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To develop a new topical lotion of Diclofenac Diethylamine 2% (w/v) containing turpentine oil and oleic acid as permeation enhancers, at various concentrations (%v/v), this study was conducted to investigate the increased permeability rate of Diclofenac lotion with objective to augment transparency and stability of the lotion and compare the in vitro and in vivo transdermal potential because transdermal delivery of Diclofenac may provide better patient compliance over oral administration. Using modified Franz-diffusion cell, the amount of DDA permeated through silicone membrane and sections of excised full thickness of rabbit skin was calculated by the UV absorbance at 276 nm. Carrageenan-induced rat paw edema model was used to investigate their in vivo performance. To explain the difference in values of flux between saturated and control may be the differential uptake of enhancer's by the SC of the skin, while flux values for all concentrations of enhancer's across rabbit skin were statistically significant (P<0.05). The formulation L4 concentration of enhancers (with 1:1 ratio) shows minimum lag time gives a picture about its rapid enhancing effect. The mode of action of these accelerants may be described by combined process of partition and diffusion, the diffusion process being dominant from the results. From this study it was concluded that these enhancers in lotion showed good stability, homogeneity and no skin irritation and had wider prospect for topical preparations. The in vivo studies gave an effective therapeutic picture of the lotions. Hence, in vitro and in vivo studies showed that the lotion could be used for effective therapy.
Lotions are liquid preparations intended for application to the skin. The inclusion of alcohol in a lotion hastens its drying and accentuates its cooling effect, whilst the inclusion of glycerol keeps the skin moist for a considerable time (BPC 1973, Winfield 2004). Transdermal delivery of Diclofenac may provide better patient compliance over oral administration however; Diclofenac is not easily absorbed on transdermal application (Kweon 2004). Diclofenac Diethylamine was chosen as a lipophilic model drug, having a MW of 260.7 and Ko/w = 4.40 (Guy 2003) and it is stated in the literature that highly lipophilic drugs with partition coefficients greater than 2 or 3 tend to remain in the stratum corneum for an extended period of time and will not penetrate well into the lower skin layers and hence their therapeutic effects will be under question. The mechanism of penetration enhancement effects of the surfactants is primarily believed to be due to the promotion of membrane-vehicle partitioning tendency of the drug (Mukherjee 2005).
Considerable research work has been focused on discovering methods to increase stratum corneum permeability. One approach was to employ chemical penetration enhancers (Barry 1995), which may increase the permeability of stratum corneum (SC) by increasing drug diffusivity within the membrane and /or by increasing drug partition from the applied formulation into the skin and/or by increasing the concentration of drug in the vehicle. The effect of these compounds on the drug cutaneous absorption profile has been quantified by permeation parameters, such as drug flux across the skin/membrane and skin/membrane drug uptake (Cordero 1997).
The enhancer components in the lotion are usually in a large molar excess of the drug and their effects are non-irritating, non-immunogenic and rapidly reversible which is especially true if the penetration enhancer permeates the skin to change the solubilizing capacity of the skin for the drug and due to their non-greasy properties, they can provide easily washable film on the skin (Shivhare 2009). The formulation based enhancer approach also ignores the physicochemical properties of the drug (one penetration enhancer fits all drugs), although there are several reports suggesting that an enhancer which is effective for a polar drug may not be effective for a lipophilic drug and vice versa (Loftsson 1989).
The aim of this study was to investigate the effect of turpentine oil and oleic acid as penetration enhancers in binary system, when used in different concentrations in the formulation of lotion, on the percutaneous absorption of DDA in vitro. Furthermore, the enhancement ratio (ER) of these penetration enhancers at different concentrations has been tabulated and contrasted.
Oleic acid, Ethyl alcohol, Methanol and Sodium chloride were all purchased from Merck Chemical Co., Germany. Turpentine oil was obtained from MS Traders, China origin. Reference standard Diclofenac Diethylamine (DDA) powder was a gift from Novartis (Pvt.) Ltd. Jamshoroo, Pakistan. Silicone membrane (Samco. USA; 400Î¼m thickness).
Preparation of Diclofenac Lotion:
2 gram Diclofenac Diethylamine was dissolved in 15 ml of ethanol in 100 ml volumetric flask and then added various concentrations (i.e.1%, 2%, 3% and 4%) of oleic acid and turpentine oil (1:1 ratio v/v) into the drug solution (L1, L2, L3 and L4) and also all other excipients and then volume was made up to the mark with ethanol. The resulting lotion as given in table 7 was then used for permeation studies.
