Research Journal of Pharmacy and Technology ISSN: 0974-360X(Online), - 0974-3618(Print) Home | About RJPT | Current Issue | Archives | Editorial Board | Instructions | Paper ‘Submi News & Conferences. | Subscription | Corrigendum | Contact Us 2 eprroriaL BOARD Editor In Chief Dr. Mrs, Monika S, Daharwal Address : RJPT House, Lokmanya Grih Nirman Society, Rohanipuram, In- front of Sector- 1, Pt. Deendayal Upadhyay Nagar, Raipur 492 010, (CG) India Email ID : editor.rjpt@gmail.com Associate Editors Dr. A.K. Jha Address : Principal, Shri Shakaracharya College of Pharma, Sciences, Bhilai CG India Email 1D jhaaak@rediffimail.com Dr. R. B. 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[HTML Paper) [MTMLPaperaspe?Joumal-Receareh Journal of Pharmacy and TechnologyPIO=2019 128 $) (ce) ‘A em: (pt, rt) Private Access Pretiminary Phytochemical Analysis and eeation and identiiatlonof ative principle components of petroleum ether extract. {tom eaves of LawsoniainermisL(AbstractVlew.aspx?PID=2019-12-48) lAbstract) (PDF Paper) {HTML Paper] [HTNLPaper.asor?Jaurnal-Research Journal of Pharmacy and Technology PI0=2019-12-8-9) ice) Pvt: 0 ct, 2 ert) Private Acess mete ogtntpptnvgcr) | ARMghs reseed | Stenap (stems sp) > Deen and woes Sop TsbeReearh hoeNsbrearch cami) 8/2/2019, 1:59 PMRIPT - The Anti-inflammatory Activity of p-methoxycinnamic aci, ‘hutp://rjptonline.org/ Abstract View.aspx”PID=2019-12-8-7 Qyp Pepseret servo 2 Interatonal Conference on Fostering Interplay Research neath senses (ICFIRNS) 2019 (News Feds) (1-M&y-2019) | 10f2 sous ese San fttomec on Teeny vane veors2018 ope: 308368 ISSN Print : 0874-3618 y sso: oat i son Already Registered eet Login The Anti-inflammatory Activity of p-methoxycinnamic acid (PMCA) in the Nanostructured lipid carrier (NLC) system using combinations of solid lipid, beeswax- oleum cacao and liquid lipid, Virgin Coconut oil (VCO) (AbstractView.aspx?PID=2019-12-8-7) Iristiana Erawae heps:/scholar google.coInscholar?g="Tristiana Erawat!’), Dew Melani ‘Mariyadi Qnaps:scholargoogle.conscholar?q~"Dewi Melani Harlyadi"), Noarma Rosita (dutps:schotargoogle.conscholar?q~"Noorma Rosita’), Tuiek Purwandh (hps/echolar google codivecholar?q="Tutiek Purwan") cr.tnepae cif nance tneyaoo ee). ann elena naitaetanse mean 20) Address: Pian Ot aH Noms Roe Tate Porat ‘rman armen Dharma og tnt, Shy does ‘Conon tar Ol No: 0.5888/0874-360K.2019.00617.6 _htips/scholar google.cai/scoar?g"105958 874. 36042018.20617.6") ‘Objective: The nanostructured lp ear (NLC) stem use the combination of besa oleum cacao a virgin acon ol (¥CO in Ws study, was the development of nanoaraulsion NE) and sod Unidnanagartcle (LN) desvery sytem, which ae used as nano carers pmethorycinnamic acid (PCA) PMCAasecunder metabolite of champheragalnga has antnflamatory eflect which is GHRCU to dissolve in wate, Several combination of sald pis beeswax-cleum cacao} and quid pis VEO) were prepared, namely | (040); (2030; an Fi (82:20) in the NLC syrtom, Material and Method: The NIGPMCA system was prepared by the high shear homogenization method, than choracteaed and subjected to aninfamvmtory 2thty tests compared with ‘anoemulkon ad solid pi nanoparticle (LN). Th oslo the research: The increasing concentration of VCO In the NLGPMCA system rested in loner viscosity, and greater entrapment eficency, nary in the HLC-PMCA system with a combination of beeswas-cleum cacao and VCO of 63:40, NUCPMCA with the combination of boeswaleum cacao and VEO of 6040 (has parce sie (236.0) = 17-15 nm) smaller han SLN-PMCA (68550 + 7259 rm) bu bigr than NE-PMCA (29.27 = 1.85 am) Deduct ofF | NLC PMA a0 400) ge than SUN MCA 3.523.659] an NE-PNCA 3352 #365) PMCAinF1(60:40) NLC, SLN and NE has the