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International Journal of Pharmacy. Photon 104 (2013) 144-164 https://sites.google.com/site/photonfoundationorganization/international-journal-of-pharmacy Original Research Article. ISJN: 8237-7516

International Journal of Pharmacy Ph ton Wound healing Activity of Niosomal Gel of Punicalagin from peels of Punica Granatum

Hanu Priya Yadav* Department of Pharmaceutical Sciences, Lovely Professional University, Phagwara, Punjab, India Article history: Received: 21 December, 2012 Accepted: 19 January, 2013 Available online: 14 April, 2013 Keywords: Punica granatum, Punicalagin, Novel Drug Delivery System, Niosomes, Vesicular system, Niosomal gel. Corresponding Author: Hanu Priya yadav* M. Pharmacy (Pharmaceutics) Email: hanupriya.yadav@gmail.com Mobile No.: +919466906727 Abstract A well known ancient fruit named as Punica granatum(family- Punicaceae) which is commonly known as Pomegranate, Anar or Dalim in North India whose therapeutic qualities have rebounded and echoed throughout the millennia. Plant based formulations have been used since ancient times and playing a role as a remedial against various human and animal diseases. Therefore, much energy has been devoted to the treatment of disease and enhancement of physical and mental health in the systems of Allopathic, Ayurvedic, Homeopathic and Unani. The interest in traditional medicines has increased in various parts of world. Punicalagin is chemically named as 2,3-(S)-

hexahydroxydiphenoyl-4,6-(S,S)-gallagyl-D-glucose and belongs to a category of hydrolysable tannin. In this vein, present investigation was an endeavor to formulate the vesicular formulation of Punicalagin and to evaluate its effect on wound healing by in vivo studies. For this, the Punicalagin was extracted, isolated and purified from peels of Punica granatum. Thus, to protect its hydrolysis, it is formulated into a nanocarrier system known as niosomeswhich is based on the preparation of niosomes by using a non-ionic surfactant in varying amounts and keeping the amount of cholesterol constant. The formulations were evaluated on the basis of evaluation parameters and thus optimized.Thebest optimized niosomalformulation i.e. F7 was then formulated as hydrogel and evaluated. This topical hydrogel was then used to check out the wound healing effect on wistar rats for 14 days. It was concluded that the % wound contracted in case of niosomal gel (88.61%) is more than the marketed formulation (75.72%), then to conventional (plain) gel (34.73%) and negative control (23.88%) after 14 days. Citation: Yadav H.P., 2013. Wound healing Activity of Niosomal Gel of Punicalagin from peels of Punica Granatum. International Journal of Pharmacy. Photon 104, 144-164.

1. Introduction

Tannins are one of the most widely occurring groups of substances in different families of higher plants. They are high molecular weight plant polyphenols and the secondary metabolites which are present in solution form in cell sap and also in distinct vacuoles. Chemically, tannins contain the mixture of complex organic substances in which polyphenols are present, generally with o-dihydroxy or o-trihydroxy groups on phenyl ring and they are devoid of nitrogen (Kokate et al., 2006). The pomegranate (Punica granatum L.)is an ancient fruit which has been widely consumed in various cultures for thousands and thousands of yearsbelongs to family Punicaceae is commonly known as Anar or

Dalim in North India (Agli et al., 2010). The Babylonians regarded pomegranate seeds as an agent of resurrection; the Persians believed the seeds conferred invincibility on the battle fields, while for the ancient Chinese, the seeds symbolized longevity and immortality (Martos et al., 2010). Pomegranate husk is a traditional Chinese medicine used as antibacterial, anti-inflammatory and hemostasis agent which is rich in phenolic compounds. Among these polyphenols, the most abundant compound is Punicalagin. Punicalagin levels in husk depend upon the area for fruit growth, processing conditions as well as storage conditions (Lu et al., 2008). Punicalagin is a potent antioxidant whose bioactivity can be explained by its ability to hydrolyze into Ellagic acid (EA) in

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vivo and across the mitochondrial membrane in vitro. Punicalagin is a herbal drug that is extracted from the peels of pomegranate. Punicalagin is the largest known molecule found in blood. It has many biological activities like antioxidant, antiulcer, antiartherogenic etc. Its mechanism of action is multiple targeting and it may acts as a prodrug inside the body as it further breaks down into two bioactive molecules i.e. Punicalin and Ellagic acid. It belongs to a category of hydrolysable tannin and a high molecular weight compound, thus it gets hydrolysed easily and unable to penetrate through the skin. Thus, it is formulated as a niosomal gel to check out the wound healing activity. Niosomes are the carriers for the drugs which are hydrophilic in nature (William et al., 2003). Thus, the aim of the study is to check out the efficacy of Punicalagin niosomal gel by performing in vivo studies for the wound healing activity. For the better performance of pharmaceutical formulations with respect to controlled release, bioavailability, storage stability and lesser side effects constitute the main motivation for research of novel drug delivery systems (Mehta et al., 1993). Novel drug delivery systems are used to improve the drug potency, control drug release to give sustained therapeutic effect, provide safety and reduces toxic effects. It may target/delivery of drugs specifically to tissue, organ or location in the body. There are various novel drug delivery strategies like liposomes, niosomes, aquasomes, microsponges, microemulsions, and solid lipid nanoparticles to enhance the topical delivery of agents. Among different carriers, liposomes and niosomes are well documented for dermal drug delivery (Shahiwala et al., 2002). Vesicles formed on hydration of mixture of cholesterol and single alkyl-chain non-ionic surfactants were first introduced by Handjani-Vila. Initially reported as a feature of cosmetic industry, they are now extensively used as drug delivery systems (Mehta et al., 1993). Niosomes may be defined as a unilamellar or multilamellar vesicles in which the aqueous solution is enclosed in highly ordered bilayers made up of non-ionic surfactants with or without cholesterol and dicetylphosphate and exhibit behaviour similar to liposomes in-vivo (Shahiwala et al., 2002). They are capable of entrapping both hydrophilic and hydrophobic drugs as shown in figure 1.

