The Influence of Sucrose Ester Surfactants And

Eid Ahmad Mustafa Masoud et al. IRJP 2012, 3 (6)

INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230 – 8407 Research Article

THE INFLUENCE OF SUCROSE ESTER SURFACTANTS AND DIFFERENT STORAGE CONDITIONS ON THE PREPARATION OF A NOVEL SWIETENIA MACROPHYLLA OIL NANO-EMULSION Eid Ahmad Mustafa Masoud1,2*, Baie Saringat Haji1 and Arafat Osama Mohammad1 1Department of Pharmaceutical Technology, School of Pharmaceutical Sciences, University Sains Malaysia, Pulau Pinang, Pinang, Malaysia 2Department of Pharmaceutical Technology, Faculty of Pharmacy, University Technology Mara, Puncak Alam, Selangor, Malaysia

Article Received on: 21/03/12 Revised on: 19/05/12 Approved for publication: 30/05/12

*Ahmad Mustafa Masoud Eid, Department of Pharmaceutical Technology, School of Pharmaceutical Sciences, University Sains Malaysia,18000, Mendin, Pulau Pinang, Pinang, Malaysia Email [email protected]

ABSTRACT Nano-emulsions are emulsions with uniform and extremely small droplet sizes, typically in the range of 20–200 nm. Nano-emulsion concept has attracted considerable attention in recent years for its application in personal care and cosmetic products also due to their ability to improve the penetration and permeation of active ingredients through the skin. A composition of sucrose ester as surfactant, glycerol and Swietenia macrophylla oil were used to produce nano-emulsion by simple mixing and applying heat to dissolve the sucrose ester in the glycerol then adding the oil to the mixture. Three different types of sucrose ester (laureate, oleate and palmitate) were used to investigate their influence on the production of nano-size emulsion. Sucrose laureate produced nano- emulsion with good droplet size, polydispersity index and negative zeta potential compared to oleate and palmitate. The oil droplet size produced using sucrose laureate was below 200 nm with polydispersity index 0.2 and negative zeta potential lower than -40 mV. Stability study was conducted for the nano- emulsion at different temperatures (4 °C, 25 °C and 40 °C) for six months. Nano-emulsion formulation was very stable at 4 °C compared to 25 °C and 40 °C while at 25 °C showed moderate stability but it was unstable at 40 °C. The nano-emulsion containing the novel Swietenia macrophylla oil was successfully prepared using sucrose laureate and the ideal storage condition for nano-emulsion was 4 °C. Keywords: Phase diagram, nonionic surfactants, nano-emulsion, sucrose ester, Swietenia macrophylla oil.

