Formulation & Evaluation Of Naringin Nanoethosome By Cold Method

Pawar et al.; JPRI, 33(42B): 139-152, 2021; Article no.JPRI.733062.3.5 Ultraviolet -visible spectroscopy [21-23]Preparation of calibration curve by U.V.Visible Spectrophotometric method:A. Determination of λmax in EthanolThe UV spectrum of Naringin was obtained usingUV Shimadzu 2450, Japan. Accurately weighed10 mg of the drug was dissolved in sufficientquantity of ethanol and volume made up to 10ml. The stock solution was diluted to get aconcentration of 100 µg/ml. The 1 ml of aliquotwas withdrawn and volume was made up to 10ml using ethanol to obtain the concentration of 10µg/ml. The resultant solution was scanned from200 to 400 nm and therefore the spectrum wasrecorded to get reading of maximum wavelength.Results are shown in Table 3 and spectrum ofnaringin in ethanol shown in Fig. 2ConstructionethanolofBeers-Lambert’splotinThe stock solution of 100µg/ml was prepared inethanol, used to prepare different dilutions in therange of 5-25 µg/ml. The absorbance of resultingsolutions ware measured at 283 nm by UVvisible spectrophotometer.The results are shownin Fig. 4B.Determination of λmax in pH 7.4 phosphatebufferPreparation of 7.4 pH phosphate buffer:0.2M of potassium dihydrogen phosphate(KH2PO4) solution was prepared by dissolving2.722 gm KH2PO4 of in 100 ml distil water inanother 100 ml of volumetric flask, solution of 0.2M sodium hydroxide (NaOH) was prepared bydissolving 0.8 gm NaOH in 100 ml distil water. 50ml of 0.2M KH2PO4solution was taken in anotherbeaker and specified volume of 0.2N NaOHsolution (22.4 ml) was added in it and the volumewas adjusted with distil water to 200 ml.Preparation of stock solution10 mg of Drug was accurately weighed and wasdiluted to 100 ml using phosphate buffer pH 7.4to get a final solution of conc. 100 μg/ml. Thissolution was used as stock solution. From thisstock solution, 2.5ml of Aliquot was withdrawnand volume made upto 10 ml with phosphatebuffer pH 7.4 to obtain the solution withconcentration 25μg/ml. The UV Spectrum wasrecorded in the wavelength range 200-400nm.Wavelength of maximum absorbance wasdetermined. Results are shown in the Table 3and spectrum of Naringin in phosphate pH7.4shown in Fig.3.Construction of Beers-Lambert’s plot in pH7.4 phosphate bufferThe stock solution of 100µg/ml was prepared inpH 7.4 phosphate buffer, used to preparedifferent dilutions in the range of 5-25 µg/ml. Theabsorbance of resulting solutions ware measuredat 291 nm by UV-visible spectrophotometer. Theresults are shown in Table 3 and Fig. 5.2.3.6 Fouriertransformsinfraredspectroscopic (FTIR) studies [24]The infrared absorption spectrum of Naringinwas recorded with KBr. The dried sample of drugwas mixed with KBr in the ratio of 1:9, thesample triturate and finally placed in sampleholder. The spectrum was run over the wavenumber 4000 to 650 cm-1 using FourierTransformInfraredSpectrophotometerShimadzu, 8400S, Japan. The spectral analysiswas done, by standard absorbance of thefunctional groups. Peaks observed are shown inthe spectrum Fig. 6.2.3.7 Differential scanning calorimetric (DSC)studies [13]DSC analysis was performed using DSCinstrument Shimadzu, DSC 60 by taking samples(2to5) samples were hermetically seals inaluminium pan and with nitrogen flow rate10ml/min conducted over a temperature range of30-2000c.DSC analysis was also performed toascertain the purity and identification of drug.The instrument was calibrated with indiumstandard. Accurately weighed samples wereplaced in open, flat bottom, aluminum samplepans. Thermograms were obtained by heating0the sample at a constant rate of 10 C/minute. Adry purge of nitrogen gas (20ml/min) was used0for all runs. Samples were heated from 35 C –0250 C. Scans were obtained from the samples.The melting point and the peak maxima wereobserved in the DSC graphs. DSC thermogramis shown in Fig. 7.2.3.8 Evaluation of nanoethosomesDrug Entrapment efficiency [4-9]The Encapsulationnanoethosomes was142efficiency (EE%) ofdetermined using the