Surfactants play an important role in the development of such formulations. A number of non-ionic surfactants have been used to prepare vesicles viz. polyglycerol alkyl ethers, glucosyl dialkyl ethers, crown ethers, ester linked surfactants, polyoxyethylene alkyl ether, brij, and series of spans and tweens. They are made up of biocompatible, non-toxic, non-immunogenic, and non-carcinogenic agents. NSVs are highly resistant to hydrolytic degradation. NSVs result from the self assembly of hydrated surfactant monomers. The surfactant molecules self-assemble in aqueous media in such a fashion that the hydrophobic tails face each other to minimize the high energy interactions between the solvent and tails (Mehta et al., 1993). Bundelkhand is very important region of India. It is unique in many aspects being the central part of the country; it is much safe like heart in our body. It is famous for the most popular tourist places (Jhansi, Khajuraho, Chitrakoot and Orchha). Bundelkhand is spread over southern Uttar Pradesh and northern Madhya Pradesh, between 2310' and 2630' north latitude and 7820' and 8140' east longitude. The region covers a geographical area of around 70,000 sq km and includes seven districts of Uttar Pradesh and six districts of Madhya Pradesh. Thus Bundelkhand comprises thirteen districts: Jhansi, Lalitpur, Jalaun, Hamirpur, Mahoba, Banda and Chitrakoot (all in Uttar Pradesh), and Datia, Tikamgarh, Chhatarpur, Panna, Sagar and Damoh (all in Madhya Pradesh). The plant diversity of Bundelkhand region encompasses several medicinal plants. Those play a vital role in the daily life of the humankind. Plants are the main source of socio-economic development as well as provide several things like food, fruits, flowers, fodder, fibre, fragrance, gum, resin, oil, spices, vegetable, dyes, rubber, wood, timber, etc. Beside these, plants are used as medicines are known as Medicinal plants. The medicinal plants play very important role in the health care of rural people mostly belong to the schedule castes and schedule tribes. Unfortunately people remain unaware of those plants due to lack of written proofs and literatures. Therefore, first priority must be given to study these plants and documented the traditional knowledge regarding the medicinal uses of plants need to be popularized, so that all round awareness be made possible.

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The present study provides the information regarding medicinal uses of plants in Bundelkhand region of India. The work on this aspect of this region has been carried out by some researchers e.g. Sharma et al. (2007), Lahri (2007), Nigam and Kumar (2005) and Ahirwar (2009, 2010 and 2011). Figure 1: Microscopic structure of a niosome

Advantages of niosomes are: It canaccommodate hydrophilic, lipophilic as well as amphiphilic drug moieties; niosomes exhibit flexibility in their structural characteristics (composition, fluidity and size) and can be designed according to the desired situation; improve the therapeutic performance of the drug, protect from the biological environment, restricts its effect to target cells, thereby reducing the clearance of drug; act as depot to release the drug slowly and offer a controlled/sustained release; increase oral bioavailability of drug; increase the stability of entrapped drug; enhance the skin penetration of drugs (Choi et al., 2005; Honeywell-Nguyen et al., 2005). They can be made to reach the site of action by oral, parenteral as well as topical routes; surfactants used are biodegradable, biocompatible and non-immunogenic; handling and storage of surfactants do not require any special conditions; the vesicle suspension being water based offers greater patient compliance over oily dosage forms (Karim et al., 2010; Muzzalupo et al., 2008). Wound healing is an important biological process that involves tissue repair and regeneration. A wound is defined as a break in the continuity of tissue from violence or trauma and is regarded as a heal of wound when there is restoration of that tissues to a normal condition (Esimone et al., 2005).Wounds are physical injuries that result in an opening or break of the skin. Proper healing of wounds is essential for the restorations of disrupted anatomical continuity and disturbed functional status of the skin.

Healing is a complex and intricate process initiated in response to an injury that restores the function and integrity of damaged tissues (Karodi et al., 2009). Classification of wound healing on the basis of the nature of the edges of healed wounds: 1. Primary healing (Healing by first intention): The edges of healed wounds are smoothly closed and no scar is left. In this closure of wound occurs within its hours of creation (Esimone et al., 2005). 2. Secondary healing (Healing by second intention):The formation of granulation tissues occur which fills the gaps between the wound edges associated with the loss of tissue significantly and leaves a little scar. In this, no formal wound closure occurs and wound closes spontaneously by contraction and re-epithelialization (Esimone et al., 2005). 3. Tertiary healing (Healing by third intention): The wound is left open for 3-5 days until a granulation bed falls before they are sutured and results in extensive scar formation. It is also known as delayed primary closure that involves initial debridement of wound for an extended period and then formal closure with suturing or by another mechanism (Esimone et al., 2005). Wound healing involves continuous cell-cell and cell-matrix interactions that allow the process to proceed in three overlapping phases. Healing requires the collaborative efforts of many different tissues and cell lineages (Karodi et al., 2009). The typical wound, after primary closure, may take over a year to fully mature; the appearance of the scar may dramatically change during this time. Thus, all wounds should be at least 1 year old before scar revision is considered. The basic principle of optimal wound healing is to minimize tissue damage and provide adequate tissue perfusion and oxygenation, proper nutrition and moist wound healingenvironment to restore the anatomical continuity and function of the affected part (Karodi et al., 2009). There are 3 phases involved in wound healing whichare described in table 1. 2. Objectives of Research 1. Punicalagin is having a lot of pharmacological activities but there is very less research work done on the formulation aspect of this molecule (Cerda et al., 2003).

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Table 1: Phases of wound healing describing stages Phases of wound healing Duration Description Inflammatory phase Proliferative phase Remodelling phase

Immediate to 2-5 days Days to 3 weeks Weeks to 2 years

1.Hemostasis Following vasoconstriction, platelets adhere to damaged endothelium and discharge ADP, promoting thrombocyte clumping, which dams the wound. 2.Inflammation The inflammatory phase is initiated by the release of numerous cytokines by platelets. This phase includes the following: -Angiogenesis -Collagen deposition -Granulation -Contraction -Re-Epithelialization After the third week, the wound undergoes constant alterations, known as remodeling. This phase includes the following: -Formation of New collagen - Scar formation

2. Punicalagin is water soluble (hydrophilic) and hence it is unable to penetrate through skin itself. Thus, a vesicular system like niosomes which act as carriers and hence will help in penetration of drug through skin and provide a prolong release. 3. Various experiments in the literature have been found which involves studying wound healing activity in vivo using whole plant extract of Punica granatum but no experiments have been concluded so far for the same activity employing the specific constituent of this plant i.e. Punicalagin. 4. Drug chosen for the study is safe as there is no toxicity reported (Cerda et al., 2003). The objective of the present study is to develop a simple, precise, accurate, and economical analytical method for the estimation of Punicalagin extracted from peels of Punica granatum. To perform the compatibility study of drug, Punicalagin with the excipients used in formulating niosomes. To develop a vesicular system like niosomes which act as carriers and hence will help in penetration of drug through skin and provide a prolong release. The best optimized niosomal formulation was then incorporated into gel and evaluated according to evaluation parameters. The gel was then applied topically on wistar rats to check out the wound healing effect for 14 days. The purpose of the present study is to: To extract, isolate and purify Punicalagin

from peels of Punica granatum. Characterisation of Punicalagin.