INTRODUCTION nutrition.12,13 The main advantage of using nano-emulsions The growing demand of consumers and the advances in for the skin is its ability to improve the penetration and knowledge about production and stability of dispersed permeation of active ingredients through the skin without the systems enable the development of differentiated vehicles need of incorporating penetration enhancer in the such as nano-emulsions, which have been effectively used to formulation.14-16 The aim of this research was to determine increase the bioavailability and improve the stability of the the impact of different surfactants on the preparation of active ingredients. The technological applications of nano- Swietenia macrophylla oil nano-emulsion and its stability emulsions have increasingly been used in various over different storage conditions. applications due to their characteristic properties, small MATERIALS AND METHODS droplet size (in the range 20-200 nm) with high interfacial Materials area, transparent or translucent appearance, high Sucrose Laurate 1695, Oleate 1570 and Palmitate 1570 were solubilization capacity, low viscosity, and high kinetic supplied by Juhalim Biotech SDN BHD (Kuala Lumpur, stability sedimentation, flocculation, and in some cases, the Malaysia). Swietenia macrophylla (SM) oil was gifted by coalescence.1-4 Nano-emulsions are mainly used in the fields Nawa Pharma Sdn Bhd (Kuala Lumpur, Malaysia). Glycerol of pharmaceuticals, cosmetic and chemical fields. However, was supplied by Sigma-Aldrich (USA). it was not until recently studied in more depth with regard to Formulation of nano-emulsion the different mechanisms of formation of nano-emulsions and Series of formulations containing various combinations of the role of surfactants, the processes at colloidal and Swietenia macrophylla oil as an oil phase with surfactant and Interfacial levels involved in their formation and the main glycerol as a liquid phase were used to produce nano- mechanisms that affect their stability mainly Ostwald emulsion. A pseudo-ternary phase diagram was constructed ripening. based on three different types of surfactants combination with Swietenia macrophylla (SM) is one of the most important glycerol and oil separately at a constant temperature. plants of the family, Meliaceae.5 S. macrophylla is located in Formulation A contained a mixture of oil, sucrose laurate and more than 40 countries including in Brazil, Bolivia, Mexico, glycerol while formulation B consisted of oil, sucrose oleate Guatemala, Peru and other central American countries.5-8 Its and glycerol and formulation C a contained mixture of oil, fruit is known as “sky fruit” because it seems to point sucrose palmitate and glycerol. The mixtures were then used upwards to the sky.8 This plant has various types of medicinal to distinguish the effect of sucrose esters on the values like anti-malarial and anti-diarrheal effects.9,10 Also emulsification of the oil. The formulations were weighed the plant extracts have been reported to possess anti-bacterial based on pseudo-ternary automatic diagram using analytical and anti-fungal activities.11 In the pharmaceutical field, nano- balance (Meller Tolledo). Both of the phases, oil and emulsions have been used as a drug delivery system through glycerol, were heated separately first at about 75 °C ± 2 °C various systemic routes mainly: oral, topical and parenteral using hot plate, then the surfactant was mixed with the hot Page 199 Eid Ahmad Mustafa Masoud et al. IRJP 2012, 3 (6) glycerol using glass rod and stirred continuously until the macrophylla oil to construct the ternary phase diagram. The surfactant was dissolved in the glycerol then the oil was three different systems showed different behavior in added to the mixture slowly and stirred for about 10 minutes producing the nano-emulsion. As a comparison between to ensure its total disappearance and the formation of them, system A shown in figure 1 which comprises sucrose emulsion. A sample of the emulsion was dissolved in distilled laurate as non-ionic surfactant, produces larger region of water to form a dilute emulsion. In order to find the efficient nano-emulsion compared to other systems due to its good region of emulsification, the formulations were observed emulsification properties. On the other hand, figure 2 shows after dilution with water and the droplet size of emulsion was the ternary phase diagram of system B containing sucrose measured in order to find the efficient region of palmitate with bad nano-emulsion properties producing small emulsification. All experiments were carried out at room region of nano-emulsion compared to other systems and less temperature of about 25 °C. The formulations that formed emulsification properties compared to sucrose laurate. non-emulsified phases were not shown in the phase diagrams However, the ternary phase diagrams of system C containing because they are out of the study scope. According to the sucrose oleate shown in Figure 3 has better nano-emulsion ternary phase diagrams, nano-emulsion (NE) region was region compared to system B with moderate emulsification marked by the transparent and fine nano droplets, whereas properties. Sucrose laurate showed the best emulsification micro-emulsion (ME) region was marked due to more properties compared to sucrose palmitate and oleate, which whitening and isotropic solutions that might contain micelle may be due to its good miscibility properties. The same solutions and coarse emulsion (CE) was the region of visibly findings were stated by Szuts et al.17, who mentioned that cloudy dispersions even by visual observation. Then nano- sucrose laurate was good in preparing solid dispersion due to emulsion region was selected. its high miscibility properties compared to sucrose palmitate Droplet size and zeta potential analysis and sucrose stearate. Droplets size and