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Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 74 No. 2 pp. 497ñ504, 2017 ISSN 0001-6837 Polish Pharmaceutical Society FORMULATION AND ASSESSMENT OF SEMI-SOLID CARRIER INCORPORATED WITH HERBAL EXTRACT OF LAWSONIA INERMIS AYSHA ASLAM1,2*, FAISAL SAMEE3, MUHAMMAD ZAMAN1, ATTIQUE UR RAHMAN MUFTI4 and ATIQ UR REHMAN1 Faculty of Pharmacy, The University of Lahore, Lahore, Pakistan Institute of Pharmaceutical Sciences, University of Veterinary and Animal Sciences, Lahore, Pakistan 3 College of Pharmacy, University of the Punjab, Lahore, Pakistan 4 Faculty of Pharmacy, University of Sargodha, Sargodha, Pakistan 1 2 Abstract: The development of an optimal microemulsion of lawsone for transdermal delivery was the core objective of the study. Effects of formulation variable including oils, surfactants and co-surfactants on the percutaneous delivery of lawsone microemulsion have also been inspected. Pseudoternary phase diagrams with oil, cosurfactant-surfactant mixtures (Smix) were constructed to recognize the microemulsion areas. Microemulsion prepared was of 5%. The in vitro transdermal penetration of microemulsion of lawsone was determined by Franz diffusion cell. These profiles indicated that the absorption and rate of penetration of lawsone microemulsion was far better than the saturated solution of lawsone. The formulation was characterized for pH, conductivity, viscosity and passed stability tests. A non-irritant formulation to skin was prepared in this way. The outcomes specify that the transdermal drug delivery ability and phase behavior of microemulsion is affected by the type of cosurfactant and surfactant. Keywords: cosurfactant, lawsone, surfactants, transdermal microemulsion Natural Orange 6. Phase is solid. Chemically it is 2hydroxy-1,4-naphthoquinone. It is almost insoluble in water, soluble in methanol and 95% ethanol. Molecular weight is 174.153 g/mol and density is 1.46 g/cm3, melting point is 195-196OC with decomposition, with an optical absorption maximum of 452 nm. Molecular formula is C10H6O3. Aqueous lawsone extracts strongly absorb UV light, and can act as effective sunscreens. The plant has many activities including therapy for malignant ulcers, jaundice and epilepsy, and for dyeing grey hair (4). The leaves are used in obstinate skin diseases, bleeding disorder (5) diuretic, ophthalmia, syphilitis, sores, amenorrhoea, scabies, headache, bronchitis, boils, spleen diseases and favors the growth of the hair. Flowers are refrigerant and used in insomnia (6) and headache (7). The bark is used in jaundice, spleen enlargement and leprosy. It is used as medicinal plant because of its antibacterial, antifungal, antihemorrhagic, hypotensive, sedative, antiamoebiasis and astringent effects (8). Many diseases are being treated with drug obtained from plant herbal products (1). Knowledge and benefits regarding medicinal drugs are available in the earliest literature. Nowadays, people are using traditional medicines for their health care needs (2). Lawsonia inermis generally known as henna belongs to Lythraceae family nurtured in warm dry climates. A source is a 2.6 m multibranched small tree which leaves are opposite, elliptical, sub-sessile and broadly lanceolate (1.5ñ5.0 cm ◊ 0.5ñ2 cm), with depressed veins on the dorsal surface. Flowers have four sepals, petals are obviate and a 2 mm calyx tube with white or red stamens inserted in pairs on the rim. Ovary is four celled style up to 5 mm long and erect; 4ñ8 mm diameter brownish capsules fruits are small having 32ñ49 seeds, opening in four splits (3). It contains tannins as active ingredients. Main chemical components include lawsone, hennadiol, scopoletin, betulin, quinone and naphthoquinone. The primary coloring matter of henna is lawsone, also known as * Corresponding author:e-mail: [email protected] 497 498 AYSHA ASLAM et al. The aim of the present study was to prepare microemulsion using herbal plant extracts and evaluate their release from the topical preparations using Franz cells. Nylon membrane was used for this purpose. Drug release from the topical preparation containing traditional remedy was investigated. EXPERIMENTAL Materials Synthetic standard of lawsone was purchased from Aldrich (Sigma-Aldrich, Germany). Olive oil, seasame oil, oleic acid, Polysorbate 80, and sorbitan monolaurate (were all from Daejung, Korea), propylene glycol (Merck, Pakistan), 