Compatibility study of Punicalagin with excipients.

To formulate physically and chemically stable niosomes of Punicalagin.

Characterization of the prepared niosomes.

Development of niosomal gel system of optimized formulation.

Evaluation of optimized niosomal gel. In vivo studies.

3. Materials Punicalagin was extracted from pomegranates which were purchased from local market in Bhiwani, Haryana (India). Peels were authenticated from National Institute of Science Communication and Information Resources, New Delhi, India. The common excipients like Methanol, Ethanol, Acetone, Potassium dihydrogen phosphate, Disodium hydrogen phosphate,Triethanolamine and Sodium chloride were obtained from Loba Chemie Pvt. Ltd., Mumbai. Carbopol 934, Diethyl ether, Ethyl paraben, Methyl paraben, and Propylene glycolwere obtained from Central Drug House Pvt. Ltd., New Delhi. Span 60 and Cholesterolwere obtained from S.D. Fine Chemicals Ltd., Mumbai, India. Framycetin sulphate (as marketed formulation) was purchased from the local market in Jalandhar, Punjab (India). 4. Methods 4.1 Extraction, Isolation and Purification of Punicalagin from peels of Punica granatum 4.1.1 Extraction and Isolation of Punicalagin from peels of Punica granatum Fruits were washed, separated from seeds and juice and cleaned to yield husks/peels. Peels of the Punica granatum were dried in

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shade and powdered them with the help of blender. Powdered husk was macerated with water and methanol for 7 days with occasional stirring. After maceration process was completed, thick husk puree was squeezed by hand and filtered through muslin cloth to yield dark brown aqueous extract. The column having Sephadex LH-20 used as stationary phase and prepared for chromatography by pre-washing in methanol and pre-equilibrated in water for 12 hr. The aqueous extract was divided into portions and adsorbed onto the Sephadex LH-20. Each column was eluted with excess amount of distilled water until sugary pale yellow elute was clear in colour. The adsorbed tannins were eluted with methanol to yield dark brown solution. The collected fractions were then passed through C18 catridges (Waters Sep-Pak Vac 20cc) and eluted with methanol to obtain the yellowish brown solution which contained pure compound, Punicalagin. The methanol was removed by Rota-evaporator in vacuo at low temp. (37C) and obtained the dark brown powder as TPT (total pomegranate tannins) (Seeram et al., 2005). 4.1.2 Purification of Punicalagin from TPT Sephadex LH-20 resin column was used to isolate the pure compound from TPT. TPT obtained was adsorbed onto a Sephadex LH-20 column that was pre-equilibrated with water: methanol (8:2 v/v) and eluted with increasing amount of methanol. The fraction was evaporated in vacuo and then re-chromatography was done by pre-equilibrating column with ethanol. Elution was done with increasing amounts of water and acetone then to ethanol: water: acetone (6:3:1 v/v/v) and finally with ethanol: acetone (1:1 v/v). The fraction was collected and then evaporated in vacuo to obtain yellowish brown powder as Punicalagin(Seeram et al., 2005). 4.2Preformulation Studies of Punicalagin 4.2.1 Organoleptic Properties The organoleptic properties include physical state, colour and odour was done by visual inspection. 4.2.2 TLC Identification Test (i) Preparation of sample- The powdered drug (0.5g) was macerated in methanol for 4 days. The extract was filtered and process was repeated thrice. The filtered extract was pooled, evaporated to dryness under reduced pressure and residue was dissolved in methanol. (ii) Preparation of Solvent system-Chloroform: Ethyl acetate: Formic acid: Methanol in a ratio

of 4: 5.2: 0.6: 0.2 was mixed to prepare solvent system. Spotting of the sample was done with the help of capillary on TLC plate. The plate was then placed in the solvent system till the saturation point was reached. (iii) Visualisation-TLC plate was dipped in a solution consisting of anisaldehyde (0.5ml), glacial acetic acid (9.5 ml), methanol (85ml) and conc. sulphuric acid (5ml) for a few seconds. It was then heated at 110C in hot air oven till coloured band appeared and Rf value was then calculated as in equation (1).

4.2.3 Chemical identification test To identify the drug chemically, a pinch of drug was taken in a test tube and 2 ml of ferric chloride solution was added to it. Presence of a colour shows the presence of tannin. 4.2.4 Characterisation of Punicalagin 4.2.4.1 UV spectroscopy Punicalagin was dissolved in phosphate buffer saline pH 7.4 and the sample was scanned at wavelength ranging from 200-400nm by UV spectrophotometer. The absorption maxima were compared with that available in the literature (Gil et al., 2000). 4.2.4.2 IR spectroscopy IR spectroscopy of Punicalagin was performed using FTIR 8400S (Shimadzu). KBr pellets of Punicalagin were prepared by applying a pressure of 8 tons in a hydraulic press. The pellets were scanned over a wave number range of 4000400 cm-1.The spectrum obtained was interpreted by the literature (Furniss et al., 1988). 4.3 Standard UV Plots 4.3.1 Determination of absorbance maxima (max) Punicalagin (10 mg) was accurately weighed and transferred to a 100 ml volumetric flask. To this, pH 7.4 PBS was added to dissolve the drug. From this solution, 1 ml of solution was pipetted out in 10 ml volumetric flask and volume was made up to 10 ml with distilled water. The sample was scanned on a double beam UV-visible spectrophotometer. An absorbance maximum of Punicalagin was determined in pH 7.4 PBS. 4.3.2 Standard plot of Punicalagin in pH 7.4 Phosphate buffer The standard plot of Punicalagin was prepared in pH 7.4 PBS. 10 mg of drug was weighed accurately and dissolved in 100 ml of pH 7.4

--- (1)

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PBS. Appropriate dilutions were made with buffer to obtain test solutions ranging from 5g/ml to 35 g/ml. The absorbance of the drug in the buffer was measured on a double beam UV-visible spectrophotometer at max of 253.6nm against the respective blank. 4.4 Compatibility Study 4.4.1 Physical characterisation of drug excipient mixture Drug and each excipient were separately passed through sieve # 20. Drug and each excipient were weighed in the ratio of 1:1 and mixed properly with 15 ml of water for injection (WFI) as shown in table 2. Different drug excipient mixtures were introduced into glass

vials containing 15 ml of water covered with rubber caps which were followed by labelling. Then the vials were kept under three different conditions, one at 5 3C (refrigerated temperature), 25 2C (room temperature) and 40 2C/ 75 5% RH. Observations were taken on 0th, 7th, 14th, 21st and 28th day for physical compatibility. 4.4.2 Chemical characterisation of drug excipient mixture For chemical compatibility, the study was carried out by taking the different drug excipient mixtures after 28 days and analyzed spectrophotometrically by UV.