size distribution of self-emulsifying system In general, all the surfactants produced nano-emulsion were determined using Malvern Mastersizer 2000 laser formulations with simple stirring. The capability of diffraction particle analyzer (Malvern instruments, UK). producing nano-emulsion was due to the temperature used to Photon correlation spectroscopy or dynamic light scattering, dissolve the sucrose ester in the glycerol. The heat treatment which is specialized in analysis of particle size of submicron, of the formulations may lead to changes in the molecular and is a valuable technique analyzing changes in light characteristics of the surfactant. Therefore, sucrose ester intensity fluctuations initiated by Brownian motion. The becomes progressively dehydrated during heating because it temperature is kept stable otherwise convection currents in is non-ionic surfactant with a hydrophilic head group. For the sample will cause non-random movements which will that reason, the surfactant molecules will have changes in the ruin correct size interpretation. interfacial tension, packing, and oil/water solubility during To observe the droplet size and size distribution, 250 µl of an heating. Same results were shown in previous studies on non- emulsion was added to 300 ml of distilled water in a 500 ml ionic surfactants that produced micro-emulsions and nano- beaker. A glass rod was used to induce gentle agitation in the emulsions formulations by the help of these changes mixture. The droplet size and size distributions of resultant facilitated at higher temperatures.18-21 In addition, a kinetic emulsion were examined by using Malvern Mastersizer 2000 energy barrier in the oil-glycerol-surfactant system prevents it laser diffraction particle analyzer. This step was conducted from moving from an emulsion to micro-emulsion and nano- for screening study of ternary phase diagram. Mean size and emulsion at ambient temperature. But as the temperature was size distributions measurements were performed in raised this kinetic energy barrier was reduced, which helped triplicates. Zeta potential was also measured using Malvern in changing the emulsion from one state to another. Same Nano Zetasizer. results were stated by Rao & McClements, 21, who used oil- Stability of nano-emulsion formulations during storage water-surfactant to produce micro-emulsion and nano- Droplet size, size distribution and zeta potential are among emulsion. the most important characteristics for the evaluation of the Different sucrose esters as non-ionic surfactants were used to stability of emulsion. Therefore, the effect of temperature and study the effect of various HLB values on the formulation storage time was studied on the optimum formulations of and the capability to achieve nano-emulsion. As the degree of nano-emulsion. The droplet size, size distribution and zeta sucrose esterification increased and/or the fatty acid chain potential were evaluated immediately after the production of length increased, the sucrose ester HLB value is reduced. Oil nano-emulsion and also after 1, 2, 4 and 6 months of storage in water dispersion required HLB value between 9 to 18, so under different temperatures 4, 25 and 40 °C. the selection of the optimum HLB value of emulsifying agent RESULTS AND DISCUSSION depends on its hydrophilicity.22 System A containing sucrose Effect of different surfactants combination on the laurate with high HLB value (HLB 16) was chosen as a non- formulation of nano-emulsion ionic surfactant to produce large region of nano-emulsion Various combinations of oil, surfactant and glycerol were formulations with smaller oil in water droplets and good studied for their potential to prepare nano-emulsion by stability. The ability of sucrose laurate to form nano-emulsion stirring method. The ternary phase diagrams of such with small droplets size and good stability was due to its combinations were presented in figures (1-3). The oil, good droplets entrapment and stabilization efficacies which is surfactant and glycerol mixtures were divided into three explained by to the low amounts of di-, tri-, and polylaurate different systems. System A (Oil, Sucrose laurate and (20%) and higher amount of monolaurate (80%) as well as Glycerol), system B (Oil, Sucrose Palmitate and Glycerol) the lauric acid short chain length. While in system B and and system C (Oil, Sucrose Oleate and Glycerol). system C sucrose oleate (HLB 15) and sucrose palmitate The development of the nano-emulsion formulation was (HLB 15) were used respectively, both of the surfactants obtained by three different combinations for Swietenia showed nanoemulsion region but it was less compared to Page 200 Eid Ahmad Mustafa Masoud et al. IRJP 2012, 3 (6) system A. that is due to their low HLB value compared with combination which contains 20% sucrose laureate 1695 and sucrose laurate and less amount of monoesters (70%). Leong 50% oil will produce a stable nano-emulsion with small et al., stated that sucrose laurate 1695 was better than sucrose droplet size and uniformity. palmitate 1570, stearate 1570 and oleate 1570 in preparing Droplet size, polydispersity index and zeta potential phytosterol nano-dispersions with small particle size below analysis than 100 nm.22 Therefore, it could be concluded that sucrose Different formulations of nano-emulsion optimized by laurate as non-ionic surfactant having high HLB value is ternary phase diagram are presented in Table 2, measured by suitable in forming nano-emulsion with small droplets with Malvern Zetasizer to determine their droplets size, high stability. polydispersity index (PdI) and zeta potential. In general, all Influence of oil and surfactants ratio on droplets size and formulations showed small mean droplet size between 101 to size distribution 171 nm, it is observed from the table that droplets size was The droplet size and size