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The 6 International Conference on Natural Products for Health and Beauty (NATPRO6) January 21-23, 2016 Organized by Khon Kaen University

Microemulsion and biological activity of Indian gooseberry extract Issara Keetaa, Sitthiphong Soradecha, Buppachart Potduanga* and Sasamol Phasukb a

Pharmaceutical and Natural Product, Thailand Institute of Scientific and Technological Research, Phathum thani 12120, Thailand b Science Center, Valaya Alongkorn Rajabhat University, Phathum thani 13180, Thailand *[email protected]

phone +66-66249108

fax +66-25779110

ABSTRACT The purpose of this study was to develop a new formulation for microemulsion delivery of the most yielded variety (56.61%) of Phyllanthus emblica fruit extracts which was antioxidant and antityrosinase effective and exhibited ellagic acid as the RP-HPLC prominent peak. Formulations were prepared by HLB 10 and HLB 12 phase diagram (HLB, Hydrophilic lipophilic balance). The amount of mixed surfactants (Tween80 and Span80), oils (mineral oil, coconut oil, grape seed oil, sunflower oil and jojoba oil) and water were varied to select suitable proportions for microemulsion preparation. The resulted microemulsions were clear and golden-yellow. The appearance, color, particle size and % entrapment efficiency of the selected P. emblica extract loaded micro emulsion was evaluated. The particle size of microemulsion exhibited in range of 150-200 nm, the smallest was sunflower oil No. 81 HLB 12 at 154 ± 0.01 nm. The % entrapment efficiency of microemulsion was in the range of 85-96%, and grape seed oil No.81 HLB 12 gave the maximum entrapment efficiency of 95.17 % ± 0.01%. All of them were stable under heating-cooling test at 4 ̊C 24 hrs and 45 ̊C 24 hrs for 6 cycles. Keywords: Microemulsion, Hydrophobic lipophilic balance, Phyllanthus emblica, antioxidant, antityrosinase, RP- HPLC

1. INTRODUCTION Microemulsion is thermodynamically stable transparent system dispersions of oil and water stabilized by interfacial film of surfactant frequently in combination with co-surfactant. The droplet size of microemulsion is usually in 20-200 nm. Microemulsion is easy to prepare without energy consumption during preparation. Microemulsion formation is reversible, it may become unstable at low or high temperature but when the temperature returns to the range microemulsion reforms. Microemulsion is used as a delivery system to improve the efficacy of a drug, thus the total dose could be minimized together with the size effects [1, 2]. Phyllanthus emblica L. (EUPHORBIACEAE), Indian gooseberry or Ma-khampom (in Thai), is a tree of small to moderate size found in tropical Southeast Asia and throughout Malaysia and East Timor. The fruit is commonly used in Asian traditional medicine as an alternative treatment of diarrhea, jaundice, skin disorders, inflammations, premature graying and the burning sensation of the body. The fruit contains vitamin C, gibberellins, lupeol, kaempferol, quercetin, emblicanin A and B, punigluconin, pedunculaginn, phyllanthin, zeatin, amlaic acid, corilagin, ellagic acid, putranjivain A, digalic acid, phyllemblic acid, emblicol and galactaric acid [3]. P. emblica fruit extract is anti-tyrosinase and antioxidant effective which is beneficial as an agent for skin-lightening and skin protection against UV induced free radicals [4]. This study was to develop a formulation for microemulsion delivery of P. emblica fruit extract by HLB 10 and HLB 12 phase diagram (HLB, Hydrophilic lipophilic balance) with varying amounts of mixed surfactants (Tween80 and Span80), oils (mineral oil, coconut oil, grape seed oil, sunflower oil and jojoba oil) and water. The appearance, color, particle size and % entrapment efficiency of the stable microemulsion was evaluated. Comparative chemical and biological properties of 5 varieties of P. emblica were also reported.