2 gram Diclofenac Diethylamine was dissolved in 15 ml of ethanol in 100 ml volumetric flask and the volume was made up to the mark with phosphate buffer saline. This was used as reference control solution (LC) without any enhancer.
In Vitro Studies
The above formulated enhancer-based lotions containing Diclofenac diethylamine were subjected to evaluate for the following parameters:
Preformulation studies are preferred to ensure the development of a stable as well as therapeutically effective and safe dosage form. It is a stage during which characterizes the physico-chemical properties of the drug and its interaction with various formulation components.
The pH of all lotion formulations was determined by using digital pH-meter.
Excess DDA was added to each solvent or co-solvent mixture and stirred with a magnetic bar for 48 hours (to attain equilibrium) in a water bath maintained at 37°C±2. Solutions were centrifuged for 30 minutes at 4000 rpm. The supernatant was then diluted and assayed by UV-spectrophotometer Agilent 2005 with software Version-2005 (Courtesy Agilent, Germany) at the wavelength of 276 nm. Experiments were performed in triplicate (n=3) and mean values with standard deviation (±SD) and coefficient of variation were calculated.
Solvent such as ethanol are very often used to increase the drug solubility in water or aqueous vehicles. The solubilisation effect is primarily dependent on the solubility (or polarity) of the drug with respect to the solvent (S) and co-solvent (CoS) system (Yalkowsky 1981). The degree of saturation (DS) was also calculated by the method used by Davis et al (Davis 1991).
Drug content determination:
A specific quantity 5 ml (100mg) of each formulated lotion was taken and dissolved in 5 ml of ethanol and then made up the volume 100ml with phosphate buffer saline (pH 6.8). The volumetric flask containing lotion was shaken for 2 hours on mechanical shaker in order to get complete solubility of drug and uniform solution. This solution was filtered and estimated spectrophotometrically at 276 nm using phosphate buffer saline (pH 7.4) as blank.
Viscosity was determined by using brookfield viscometer. Viscosity measurements were carried out at room temperature (25°C±2) using a Brookfield viscometer (Model RVTDV II, Brookfield Engineering Laboratories, Inc, Stoughton, MA).
It was determined by wooden block and glass slide apparatus. 5ml (100 mg) of lotion was added to the pan and the time was noted for upper slide (movable) to separate completely from the fixed slides (Gupta 1999).
Spreadability was then calculated by using the formula:
S = M.L / T
S = Spreadability
M = Weight/Volumes tide to upper slide
L = Length of glass slide
T = Time taken to separate the slide completely from each other
All formulated lotions were tested for homogeneity by visual inspection after the lotions have been set in the container. They were tested for their appearance and presence of any aggregates (Gupta 2010).
Accelerated Stability Studies
All the formulated lotions were subjected to a stability testing for three months as per ICH norms at a temperature of 40°C±2. All selected formulations were analyzed for the change in appearance (color & odor), pH, phase separation and drug content (ICH 2003).
Diffusion studies through Rabbit skin and Silicone membrane:
Diffusion studies of the lotion across rabbit skin and silicone membrane were performed using Franz-type diffusion cells (made of Germany at SOP, London) that have a receptor phase of ~4.5 ml and a diffusion area of ~0.85cm2. The full thickness rabbit skin was taken from the abdominal surface and hairs were carefully cut as short as possible using scissors, without damaging or scratching the skin surface. Skin/or sheets of silicone membrane were cut according to the diameter of the diffusion cell and the half cell was held fast by a clamp stretching of the skin/or membrane as evidenced by distortion or expansion of the circular outline was corrected and the half cell were held fast by a clamp. The skin was placed in a normal saline solution before mounting on to the diffusion cell (Shah 2005). Both rabbit skin and silicone membrane were soaked overnight in the receptor solution i.e. PBS. The skin/ or membrane was then placed between the two compartments of the diffusion cells using Silicone grease (Dow, USA) to produce a leak-proof seal between the membrane and the two compartments of the diffusion cell. The receptor compartment was filled with PBS at pH 7.4 and each lotion (1 ml) was placed in the donor compartment. To remove air bubbles and prevent the buildup of air pockets in the receptor phase, PBS was degassed in an ultrasonic bath. To prevent evaporation from the receptor compartment, the cell arm was covered with a glass lid. Uniform mixing of the receptor solution was obtained with a magnetic stirrer that was placed in the receptor compartment. The diffusion cells were placed on a stirring bed immersed in a water bath at 35°C±2. After one hour interval, the receptor solution was completely removed and refilled with fresh pre-thermostated PBS. Sink conditions were met in all cases. From the side arm of the receptor compartment, 0.5 ml of the sample was drawn each time at an interval with the help of 1 ml syringe (Sun, Korea) and correcting the receptor half cell volume with pre-thermostated PBS. The sample taken from the receptor cell was run on U.V. spectrophotometer (Agilent2005; software version 2005) at the wavelength of 276 nm. The diffusion experiments were performed under occluded conditions by covering the donor compartment with Para film. All experiments were performed at 35°C±2 in ±SD (n=5) and sampling time was 0-3 hours with predetermined intervals for silicone membrane while 24 hours for rabbit skin studies.