distribution were studied for all decreased with an increase in the oil concentration, also all formulations at the preliminary investigation using Master the formulations showed low PdI in a range between 0.123 to Sizer Malvern Instrument. Figures 1-3, show different nano- 0.498. In addition zeta potential was between -43.1 to -27.4 emulsion regions when different surfactants have been used. mV. Their droplets size and size distributions were different. In general, all the formulations have high negative value of System A containing sucrose laurate as surfactant led to the zeta potential charge above 30 mV, which indicates that formation of oil droplets with the smallest size and size nano-emulsion formulations are stable.24 Formulation B distribution compared to other systems B and C. For showed slightly low zeta potential charge and high PdI, Swietenia macrophylla oil in system A the smallest droplet which indicates that this formulation might be unstable over size was 114 nm with uniformity 0.163. But system B storage because it has a low amount of surfactant which is not containing sucrose palmitate showed significant higher enough to make it stable. In summary, formulations droplets size and size distribution compared to system A. The containing 50% oil showed smaller droplets size and zeta smallest droplet size was 365 nm and 0.302 uniformity. potential compared to formulations containing 36% oil, as While system C having sucrose oleate showed better results well in a comparison between the formulations containing compared with system B but still not as small as system A. 50% oil, formulation D with the optimum amount of Its droplet size was 221 nm and uniformity 0.291. surfactant showed the smallest droplets size, lowest PdI and Table 1 show the chosen points of nano-emulsion the best zeta potential. Also it is known that large amount of formulations for Swietenia macrophylla oil from system A, surfactants cause skin irritation, therefore, formulation D will their droplets size was less than 200 nm. Swietenia be the optimum formulation compared to other nano- macrophylla oil has good droplets size and droplets emulsion formulations. distribution. In addition, sucrose laurate led to production of Influence of different storage temperatures on the nano-emulsion formulations with various oil concentrations droplets size, polydispersity index and zeta potential between 36 to 50%. From the ternary phase diagram it can be One of the most important characteristics for evaluation of observed that when the oil concentration in the formulations the stability of emulsion system is particle size distribution. increased from 20% to 50%, their droplets size was decreased Therefore, the particle size parameters and the surface and form more stable nano-emulsions. This may be attributed electrical charge (zeta potential) were evaluated immediately to the fact that sucrose ester was bounded to the surface of after the production of nano-emulsion and over six months the oil droplets when the viscosity of the formulations was (1, 2, 3, 4, 5 and 6 months) of storage at three different increased and therefore will be less available to participate in temperatures (4, 25 and 40 °C), the evaluation was carried for the formation of a gel phase. The same findings were the formulations having droplets size below 200 nm and reported by Murakami et al. 23 and Rao & McClements, 21, when separation was not visible. The measurement of the who stated that the viscosity has an influence in forming droplets size initially and during storage gives an indication stable colloidal dispersion formulation with small droplets about the system stability.25-27 size using sucrose ester as non-ionic surfactant. But as the oil Nano-emulsion formulations were stored at three different concentration goes up more than 50% the droplets size was temperatures to test the system under anxiety condition. In increased then separation happened due to less amount of general nano-emulsion formulations for Swietenia surfactant available to form colloidal dispersion and stabilize macrophylla oil as shown in Figures 4-6, remained in their the system. nano-size range below 200 nm when stored at 4 °C. Also The In general, the formulations B, D and F that contain 50% oil polydispersity index remain in the same magnitude as the concentration showed smaller droplets size with good values obtained immediately after production and the droplets uniformity compared to formulations A, C and E that contain still showing negative charge at their surface <-30 mV. An 36% oil concentrations. Also in a comparison between the exception was formulation B, which showed an increment in formulations with 50% oil, it is obvious that formulation B the size above 200 nm and instability, because the initial contain 15% surfactant has larger droplet size and uniformity reading for this formulation showed high polydispersity compared to formulations D with 20% surfactant and F with index, above that 0.3, and low negative zeta potential, below 25% surfactant. The high uniformity for formulation B which -30 mV, therefore, it was unstable during storage. However, was higher than 0.3 makes it unstable due to Ostwald when nano-emulsion formulations were stored at 25 °C ripening when compared to other formulations which have Figures 7-9, there were a slightly significant increase in the uniformity below 0.3. On the other hand, both formulation D droplets size above 200 nm. Also at 40 °C Figures 10-12, all and F showed almost similar droplet size and uniformity. the formulations showed a significant precipitation with However, formulation D is better than F because it has less increase in the droplets size, polydispersity index and amount of surfactant. Therefore, it can be concluded that the decrease in their negative zeta potential. Page 201 Eid Ahmad Mustafa Masoud et al. IRJP 2012, 3 (6)

