Formulation and Evaluation of Herbal Emulgel of Pothos scandens Linn for Burn Wound Healing Activity

Transcription

1 Formulation and Evaluation of Herbal Emulgel of Pothos scandens Linn for Burn Wound Healing Activity Mohammed Haneefa K.P 1*, Abid 2, Shahima Hanan K 3, Guru Prasad Mohanta 4,Chandini Nayar 5 1, 2, 3 Alshifa College of Pharmacy, Poonthavanam Post, Kerala, India *Research Scholar, School of Chemical & Biotechnology, SASTRA University, Tamil Nadu, India 4 Department of Pharmacy, Annamalai University, Tamil Nadu, India 5 School of Chemical & Biotechnology, SASTRA University, Tamil Nadu, India Abstract The main aim of this work was to formulate the leaf extract of Pothos scandens in to an emulgel and investigate their burn wound healing activity. Ethanolic extract of dried leaves of Pothos scandens were subjected to preliminary phytochemical evaluation and wound healing activities studies. Emulgel formulations were prepared using three types of gelling agents: Carbopol 934, Carbopol 940 and HPMC K 4 M. The influence of the type of the gelling agent on the drug release from the prepared emulgel was investigated. The prepared emulgel were evaluated for their physical appearance, ph, viscosity, spreadability, in vitro drug release, pharmacological activity and stability. It was finally concluded that the formulation F1 with 1%w/w Carbopol 940 was found to be more promising formulations as it shows better physicochemical characteristics and higher pharmacological activity compared to other formulations. Herbal emulgel of ethanolic extract of Pothos scandens shows significant improvement in burn wound contraction and hence this is a promising candidate in burn wound healing Keywords: Pothos scandens, Burn wound healing, Emulgel, Carbopol 934, Carbopol 940, HPMC K 4 M. INTRODUCTION: Topical preparations are used for the localised effect at the site of their application by virtue of drug penetration into the underlying layer of skin or mucous membrane. Topical drug administration is a localized drug delivery system anywhere in the body through ophthalmic, rectal, vaginal and skin as topical routes. The main advantage of topical delivery system is to bypass first pass metabolism 1, 2.When gels and emulsions are used in combined form the dosage forms are referred as Emulgel. The presence of a gelling agent in the water phase converts a classical emulsion into an emulgel. Direct (oil-in-water) system is used to entrap lipophilic drugs whereas hydrophilic drugs are encapsulated in the reverse (water-in-oil) system 3. Emulsions possess a certain degree of elegance and are easily washed off whenever desired. They also have a high ability to penetrate the skin. Emulgels for dermatological use have several favourable properties such as being thixotropic, greaseless, easily spreadable, easily removable, emollient, non-staining, water-soluble, longer shelf life, bio-friendly, transparent & pleasing appearance 4. Pothos scandens ( Family- Araceae ) is a climbing shrub having adventitious aerial roots. The internodes of the plant are cm and its leaves are very variable. The leaves are obovate, elliptic or lanceolate and coriaceous, having a bright green colour. Pothos scandens has been used in herbal medicine for the treatment of skin disease, boils, swellings, wound ulcers, dropsy, menorrhagia, vomiting, flatulence and burning sensations. The extract is also reported regarding its usage in the treatment of cuts and wounds 5,6,7,8. The present study was conducted to formulate herbal emulgel of Pothos scandens using gelling agents like Carbopol 934, Carbopol 940 and HPMC K4M. The prepared emulgels were evaluated for physicochemical as well as for pharmacological activity. MATERIALS AND METHODS Materials Carbopol 934, carbopol 940 and HPMC K4M were obtained from Loba Chem. Pvt Ltd, Mumbai. Methyl paraben sodium and propyl paraben sodium were obtained from Hi Media laboratories. Glycerol and triethanolamine were obtained from Nice chemicals Pvt. Ltd, Mumbai. Plant materials and extract preparation P.scandens Linn. (Family: Araceae) were collected from local areas of Perinthalmanna, kerala and was authenticated by Prof. M. Sabu, Head of Department of Botany, University of Calicut. The foreign, earthy matter and residual materials were removed carefully from the leaves and then cleaned and dried in the shade. It was then powdered and used for extraction. Powdered herb was placed inside a thimble