Solvent uptake studies:
The extent to which a vehicle can modify the membrane depends on the amount of its uptake by the membrane, and historically this has been borne out by experimental results. Factors influencing the solvent uptake include solubility parameter and molecular weight.
The uptake of the selected vehicles into silicone membrane and rabbit skin was evaluated in this study. The uptake of vehicles into the silicone membrane was experimentally determined by a gravimetric method. Silicone membrane was cut to an appropriate size (~1cm2) and weighed using a balance (10mg accuracy). They were then placed in a sample bottle containing the vehicle and soaked for 24 hours. The membranes were blotted dry with tissue paper and re-weighed. The experiments were performed in triplicate, at room temperature. The amount of solvent taken up by the membrane was expressed as a weight percent. The solvent uptake is expressed in the following equation as:
The experiments were performed at 35°C±2 in triplicate.
The factor of difference value (FoD):
The flux (J) values calculated from the present permeation study of saturated formulations of DDA has been compared (rabbit skin permeability data vs silicone membrane data) by means of the factor of difference value (FoD) described by the following Dick and Scott equation;
Where JRS and JSM denotes maximum flux value (J) through rabbit skin (RS) and silicone membrane (SM). This study suggested that the artificial membrane model represents a significant prediction for the human skin behaviour if its associated FoD value is less than 3 (Dick 1992).
In Vivo Studies
Primary skin irritation test:
Primary test for irritation was performed on human volunteers. For each ratio of lotion, eleven volunteers were selected and small amount of formulated each lotion was applied on an area of 2 square inch to the back of hand. The volunteers were observed for lesions or irritation.
Draize's skin irritation test:
Modified Primary skin irritation and corrosion based on scoring method described by Draize et al. (Draize, 1944) are evaluated. Scores as assigned from 0 to 4 based on the severity of erythema or oedema formulation and this scorings approach explains for cutaneous toxicity for a transdermal system. The procedure involves applying 0.5ml of a test lotion to skin of a restrained, conscious rabbit that was shaved and abraded (several layers of skin are removed with sticky tape), then covered with plastic sheeting (Animals in product testing 2009) and leaving it for four hours. The animals were observed for up to 14 days, for signs of erythema and oedema in the skin test, and redness, swelling, discharge, ulceration, hemorrhaging, cloudiness, in the tested skin. The animals are euthanized after testing (Carbone, 2004).
Hot tail-flick test (Tolerance test):
The tail-immersion version of the tail-flick test was performed according to a previous report with modifications (Sewell 1976).The rat was held in a cloth restrainer during testing. This method of restraint is a less stressful means of containing rats during tail-flick testing and has been shown to reduce variability in response latencies compared with commercial restrainers (Rice 1977). The tails of mice (2-5cm) were immersed in warm water kept constant at 53±1 °C, and the reaction time was measured as the time taken for the rat to deflect their tails. The first reading was ignored, and the reaction time was taken as the mean of the next 2 readings. The latent period of the tail-flick response was taken as the index of antinociception and was determined at 30, 45 and 60 min after the application of relevant lotion formulations. The cut off time was 20 seconds. In this study 5 different groups of rats, each of which consisting of 3 animals were used and each of the formulations in table 3 was assessed with each group.