Table 2: Composition for compatibility study of Punicalagin with excipients S. No. Drug+ Excipient+ WFI Ratio of drug and excipients 1. Drug + WFI 1:1 2. Drug+ Span 60+ WFI 1:1 3. Drug+ Cholesterol+ WFI 1:1 4. All excipients+ WFI 1:1 5. Drug+ all excipients+ WFI 1:1 (WFI=Water for injection) 4.5 Preparation and Purification of Niosomes 4.5.1 Preparation of niosomes by ether injection method-Niosomes were prepared using Ether injection method (Karki et al., 2008). Drug, surfactant and cholesterol were used in the ratios as indicated in Table 3. Mixture of surfactant (Span 60) and cholesterol dissolved in a mixture of diethyl ether and chloroform was slowly introduced through 20-gauge needle into warm aqueous solution of Punicalagin maintained at 20 C. Evaporation

of ether leads to formation of single layered vesicles. 4.5.2 Purification of niosomes by centrifugation method-The prepared niosomes were separated from unentrapped drug by centrifugation method (Hao et al., 2002). Niosome suspension was centrifuged at 40,000 rpm (1,37,088 G) for 30minutes. Clear supernatant was removed by pipetting and remaining sediment i.e. niosomes containing only entrapped drug were obtained for further evaluation.

Table 3: Composition of niosomal formulations of Punicalagin S.No. Formulation Surfactant used Surfactant: Cholesterol: Drug 1. F1 Span 60 1:1:1 2. F2 Span 60 2:1:1 3. F3 Span 60 3:1:1 4. F4 Span 60 4:1:1 5. F5 Span 60 5:1:1 6. F6 Span 60 6:1:1 7. F7 Span 60 7:1:1 8. F8 Span 60 8:1:1 9. F9 Span 60 9:1:1 10. F10 Span 60 10:1:1

4.6 Evaluation of niosomes 4.6.1 Optical microscopy-The morphology of prepared niosomes was done by optical microscopy. The photomicrographs of the preparations were obtained with the help of photomicroscope at 1000 X. 4.6.2 Micromeritics studies-For the micromeritics study, the niosomes were probe sonicated and then the vesicle size and size

distribution profile were determined using dynamic light scattering (DLS) method (Malvern Instruments Ltd, Worcestershire, UK). Particle size analysis was done by using particle size analyser. 4.6.3 Entrapment efficiency Ultracentrifugation technique was adopted for the removal of the unentrapped drug. The centrifuged niosomes were lysed with 0.1% v/v

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Triton X-100 and left for 1 hour and filtered to separate drug from vesicles. Thereafter dilutions were made and solution was analyzed spectrophotometrically employing ultraviolet-visible spectrophotometer.The entrapment efficiency was calculated by using following equation:

--- (2)

4.6.4 Morphology and structure of vesicles-The prepared and optimized formulations were characterized for morphology (i.e. shape and lamellarity) employing Hitachi TEM analyzer. 4.6.5 Zeta potential analysis- Zeta potential of niosomal preparation is related to the stability of niosomes. Zeta potential indicates the degree of repulsion between adjacent similarly charged particles for small molecules and particles, a high value of zeta potential confers stability, i.e. the solution or dispersion will resist aggregation. Zeta potential for niosomal formulation was performed using Zeta sizer Beckman coulter instrument. 4.6.6 Stability studies of Punicalagin containing niosomes Physical stability studies were carried out to investigate the leaching

(leakage) of drug from niosomes (in a suspension form) during storage. The optimized niosomal formulation (F7) composed of Span 60 and cholesterol in 7:1 molar ratio were sealed in 20 ml glass vials and stored at refrigeration temperature (5 3C) for a period of 2 months. Samples were withdrawn at definite time interval of 15 days and the percentage entrapment of the drug was determined as described previouslyafter separation from unentrapped drug. 4.7 Development of topical hydrogel: For the development of topical hydrogel, an appropriate quantity of carbopol 934 (1% by weight) was added to 100 ml distilled water. It was then left to swell and hydrate at room temperature for two to obtain a homogeneous mixture. The propylene glycol was used to dissolve ethyl and methyl paraben and this solution mixture was incorporated into the homogeneous mixture. Then, it was stirred for 60 min at 800 rpm, followed by addition of few ml of triethanolamine drop wise to neutralize the formulation. Mixing was continued until gel appeared with desired consistency.The pellets from optimized formulation (F7) equivalent to 1% w/w containing 19.5 mg drug were incorporated in carbopol gel. It was then mixed to prepare niosomal gel whose composition is given in table 4.

Table 4: Composition for optimized niosomal gel

Ingredients employed Quantity (% w/w) Pellets from niosomal dispersion 19.5 mg equivalent to 1% Carbopol 934 1% Propylene glycol q.s. Methyl paraben q.s Ethyl paraben q.s. Triethanolamine q.s. Distilled water q.s. to 100 ml

4.8 Evaluation of gel 4.8.1 Rheological behavior of developed system The gelled system was evaluated for rheological behavior using Brookfield viscometer. Viscosity of the gel was determined at various rpm and by using different types of spindles. 4.8.2 Homogeneity-The developed gel was tested for homogeneity by visual inspection after the gel had been set in the container. It was tested for their appearance and presence of any aggregates. 4.8.3 Consistency The measurement of consistency of the prepared gel was done by dropping a cone

attached to a holding rod from a fix distance of 10 cm in such way that it should fall on the centre of the glass cup filled with the gel. The penetration by the cone was measured from the surface of the gel to the tip of the cone inside the gel. The distance travelled by cone was noted down after 10 seconds. This process was carried out in triplicate. 4.8.4 pH-The pH of the gel formulation was determined by using digital pH meter and readings were taken in triplicate. 4.8.5 Determination of gel strength-The method by which the properties of polymeric system may be conveniently determined is texture profile analysis. A TA-XT2 Texture analyzer with probe was used to determine the