made from thick filter paper, which was loaded into the main chamber of the soxhlet extractor. The soxhlet extractor was placed onto a flask containing the extraction solvent until it get exhausted. The extract is filtered and concentrated under reduced pressure. It was stored at C until use. Animals A total of 78, 12-week old healthy wistar strain rats weighing g of either sex, bred locally in the animal house of Al Shifa College of Pharmacy, Perinthalmanna, were selected for the wound healing studies and 9 albino rabbits (average wt 2.2 kg) were used for primary skin irritation test. They were housed under controlled conditions of temperature (23 ± 2 ) 0 C, humidity (50 ±5) 0 C and hours of light and dark cycles. The animals were housed individually in polypropylene cages containing sterile paddy husk bedding and free access to food and water ad libitum. The study was conducted after obtaining 63

2 the approval from Institutional Animal Ethical Committee.(Reg No: 1195/ac/08/CPCSEA). Acute toxicity studies Acute toxicity studies were performed for the extract as per OECD guidelines (Class method). Three healthy albino rates average weight 150 g were used for the study. A maximum dose of 2g/kg body weight was administered orally.animals were observed individually after dosing once during the first 30 minutes, periodically during the first 24 hours, with special attention given during the first 4 hours, and daily thereafter, for a total of 14 days. Wound healing studies of extract 9,10 Rats were anesthetized with ketamine + xylazine (50 mg/kg+5 mg/kg) and the hair on the back was clipped with electric clippers. Burn wounds were created by using a device with an iron piece and a wooden handle placed on the back of the rat. It was heated to red hot over flame and was placed in contact with the back of anaesthetized rat up to 10 seconds without any pressure. After this, each animal was placed in a separate cage for full recovery from anaesthesia before being returned to holding rooms. The wound of the test animal was applied with extract in glycerol and the control group I was applied with glycerol as the solvent control and control group 2 were left untreated. The application was repeated daily for the next 20 post-operative days and the following parameters were studied. (1) Epithelization period: It was monitored by noting the number of days required for scar to fall away, leaving no raw wound behind; (2) Wound contraction: To monitor this, progressive changes in wound area were followed planimetrically. Leaving the wounding day, wounds were traced on a transparent paper on an alternate day. The animal was restrained in proper position during tracing. The tracings were then transferred to 1 mm2 graph sheet. From this, wound areas were read and the percentage of wound contraction was calculated taking the initial size of wound (250 mm 2 ) as 100%. % Wound contraction initinal wound size inal wound size = 100 initial wound size Formulation of Emulgel 11 Different formulations were prepared using varying amount of gelling agent and penetration enhancer. The method only differed in the process of making gel in different formulations. The preparation of emulsion was same in all the formulations. The gel bases were prepared by dispersing Carbopol 934 and Carbopol 940 in distilled water separately with constant stirring at a moderate speed using mechanical shaker. Formulations F1, F2 and F3 were prepared by carbopol 934 and F4, F5 and F6 by carbopol 940 as gelling agent. In formulations F7, F8 and F9 the gel were prepared by dispersing HPMC K4M in heated distilled water (80 C), and the dispersion was cooled and left overnight. The ph of all the formulations was adjusted to using tri ethanol amine (TEA). The oil phase of the emulsion was prepared by dissolving Span 20 in light liquid paraffin while the aqueous phase was prepared by dissolving Tween 20 in purified water. Methyl and Propyl parabens were dissolved in propylene glycol and mixed with the extract dissolved in ethanol, being hydrophobic was dissolved in oil phase. Clove oil was also mixed in oil phase. Both the oily and aqueous phases were separately heated to 70 to 80 C, then the oily phase was added to the aqueous phase with continuous stirring until it got cooled to room temperature. The obtained emulsion was mixed with the gel in 1:1 ratio with gentle stirring to obtain the emulgel. The composition of different formulations has been