Pawar et al.; JPRI, 33(42B): 139-152, 2021; Article no.JPRI.73306dialysis technique for separating the nonentrapped naringin, 1 ml of naringin-loadednanoethosome dispersions were dropped into adialysis bag (molecular weight cut off: 10 kDa)immersed in 50 ml of methanol solution andshook at 50 rpm. 1 ml of ethanol solution wastaken out after 2 h and make a appropriatedilution and then analyzed by UV to account thequantify the unentraped drug (Wf). Then 1 ml ofnaringin -loaded nanoethosome dispersions weredemulsified with 25 ml methanol and theobtained solution was detected as total drug indispersion (Wt). The encapsulation efficiency(EE%) could be counted by the followingequations. results are shown in Table 5.Drug entrapment ef iciecyActual drug content in nanoethosome 100Theoretical drug contentActual drug content [13,18]Precisely weighed equivalent quantity (10 mg) ofnanoethosomes containing drug was kept in 100ml of phosphate buffer pH 7.4 solutions for anhour with continuous stirring. Filtered sampleswere further analysed at 283 nm next to blankusing UV-visible spectrophotometer (Shimadzu,UV-2600)Actual Drug content (%) (Nact / Nms) * 100values are observed in monodisperse sample,whereas higher PDI value indicates particle sizedistribution and the polydisperse nature of thesample.PDI can be calculated by using followingequationPDI ΔD/DaveWhere, D is distribution donated by SD and Daveis the average particle size.Vesicle morphology [27-28]Morphology of the vesicles was visualized byscanning electron microscope (SEM). A drop ofnanoethosome dispersion was placed on acarbon coated grid to leave a thin film, before thefilm got dried on the grid, it was negativelystained with 1% phosphotungstic acid. A drop ofstaining solution was added on the film, and theexcess of the solution was drained off with a filterpaper. The grid was allowed to air dry, andsample was viewed by SEM at 80 kV.Morphology of vesicle are shown in Fig. 10.3. RESULTS AND DISCUSSION3.1 Preformulation StudiesWhere Nact actual naringin content in weighedquantity of nanoethosomes,Nms weighedquantityofnanoethosomes andNthe theoretical naringin content innanoethosomesOrganoleptic propertiesParticle size and zeta potential determination[25-26]Melting Point1 ml of nanoethosomal suspension was dilutedby 10 ml of double distilled water and thenvesicle size and zeta potential were determinedby a dynamic light scattering particle sizeanalyzer Malvern Zetasizer, (Malvern InstrumentLtd.).Meanvesiclesizeandzetapotential graphical representation given in Fig. 8and 9.Polydispersibility index [27]The Polydispersity index (PDI) is an index ofwidth or spread or variation within the Particlesize distribution. PDI can be determined bydynamic light scattering instrument. Lower PDIThe sample of drug received was studied for itsorganoleptic characters which shows light yellowcolour, odourless in nature and amorphous innature.Melting point of pure Naringin is found in 167171 c which is found to be nearly to standardmelting range of naringin. That indicates thepurchased samples obtained were of purequality.SolubilityThe drug naringin was found practically insolublein distilled water, while the solubility of pureNaringin in ethanol, water and in phosphatebuffer pH 7.4 was found to be 2.7, 11.8 and 11.2mg/ml respectively. It indicates that the drug issoluble in ethanol, methanol as well as sparinglysoluble in phosphate buffer ph 7.4.143