It is postulated that when the temperature is increased, the oil 9. Maiti A, Dewanjee S, Mandal SC, Annadurai S. Exploration of droplets movement in the emulsion is also increased. Then Antimicrobial Potential of Methanol and Water Extract of Seeds of Swietenia Macrophylla (Family: Meliaceae), to Substantiate Folklore the droplets will be forced to make physical contact and due Claim. Iranian Journal of Pharmacology and Therapeutics 2007; 6:99- to their low negative zeta potential their mutual repulsion is 102. overcome, which cause higher tendency to flocculate and 10. Munoz V, Sauvain M, Bourdy G, Callapa J, Rojas I, Vargas L, Tae A, coalescence.28,29 In addition, the significant difference Deharo E. The search for natural bioactive compounds through a multi disciplinary approach in Bolivia. Part II. Antimalarial activity of some between the formulations stability stored at 25 °C and those plants used by Mosetene Indians. Journal of Ethnopharmacology 2000; stored at 40 °C, may be due to the change in the optimum 69:127-137. curvature of the surfactant monolayer and the type of 11. Majid MA, Rahman IMM, Shipar MAH, HelalUddin M, Chowdhury R. emulsion structure formed because of the dehydration of the Physico-Chemical Characterization, Antimicrobial Activity and Toxicity Analysis of Swietenia mahagoni Seed Oil. International Journal of hydrophilic surfactant head group at high temperatures. The Agriculture and Biology 2004; 6:350–354. 21 Same findings were reported by Rao & McClements, , who 12. Tamilvanan S. Oil-in-water lipid emulsions: implications for parenteral studied the Influence of storage temperature on the colloidal and ocular delivering systems. Progress Lipid Research 2004; 43: 489- dispersions of lemon oil using sucrose palmitate as non-ionic 533. 13. Schmidt E, Dobler C, Nissing F, Runkel. Influence of hydrophilic surfactant. surfactants on the properties of multiple W/O/W emulsions. Journal of The differences between the evaluated parameters were not Colloid and Interface Science 2009; 338:184–192. significant at 4 °C of the storage for six month, which means 14. Amselem S, Friedman D. Submicron emulsions as drug carriers for that the systems were physically stable under these storage topical administration, in: Submicron emulsions in drug targeting and delivery. Benita S, editor. London; 2004. conditions. Over the period of six months the mean droplets 15. Shah P, Bhalodia D, Shelat. Nanoemulsion: A pharmaceutical review. size remained lower than 200 nm for all the formulations Systematic reviews in pharmacy 2010; 1:24-32. except formulation B as mentioned above. The relatively 16. Sharma JN, Bansal M, Visht S, Sharma PK. Kulkarni. Nanoemulsion: A small, nano-droplets distribution displayed a satisfactory new concept of delivery system. Chronic Young Scientist 2010; 1:2-6. 17. Szuts A, Lang P, Ambrus R, Kiss L, Deli MA, Szabo-Revesz P. long-term stability (PdI <0.3). After six months of storage at Applicability of sucrose laurate as surfactant in solid dispersions room temperature, all nano-droplets still revealed a negative prepared by melt technology. International Journal of Pharmaceutics charge on their surface (zeta potential <-30), but their size 2011; 410:107-110. was more than 200 nm. In conclusion, in order to keep nano- 18. Szuts A, Lang P, Ambrus R, Kiss L, Deli MA, Szabo-Revesz P. Applicability of sucrose laurate as surfactant in solid dispersions emulsion formulations stable, they need to be stored at 4 °C. prepared by melt technology. International Journal of Pharmaceutics CONCLUSION 2011; 410:107-110. In conclusion, Nano-emulsion was prepared using sucrose 19. Anton N, Gayet P, Benoit JP, Saulnier P. Nano-emulsions and ester as surfactant blended with glycerol and Swietenia nanocapsules by the PIT method: an investigation on the role of the temperature cycling on the emulsion phase inversion. International macrophylla oil. The nano-emulsion properties were affected Journal of Pharmaceutics 2007; 344:44-52. by the nature and the concentration of sucrose ester 20. Anton N, Vandamme TF. The universality of low-energy nano- surfactant. Sucrose laureate surfactant showed better nano- emulsification. International Journal of Pharmaceutics 2009; 377:142- emulsion properties with small droplets size, low 147. 21. Rao JJ, McClements DJ. Stabilization of phase inversion temperature polydispersity index and high zeta potential negative value nanoemulsions by surfactant displacement. Journal of Agricultural and compared to sucrose oleate and palmitate. In addition, the Food Chemistry 2010; 58:7059-7066. properties of nano-emulsion were affected by different 22. Rao J, McClements DJ. 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Table 1 Chosen point of nano-emulsion containing Swietenia macrophylla oil from the ternary phase diagram measured by Master Sizer. Formulation Sucrose Laurate (%) Glycerol (%) SM oil (%) Droplet size (nm) ± Uniformity ± SD SD A 19.2 44.8 36.0 183 ± 1.3 0.297 ± 0.001 B 15.0 35.0 50.0 174 ± 1.1 0.554 ± 0.008 C 25.6 38.4 36.0 132 ± 1.4 0.299 ± 0.003 D 20.0 30.0 50.0 114 ± 0.7 0.163 ± 0.006 E 32.0 32.0 36.0 129 ± 1.1 0.262 ± 0.002