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The 6 International Conference on Natural Products for Health and Beauty (NATPRO6) January 21-23, 2016 Organized by Khon Kaen University

2. MATERIALS AND METHODS Plant materials Fruits powder of P. emblica was provided by the Agricultural Technology Department of Thailand Institute of Scientific and Technological Research (TISTR). Preparation of the extracts Five varieties of P. emblica fruit powder, 500 g each, was separately macerated with ethanol-water for 3 nights, filtered, rinsed and evaporated at 45 °C under reduced pressure using a rotary evaporator. RP-HPLC of the extract RP-HPLC at 285 nm of the most yielded P. emblica extract was carried out on a X-terra RP18 (3.9 mm x 150 mm, 5 µ) column, using 0.2% formic acid (A) and acetonitrile (B) as mobile phase at 0.6 ml/ min, 30˚C. Total flavonoid content assay Total flavonoid content was measured by Aluminium chloride colorimetric assay. One milliliter of sample or standard solutions of rutin was added to a 10 mL volumetric flask containing 4 mL of distilled water. At T0 (start time), 0.3 mL of 5%NaOH was added and after 5 min (T5) add 0.3 mL of 10%AlCl3. At 6 min (T6), 2 mL of 1M NaOH was added, and then added water to make 10 mL and mixed well. The absorbance was measured at 510 nm versus a blank. Total phenols and tannin assay Total phenol and tannin contents were determined by Folin-Ciocalteau assay using gallic acid and ellagic acid as the standard solution respectively. Sample or standard solution 0.1 mL was added to a 25 mL test tube containing 8.4 mL of distilled water. Folin-Ciocalteau reagent 0.5 mL was added to the mixture and shaken for 5 min before adding 1 mL of 20%Na2CO3 solution. The mixture was shaken before incubated in the dark for 1 hour at room temperature. The absorbance was measured against the reagent blank at 760 nm. Antioxidant assay Antioxidant activity of P. emblica extracts were determined by DPPH radical scavenging micro-plate method as described by Potduang et al. [5]. Mixed 0.1 mL each of absolute ethanol solutions of the extract and 0.06 mM DPPH (2, 2diphenyl-1-picrylhydrazyl) in a micro-plate for 30 min. Measured the 517 nm absorbance using a micro-plate reader. All samples were run in triplicate compared to BHA. Calculated from the equation: % Scavenging = 100 × [C-(A-B)] / C The absorbance are represented as A = the reaction mixture, B = blank (ethanol) and C = control (DPPH in ethanol). Calculated the EC50 using the resulted linear equation of %scavenging vs. log concentration. Antityrosinase assay Antityrosinase activity of P. emblica extracts were determinded by the Dopachrome micro-plate method as described by Potduang et al. [6]. The solutions of 50 μL extract in 20% ethanol, 50 μL of 314.8U/mL mushroom tyrosinase enzyme in buffer and 100 μL of 0.02 M sodium phosphate buffer (pH 6.8) were mixed. After 10 min, added 50 μL of 0.34 mM L-Dopa (Sigma) in buffer, mixed well and measured the absorbance at 492 nm by a micro-plate reader. Re-measured the absorbance after 2 min. All samples were run in triplicate compared to Kojic acid. Calculated from the equation: % Tyrosinase inhibition = [(A-B) - (C-D)]×100/ (A-B) The absorbance differences are represented as A = control (L-Dopa + enzyme), B = blank 1 (L-Dopa), C = reaction mixture (enzyme + sample + L-Dopa) and D = blank 2 (sample + L-Dopa). Calculated the IC50 using the resulted linear equation of %tyrosinase inhibition vs. log concentration. Preparation of P. emblica extract loaded microemulsion The O/W microemulsions were prepared using pseudo-ternary phase diagram at room temperature. HLB 10 and HLB 12 were used to calculate the condition of microemulsion form. Different oils (mineral oil, coconut oil, grape seed oil, sunflower oil and jojoba oil) and surfactants (tween 80, span 80) were used. 0.5 g P. emblica extract were mixed with 10 g water, and then made to 100 g without heating by mixing with various mixtures constituted by oil, surfactant and water in varying constant percentages from 0 to 100.

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The 6 International Conference on Natural Products for Health and Beauty (NATPRO6) January 21-23, 2016 Organized by Khon Kaen University Evaluation of microemulsion The organoleptic of microemulsion was observed including color, odor, and cloudiness. Stability assessment The stability of microemulsion was assessed under extreme by heating at 60˚C for 5 hours in an oven for separation check [7], and by heating-cooling assay at 4˚C 24 hrs and 45˚C 24 hrs for 6 cycles.