Pawar et al.; JPRI, 33(42B): 139-152, 2021; Article no.JPRI.733063.2 Ultraviolet-VisibleStudySpectroscopy8.1.4.1: Determination of λmax of Naringin inethanol and 7.4 phosphate bufferThe UV spectrum of Naringin solution in ethanoland phosphate buffer pH 7.4 exhibitedwavelength of absorbance maximum at 283.73nm, 291.09 respectively. This is near to thereported value. However, keeping in mind theprobable concentrations likely to be encounteredwhile carrying out In-vitro release studies andconsidering the predicted theoretical λmaxinvolved, the working λmax was decided as283.73nm in ethanol and 291.09 in phosphatebuffer pH7.4. The spectrum of Naringin is shownin (Fig.2 and 3).0endothermic peak at 167 c corresponding to themelting of the pure drug and naringinnanoethosome showed DSC thermogram atrange of 159.69 which shows that slightly shiftingof drug in peak which is due to amorphization ofnaringin in nanoethosomal matrix. Dsc resultindicating that there is absence of incompatibilitybetween drug & excipients.4. EVALUATIONOFNANOETHOSOMESNARINGINEvaluations of prepared nanoethosomes ofNaringin were carried out for Actual DrugContent, entrapment efficiency and vesicle sizeand morphology study.4.1 Actual Drug Content3.3 Construction of Beers-Lambert’s PlotThe calibration curve (Fig. 4 and Fig. 5) wasfound to be linear in the concentration range of 2to 25μg/ml (Table 3) having coefficient ofregression value R2 0.998 and Slop y 0.053x 0.002 in ethanol and in pH 7.4 phosphatebuffer having coefficient of regression value R2 0.994 and y 0.0108x 0.008.3.4 FITR Spectroscopy of Naringin4.2 Entrapment EfficiencyThe powdered mixture of Naringin and KBr wastaken in a sampler and the spectrum wasrecorded by scanning in the wavelength region of4000- 700 cm-1 using FTIR spectrophotometer.The FTIR spectrum of naringin was shown in(Fig. 6 ) and principle peaks obtained at wavenumber 1818 cm-1 for aromatic C O, 3416 cm -1-1for O-H stretching, 1361 cmindicates thatpresence phenol ring, 2957-2859 cm -1 foraromatic & aliphatic C-H stretching. The naringin1ethosome shows peaks at 3416 cm ,1361 cm1-1-1,1818 cm , and 2957 cm which confirmed thatno changes in drug peak at both spectra.Absorption bands shown by Naringin andNaringinnanoethosomesshowsallcharacteristics of the groups present in itsmolecular structure. The presence of absorptionbands corresponding to the functional groupspresent in the structure of Naringin confirms theidentification and purity of purchased Naringinsample.3.5 Differential Scanning(DSC) StudiesThe uniform dispersion of drug in thenanoethosome can be determined by drugcontent analysis. It was observed that around59.6 to 83.9 % drug can be incorporated in thenanoethosome F4 batch showing uniformdispersion of drug in the nanoparticle.DrugcontentanalysisareshowninTable 4.CalorimetricDSC thermogram for Naringin is shown in theFig. 7. DSC studies indicated a sharpAfter preparing nanoethosomal dispersion,unentrapped drug is separated by dialysismethod described above and the drug remainedentrapped in nanoethosome is determined bySpectrophotometric method.% Entrapment efficiency (% EF) (Amountof drug entrapped/ total amount of drug) x100Nanoethosome consisting lipid & surfactantsdisplayed diverse % EE. Maximum %EE(83.48%) was obtained from nanoethosomeprepared with SPC (F4). The results could beexplicated as that the entrapment of lipophilicdrug into lipid vesicle was facilitated by drugdistribution coefficient between lipid phase andaqueous solution. From results obtained, theaffinity of surfactant to lipid is decrease. Basedon affinity of surfactant with lipid and stronglipophilic of a drug (logP 3.31), naringin wouldbe more prevalent dispersing in double layer lipidestablished by PEG and soya lecithin in ratio of100:500 (F4).The entrapment efficiency wasshown in Table.5144