equivalent at Inflammatory activ based on the observations ofthe PMN cells number in er skin mice with edema. Conclusion: The NLC PHCAwsth the combination of beeswax oleum cacao and ¥CO of 640 ws the best forma erwonos: sntinlameatry activity, beeswax, nanostructure lipid cantar (NLC, oleum e330, pmethoujinnamie acd (PMA virgin coconut UCD), ‘Wstiana Erowat, Dew Melani Horyadl, Noorms Rost, Tue Purwant. The Antnfiammatory Ativty of prmethorycnnamic acid (PHA) inthe Nanostctured pid cater (NLC) stem using combination fsb ip, ‘beesnaxoleum £300 an4 ligid Hpi, gl Coconut ofl VCO), Research 3. Phorm. ond Tech 2018, 128 26193625. {73 ew oy prtouraperasptours-Reench onal of hamacy ad TechnlogO-2181287) [POF PAncR How 822019, 2:36 PMRIPT - The Anti-inflammatory Activity of p-methoxycinnamic aci ‘http://¢jptonline org/ Abstract View aspx? 2 sone see pte tiptonieeor/) | Alighsrurd | Stamp (stenapase) Tyce 20f2 8/2/2019, 2:36 PMResearch J. Pharm. and Tech 12(8): August 2019 ISSN 0974-3618 (Print) RESEARCH ARTICLE www.rjptonlineorg. ARIPT ‘The Anti-inflammatory Activity of p-methoxycinnamic acid (PMCA) in the Nanostructured lipid carrier (NLC) system using combinations of solid lipid, beeswax-oleum cacao and liquid lipid, Virgin Coconut oil (VCO) ewi Melani Hariyadi, Noorma Rosita, Tutiek Purwanti Department of Pharmaceutics Faculty of Pharmacy, Airlangga University, Surabaya, Indonesia, "Corresponding Author E-mail: era_ffua@yahoo.co.id, tristiana-e-m@ff-unair.ac.id ABSTRACT: ‘Objective: The nanostructured lipid carrer (NLC) system used the combination of beeswax-oleum cacao and virgin coconut oil (VCO) in this study, it was the development of nanoemalsion (NE) and solid lipid nanoparticle (SLN) delivery systems, which are used as nano-carriers of p-methoxycinnamie acid (PMCA). PMCA a secunder metabolite of champheria galanga has antinflamatory effect which is difficult to dissolve in water. Several combinations of solid lipids (beeswax-oleum cacao) and liquid lipids (VCO) were prepared, namely F I (60:40); F If (70:30); and F TH (80:20) in the NLC system. Material and Method: The NLC-PMCA system was prepared by the high shear homogenization method, then characterized and subjected to anti- inflammatory activity tests compared with nanoemulsion and solid lipid nanoparticle (SLN). The results of the research: The increasing concentration of VCO in the NLC-PMCA sysiem resulted in lower viscosity, and sreater entrapment efficiency, namely in the NLC-PMCA system with a combination of beeswax-oleum cacao and VCO of 60:40, NLC-PMCA with the combination of beeswax-oleum cacao and VCO of 60:40 (F 1) bas particle size (236,00 + 17.15 nm) smaller than SLN-PMCA (665.60 + 72.59 nm) but biger than NE-PMCA, (29.27 + 1.85 nm), Occlucivity of F INLC-PMCA (41.50 + 4.00 %) higher than SLN-PMCA (33.52 = 3.65 °%) and NE-PMCA (33.52 4 3.65 %). PMCA in F I (60 : 40) NLC, SLN and NE has the equivalent anti- inflammatory activity based on the observations of the PMI cells number in car skin mice with edema Conclusion: The NLC-PMCA with the combination of beeswax-oleum cacao and VCO of 60:40 was the best, formula KEYWORDS: anti-inflammatory activity, beeswax, nanostructured lipid carrier (NLC), oleum cacao, p- ‘methoxycinnamic acid (PMCA), virgin coconut oil (VCO), it was determined that PMCA INTRODUCTION: In a previous study p-methoxyeinnamic acid (PMCA), which is difficult to dissolve in water, was formulated in the nanoemulsion [1]. Tk was known the first colloidal system based on nanotechnology that successfully carries active ingredients through the skin barrier for topical use is the nancemulsion system [2,3]. The PMCA nanoemulsion system using a combination of surfactants (Tween 80 and Span 80), ethanol 96% o« surfactant, and several plant oil phases, namely VC‘ ‘corn oil, and soybean oil [3]. Received on 21.02.2019 Modified on 11.03.2019 Accepted on 06.04.2019 © RIPT All right reserved From the study, ‘nanoemalsion that uses VCO as an oil phase produces the smallest droplet size. This is because VCO contains faty acids with C atomic chains, which are shorter than the fatty acid content in corn oil and soybean oil. The smaller size of nanocmulsion droplets results in the greater rate of release of drug ingredients from the carrier system and the tate of penetration of drug ingredients into the skin, so the resulting effectiveness is, also higher. However, being in storage for more than 2 months, PMCA nanoemalsion preparations. tend to coalescence, so the size of the nanoemulsion droplets grows larger [4]. Than the PMCA formulated in Solid Lipid Nanostructure (SLN) systems, the liquid lipid is replaced with solid lipid using cetylalcohol and Tween '80 as surfactant can inhibit release and extend their anti- 3619Research J Pharm. and Tech 12(8) August 2019 inflammatory effects [5] However, the use of solid lipids that are too ordered on the storage the active ingredients {ends to be pushed out, so a combination of solid lipids and liguid lipids is needed to overcome this. The improvement of the SLN system is the NLC system, which consists of a certain amount of solid lipid and liquid lipid matrices. NLC remains in its solid form by controlling the levels of liquid lipids added to the formulation, so the controlled drug release properties for NLC can be achieved [6]. Liquid lipids in NCL can reduce crystalline formation in solid lipids thus increasing system stability on storage [7,8]. Various liquid oils and solid lipids can be used as NLC components, among others for example combination of olive oil and palmitic acid (1.8:4.8) as NLC system to deliver ubiquinone (Q10) as an antiaging [9]. In this research the NCL system as PMCA carrier used ‘VCO as liquid lipid combined with combinations of ‘oleum cacao and beeswax as solid lipid in various ratios and Tween 80 and Span 80 as stabilizer. Furthermore, in the NLC-PMCA system formed characterization and in vivo effectiveness tests are carried out, MATERIALS AND METHODS: Materials: ‘The materials used in the study which unless otherwise stated have pharmaceutical grade purity are p- methoxycinnamic acid (Sigma Aldrich), Tween 80 (Sigma Aldrich), Span 80 (Sigma Aldrich), VCO (UD. Cocos Coconut), beeswax (PT. | Kumniajaya Muktisentosa), oleum cacao (Coffee and Cacao Research Center, Jember-Indonesia), Popylen glycol (PT, Brataco), Sodium benzoate (PT. Brataco), Ethanol 96% pa (E Merck), Na acetate pa. (E Merck), and ‘Acetic acid p.a. (E Merck) Sample preparation: NLC-PMCA system with different ratios of oleum ceacao-beeswax as solid lipids and VCO as liquid lipids, namely F I (60:40), F I (70:30), and FIIL (80:20) and SLN-PMCA as shown in Table 1, were prepared using high shear hot homogenization method by Ulua-Turrax High Shear Homogenizer IKA T-25. The NLC-APMS system is made by melting becswax at 70 °C. After ‘Table 1 Material Composition in NLC-PMCA, SLN-PMICA and NE-PMCA. beeswax melts, add oleum cacao to beeswax and let the oleum cacao" melt, The APMS which has been dissolved in VCO added into the mixture of beeswax- oleum cacao. And Tween 80 and Span 80 heated at 70 °C mixed withthe lipid phase. At the same time sodium, benzoate, propylenglycol and phosphate buffer pH 4.2 = 0.02 were heated at 70°C as water phase, Then the water phase is slowly dispersed into the lipid phase which is stirred with Ultra-Thurax High Shear Homogenizer at a speed of 5000 rpm until all