2-propanol, ethanol (BDH, England). All the chemicals used were of analytical grade. Plant material extraction Sample of henna leaves were collected in June 2012. The leaves were air-dried and powdered. The 500 g powdered leaves were soaked in 1500 mL of 96% alcohol and 1500 mL of distilled water (alcohol : distilled water 1 : 1) (by maceration) in 5000 mL beaker for 14 days and filtered through Whatmann filter paper No. 41. Solubility study Addition method was used to check the solubility of L. inermis by adding ethanolic plant extract in cosurfactants, oils and surfactants in stoppered vial having 5 mL capacity through magnetic stirring for 1 h at 25OC. Preparation of microemulsion The microemulsion was prepared as described by Chen (9). A surfactant mixture was prepared by manually mixing Tween 80 and ethanol as surfactant and cosurfactant, respectively, in a 1 : 1, v/v ratio. Surfactant mixture was added to 0.5 g of oleic acid (oil) and mixed vigorously with magnetic stirring. Plant extracts (0.5 g) were added to the surfactant mixture and then into oleic acid and stirred until completely dissolved. In order to check transdermal drug delivery potential, saturated aqueous solution of drug was compared with the microemulsion formulation. Excess amount of L. inermis extract was dissolved in distilled water to prepare the saturated aqueous solution of the drug. Membrane filter was used for filtration of solution. Preparation of apparatus and sampling Franz diffusion cells (PermeGear, Bethlehem, PA) having receptor section with a size of 12.0 mL and a surface area of 1.767 cm2 were used. Hydro alcoholic solution 70 : 30, v/v was used as the receptor medium. Equilibration of the skin was done by soaking it in hydro alcoholic solution for 12 h at 4OC (10). Receptor medium was poured into the receptor section and then the skin was placed in such a way that stratum corneum side of the skin face the donor section. A clamp was used to join two halves of the cells. Trapping of air should be avoided beneath the skin (11). In case of trapping of bubbles, sampling port was slanted horizontally until the bubble escaped. Water bath was used to maintain the temperature of the receptor solution at 32 ± 0.2OC with a Teflon-coated magnetic bar for stirring of solution at 600 rpm during the experiment. The donor section was sealed with aluminum foil and contained 1.0 mL of test microemulsion (12). A long needle was fixed to a syringe and was used to withdraw samples at fixed intervals (0.5, 1, 2, 3, 4, 5, 6, 12 and 24 h). Syringe filter (0.45 µm) was used to filter the solution analyzed by HPLC at 260 nm against a blank (13). The same amount of fresh hydro alcoholic solution was added to the receptor section after the removal of the trial sample. The experiments were performed three times. Water titration method Water titration method is mostly used to construct phase diagrams for microemulsions at room temperature (14). The diagram was plotted by ProSim Ternary Diagram software. Characterization of microemulsions HPLC analysis HPLC system equipped with a column oven (Varian Metathermô), an ultraviolet (UV) diode array detector (Varian ProStarô) and a ternary pump (Varian 9010) was used for chromatographic analysis. Reverse-phase Supelcosil LC-PAH C18 chromatographic column (5 µm, 150 ◊ 3 mm) was used for the separation of HNQ. Registered spectral range was 190ñ400 nm with λmax at 260 nm. Mobile phase: 1 mL acetic acid : methanol in volume ratio 20 : 80; flow rate: 0.8 mL/min; column and detector temperature: 40OC. Autosampler injection was of 5 µL. Preparation of stock solution: Ten milligrams of lawsone was weighed and dissolved in methanol and the final volume was made up to 10 mL in a volumetric flask (stock solution 1 mg/mL). This prepared solution (100 µL) was taken and the volume made up with methanol to give 900 µL solution (0.1 mg/mL). Further dilution 499 Formulation and assessment of semi-solid carrier incorporated... of the solution was done by adding 900 µL of methanol in 100 µL of this solution to give concentration of 0.01 mg/mL. In the same way 0.001 and 0.0001 mg/mL solutions were prepared by serial dilutions, respectively, and their peak areas were determined by HPLC at 260 nm. Microemulsions were inspected visually for the following characteristics: ● Visual clarity ● Color ● Transparency ● Homogeneity ● Phase separation Rheological measurements RVDV III Brookfield