Male rats (150±5gm) kept at the laboratory Animal house of the Institute of Biotechnology, were used. The animals were maintained under standard environmental conditions and had free access to standard diet and water. Anti-inflammatory activity was measured using carrageenan induced rat paw oedema assay (Adeyemi 2002, Ratheesh 2007). Groups of 6 rats were given a dose of the lotion (1ml of formulated each lotion was applied on an area of 2 square cm of rat paw). Just before administration of lotion, 0.1 ml, carrageenan suspension (1%) in 0.9% NaCl solution was injected into the sub-plantar tissue of the right hind paw. The linear paw circumference was measured at hourly interval for 3 h (Bamgbose 1981). The thickness of paw edema induced by carrageenan was measured by using a standard screw gauge after application of lotion. Anti-inflammatory activity was measured as the percentage reduction in oedema level when drug was present, relative to control (Duffy 2001).
A questionnaire containing six questions was prepared and given to each volunteer (total of eleven volunteers) for sensory evaluation of all developed lotions and average points were calculated from the points assigned (Nine values from -4 to +4 indicating very bad to Excellent respectively) by each volunteer for each question and the results were given in Table 5.
Statistical significance was determined between the sample means of the treatment groups using the one-way ANNOVA. A probability of p<0.05 was considered statistically significant. All results are presented as the mean ± SD, unless otherwise stated. The minimum standard deviation values assured that the process used for preparing the delivery system is capable of giving reproducible results which is further confirmed by earlier studies data (Jayaprakash 2010).
Pre-formulation studies and pH determination has been done and shown in Table 1 and 2 respectively. All other physical parameters like viscosity, spreadability, homogeneity and stability were also performed as given in table 2 and 4.
The solubility of Diclofenac diethylamine (DDA) in the vehicles used was determined (Table 3) was ~2.5 fold more soluble in Turpentine oil than water and 4-fold high in OA and between water and pure TO. It is not clear why the solubility in TO is low, as it contains fatty acids, capable of participating in hydrogen bonding. The solubility of DDA in distilled water was 42.282± 0.588mg/ml, at 32°C, which is in line with values reported in the literature (Roy 1996).
In the present study, co-solvent mixtures of DDA were made from saturated solutions of enhancers in water as OA:water and TO:water, mixture at 20:80; 40:60; 60:40; 80:20 and100:00 ratio (v/v) respectively as given in table 3 and the degree of saturation (DS) was calculated by dividing the amount dissolved in the mixture by the solubility at equilibrium in the same co-solvent mixture. The DS values 1.2 and 1.1 of DDA saturated solution for Oleic Acid/Water and Turpentine Oil/Water at 40:60 ratios (%v/v) were observed respectively.
Solubility Enhancement Ratio ( ERsol ) of DDA in both solvents have been determined as:
ERsol = Ct /Cs
Where Ct is concentration of DDA in presence of enhancer and Cs is concentration of DDA in absence of enhancer (control). The ERsol values 9.776 and 2.485 were observed for OA and TO respectively.
The highest uptake was observed for PBS (Table 8) which confirms the idea introduced above as the lipophilic solvents have solubility parameters closest to that of the membrane. The solubility parameter of silicone membrane is reported in the literature to be 7.5 (cal/cm3)1/2 by Cross et al (Cross 2003). In this case the vehicles with low logP values, it would be difficult to overcome poor solubility and it is unlikely that a high flux will be achieved even if the solvent flux is high. It is a case of balancing a number of factors to achieve the optimum topical vehicle.
FoD of formulations of DDA across Rabbit Skin vs Silicone Membrane:
In this study, Factor of Difference ranged from 0.43 to 1.51 (table 9), showing that the flux values determined by using silicone membrane (SM) were in the same order of magnitude as that of flux values calculated with rabbit skin for permeation for 3 hours study.
I-Permeation studies through Rabbit Skin:
The effect of Turpentine oil and oleic acid in amount of 1%, 2%, 3% and 4% (v/v) within the Diclofenac lotion on the permeability rate of drug through rabbit skin is shown in table 10 and figure 2 which explain all the permeation parameters with associated (± SD). The lag time was of in the range 15 to 106 minutes. The enhancers might affect fluidity of stratum corneum structure and drugs could be permeated better through the rabbit skin. The famous penetration enhancer OA penetrates into the stratum corneum, decompressing it and reduces its resistance to drug penetration (Naik 1995). Figure 3 explains the enhancing ratio ER (J) observed in the order as L1 <L2 <L3 <L4 which is comparable with the earlier work and enlisted the values in the table 11. The input rate obtained was 0.753, 0.899, 0.925, 0.949 and 0.083 which is almost 9-12 folds higher than for control.