discussed. Evaluation of Emulgel 1. Physical Examination 12 The prepared emulgel formulations were inspected visually for their colour, homogeneity, consistency, grittiness and phase separation 2. Estimation of drug content 13 Preparation of standard solution Take 100 mg of crude extract and dissolved in 100 ml of ethanol (Stock solution 1). From this stock solution 1ml was taken and diluted to 10 ml [stock solution - 2]. From stock solution - 2 take volume 2,4,6,8 and 10ml separately and made up to 10ml to get concentration range 20, 40, 60, 80, 100mcg/ml respectively. The absorbance was measured at 270 nm against ethanol as blank. Preparation of test solution of Pothos scandens Linn emulgel 1 gm each formulation containing approximately 40 mg of drug was taken in a 50 ml volumetric flask and diluted with ethanol and shaken to dissolve the drug in ethanol. The solution was filtered through whatmann filter paper, 0.1 ml of the filtrate was pipette out and diluted to 10 ml with ethanol. The content of the drug was estimated spectrophotometrically by using standard curve plotted at 270 nm 3. Measurement of ph 14 The ph of emulgel formulations was determined by using digital ph meter. 1gm of gel was dissolved in 100 ml of distilled water and it was placed for 2 hr. The measurement of ph of each formulation was done in triplicate and average values were calculated. 4. Viscosity 15 Viscosity of the emulgels was determined using Brookfield viscometer. Spindle type, model LVDV-E at 10 rpm. 200 grams of the gel was taken in a beaker and the spindle was dipped in it for about 5 minutes and then the reading was taken. 5. Spreadability 16,17 Spreadability denotes the extent of area to which the emulgel readily spreads on application to skin or the affected part. The bioavailability efficiency of a emulgel formulation also depends on its spreading value. The 64

3 Spreadability was expressed in terms of time in seconds taken by two slides to slip off from the emulgel which was placed in between the slides, under certain load. Lesser the time taken for separation of the two slides, better the spreadability. Two sets of glass slides of standard dimensions were taken. The herbal emulgel formulation was placed over one of the slides. The other slide was placed on the top of the emulgel, such that the emulgel was sandwiched between the two slides in an area occupied by a distance of 7.5 cm along the slide. 100gm weight was placed upon the upper slides so that the emulgel between the two slides was pressed uniformly to form a thin layer. The weight was removed and the excess of emulgel adhering to the slides was scrapped off. The two slides in position were fixed to a stand without slightest disturbance and in such a way that only the upper slide to slip off freely by the force of weight tied to it. A 20 gm weight was tied to the upper slide carefully. The time taken for the upper slide to travel the distance of 7.5 cm and separated away from the lower slide under the influence of the weight was noted. The experiment was repeated by three times and the mean time taken for calculation. Spreadability was calculated by using the following formula: = Where, S Spreadability m Weight tied to the upper slide (20gm) l - Length of the glass (7.5 cm) t - Time taken in seconds 6. Permeability studies 18 The in vitro diffusion studies of the emulgels were performed using dialysis membrane. The membrane was soaked in phosphate buffer ph for 6 8 hr & was clamped carefully to one end of the hollow glass tube. Phosphate buffer of ph was used for in vitro release as a receptor medium. The emulgel sample was applied on the membrane and then fixed in between donor and receptor compartment of glass tube. The receptor compartment contained phosphate buffer (100ml) of ph. The temperature of diffusion medium was thermostatically controlled at 37 0 C ± 1 0 C by surrounding water in jacket and the medium was stirred by magnetic stirrer at 500rpm. The sample were withdrawn at predetermined intervals and was replaced by equal volume of fresh fluid. The samples withdrawn were spectrophotometrically estimated at 270nm using phosphate buffer as blank. Pharmacological studies of formulation Primary skin irritation test 19,20 This study employed nine rabbits (white, average weight of 2.2 kg) to test for the skin irritation. They were kept carefully following an acclimation period of 7 days to ensure their suitability for the study. Test