F 25.0 25.0 50.0 120 ± 1.7 0.208 ± 0.004 All data are presented as mean ± SD, (n= 3).

Table 2 Nano-emulsion formulations for Swietenia macrophylla oil measured by Malvern Zetasizer Instrument. Formulation Sucrose Laurate (%) Glycerol (%) SM oil (%) Droplet size (nm) ± PdI ± SD Zeta Potential (mV) ± SD SD A 19.2 44.8 36.0 171 ± 0.7 0.263 ± 0.006 -33.1 ± 0.9 B 15.0 35.0 50.0 167 ± 1.3 0.498 ± 0.001 -27.4 ± 1.6 C 25.6 38.4 36.0 124 ± 1.1 0.264 ± 0.004 -36.4 ± 1.3 D 20.0 30.0 50.0 101 ± 1.2 0.123 ± 0.002 -43.1 ± 0.8 E 32 32.0 36.0 116 ± 0.9 0.214 ± 0.001 -37.6 ± 1.2 F 25.0 25.0 50.0 111 ± 1.4 0.179 ± 0.003 -39.2 ± 1.7 All data are presented as mean ± SD, (n= 3).

Figure 1 System A; Swietenia macrophylla oil, Sucrose laurate and Glycerol.

Figure 2 System B; Swietenia macrophylla oil, Sucrose Palmitate and Glycerol.

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Figure 3 System C; Swietenia macrophylla oil, Sucrose Oleate and Glycerol.

Figure 4 Droplet size measurement of nano-emulsion after production and subjected to different storage duration: 1, 2, 3, 4, 5 and 6 months of storage at 4 °C.

Figure 5 Polydispersity Index (PdI) measurement of nano-emulsion after production and subjected to different storage duration: 1, 2, 3, 4, 5 and 6 months of storage at 4 °C.

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Figure 6 Zeta potential measurement of nano-emulsion after production and subjected to different storage duration: 1, 2, 3, 4, 5 and 6 months of storage at 4 °C.

Figure 7 Droplet size measurement of nano-emulsion after production and subjected to different storage duration: 1, 2, 3, 4, 5 and 6 months of storage at 25 °C.

Figure 8 Polydispersity Index (PdI) measurement of nano-emulsion after production and subjected to different storage duration: 1, 2, 3, 4, 5 and 6 months of storage at 25 °C.

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Figure 9 Zeta potential measurement of nano-emulsion after production and subjected to different storage duration: 1, 2, 3, 4, 5 and 6 months of storage at 25 °C.

Figure 10 Droplet size measurement of nano-emulsion after production and subjected to different storage duration: 1, 2, 3, 4, 5 and 6 months of storage at 40 °C.

Figure 11 Polydispersity Index (PdI) measurement of nano-emulsion after production and subjected to different storage duration: 1, 2, 3, 4, 5 and 6 months of storage at 40 °C.

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Figure 12 Zeta potential measurement of nano-emulsion after production and subjected to different storage duration: 1, 2, 3, 4, 5 and 6 months of storage at 40 °C.

Source of support: Nil, Conflict of interest: None Declared

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