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gel strength as shown in figure 2. The experiment was done by placing the gels in standard beaker below the probe. In this, an analytical probe is then immersed into the sample. The Texture Analyzer was set to the compression mode with a test-speed of 1.0 mm/s. An acquisition rate of 500 points per second, a trigger force of 5 g and a probe of P/O.5R; Dia cylinder Delrin radi was used for the gel testing. The study was carried out at room temperature and readings were taken in triplicate. The force required to penetrate the gel was measured as gel strength in terms of g. Figure 2: Texture analyser

4.8.6 Spreadibility Spreadibility, in simple terms, is the ease with which a spread can be applied in a thin, even layer to skin. Firmness of a gel/spread may be measured by the amount of deformation under a given force. In this process, TAXT2 texture analyser with spreadibility rig was used (as shown in figure 3) in which gel was filled in cone and extra gel was scrapped by tissue paper. The test mode was compression, the test speed was 3mm/sec and acquisition rate of 500 points per seconds were the test conditions during the process. The whole process was carried out at room temperature and readings were taken in triplicate. Figure 3: Spreadibility Rig

4.8.7 In vitro drug release through cellophane membrane To characterize the optimized formulation, the in vitro drug permeation studies were carried out using Franz diffusion cell. Degassing of the receptor solution was done to prevent the

formation of bubbles beneath the membrane. By circulating water, the temperature of the cell was maintained at 37 C. Membrane was mounted between the donor and receptor compartments of Franz diffusion cell and excess part of the membrane was trimmed off. The prepared formulations of punicalagin i.e. Niosomal gel that contained niosomes of Punicalagin and Plain gel that contains only Punicalagin, each equivalent to 19.5 mg of Punicalagin, were applied onto the membrane in the donor compartment and covered with aluminum foil to prevent contamination and evaporation. The receptor solution was pumped by peristaltic cassette pump continuously through the receptor compartment and drained into sample collection test tubes located in the fraction collector. Aliquot (5 ml) of dissolution medium was withdrawn from the sampling port at different time interval for 24 hrsand wheneverthe sample was withdrawn, an equal volume of fresh dissolution medium was added to the cell to maintain a constant volume. The amount of punicalagin permeated through membrane was analyzed spectrophotometrically. 4.8.8 Ex vivo release studies through skin The depilated skin from the dorsal region of the sacrificed albino wistar ratwas taken for determining the permeation rate. The adipose tissue and other fatty tissues were separated from same and washed with saline and kept in PBS pH 7.4 (Dew et al., 2012; Qiu et al., 2008). Niosomal gel equivalent to 19.5 mg of Punicalagin was applied to the albino wistar rat skin in a vertical Franz diffusion cell. Other side of the skin was in contact with the dissolution medium. Franz diffusion cell was placed on a magnetic stirrer and temperature of media was maintained 37 0.5C. It was agitated at 100 rpm. The dissolution medium was 100 ml of PBS pH 7.4. Aliquot (5 ml) of dissolution medium was withdrawn from the sampling port at different time interval for 24 hrs and the amount of punicalagin permeated through membrane was analyzed spectrophotometrically. Whenever sample was withdrawn equal volume of fresh dissolution medium was added to the Franz diffusion cell to maintain a constant volume. 4.8.9 Skin retention studies In this study, the skin tissue mounted on the diffusion cell was removed and washed thrice with saline solution, followed by blotting between tissue paper to remove any adhering formulation from the surface. Subsequently,

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the skin tissue was cut into small pieces and homogenized with 10 ml of methanol for extracting punicalagin and quantified for drug content spectrophotometrically. 4.8.10 Analysis of release mechanism The release kinetics of Punicalagin from niosomal and plain gel was evaluated considering five different models including zero order, first order, Higuchis model, Hixson Crowell model and Korsmeyers Peppas model. The obtained data were analysed to determine the order of release. The selection wasbased on the comparisons of the relevant correlation coefficients and linearity test. The regression coefficient R2 value closer to 1 indicates the model fitting of release mechanism (Costa et al., 2001; Dash et al., 2010). 4.9 Wound healing activity In the present study, excision wound model was employed to study the rate of wound contraction. Male albino wistar rats weighing 200-250 g were used for evaluation of wound healing activity. The animals were divided into four groups, each group having six animals. Group-I: Negative Control (rat with wound + no treatment). Group-II: Rat with wound+ Niosomal gel. Group-III: Rat with wound+ Gel of crude drug. Group-IV: Rat with wound+ Marketed formulation (1% w/w Framycetin sulphate). Procedure Animals were anesthetized prior to and during creation of the wounds. The rats are inflicted with excision wounds. The dorsal fur of the animals is shaved and the wound was created on the back of the animals. The excision

wound of circular area 300 mm 2 and 0.2 cm depth was created along the markings using toothed forceps, a surgical blade, and pointed scissors. The entire wound was left open. The animals were divided into four groups of six each. The animals of group 2 were topically treated with the niosomal gel of the drug Punicalagin. Group 3 animals were treated with gel having crude drug Punicalagin. Group 4 animals were treated with the marketed formulation Framycetin sulphate (1%w/w) as standard. The niosomal gel, plain gel and marketed formulation were applied once a daily to all animals of four groups. The wound closure rate was assessed by tracing the wound on days 0, 2, 4, 6, 8, 10, 12, 14 post-wounding using transparency sheets and a permanent marker. The wound areas recorded were measured using a graph paper (Adiga et al., 2010; Bele et al., 2009). Determination of percentage of wound contraction For the determination of the wound contraction, excision wounds were traced on a transparent paper having a scale and the change in wound size at two days interval was calculated as the percentage of wound area that healed. The rate of wound contraction was expressed in terms of the percentage of wound area (% wound contraction) that had healed as shown in equation 3.

5. Results 5.1 Preformulation studies 5.1.1 Organoleptic properties

Table 5: Preformulation parameters and their observation

Preformulation property Observations Physical state Solid as powder Colour Yellowish brown (shown in figure 4) Odour Pleasant smell

Figure 4: Punicalagin

5.1.2 TLC Identification test Purple pink colour was appeared and Rf value

was observed 0.53 (whereas reported value of Rf is 0.58 in ICMR) as shown in figure 5. Figure 5: TLC showing detection of Punicalagin

--- (3)

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5.1.3 Chemical identification test: On addition of ferric chloride to small amount of drug, a blue colour was appeared that indicated the presence of hydrolysable tannin. 5.1.4 Characterisation of Punicalagin 5.1.4.1 UV spectroscopy The absorbance maximum (max) of Punicalagin in PBS pH7.4 was determined which are shown in figure 6. The max of Punicalagin in phosphate buffer pH 7.4 was found to be 253.6 nm. Figure 6: UV absorption spectra of Punicalagin in phosphate buffer pH 7.4

5.1.4.2 IR spectroscopy Figure 7: IR spectra of Punicalagin

5.2 Standard UV plot of Punicalagin in pH 7.4 Phosphate buffer Standard plot of Punicalagin was found to be linear with R2 = 0.9978; showing proportional increase in the absorbance with concentration which is shown in figure 8 and data is represented in table 7. Absorbance range of Punicalagin was found to be 0.154 to 0.855 nm.