Droplet size determination The droplet size of microemulsion was investigated in triplicate, using Mastersizer 2000 which measures particles in wide range from 0.02-2,000 µm. The measurements were conducted in distilled water with 10% laser obscuration. Entrapment efficiency assessment The 1.5 g of microemulsion was dissolved in 5 ml methanol, vortexed for 2 min and centrifuged at 6,000 rpm for 10 min at 10 °C. Discarded the clear solution, and then added 5 mL methanol and sonicated for 15 min. The absorbance at 282 nm was measured and used to calculate from the equation [8, 9]: % Entrapment efficiency = [Amount of drug encapsulated/ Total loading amount]×100 Data analysis All experimental measurements were triplicate performed. Result values were expressed as mean value ± standard deviation. Statistical significance in this study was examined using analysis of variance (ANOVA). The value of p < 0.05 was considered statistical significant.

3. RESULTS The ethanol-water extract of 5 varieties of P. emblica yielded 5.67 ± 0.01 - 56.61 ± 0.07 %. The extract No. 5 gave the maximum yield (p < 0.05) of 56.61 ± 0.07 %, in comparison to other P. emblica extracts. The extracts showed the antioxidant effects at the EC50 range of 2.04 ± 0.01 - 3.22 ± 0.01 µg/mL with the significantly difference of p < 0.05. They exhibited antityrosinase activity at the IC50 range of 2.78 ± 0.08 - 4.29 ± 0.04 mg/mL, but the IC50 of antityrosinase was not significantly different at p < 0.05. The range of chemical constituents in the P. emblica fruit extracts were total phenolic compounds at 222.82 ± 0.05 -518.56 ± 0.01 mg gallic acid equivalent/g sample, total flavonoids at 86.75 ± 0.05 - 122.10 ± 0.02 mg rutin equivalent/g sample and total tannins at 110.11 ± 0.25 - 928.52 ± 0.02 mg ellagic acid equivalent/g sample as shown in Table 1. The difference in total flavonoids, total phenols and total tannin of extracts were significant at p < 0.05. Table 1. Comparative %yield, biological activities and chemical properties of P.emblica extracts Varieties 1 2 3 4 5

% yield 33.63 ± 0.73a 30.13 ± 0.12a 43.33 ± 0.35a 5.67 ± 0.01a 56.61 ± 0.07a

Antioxidant EC50 (µg/ml)

Antityrosinase IC50 (mg/ml)

2.14 ± 0.02a 2.04 ± 0.01a 2.22 ± 0.01a 3.11 ± 0.30a 2.19 ± 0.01a

3.48 ± 0.74a 2.78 ± 0.08a 3.63 ± 0.28a 4.29 ± 0.04a 3.05 ± 0.37a

Total flavonoids (mg rutin quivalent/ g sample) 122.10 ± 0.02a 88.45 ± 0.02a 86.75 ± 0.05a 101.65 ± 0.02a 103.60 ± 0.07a

Total phenols (mg gallic acid equivalent/ g sample) 333.03 ± 0.03a 314.68 ± 0.01a 337.86 ± 0.01a 222.82 ± 0.05a 518.56 ± 0.01a

Total tannin (mg ellagic acid equivalent/ g sample) 928.52 ± 0.02a 796.13 ± 0.44a 917.71 ± 0.53a 638.43 ± 0.08a 110.11 ± 0.25a

Different superscript in the same column indicated significant differences (P < 0.05) RP-HPLC chromatogram of P.emblica extract showed a prominent peak of compound at 13.059 min which was the same peak as standard ellagic acid (13.422 min) when spiked, as shown in Figure 1.

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The 6 International Conference on Natural Products for Health and Beauty (NATPRO6) January 21-23, 2016 Organized by Khon Kaen University

1 Figure 1. RP-HPLC chromatograms of P. emblica (A) and standard ellagic acid (B)

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The 5 variety of P. emblica extract which gave the most yielded extract of 56.61 ± 0.07 % was chosen to prepare microemulsions using pseudo-ternary phase diagrams (HLB 10 and HLB 12) at room temperature. The suitable ratio of microemulsions which were non-separated after heating test and 6 cycles of heating-cooling test are shown in Table 2. Table 2. The suitable ratio of microemulsion HLB

Type of oil Grape seed oil

10 Sunflower oil

Mineral oil

12

Grape seed oil

Sunflower oil

Sample No.