viscometer was employed to determine viscosity at various shear rates and different torque values and results were presented in cP. The Rheocalc V2.6 software was used for calculation. The temperature was maintained at 25.2OC throughout the experiment. Droplet size analysis Zetasizer nano S (Malvern Instruments, Worchestershire, UK) was used to conclude the droplet size of the microemulsion employed. Light scattering was observed at 25OC. pH measurements WTW inoLab, Germany pH meter was employed to measure pH values of microemulsion at 25OC. Table 1. In vitro physico-chemical parameters of formulation. Table 2. Known concentrations of standard of lawsone with corresponding HPLC area values. No. Parameters Observation 1. Clarity Clear 2. pH 6.4 3. Conductivity 0.25 µS/cm 4. Viscosity 432-440 cp 5. Flux 6.7 (µg/cm2/h) Conductivity measurements WTW Cond Multi 197i (Weilheim, Germany) conductometer was employed to measure conductivity (σ) of microemulsion and results are presented in µS/cm (15). Concentration mg/mL Area a.u. 1 1 9844415 2 0.1 755025 3 0.01 60043 No. Figure 1. Pseudoternary phase diagram of L. inermis based microemulsion 500 AYSHA ASLAM et al. Figure 2. Known concentrations of standard of lawsone with corresponding HPLC peak area values (a.u.) Figure 3. HPLC chromatogram of lawsone Figure 4. Average of percentage release vs. time interval Formulation and assessment of semi-solid carrier incorporated... Table 3. Average of percentage release vs. time interval. No. Time h Average drug release % 1 0.5 8 2 1 15.5 3 2 27 4 3 37 5 4 50 6 5 66.5 7 6 70.5 8 12 81 9 24 93.5 501 Thermodynamic stability tests The selected microemulsion formulation was subjected to stress tests which are as follows (16): ??????? Heating/cooling cycle The formulation was stored for 48 h at 4OC (refrigerator temperature) and 40OC. The formulation was found to be stable at these temperatures. Freeze thaw cycle The formulation was stored for 48 h between 50OC and +25OC (room temperature). The formulation remained stable at these temperatures also. Figure 5. Graphical representation of Korsmeyer Peppas model Figure 6. Graphical representation of first order model 502 AYSHA ASLAM et al. Figure 7. Graphical representation of zero order model Table 4. 'R2' and 'n' values for zero order, first order and Korsmeyer Peppas (KP) model. Zero order First order KP model R2 K R2 K R2 N 0.993 10.661 0.4746 0.076 0.9205 0.66 MS F Significance F 15.75831 0.005395 Table 5. One way ANOVA of permeation data of microemulsions. Df SS Regression 1 4983.963 4983.963 Residual 7 2213.926 316.2752 Total 8 7197.889 Table 6. Regression statistics applied on the formulation data. Regression statistics Multiple R 0.832118 R2 0.69242 Adjusted R 2 0.64848 Standard error 17.78413 Observations 9 males/4 females. Sample (0.1 g) was retained on 1 ◊ 1 cm2 gauze dressing and applied onto the skin of inner arms directly with stretch adhesive tape (Paragonô) to fix it. Erythema was measured with Mexameterô (Courage and Khazaka, Germany) after 8 h. Before use of the formulation, the skin of inner arms was measured for control studies (17). Stability studies No phase separation occurred when centrifuged at 6000 rpm for 30 min. Calculation of flux (J) The rate of permeation per unit area or the rate of transfer is called flux (J) and is represented by a vector in space and the results are presented in µg/cm2/h (18). Skin irritation study Ten human volunteers were selected and formulation was tested for 8 h. The volunteers were within the age range of 22-29 years and include 6 Statistical analysis The collected data were analyzed by appropriate statistical procedure - analysis of variance (ANOVA) (19). 503 Formulation and assessment of semi-solid carrier incorporated... Table 7. Summary of the statistical parameters of microemulsion. Parameters Coefficients Standard error t Stat p-value Lower 95% Upper 95% Intercept 28.480 8.014 3.55 0.009 9.530 47.431 Time 3.350 0.844 3.96 0.005 1.354 5.346 y = 28.48 + 3.35x RESULTS The solubility of lawsone in different oils, cosurfactants and surfactants was determined. Pseudoternary phase diagram 1 : 1 was constructed in which oil phase is oleic acid, surfactant is Tween 80 and cosurfactant is ethanol. Instead of using pure water, hydro alcoholic extract was used. Pseudoternary phase diagram is given in Figure 1. The microemulsion formulation was evaluated with respect to clarity, pH, viscosity, electrical