phases of water are ‘exhausted then continued by adding stirring speed to 16,000 rpm for 2 minutes. The next process is. the cooling process, which is carried out with a speed decrease at 500 rpm stirred using a magnetic stirrer to reach room temperature (25°C), For NE-PMCA preparation, all components were mix by magnetic stirrer at room temperature, 500 rpm, 5 minutes, than increase mixing speed to 1000 rpm, 10 minutes. ‘Sample characterization: (Characterization of the sample includes: 1) the pH value inspection using pH-meter, 2) viscosity measurement using a cone and plate viscometer, 3) particle size measurement and polidispersity index using Delsa™ Nano Submicron Particle Size and Zeta Potential Dynamic Light Scattering, 4) and the melting temperature using Differential Scanning Calorimeter (so), Entrapment efficiency (%EE): Measurements of percent entrapment efficiency (EE) were carried out by diluting 100 mg NLC with acetate buffer pH 4.20 + 0.2 then centrifuged 2500 xpm for +45 minutes and filtered using filter paper. The supernatant was pipetted 2.0 ml and added acetate buffer pH 4.20: 6.2 to 10.0 ml in the measuring flask. The solution was filtered again using Whatman Millipore filter paper 0.22 jum. Then the solution was measured at the maximum wavelength of the PMCA which is then compared with the absorption of standard solutions. The data obtained is the concentration of PMCA in the NLC water phase. The calculation of % EE (percentage of entrapment eificieney) used the following equation (1) ‘Name “oncentation (bi) Eee Matertas FH 70:30) Fil @0:20) SIN NE DMCA. 020, 020, 020 020, ‘Oleum Case 396, 495 Beciwas 132 Ls = VCO. 7 132 Tween 80 So a8 ‘Span 80 hase Tet Propylene glycol 330, 3350 Sodium benzoate a1 010, 010 Buffer Acetate pil ‘Ad 100, ‘Aa 100 Tad 100Research J. Pharm. and Tech 12(8): August 2019 Woica drug — Wrree durg EE (*%)=— «100% Wo drug Notes: Wil ang! The amount of active ingredients used Weer dn: The amount of free active ingredients that are in the water phase Furthermore, the average, SD, and CV calculation were calculated from the efficiency of PMCA entrapment in the NLC systern [7] Octusivity test: ‘The occlusivity test was carried out by in vitro method, Amaout of $ ml water was puted into each vial (10 ml) with the same diameter. Previously, the empty and dry vials and the vial that filled with water were weighed ‘The vial that filled with water were caped using a ‘membrane filter (cellulose filter, with 0.45 mm pore, ‘Whatman no, 4), and than tie with a rope and make sure there is no gap between water and membrane. Then put 10 drops of Isopropyl myristate to saturate the ‘membrane and leave it overnight. Sample (0.05 grams) ‘was applied evenly to the membrane then observed for a week. After a week, the water that has been lost were calculate using the following equetion (2) [10] B x 100% 2) A Notes: ‘A= Amount of water lost in control B= Amount of water lost in the sample Anti-inflammation effectiveness test: ‘The effectiveness test of PMCA in the NLC system uses ‘mice as experimental animals. Measuring the ability to reduce the thickness of the ear skin of mice with edema and the number of polymorpho nuclear (PMN) cells, there were 5 animal treatment groups namely; positive control group, negative control group, group with NLC- PMCA treatment, group with SLN-PMCA treatment, and group with NE-PMCA. treatment. PMCA, effectiveness test was carried out on the skin of mice ears which had been induced inflammation by croton oil (2.5% in acetone) for as much as 10 ql using micropipette. One hour after being dripped with test samples. Then, 9 hours afer treatment, the histological preparations of mice car were made