Pawar et al.; JPRI, 33(42B): 139-152, 2021; Article no.JPRI.733064.3 Vesicle SizeDeterminationandZetaPotentialVesicle SizeParticle size analysis was done by MalvernZetasizer particle analyzer.The vesicle size ofnanoethosome varies with different ratio of lipiduse. One from each batches are subjected forvesicle size determination, depending on %entrapment efficiency.There were osome consisting different concentrationof SPC. Nanoethosome containing SPC andPEG expressed a mean particle size of 145.5nm. In general, particle size increased with druglipid ratio increased from 1:5, 1:7 and 1:2. andGraphical representation of size determinationgiven in Fig. 8.4.5 Poly Dispersibility Index: (PDI)PDI is an index of width or spread or variationwithin the particle size distribution. Monodispersesample have lower PDI value, whereas PDI ofhigher value indicates a wider particle sizedistribution and polydisperse nature of sample.PDI can be calculated by the following equationPDI Δd/davg. Where, Δ dis the width ofdistribution denoted by SD and davg. Is theaverage particle size denoted by MV (nm) inparticle size data.PDI Δd/davg 0.4As depictedin Fig. 8 the nature ofnanoethosome formulation for the optimizedbatch shows Mid-8Range monodisperse. Thepolydispersity indices of nanoethosomes wereshown at 0.354 Therefore it can be stated thatthe polymer based nanoparticle prepared by coldmethod have exhibited a homogeneous sizedistribution.4.6 Zeta Potential AnalysisThe zeta potential of optimized batch was foundto be -9.31 mV which is in good agreement withliterature due to the net charge of the lipidcomposition in the formulations. PC is a zwitterionic compound with an isoelectric point [pI]between 6 and 7. Under experimental conditionsof pH 7.4, where the pH was higher than its pI,PC carried a net negative charge. The edgeactivator used was anionic edge activator, andthe anion form of naringin was also thepredominant form at pH that pH. Therefore, anegative charge in all formulations wasobserved.Alsothenegatively chargednanoethosome formulations may improve skinpermeation of drugs in transdermal delivery. Theskin also has slight negative charge. Therefore,the negative zeta potential of the nce improved naringin permeation throughporcine skin due to electrostatic repulsionbetween the same charge of the skin surface andthe optimized formulation. Peak of zeta potentialis displayed in Fig. 94.7 Vesicle MorphologyThe morphology of naringin nanoethosomaldispersion (F4) was as shown in Fig. 10 Fromthe micrograph, it could be observed thatunilamellar vesicles with an intact bilayermembrane were formed. The particle size of thevesicles was about 145.9 nm.Fig. 1. Component of ethosomal system145

Pawar et al.; JPRI, 33(42B): 139-152, 2021; Article no.JPRI.73306Fig. 2. UV-visible spectrum of Naringin in EthanolFig. 3. UV-visible spectrum of Naringin in phosphate buffer PH 7.4calibration curve of naringin inEthanol0.6Absorbance0.50.40.3y 0.053 0.007R² 0.9980.20.100246Concentration810Fig. 4. Calibration curve of naringin in ethanol14612

Pawar et al.; JPRI, 33(42B): 139-152, 2021; Article no.JPRI.73306calibration curve of naringin inphosphate buffer 7.40.6absorbance0.50.40.3y 0.108x 0.008R² 0.9940.20.100123concentration45Fig. 5. Calibration curve of naringin in phosphate buffer PH7.4Fig. 6. FTIR spectra of naringinFig. 7. DSC Thermogram of Naringin1476

Pawar et al.; JPRI, 33(42B): 139-152, 2021; Article no.JPRI.73306Size Distribution by16141210110101001000SizeRecord 627: Nanoethosome NaringinFig. 8. Particle size distribution of optimized batchZeta Potential2000150010005000-010Record 628: Nanoethosome NariginFig. 9. Zeta potential of optimized batchFig. 10. Vesicle morphology by using scanning electron microscopy14820