animals were kept within a limited-access rodent facility with environmental conditions set to a temperature of 25 ±2 ºC, a humidity of 60-90% RH and a 12-h light / 12-h dark cycle. Animals were provided ad libitum access to a commercial rabbit-diet and drinking water was supplied to each cage. The area on the back of each rabbits was shaved prior to the experiment. The back was divided into five marked areas for the topical application of the gel containing various concentrations of extract. Test product was placed on each area for 24 hours using adhesive tape. Scoring of the erythema and edema was performed at 24 and 72 hours with Draize technique. The positive control of this experiment is 98% lactic acid. Wound healing studies of the formulation 9,10 Wound healing studies of formulation were prepared similar to wound healing studies of the extract in which the control groups of rats were applied with dummy gel. The application was repeated daily for the next 20 post operative days and the following parameters were studied, epithelisation period and wound contraction. Stability studies of formulation 18 Stability studies were carried out on formulations according to International Conference on Harmonization (ICH) guidelines.short term accelerated stability study was carried out for the period of 3 months for the formulations. The samples were stored at different storage conditions of room temperature (25 ± C), elevated temperature such as 40 C at 75% RH. Samples was withdrawn on weakly interval and analyzed for visual appearance, clarity, ph, spreadability and viscosity. All the emulgel formulations were stored for a period of 12 weeks. Samples were withdrawn at 1,3,6,9 and 12 week and assessed for drug content. At the end of 12th week they were evaluated for physical parameters and integrity of the product. RESULTS AND DISCUSSION Acute toxicity studies Study revealed that there was no mortality and hence the extract was found to be safe for administration. After administration there was no significant change in the body weight of the animals. Wound healing studies of extract Table 1 shows the effect of P. scandens leaf extract administered topically on wound healing in rats with burn wound. Wound contraction studies in Pothos scandens Alcoholic extract revealed that 4 % of Alcoholic extract have maximum wound healing activity. In this model, P. scandens treated animals were found to epithelise in 22 days while the solvent control and the untreated rat s epithelise with 35 and 40 days respectively. On the 20th day, the percentage wound area reduction in solvent controls, untreated rats and the extract treated rats were 64 %, 54% and 95% respectively. Formulation and evaluation of gel Herbal emulgels of P.scandens Linn were prepared successfully by using different concentration of Carbopol 934, Carbopol 940 and HPMC K 4 M. It was found that all the formulations were light green in colour and the physical evaluations were given in Table 2. The P. scandens alcoholic extract shows λ max at 270 nm. All the formulations were found to be neutral (ph 6.2 to 7.2) and drug content was found to be in the range of 89%-100 % w/w (Table 2). Viscosities of all the formulations are given in Table 3. 65

4 Conc. of the alcoholic extract (%w/v) Solvent control Table 1- Wound healing effect of Pothos scandens extract in burn wound model. Day 4 Day 8 Day 12 Day 16 Day 20 Epithelization time (In days) 3.10± ± ± ± ± ± % 8.61± ± ± ± ± ± % 5.25± ± ± ± ± ± % 7.46± ± ± ± ± ± % 5.00± ± ± ± ± ± % 5.98± ± ± ± ± ± 2.4 untreated control Values are mean ±SD of six readings Formulation F1 F2 F3 F4 F5 F6 F7 F8 F9 2.90± ± ± ± ± ± 1.06 ph Excellent ++ Good Table 2-Characteristics of P. scandens gel formulations. Viscosity Spreadability gm Drug content (cps) cm/sec (%w/w) Extrudability ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Table-3- Regression co-efficient (R 2 ) values of kinetic models for formulation F1and F4 Formulation code Zero Order First Order Higuchi Model Peppas Model F F Nature of gel Table-4 Wound healing effect of formulations in burn wound model. Day 4 Day 8 Day 12 Day 16 Day 20 Epithelization time Control of Carbopol ± ± ± ± ± ±1.05 F1 7.96± ± ± ± ± ±1.25 F2 8.92± ± ± ± ± ±1.02 F3 6.25± ± ± ± ± ±1.54 Control of Carbopol ± ± ± ± ± ±1.28 F4 9.14± ± ± ± ± ±0.84 F4 ± ± ± ± ± ±0.32 F6 2± ± ± ± ± ±1.03 Spreadability denotes the extent of area to which the gel readily spreads on application to skin