afer the mice were sacrificed and then stained with Hemato-Eosin (HE), ‘The histological preparations were then observed under 4 microscope. Measurements were made on (edema) skin thickness and the amount of inflammatory cell infiltration of polymorpho nuclear (PMN) or neutrophils, namely the main inflammatory cells in acute inflammation [4]. Percentage reduction in edema thickness (PTE) was calculated based on the cequation(3): TRY)-TS) * 100% (3) T (KHOR) ‘skin thickness decrease T (K +) = thickness of skin that had edema (positive control) ‘T (S) = thickness skin that had edema after sample was applied T (K.)= thickness of healthy skin (negative control) ‘The calculation of PMN cells on histological preparations of mice without treatment (K-), after induction of croton oil (K+), after treatment with NLC- PMCA (NLC), after treatment with SLN-PMCA (SLN), and after treatment with nanoemulsion PMCA (NE) were observed using an Olympus CX21 microscope at 400x magnification. RESULT! 1. The pH Value and Viscosity: ‘The measurement results of the pH value and viscosity of the NLC-PMCA system with various ratios of solid lipid combination of beeswax-oleum cacao with VCO as liguid lipid, namely FI (60:40); F MT (70:30); F TM (80:20); SLN-PMCA and NE-PMCA by pHl-meter and the cone and plate viscometer can be seen in Table 2 Based on the data pH in Table 2 all of the formula has pH around 4, and the results of the viscosity statistical {est using one-way ANOVA with 95% confidence level, obtained results a significance value of 0.002 smaller than 0.05 and the results of the Tukey's HSD test, it can be concluded that NE-PMCA has the lowest viscosity, and for the NLC-PMCA the F T (60:40) has the lowest viscosity. 2. Particle Size and Particle Size Distribution: Particle size measurement results of the NLC-PMCA system with various ratios of beeswax-oleum cacao as solid lipid. with VCO as liquid lipid, namely: FI (60:40); F I (70:30); F Ill (80:20), SLN-PMCA and NE-PMCA using the Delsa™ Nano Submicron Particle Size and Zeta Potential Dynamic Light Scattering can be seen in Table 3. The results of particle size examination were then subjected to a one-way ANOVA statistical test with 95% confidence degree and Tukey's HSD test. A significance value of 0.239 was obtained, which was more than 0.05. This means that there was no differences in particle size for all NLC-PMCA formulas. And the particle size distribution or polydispersity index (PL) measurement results of all systems were below 0.3, its mean the particle size each formula homogen. 3621Research J Pharm. and Tech 12(8) August 2019 Table 2: pH value and visisity of NLC-PMCA System with Various ratios of Beeswax-Oleum Cacao 4 Solid Lipid with VCO AF (030); and FL iscsi ep ‘Average | 5D Enea —[4a Pore [gon 17 F030) 4s! 02] 163.10 ona Fuilqwo30)— [436 [oni —[ ios 1056 uN tax ——[ oor [ans Tas tre 401 [oos Pats ——T Table 3: PartcleDropet Stes und Polydlspersy Inder (PI) of NLCPMCA System with ‘eo Cacao a5 Soll Lipid with VCO. as Ligeld Lip - coal FI (0:308 and FID Z ae Se | Formula | Parle Genera Saran vi Figure 2: Thermogram of meting temperature of NLC-PMCA ____| average formula (780) Figo — | 33a00 FMCo39)—[ 341-01 = FL @0-20) | 25808 aiaeaae 1 SOY 58.0 “Le | NE 2a | a | 3. Melting Point and Reerystallization Index: i | ‘The mesurement of the melting point and * | reerysiallization index was carried out on the NLC- | PMCA system with various ratios of beeswax-oleum | | cacao as Solid lipid with VCO as liquid lipid, namely: FI, | (60:40); F IT (70:30); and F III (80:20) using Differential \ | Scanning Calorimeter (DSC) compared to oleum cacao,“ | beeawny, and PMCA. The result of the thermogrim | 9 8" ° 8 8 8 = ee ‘melting point ofthe formulas ean be seen in Figures 1,20 2 Se See ee eMETRR Sok ovis and 3, while the melting point parameters of each formula can be seen in Table 4, From the peaks that appear in the thermogram of each sample, the re- crystallization index was calculated based on the following equation (4) ‘AH NLC RL Figure 3: Thermogram of melting temperature of NUC-PMCA formula 1H (80:20) Table 4: Melting Points of PMCA, Oleum Cacao, Beeswax, and NLC-PMCA. (ae ueTER ‘SAR S100 Figure 1: Thermogram of melting temperature of NLC-PMCA format T (60:40) ‘Materials Meine pat [AN ed | TCH ©) PRCA 173.00 af Beeswax 59.99 =30046 ‘Olea Casa 33S. alisi7 [= | Fomula (0:40) [36.85 st 0st [os st 88.97 Fomua li os0) [S801 sas 007 / | 103 44 Ei / Formula WT(RO30)_|_104.13 Taies [= 4, Entrapment Efficiency: The resulis of the determination of PMCA recovery in the NLC, SLN system with different ratios of solid lipids and liquid lipids ean be seen in Table 5, it was known that the percentage of recovery of all formulas were > 94%, and the results of the calculation of the % CV value of active materials showed that the % CV value of all formulas were less than 6%, so it can be said that all NLC-PMCA formulas were homogencous. ‘The determination of the PMCA in NLC and SLN system entrapment efficiency, the results can be seen in 3622esearch J Pharm. and Tech 12(8) August 2019 Statistical test with 95% confidence degree followed by the Tukey’s HSD test with a significance value of 0.020 0.05. Thus, it can conclude that F 1 (60:40) had a higher entrapment efficiency than F Il (70:30) and F Ul (80:20). 5. Occlusivity test: ‘The result of Fl NLC-PMCA (41.50 + 4.00 %), SLN- PMCA (33.52 # 3.65 %) and NE-PMCA (20.30 + 239 °%) occlusivites after one week obsevation known the Fl NLC-PMCA system has higer occlusivity. 6, Anti-inflammation effectiveness test: To determine the PMCA activity in the NLC system, a test was conducted on the reduction in skin thickness that induced edema in the ear skin tissue of the mice, as well asthe observation of the number of PMN cells, The NLC-PMCA formula tested for activity was F 1 (60:40) because it had the highest entrapment efficiency. The NLC-PMCA activity test was compared. with nanoemulsion system (NE:PMCA) and_ solid lipi nanoparticle system (SLN-PMCA). a. Decrease of Skin Thickness at The Ears Mice with Edema: ‘The results of the PMCA activity test in the NLC system fon the thickness reduction of the car mice skin with edema can be seen in Table 6 and Figure 4. The results of a one-way ANOVA statistical test with 95% confidence degree and Tukey's HSD test on the reduction inthe thickness of the skin with edema in the car skin tissue of mice observed in 5 visual fields with a magnification of 100x, which found a significance value of 0.623 > from 0.05. Skin thickness of mouse cars after exposure with NLC-PMCA, SLN-PMCA and NE- PMCA were not significantly different from the negative control and significantly different from the positive controls. So it ean be concluded that NLC-PMCA ean provide the same anti-inflammatory activities as SLN- PMCA and NE-PMCA. b. Number of PMN Cells: ‘The results of the calculation of the number of PMN cells contained in histology preparations in S visual fields with magnifications of 400x can be seen in Table 6 and Figures. It was known that NLC-PMCA can reduce the number of PMN cells in the ear mice skin with edema better than SLN-PMCA and nanocmulsi- PMCA, but still far compared to the negative control Table 5: Recovery Percentage and Entrapment Fiiency of PMCA in NLC and SLN Formula | Recovery (%) Entrapment _| emieieney ‘Average [SD [EV | Aveage | SD | ay Figo, | east fois foie [aay a0 “Peso Po P3936. 