Table 6: IR spectra showing the peaks of functional groups present in Punicalagin Wave number cm-1 The peak corresponds to Observed value *Reference value 1589 1560 C-O 3419 and 3720 3400-3550 O-H 1683 1680 C=O conjugated with aliphatic C=C 1261-1242 1270 -1230 -O- (Aralkyl ether) 1112 and 1350 1100 -1300 COOR 1456-1589 1450 -1600 Aromatic ring

*Reported peaks in literature (Furniss et al., 1988) Table 7: Absorbance data for calibration curve of Punicalagin

Concentration (g/ml)

Absorbance* Average SD % RSD

Abs 1 Abs 2 Abs 3

0 0 0 0 0 0 0

5 0.154 0.156 0.153 0.15433 0.00152 0.9897

10 0.253 0.255 0.253 0.25366 0.00115 0.4552

15 0.395 0.396 0.394 0.395 0.001 0.2531

20 0.496 0.493 0.492 0.49366 0.00208 0.4216

25 0.593 0.599 0.601 0.59766 0.00416 0.6965

30 0.736 0.743 0.74 0.73966 0.00351 0.4747 35 0.855 0.857 0.854 0.85533 0.00152 0.1785

*Each value is average of three determination, SD = Standard Deviation, %RSD = Percent Relative Standard Deviation 5.3. Compatibility Study 5.3.1 Physical characterisation of drug excipient mixture The compatibility study of Punicalagin with various excipients showed that there was no

colour change as well as there was no microbial growth occurred in the solution as well as in powder form as shown in table 7. This showed that drug is physically compatible with the excipients.

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Figure 8: Standard plot data of Punicalagin in PBS pH 7.4

5.3.2 Chemical characterisation of drug excipient mixture For chemical compatibility studies, different drug-excipient mixtures were analyzed by UV spectroscopy that showed that there is no change in max i.e. 253.6 nm which are shown in figure 9. Figure 9: Compatibility study data of Punicalagin with different excipients done and by UV spectroscopy (a) Drug at 0th day (b) Drug at 28th day (c) Drug+ Span60 at 0th day (d) Drug+ Span60 at 28th day (e) Drug+ Cholesterol at 0th day (f) Drug+ Cholesterol at 28th day (g) Drug+ Mixture of all excipients at 0th day (h) Drug+ Mixture of all excipients at 28th day (a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

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Table 8: Compatibility study data of Punicalagin with different excipients done at 0th, 7th, 14th and 28th day by physical observation

Physical compatibility study of Punicalagin with different excipients Sample (Drug+Excipients) Ratio

Appearance at 0th day

Observation at different day and condition A (7th day); B (14th day); C (28th day) 5C3C 25C 2C 40C 2C

(75% 5%RH)

A B C A B C A B C Drug + WFI 1:1 Yellow

colour

Span 60 1 White colour Cholesterol 1 White colour Carbopol 934 1 White colour Drug + Span 60 + WFI 1:1:1 Slight yellow

Colour

Drug + Cholesterol + WFI 1:1:1 Slight yellow Colour

Drug + Carbopol 934 + WFI

1:1:1 Slight yellow Colour

Drug+Span60+ Cholesterol+ Carbopol 934+WFI

1:1:1:1:1 Slight yellow Colour

same as original Figure 10: Photomicrographs of different formulations at 1000 X (a) F1 (b) F2 (c) F3 (d) F4 (e) F5 (f) F6 (g) F7 (h) F8 (i) F9 (j) F10 (a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j)

5.4 Evaluation of niosomes 5.4.1 Optical microscopy: Single unilamellar niosomes were observed at 1000X by optical microscopy. The niosomes were found to be spherical in shape. 5.4.2 Micromeritics studies: The average particle sizes of the sonicated niosomes were found to be in nanometer

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Table 9: Values of Average particle size and Polydispersity index (P.D.I) for different formulations Formulation Particle size range (in nm) P.D.I. range F1 334.8 0.548 F2 279.6 0.227 F3 291.7 0.214 F4 278.7 0.236 F5 240.6 0.304 F6 336.8 0.270 F7 259.5 0.226 F8 294.1 0.324 F9 440.7 0.505 F10 625.2 0.438

Figure 11: Graphs showing (a) Particle size (b) PDI range for all niosomal formulations

rangewhich lies in range of 240.6 nm 625.2 nm. The PDI value which characterizesthe uniformity of vesicles in suspension that lies in range of 0.226- 0.548. 5.4.3 Entrapment efficiency According to the entrapment efficiency calculated, it was found that F5, F6 and F7 has greater entrapment efficiency as compared to other formulations whereas F7 has maximum entrapment efficiency of 65.93% when compared with F5 and F6 as shown in table 10 and figure 12. Thus F7 was selected for formulating gel. Table 10: Entrapment efficiency of niosomal formulations

S. No. Formulation name

% drug entrapment*

1 F1 28.97 0.06 2 F2 32.18 0.06 3 F3 35.29 0.06 4 F4 44.96 0.04 5 F5 56.68 0.12 6 F6 59.00 0.06 7 F7 65.93 0.06 8 F8 55.70 0.08 9 F9 46.73 0.12 10 F10 42.50 0.13

*Average of three determinants S.D. 5.4.4 Morphology and structure of vesicles The transmission electron micrographs of unilamellar niosomes of optimised formulation (F7) composed of span 60 and cholesterol in

7:1 molar ratios are shown in figure 13 (a) and (b). They reveal the presence of well identified and nearly perfect spheres. In figure 13 (a), the size of niosomes lies in range of 37.6 nm to 43.6 nm whereas in 13 (b), size of niosomes lies in range of 60.3nm to 358 nm. Figure 12: Comparison of entrapment efficiency of all formulations

5.4.5 Zeta potential analysis Zeta potential of the optimized formulation (F7) as shown in figure 14 which was found to be -27.2mV depicts the stability of niosomal formulation. 5.4.6 Stability studies: Drug leakage study Physical stability studies were carried out to investigate the leaching of drug from the niosomes of optimized formulation (F7) during storage at refrigerator temperature. The percentage of entrapment efficiency of Punicalagin after a period of 2 months got reduced to 25% as shown in table 11.