% oil

% surfactant

% water

sample No.77 sample No.78 sample No.79 sample No.77 sample No.78 sample No.79 sample No.75 sample No.76 sample No.77 sample No.78 sample No.79 sample No.80 sample No.81 sample No.76 sample No.77 sample No.78 sample No.79 sample No.80 sample No.81 sample No.76 sample No.77 sample No.78 sample No.79 sample No.80 sample No.81

25 20 15 25 20 15 35 30 25 20 15 10 5 30 25 20 15 10 5 30 25 20 15 10 5

65 70 75 65 70 75 55 60 65 70 75 80 85 60 65 70 75 80 85 60 65 70 75 80 85

10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

Table 3 shows droplet size and percentgae of entrapment efficiency of the stable microemulsions containing P. emblica extract with different oil i.e., grape seed oil, sunflower oil and mineral oil. The results indicated that only 6 samples of the microemulsions had the droplet size within the range of 154 ± 0.01 to 199 ± 0.01 nm. Sunflower oil gave the significant (p < 0.05) smallest size (154 ± 0.01 nm) microemulsion. Grape seed oil gave the significant (p < 0.05) maximum % entrapment efficiency of 95.170 ± 0.01 %.

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The 6 International Conference on Natural Products for Health and Beauty (NATPRO6) January 21-23, 2016 Organized by Khon Kaen University

Table 3. Particle size and entrapment efficiency of the stable microemulsions Sample

Size (nm)

% Entrapment

Sunflower oil, No. 81 HLB12

154

94.960

Grape seed oil, No.81 HLB12

163

95.171

Mineral oil, No 75 HLB12

184

90.776

Mineral oil, No 76 HLB12

199

91.159

Mineral oil, No 80 HLB12

156

93.366

Mineral oil, No 81 HLB12

155

89.554

4. CONCLUSIONS th

The 5 variety of P.emblica extract which gave the most yielded extract of 56.61% was chosen to prepare microemulsions. The extract exhibited antioxidant effects at EC50 = 2.44 µg/ml, and antityrosinase activity at IC50 = 3.04 mg/ml. Its total phenol was 518.66 mg gallic acid equivalent/g sample, total flavonoids was 103.60 mg rutin equivalent/g sample and total tannins was 110.11 mg ellagic acid equivalent/g sample. Sunflower oil gave the smallest size microemulsion of 154 nm. Grape seed oil gave the microemulsion of maximum entrapment efficiency at 95.17 %

ACKNOWLEDGEMENTS This work was supported by Thailand Institute of Scientific and Technological Research (TISTR), under the advice of Mr. Sitthiphong Soradech and Dr. Buppachart Potduang of the Pharmaceutical and Natural Products Department. We are grateful to thank all members of the institute who in one way or another help this project to succeed.

REFERENCES 1. 2. 3. 4.

5. 6. 7. 8. 9.

Azeem A, Rizwan M, Ahmad FJ, Khan ZI, Khar RK, Aqil M, et al. 2008. Emerging role of microemulsions in cosmetics. Recent Pat. Drug Deliv. Formul. 2, 275-289. Singh PK, Iqubal MK, Shukla VK, Shuaib M. 2014. Microemulsion: Current Trends in Novel Drug Delivery System. J. Pharm. Chem. Biol. Sci. 1, 39-51. Phyllanthus emblica L. Available at http://www.globinmed.com/index.php?option=com_content&view= article&id=79255:phyllanthus- emblica-l&catid=718:p&Itemid=150, accessed 2 December 2015. Penning A. 2012. Beauty in layers: Multitasking ingredients. GCI Magazine, 5 pages. Available athttp://www.gcimagazine.com/marketstrends/segments/antiaging/156350405.html?page=3, accessed December 2015. Potduang B, Chongsiriroeg C, Benmart Y, Giwanon R, Supatanakul W, Tanpanich S. 2007. Biological Activities of Schefflera leucantha. Afr. J. Trad. CAM. 4(2):157–164. Potduang B, Meeploy M, Giwanon R, Benmart Y, Kaewduang M and Supatanakul W. 2008. Biological activities of Asparagus racemosus. Afr. J. Trad. Complement. Altern. Med. 5(3), 230-237. Rukmini A, Raharjo S, Hastuti P, Supriyadi S. 2012. Formulation and stability of water-in-vergin coconut oil microemulsion using ternary food nonionic surfactants. Int. Food Res. J. 19(1), 259-264. Saranyu Songkho, Wiwat Pitchayakorn and Juraithip Hwangsinthaweekul. 2010. Plauoi-extract in microemulsion for treatment of dermatitis. The Thailand Research Fund. Siriphan P, Chutimaworapan S, Chaichantipyut C. 2008. Antioxidant evaluation and formulation development of Zingiber officinale extract loaded in solid lipid nanoparticles for skin delivery. Thesis (M.Sc.) Chulalongkorn University.

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