or the affected part. The bioavailability efficiency of a emulgel formulation also depends on its spreading value. Maximum spreading value was found for formulation with 1% carbopol 934. The extrudability reflects the capacity of the gel, to get ejected in uniform and desired quantity when the tube is squeezed. The results of extrudability are shown in Table 2. Permeability studies The results obtained in in vitro release studies were plotted in different kinetic models. Regression coefficient (R 2 ) values of different kinetic models are shown in Table 3. This indicated that the release data was best fit with Higuchi model kinetics because the value of R 2 is greater in this model. Higuchi equation explains the diffusion release mechanism, so formulations follow the diffusion mechanism of drug release. 66

5 Stability studies The formulated emulgels were subjected for stability studies. No colour fading was observed for all prepared gels. The ph of all formulations were not affected and found to be within the range of The viscosity and spreadability of all emulgels was found to be same especially at ambient, 4-5 O C and 37 0 C temperature. The drug content was found to be in the limit % for all emulgel formulation at all temperature conditions. Pharmacological studies of formulation Primary skin irritation test The skin irritation studies revealed that all formulations were non-sensitizing and safe for use. Wound healing activity studies of the formulation In this model, formulation with carbopol 940 treated animals shows better healing compared to carbopol 934 treated rats. Formulations with 1% carbopol 940 treated rats were found to epithelise in 20 days while the control group epithelise with 30 days. Results are shown in Table 4. Statistical analysis Data obtained from pharmacological experiments are expressed as mean ± SD. Differences between the control and the treatments in these experiments were tested for significance using student t test. Differences were considered significant if P > CONCLUSION The aim of the study was to formulate and evaluate Pothos scandens Linn leaf extract in to a emulgel for burn wound healing activity. The leaves were successfully extracted with ethanol and water. The phytochemicals present in the extract were identified by qualitative phytochemical screening, which reveals the presence of alkaloids in chloroform extract, alkaloids, saponins, and carbohydrates in aqueous extract and alkaloids, proteins and flavonoids in the alcoholic extract. The preliminary pharmacological study showed that the ethanolic extract of Pothos scandens Linn leaf possess maximum wound healing effect in rats and the effects produced was maximum in 4% alcoholic extract and this concentration was used for the formulation. The extract was formulated in to 9 different emulgel formulations by changing the concentration of Carbopol 934, Carbopol 940 and HPMC K4M. All the formulations were found to be neutral (ph to 7.0) and drug content was found to be in the range of %w/w. On physical evaluation of all the formulations, F1 (1% Carbopol 934) and F4 (1% Carbopol 940) was found to be optimum in terms of gel consistency, viscosity, spreadability and extrudability. The invitro release studies were conducted and results are shown in Tables & Graphs Maximum release was shown by F1 and F4, which follows Higuchi diffusion model. Stability studies revealed that there was no significant difference in the physical and chemical parameters. Thus the formulations were found to be stable for six weeks. Pharmacological evaluation of gels revealed that all formulations are non sensitizing and safe for use. Pharmacological studies of the formulations showed that F1 (1% Carbopol 934) has greatest pharmacological activity. It was finally concluded that the formulations F1 was found to be more promising formulation as it shows better physicochemical characteristics and higher pharmacological activity compared to other formulations. REFERENCES 1. C. Surver and F.A. Davis, Bioavailability and Bioequivaance, In: K.A Walter (eds.), Dermatological and Transdermal Formulation, Marcal Dekker, New York, 2002, pp , Sharma S. Topical drug delivery system. Available from: Section/science-news/. [Cited in 2011 Aug 9]. 3. Kuller R, Saini S, Seth N, Rana AC, Emulgel: A surrogate approach for topical used hydrophobic drugs. Int J Pharm Bio Sci, 1(3): , (2011). 4. 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