II-Permeation studies through silicone membrane:
The permeation of Diclofenac lotion through silicone membrane, using TO/OA vehicles of varying concentrations (1%, 2%, 3% & 4% v/v) was evaluated and enlisted in table 11. The flux values with associated standard deviations (±SD), the permeation parameters tlag, D, kp and K are also illustrated in figure 4. The lag time was observed in the range of 48 to 98 minutes. There is no significant difference (P >0.05) between all permeation parameters of the lotion, these values almost behaving as increase with the increase in the concentration of enhancer solution from L1 to L4. Figure 5 explains the enhancing ratio ER (J) and the values were observed in the order as L1 <L2 <L3 <L4 which is comparable with the earlier work. The input rate obtained was 0.0519, 0.0744, 0.0746, 0.0882 and 0.0297(µg/min) which is almost 2-4 folds higher than for control. Skin irritation studies revealed that the lotion has no erythema and oedema observed during study period. The in vivo studies gave a picture of effective therapeutic lotion and results are shown in table 6.
For some time increasing the lipid solubility or its surrogate, the partition coefficient (K) between a lipid and water, has been the standard working paradigm for increasing permeation of the skin, and the permeability coefficient (kp = distance/time) has been the quantitative measure of the results. The shorter chain and more water soluble alcohols exhibiting lower (K) values gave the greater flux values (J = amount/area _ time; the more clinically relevant measure of permeation) and D values, regardless of whether they were applied neat or in an aqueous vehicle as in this study while Kp showed opposite trends for the two applications.
The skin permeation rates of the drug calculated from the permeation profiles of each formula are shown and among these formulations tested, formula (L4) which was composed of 2% DDA, 4% (v/v) Turpentine oil and Oleic acid showed the highest permeation rate (6.04µg/cm2/min). The content of enhancers' concentration in Diclofenac lotion affected the skin permeation rate of DDA significantly. As the content of enhancers' concentration was decreased from 4% (v/v), L4 to 1% (v/v), L1 the skin permeation rate of DDA also decreased i.e. it may be due to thermodynamic activity of drug in the lotion as DDA is poorly water soluble (42.282± 0.588 mg/ml) and yet solublised in the enhancers' mixture (Kweon 2004). The reported data in this study (Figure 7, 8) showed that K is decreasing and D is increasing from L1 to L4, hence permeation through rabbit skin is diffusional although partitioning is occurring in the skin as the earlier studies confirmed the deposition of DDA into the skin (Green 1988).
It was also found that the skin permeation of the DDA in Diclofenac lotion was significantly influenced by the content of ethanol and enhancers' mixture due to increasing solubility of DDA. The literature supports our data that skin permeation rate of DDA was increased by 9.7-folds (Walker 1991). It is possible that, with increasing the content of alcohol, the size of internal phase of the lotion may be decreased making the surface area of the droplet increased significantly. The influence of alcohol in aqueous solutions upon the transport behaviour of several permeants across the skin has been evaluated earlier (Obata 1991, Takayama 1991).
It has been reported that alcohol may alter or form additional pore/polar pathways in the stratum corneum as a result of combination of changes in protein conformation, reorganization within the lipid polar head region or lipid extraction and also induced the reduction in the barrier property of SC (Bommannan 1990). The content of oil also showed similar effects on the skin permeation of DDA but its mechanism is different from that of surfactants/cosurfactants. Solvents used in this study enter the SC, changed its solution properties by altering the chemical environment and thus reduced the barrier capacity of the cutaneous layer (Barry 2001) as in L4 flux is 6.04± 0.003 (µg/cm2/min) and 0.112± 0.005 (µg/cm2/min) in rabbit skin and silicone membrane respectively whereas in L1, it is 4.80± 0.031 (µg/cm2/min) and 0.066± 0.002 (µg/cm2/min) respectively. A dermally applied lotion may be expected to penetrate the stratum corneum and remained in the whole horny layer (figure 6) and may simultaneously alter both lipid and the polar pathways as previously suggested in the double labeling studies (Francoeur 1990) and the freeze-fracture electron microscopic studies.