Particle size range

Formulations (a)

P.D.I. range

Formulations (b)

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Figure 13: TEM micrographs (a,b) of optimized niosomes (F7)

Figure 14: Zeta potential analysis for optimized niosomes (F7)

Table 11: Stability data of niosomal formulation F7

Days 53C* 0th day 65.66 0.14 15th day 62.66 0.03 30th day 60.16 0.04 45th day 52.20 0.11 60st day 49.63 0.10

*Average of three determinations S.D 5.5 Evaluation of topical gel of niosomes containing Punicalagin

5.5.1 Table 12: Physicochemical properties of topical hydrogel

Characteristics Observation Homogeneity Good Grittiness No Clarity Clear pH 7.03 0.208 Consistency 6 mm

5.5.2 Rheological behavior of developed topical gel The viscosity of the niosomal gel was determined at room temperature by using different types of spindle and at different speeds which is shown in table 13. The viscosity of the niosomal gel was found to be 38400 cps by spindle 63 and at 3 rpm which can be calculated by the following formula

Table 13: Viscosity of the formulated topical gel Speed of spindle (rpm)

% Torque reading Viscosity (in cps) Spindle 61 Spindle 62 Spindle 63

1 - - - - 1.5 - - - - 2 - - - - 2.5 - - - - 3 - - 96% 38400

Table 14: Comparison of gel strength data for niosomal gel and plain gel Sample name Gel strength Average S.D. Coefficient of

variation 1 2 3 Niosomal gel 20.24 21.22 22.23 21.23 0.993 4.677 Plain gel 19.39 20.42 21.10 20.30 0.860 4.237

5.5.3 Determination of gel strength The force required to penetrate the gel was measured as gel strength in terms of g which is shown in table 14. The gel strength of niosomal gel and plain gel was carried out and the results were found to be 21.23 0. 5.5.4 Spreadibility

Spreadibility is the ease with which a spread of a formulation can be applied in a thin, even layer to skin. According to graph as shown in figure 17 and 18, the firmness of niosomal gel and plain gel (without niosomes) in terms of force is 161.66 g and 192.55 g respectively as shown in table 15. Similarly, the work of shear which was calculated for niosomal and plain gel (without niosomes) was found to be 145.91 g.sec and 175.43 g.sec respectively as shown

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Table 15: Firmness data for niosomal and plain gel Sample name

Firmness in terms of force (g) Average S.D. Coefficient of variation 1 2 3

Niosomal gel

160.43 165.70 158.83 161.66 3.5 2.2

Plain gel 199.80 191.33 186.53 192.55 6.7 3.4 Table 16: Work of shear data for niosomal and plain gel

Sample name Work of shear (g.sec) Average S.D. Coefficient of variation 1 2 3

Niosomal gel 144.47 150.51 142.75 145.91 4.0 2.7 Plain gel 170.45 172.81 183.05 175.43 6.7 3.8

Figure 17: Graph representing the spreadibility of niosomal gel

Figure 18: Graph representing the spreadibility of plain gel

in table 16 and graphs are shown in figure 17 and 18. Therefore, from the above data it is concluded that niosomal gel require less force to spread as compared to plain gel. 5.5.5 Drug release study The amount of drug permeated from niosomal as well as plain gel for in vitro and ex vivo study is shown in table 17 and 18 respectively. The drug release from niosomal gel is much more as compared to plain gel when compared in vitro and ex vivo study. Thedata obtained from in vitro and ex vivo study for niosomal gel and plain gel was statistically analysed by one way ANOVA using a software, Graph pad it was concluded that the test is significant with p < 0.05 that means there is a difference in the release pattern of niosomal gel when compared with plain gel by in vitro and ex vivo method. The ***p value comes out to be 0.0001 by Bonferronis multiple comparison test.

Figure 19: Comparison between the amounts of drug permeated in vitro from niosomal and plain gel in vitro

Figure 20: Comparison between the amounts of drug permeated in vitro from niosomal and plain gel in vitro

5.5.6 Skin retention studies The amount of drug retained in skin from niosomal gel was found to be 211.27g/cm whereas in case of plain gel, the amount retained was 149.35 g/cm as shown in table 19. 5.6 Analysis of kinetic release models To ascertain the drug release mechanism and release rate, the ex vivo drug release data of prepared formulations were fitted with various release models. The models selected were zero order, first order, Higuchi, Korsemeyer Peppas and Hixson Crowell modelwhich are shown in table 20 and figure 21 - 25. R2 value in case of zero order release was found to be higher than the other kinetic release models in niosomal gel of the optimized formulation F7 as well as in the plain gel thus suggesting that the formulation followed zero order which is best and suitable for the topical drug delivery.

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Table 17: Comparison of the amount of drug permeated in vitro from niosomal and plain gel Time in hrs Amt permeated from plain gel (g/cm2) Amt permeated from niosomal gel (g/cm2) 0 0 0 1 138.64 216.12 2 271.34 344.09 3 533.68 617.47 4 764.64 806.65 5 1182.91 1379.72 6 1599.99 1828.76 7 3494.84 4798.62 8 3792.20 5194.99 9 3978.33 6087.26 10 4279.31 6380.32 11 4320.64 6782.71 12 4364.20 6831.96 14 4642.45 7357.64 16 5507.34 7143.61 18 5583.27 7528.79 24 5634.41 7855.43

Table 18: Comparison of the amount of drug permeated ex vivo from niosomal and plain gel

Time (in hrs) Amount of drug permeated from plain gel (g/cm2)

Amount of drug permeated from niosomal gel (g/cm2)

0 0 0 1 300.49 233.34 2 389.63 434.49 3 644.79 569.49 4 751.89 864.33 5 943.05 1252.47 6 1525.95 1720.29 7 4947.22 5541.60 8 5100.98 5754.60 9 5237.70 6262.89 10 5308.12 6650.65 11 5614.27 8667.28 12 5844.13 8601.16 14 6078.82 9282.67 16 6128.58 9154.74 18 6504.29 9626.01 24 6545.10 10038.74

Table 19: Comparison of niosomal and plain gel for skin retention study

Formulation Absorbance Dilution factor Conc.(g/ml) D.F.*Conc. Amount retained (g/cm)

Niosomal gel

0.263 50 10.26778 513.3891 211.2712

Plain gel 0.885 10 36.29289 362.9289 149.3534 Table 20: Comparison of K and R2 values for all kinetic models