conductivity and flux. Viscosity was measured using spindle no. 2 with speed of 20 rpm. Values of viscosity, pH, conductivity and flux are given in Table 1. In vitro release studies of natural constituent of L. inermis extract from microemulsion formulation were done by combination of Franz cells and HPLC determination technique using C18 column. Standard curve was constructed against known concentration of lawsone standard vs. peak area of HPLC chromatogram. Values for standard curve are given in Table 2 and graphical representation is given in Figure 2. Regression value of standard curve is highly applicable R2 = 0.9995. Average percentage release of active constituent of formulation by using vertical diffusion cells at different time intervals was given. Values of graph are given in Table 3 along with the graphical representation in Figure 4. Different models were applied at time and average percentage release. Following models were applied and graphs were plotted. 1. Korsmeyer Peppas model 2. First order model 3. Zero order model Graphical representation is given in Figures 57. The data of the formulation show results according to zero-order model. Calculation of ëR2í and ëní value for each model is given in Table 4. The collected data were analyzed by appropriate statistical procedure which is analysis of variance (ANOVA) or by any suitable statistical procedure fit for data. The details are given in Table 5. The data show that results are significant. The results were analyzed by one-way analysis of variance (ANOVA). The value of p = 0.009295 (Table 5), therefore it showed significant result. The transdermal flux of microemulsion formulation significantly increases. Tables 5-7 summarize the statistical parameters of microemulsion. The calculated value for Y intercept is 28.48 + 3.35 x. It shows that an increase in time will increase the permeation rate of the extract. DISCUSSION Microemulsion was prepared by using Tween 80, ethanol and oleic acid. Four different formulations were prepared. Pseudoternary diagram was constructed, in which shaded area represents the microemulsion range whereas non shaded area represents unclear range. Phase diagrams were used to determine concentrations required to prepare microemulsion. All the four formulations were prepared at 1 : 1 ratio. Out of these four formulations one was selected on the basis of clarity, pH, conductivity, flux and viscosity. The high conductivity of microemulsion 0.25 µS/cm showed that it was O/W type microemulsion. Microemulsion had a pH of 6.4 which was the indication that it could be nonirritating to skin and will be best for topical application. The viscosity was found to be 432-440 cP. The flux value 6.7 µg/cm2/h showed the highest permeation rate through skin. Surfactants are known to enhance the permeability and flux. Same effect of the non-ionic surfactant was noticed here in the current study that addition of non-ionic surfactant, Tween 80 has increased the flux greatly. They increase the permeation by reducing the barrier characteristics and crystallinity of lipid bilayer thatís why they are commonly and effectively used in topical formulations. Various formulated formulations were evaluated by various stated parameters and finally the best formulation was selected. The permeation profiles followed zero order kinetics. The permeation experiments were analyzed by one-way analysis of variance (ANOVA) at the level of p < 0.05. The value of p = 0.009295 which is smaller than 0.05, therefore it showed significant result. The 504 AYSHA ASLAM et al. micro emulsion formulation significantly increased the transdermal flux in comparison to the saturated aqueous drug solution which was used as the control. CONCLUSION Microemulsion containing lawsone was formed effectively. Instead of simple aqueous solution of drug, prepared microemulsion is estimated to boost the transdermal drug delivery potential of this drug. Hazard of side effects can be overwhelmed in this way and recurrent application will be possible. Acknowledgment To Mr. M. Nadeem Irfan Bukhari, for the technical support in the Laboratory of Pharmaceutics, University College of Pharmacy, University of the Punjab, Lahore, Pakistan. REFERENCES 1. Biswas K., Chattopadhyay I., Banerjee R.K., Bandyopadhyay U.: Curr. Sci. 82, 1336 (2002). 2. Goyal B., Goyal R., Mehta A.: Pharmacogn. Rev. 1, 143 (2007). 3. 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