9430 [1.09 [ie [37.80 | x02 93.36 [2395 [4s6 [2s ‘Table 6: The Ear Mice Skin Thickness after 9 Hours Treatment and The Number of PMN Calls in Histological Preparations of ‘Mice Ear Skin Treated with NLC-PMCA, SLN-PMCA, and NE- PMCA Compared to Postive Control and Negative Control. ‘Observation Using Olympus X21 Microscope, Wilh 100% and 400s Magaiications ‘Treatment | Skiw thiclness | Number of PMN Call per field of | per'field of view | ‘lew averages | average SD (%) L SD um) = Postine coal [33544 = 362 | 16662333 ‘Negative contol [ 1390.6 4086 [5.6207 SLCPMCA | 1as4963554 —[as.6=257 [stypaca [arms see | 03.3172 [NEMcA 1s0> 1369 [113.3=483 control (K:), postive control (KC +), with NLC-PMCA (NLC) treatment, with SLN-PMCA (SLN) treatment, and with ‘nangemulsion-PMCA (NF). treatment. Observed vslng Olympus ‘CAE mleroscope with 100x magnleaion [PMN cells i the ears of mice without treatment (K-), after induced by croton all (K after treatment with NLC- PMCA (NLC), after treatment wits SLN-PMCA (SLN), and after Figure treatment with nanoemulsion PMCA (NE ) Observed. with ‘Olympus CX2E mlcroscope at 400% magnification 3623‘Research J. Pharm, and Tech 12(8): August 2019 DISCUSSION: In this NLC-PMCA study VCO was used as liquid lipid because it was determined that PMCA nanoemulsion that uses VCO as an oil phase produces the smallest droplet size. This is caused by VCO contains fatty acids with C atomic chains which are shorter than the fatty acid content in corn oil and soybean oil. The smaller size of nanoemulsion droplets results in the greater release rate of active ingredients from the carrier system and the penetration rate of active ingredients into the skin, so the resulting effectiveness is also higher [4]. As stabilizers, ‘Tween 80 and Span 80 were used, which are nonionic surfactants, safer than other types of surfactants in the event of skin irritation [11,12]. NLC remains in its solid form by controlling the levels of liguid lipids added to the formulation, its can controlled drug release properties for NLC can be achieved.° The choice of constituent materials and ratios between solid lipids and liquid lipids are important factors in the NLC formulation. To decreased ordemess of the solid lipid in this research also used combination of beeswax and coleum cacao. ‘The data pH value in Table 2 itis known that all NLC- PMCA systems that formed had pH values that are slightly below the pH range of the skin at 4.5-6.5 so it docs not causes irritation, From the viscosity data, itis known that F I (60:40) has the lowest viscosity, F'T has the highest VCO content, which is the highest tiquid lipid. An increase in liquid lipid consetration decreases the viscosity of NLC-APMS, but has no effect on particle size and particle size distribution or polydispersity index (PI). All formula have particle size <500 nm, it was known a good NLC system has particle size up to 500 nm [7]. And then in Table 3, the results of particle size distribution or polydispersity index (PI) of NLC-PMCA systems were below 0.5 indicated the absence of particle aggregation, and the presence of a homogeneous particle size distribution [13,14] Based on the results of the melting point examination with DSC in Figures 1, 2, 3 and Table 4 it can be seen that the F TIL has a different thermogram form than F I and F TI, In F TIT there is only one peak, this can be caused by the decreasing amount of liquid lipids the possibility of molecular interaction between oleum cacao and beeswax would occur, so there was endothermic peak of each solid lipid (oleum cacao and beeswax) an appeared. In F 1 and F Tl, there was a shift in endothermic (oleum cacao and beeswax) peaks (56.85 °C