Lam.) f

Ravindran et al. / Formulation and Evaluation IJPCR, Volume 8, Issue 9: September 2016 Page 1306 Tween 80 Figure 1: Fruits of Dimocarpus longan. Formulation 1 Formulation 2 Figure 2: Formulated antioxidant creams The added to a free radical Chemicals 2,2 -Diphenyl-1-picryl hydrazyl (DPPH) was obtained from Sigma Aldrich Co, St Louis, USA .

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