Pawar et al.; JPRI, 33(42B): 139-152, 2021; Article no.JPRI.73306Table 1.Variables in Box-Behnken design for preparation of naringin nanoethosomesFactorLevel Used,Actual (Coded)Medium(0)High( 1)Low(-1)Independent variablesX1 soya phospholipid (gm)X2 polyethylene glycol(ml)X3 Ethanol(ml)200410500520700630Dependent variablesY1 Entrapment efficiency(%)Y2 Particle Size (nm)Table 2. Formulation of naringin nanoethosome.Formulation codeF1F2F3F4F5F6F7F8F9FactorX1SPC( mg) 1 1 1000-1-1-1FactorX2PEG(ml) 10-1 10-1 10-1FactorX3Ethanol(ml) 1-10-10 10 1-1Drug entrapmentEfficiency (%)Y1Particle size(nm)Y279.91%79.83 %75.83 %83.60 %80.66 %80.16 %73.44 %73.40 %74.05 le 3. Absorbance’s of different concentration of NaringinIn EthanolSr. No.12345Concentration inμg/ml (ppm)246810Absorbance0.1120.2240.3120.4350.534pH 7.4 phosphate BufferConcentration in Absorbanceμg/ml (ppm)50.121100.244150.389200.494250.598Table 4. Actual drug content and Drug Loading of Naringin F4F5F6F7F8F9Actual drug content%70.3 %68.89 %71.78 %83.67 %80.4 %81.2 %63.5 %59.6 %70.45 %149Drug loading%75.7 %75 %73.9 %78.5 %78 %77.77 %63.23 %60 %71.42 %

Pawar et al.; JPRI, 33(42B): 139-152, 2021; Article no.JPRI.73306Table 5. Entrapment Efficiency of Naringin NanoethosomeBatch CodeF1F2F3F4F5F6F7F8F9Absorbance at283 125.9122.21Unentrapeddrug (%)27.9134.1125.9116.5117.6120.8125.5125.9122.48% Entrapment 74.5877.52the producing company rather it was funded bypersonal efforts of the authors.5. CONCLUSIONIt has been nearly twenty years since theinvention of ethosomes and through this erathese nanocarriers have proved their distinctiveability to deliver therapeutic agents. From allabove observations and results obtained, it canbe concluded that all the formulations ion study of drug and excipientsshows purity of drug and excipients. .Thecompatibility study of drug and excipients wasperformed and it was found that the drug iscompatible with excipients used in formulation.Nanoethosomes of Naringin were successfullyformulated by cold method and examined forentrapment efficiency, drug content, size andshape of vesicle and morphology. The resultsobtained from study confirms, maximumentrapment efficiency and drug content werefound of F4 batch, hence it was taken asoptimized batch. It shows % entrapmentefficiency of 83.48%, percent drug content of95.16%. Also the average mean diameter ofoptimized batch of nanoethosomes was found tobe 145.7 nm. The zeta potential was found to be-9.2 mV. The vesicles were found as unilamellar.Hence it can be concluded that aboveformulation can be effectively used innanoethosomal delivery.CONSENTIt is not applicable.ETHICAL APPROVALIt is not applicable.COMPETING INTERESTSAuthors haveinterests i H, Aswathi R, Hegde RS,Managuli K, Bhaskar. Naringin nanoethosomal novel sunscreen creams:Development and performance evaluation.Published by Elsevier; 2020.2.Touitou E. Inventor. Composition ofapplying active substance to or through theskin. US patent. 1996;5:540-934.3.Patel S. Ethosomes: A promising tool fortransdermal delivery of drug, PharmaInfo.Net. 2007;5(3).DISCLAIMER4.The products used for this research arecommonly and predominantly use products in ourarea of research and country. There is absolutelyno conflict of interest between the authors andproducers of the products because we do notintend to use these products as an avenue forany litigation but for the advancement ofknowledge. Also, the research was not funded byTouitou E, Dayan N, Bergelson L, Godin B,Eliaz M. Ethosomes novel ion and skin penetra

Formulation & Evaluation of Naringin Nanoethosome by Cold Method Ashish Y. Pawar1*, Khanderao R. Jadhav2, Komal Naikwade1 . Finally, the formulation is stored under refrigeration [3,4,9,10]. 2.3 Characterization 2.3.1 Preformulation studies [14-16] Preformulation is that the initiative in designing or