and 55.01°C) and a decrease in enthalpy between single solid lipids with NLC-PMCA (5.87 J/g and 1.43 Jig). This can be due to differences in composition between single solid lipids (oleum cacao and beeswax) in the formulas. In the calculation of the recrystallization index for F I and F Il, a decrease in the value of RI (0.326 and 0.074%) was obtained. This shows a decrease in the order of the crystal lattice, From these results, it can be concluded that the addition of PMCA and VCO can affect endothermic conditions (melting point and enthalpy). The entrapment efficiency of FU higer than FI and F HL its caused by the use of a combination of sold lipids and liguid lipids in NLC system can reduce crystallinity in the crystalline matrix and form irregular matrix, thus creating a space where drug molecules ean be entrapped in the system [15,16]. Higher content of liquid lipid in NLC-PMCA also occured the less ordered matrices, supported by the recrystallization index data. The lower of reerysallization index of an ingredient ovcered the less ordered the erystal lattice is, the crystal space ean sccommodate larger drugs, so the resulting entrapment efficiency would be greater {17,18}, The addition of liquid lipids also increased stability caused the crystallization process to be inhibited, so drug expulsion daring storage ean be minimized [17,19]. NLC-PMCA ean produce ant-inflammatory activity equivalent to SLN-PMCA and NE-PMCA To seek improvement in the delivery system so as to increase the effectiveness of the PMCA as an anti- inflammatory, the rate of release and penetration of the system needs (0 be determined. An active material can hhave the desired effect if it can be released from the system, and then penetrate into the skin to the site of action. Easier the active material detaches from the system, the faster penetration will be and the faster effect will be too. CONCLUSIONS: Based on the results of the characterization of the NLC PMCA system with ratios of beeswax-oleum cacao solid lipid and VCO lipid of 60:40, 70:30, and 80:20 which included organoleptic, pH, viscosity, particle size, particle size distribution, melting point, entrapment efficiency, and activity tests, in this research it can be concluded that: The increase of the amount of VCO in the NLC-PMCA system results in smaller particle size, lower viscosity, and greater entrapment efficiency, namely in the combination of beeswax-oleum cacao solid lipid and VCO liquid lipid of 60:40. NLC-PMCA with a combination of beeswax-oleum cacao solid lipid and VCO liquid lipid of 60:40 had equivalent anti- inflammatory activity as SLN-PMCA and nanoemalsi- PMCA, based on the observations of the PMN cells number in ear skin of mice with edema. The NLC- PMCA with the combination of beeswax-oleum cacao ‘and VCO of 60:40 was the best formula. 3624esearch J Pharm, and Tech ACKNOWLEDGEMENT! The authors would like to thank the Minister of Research, Technology, and Higher Education who funded this research, Universitas Airlangga and the Faculty of Pharmacy for providing laboratory facilities for the studies, and to students Alifiah Sakinah and Sarah Nurul Iman, under the writers’ supervision who helped conduct this research. ETHICAL CLEARENCI All animal experiments were conducted with the permission from Animal Care and Use Committee (ACUC) of Veterinary Faculty, Airlanggn University, Surabaya-Indonesia. (Reference number, 2KE.0S1.01, 2018) CONFLICT OF INTEREST: No conflict of interest is associated with this work. AUTHOR CONTRIBUTION: We declare that this work was done by the author(s) named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors. 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