Formulation name

Zero order kinetics

First order kinetics

Higuchi model Korsemeyer Peppas model

Hixson Crowell model

K R2 K R2 K R2 K R2 K R2 Niosomal gel of F7

749.91 0.938

-0.110 0.508 2885.9 0.845 -1.42 0.434 -0.925 0.710

Plain gel 489.88 0.882

-0.099 0.459 1907.1 0.813 -1.37 0.338 -0.761 0.637

5.7 Determination of wound contraction To check out the effectiveness of the niosomal gel of Punicalagin, in vivo studies were conducted and concluded that the after 14

days, the % wound contracted in case of niosomal gel (88.61%) is more than the marketed formulation (75.72%), then to

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conventional gel (34.73%) and negative control (23.88%). Thus the niosomal gel containing Punicalagin is very effective as compared to the conventional/plain gel as well as to the marketed formulation. Figure 21: Analysis of release kinetics by Zero order kinetics

Figure 23: Analysis of release kinetics by Higuchi model

Figure 24: Analysis of release kinetics by Korsemeyer Peppas model

Figure 25: Analysis of release kinetics by Hixson Crowell model

Figure 26: Comparative evaluation of % wound contraction

2 4 6 8 10 12 140

10

20

30

40

50

60

70

80

90

100

negative control marketed formulation plain gel niosomal geldays

% c

ontr

actio

n

Conclusion Punicalagin was extracted, isolated and purified from the peels of the Punica granatum which was identified by carrying out chemical identification test, thin layer chromatography (TLC), UV spectrophotometry and FTIR. Compatibility studies of Punicalagin with the excipients used during the work were conducted for 28 days and observed that drug is compatible with the excipients physically and chemically. On the basis of the findings, it can be stated thatthe formulation F7 is the best formulation from all the other formulations which were prepared by different compositions of Span 60. The F7 formulation has greater entrapment efficiency as compared to other formulations, as F7 showed 65.93% whereas F5 and F6 showed 56.68% and 59% respectively. Also, F7 has particle size of lowest value as compared to others i.e. 259.5 nm as shown in figure 28. TEM, zeta potential and stability studies were conducted for the best formulation F7. TEM studies showed that niosomes in F7 are nearly perfectly spheres whereas zeta potential study showed the stability of formulation F7. The optimized formulation was formulated into a topical hydrogel and various evaluating parameters like consistency, pH, viscosity, spreadibility, gel strength, drug release study (in vitro and ex vivo) and skin retention study was done. In in vitro and ex vivostudy, it was observed that amount of drug permeated from niosomal conventional gel was done by applying kinetic models to them and concluded that both formulations followed zero order kinetics as R2 value came to be higher as compared to other models which is best kinetic model for the transdermal drug delivery system. To check out the effectiveness of the niosomal gel of Punicalagin, in vivo studies were conducted on the wistar rats for the wound healing activity and it is concluded that the after 14 days, the % wound contracted in case of niosomal gel (88.61%) is more than the marketed formulation (75.72%), then to conventional gel (34.73%) and negative control (23.88%). So it can be stated as niosomal gel of Punicalagin is best as compared to others. This research work has been conducted as there is very less research work done on the aspect of its formulation part as well as on the particular constituent extracted from the Punica granatum. The special finding of this report is that research work concluded the effectiveness of the formulation i.e. niosomal gel incorporated by the particular constituent i.e. Punicalagin. Only using the extract of

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either the whole plant or any plant part has been used in previous researches but not particular constituent. This article gives an idea that further studies can be conducted on the formulation aspect by incorporating the particular constituent for different activities. The future studies can be carried out by incorporating this drug into different vesicular system and their comparison or by using

different non-ionic surfactants in different ratios or by changing the amount of cholesterol as well. Further studies can be conducted by designing the different formulations for different activities. The main aim of this study is to check out the effectiveness of niosomal gel containing Punicalagin for the wound healing activity which has been carried out successfully.

Table 21: Mean area closure of Excision wound area on following post wounding days

Group name

Wound area measured* 0th day 2th day 4th day 6th day 8th day 10thday 12thday 14thday

Negative control (Group I)

301.3 7.5

289.1 6.6

280.5 9.1

266.3 8.2

255 7.4

245 6.6 235.1 6.8 223.3 8.7

Plain gel (Group II)

306.1 11.4

285.8 11.5

266.6 9.8

255.6 12.5

237.3 12.8

220.1 15.0

210 15.1 200 15.1

Niosomal gel (Group III)

298.6 10.5

259.8 8.3

230.3 9.0

178.8 10.3

94.66 3.9

57.66 5.9

48.3 6.8 34 6.6

Standard (Group IV)

301.3 7.5

270.5 8.5

243.6 6.8

203.1 5.8

130.8 6.1

103.5 5.0

93.1 5.3 73.1 6.9

*Values are expressed as Mean SD, n=6 in each group Table 22: Mean Percentage closure of Excision wound area on following post wounding days

Group name

% Wound contraction* 0th day

2th day

4th day 6th day 8th day 10th day

12th day 14th day

Negative control (Group I)

0 4.02 1.0

6.9 0.89

11.61 1.2

15.37 1.1

18.69 1.3

21.95 1.3 25.88 2.0

Plain gel (Group II)

0 6.65 0.39

12.89 0.91

16.51 1.6

22.51 1.6

28.13 2.8

31.46 2.9 34.73 3.0

Niosomal gel (Group III)

0 12.98 1.7

22.86 1.9

40.16 1.3

68.67 1.9

80.67 1.9

83.81 2.2 88.61 2.1

Standard (Group IV)

0 10.23 0.88

19.13 0.86

32.56 1.14

56.57 1.9

65.63 1.8

69.06 1.9 75.72 2.1

*Values are expressed as Mean SD, n=6 in each group

On the 0th day 1. Negative control (1) (2) (3)

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2. Marketed Formulation (4) (5) (6)

3. Plain gel (7) (8) (9)

4. Niosomal gel (10) (11) (12)

On the 14th day 1. Negative control Figure 27: Contraction of wound on 0thand 14th day (lxxxv) (lxxxvi) (lxxxvii)

2. Marketed Formulation (lxxxviii) (lxxxix) (lxxxx)

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3. Plain gel (lxxxxi) (lxxxxii) (lxxxxiii)

4. Niosomal gel (lxxxxiv) (lxxxxv) (lxxxxvi)

Figure 28: Particle size distribution and Particle size analysis of F7

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1993. In vitro activities of free and liposomal drugs against Mycobacterium avium M. Intracellulare complex and M. Tuberculosis. Antimicrobial Agents and Chemotherapy, 37(12), 2584-2587.