The lyophilic domain of the lotion can interact with the stratum corneum in many ways. DDA dissolved in the lipid domain of the lotion can directly partition into the lipids of the stratum corneum or the lipid vesicle themselves can intercalate between the lipid chains of the stratum corneum, thereby destabilizing its bilayer structure. In effect, these interactions will lead to increase the permeability of the lipid pathway to DDA. The binary combination of TO and OA influences the penetration in accordance with earlier studies in which a synergistic effect with these binary solvents were observed (Ho 1998, Ota 2003, Potts 1991). The topical diclofenac studies showed similarity in study design, duration and outcome assessments. Future research is required as there are no long term studies looking at the efficacy and safety for chronic (>12 weeks) use of topical diclofenac. It would be beneficial to have more evaluation and clinical trial comparison of the different topical diclofenac formulations to each other (Nair 2010).
The anti-inflammatory activity of formulated lotions was evaluated by carrageenan-induced rat paw oedema method and results showed that the L4 gave 74% of inhibition on carrageenan-induced rat paw oedema at third hour which indicated that this ratio have significantly maximum anti-inflammatory activity as compare to control and other formulations (Singh 2009). The present results also indicate the efficacy of L4 as effective therapeutic agent in anti-inflammatory conditions. Simple tail-immersion test was assessed which is rapid to evaluate animal response to painful condition. Although all the formulations have significant differences in response time and the most prolonged response belonged to L4 which is in agreement with the results reported earlier (Kang 2001, Mohammadi-samani, 2010).
On the other hand, there is a general experience that hydration of the skin plays an important role in the percutaneous uptake of DDA. When the aqueous fluid of the sample enters the polar pathways, it will increase the interlamellar volume of stratum corneum lipid bilayers, resulting in the disruption of the interfacial structure. Since some lipid chains are covalently attached to corneocytes, hydration of these proteins will also lead to the disorder of lipid bilayers (Idson 1978, Bouwstra, 2003). Similarly, swelling of the intercellular proteins may also disturb the lipid bilayer; a lipophilic drug like DDA can then permeate more easily through the lipid pathway of the stratum corneum. The greater drug penetration enhancing activity of lotion may be attributed to the combined effects of both lipophilic and lipophobic domains of lotions. The results revealed that lipophilic enhancers are more effective than lipophobic ones (Yamada 1987).
Comparison of formulations of DDA across Rabbit Skin vs Silicone Membrane:
In this study, FoD ranged from 0.43 to 1.51 (table 9), showing that the flux values determined by using silicone membrane (SM) were in the same order of magnitude as that of flux values calculated with rabbit skin or/ and human epidermis as illustrated in figures 9 and 10 for permeation study after 3 hours using OA and TO respectively. The enhancer might affect fluidity of stratum corneum (SC) structure and DDA could be permeated better through the rabbit skin. Moreover, the FoD values did not appear to be related to any physicochemical properties of the present study enhancer's solution under permeation study as reported in above results details, this suggests that the difference between both the membranes i.e. rabbit skin and silicone membrane, have no direct implications concerning the diffusion of DDA, this fact confirms the validity of the model in this permeation study. Thus, considering all this discussion together with the FoD range, this animal model (Rabbit skin) and silicone model membrane can be regarded as predictive of human skin permeability (Cilurzo, 2007).
Conclusion: we could conclude that the enhanced permeation of DDA may not be only by the partitioning of the drug into the stratum corneum but also by modifying intercellular lipids, disrupting their highly ordered structure and thus increasing the diffusion of DDA through the membrane with increased solubility in the presence of OA and TO and it is important to observe the increased amounts of DDA in the skin may also be retention of the drug by the skin. From the diffusion coefficient (D) values it can be seen that use of enhancers has decreased the resistance to the diffusion of drug. The benefit of penetration enhancement in this study was counterbalanced by the fact that at this range of concentration, the use of these solvents cannot harm the skin. Since terpenes are relatively safe compounds, their incorporation in low concentrations into topical formulations could be recommended (Mohammadi-samani 2010, Nokhodchi 2007). The in vitro and in vivo studies showed that the lotion could be used for effective therapy.
Declaration of interest:
The authors report no conflicts of interest.
The authors are greatly thankful to Bahauddin Zakariya University, Multan for financial support and HEC for award of training scholarship (IRSIP) for PhD scholars who helped the author to do work at London School of Pharmacy in skin Lab # 439.