INTRODUCTION:

A burn can be defined as a diseased state when there is an injury to the skin primarily caused by direct heat or radiation, radioactivity, electricity, friction or contact with chemicals. It is the fourth most common type of trauma leading to infection, scarring, and sometimes even death¹. As per the latest WHO data, burn takes millions of lives every year and puts a public health burden. The skin is one of the largest organ of the human body which act as a first-line defense against mechanical injuries and microbial infections.

It consists of an epidermis which is formed of a stratified epithelium along with a basal-cell layer made up of keratinocytes and a dermis. The epidermis forms a protective covering against the environmental exposures, and the dermis contains the connective tissue, including cross-linked collagen protein, which provides flexibility and elasticity to the skin. Dermis also includes an extracellular matrix composed of proteoglycans which maintain hydration in the skin. It also contains skin appendages such as hair follicles, sweat glands, oil glands, nerves and blood vessels².

Burn wounds are classified based on the type of causative agents like heat, chemicals, electric shock and depth of injury caused by them. These agents cause coagulative necrosis by inducing tissue damage through energy transfer and directly damage cellular membranes³. After a burn injury takes place, coagulation of blood vessels occurs in order to maintain the homeostasis. Interestingly, the burn wound and systemic infections are in correlation with the size of the injury and the risk of subsequent infections. Based on the size and extent of wound inflicted, burns are categorized as first degree burns which are marked by non-blistered and reddened skin, second-degree burns are characterised by prominent blisters and affect dermis as well up to some extent, and lastly, the third-degree burns damage a widespread thickness of the skin having whitish leathery appearance⁴. Microcirculation, circulating cells, immune cells, mast cells and the growth factors and cytokines released by them regulate the wound healing process⁵.

Among the burn wound models like direct heating by metal, electricity induced, and hot water-induced models, the hot water model has achieved widespread applications. As burns caused by hot liquids are among the most frequent causes of burns in children and the elderly, this model can provide more relevance in the drug development for wounds⁶. Rats can bear a total body surface area of up to 60%, which helps mimic clinical burn conditions with similar pathophysiological features but variability in their skin thickness as compared to human pose a limit in the burn research^6,7.

There are many available topical agents which are applied for burn wound management. Drugs like Silver sulfadiazine (SSD), silver nitrate, etc., are commonly used as a standard topical therapy for the treatment of burn wounds with antibacterial properties⁸. The available agents have shown to possess several side effects such as, neutropenia, erythema multiform, methemoglobinemia, leukopenia and renal toxicity which indirectly affects the process of wound healing⁹. Therefore, there is an urgency for finding out new agents for the treatment of burn wounds in with lesser adverse effects and better efficacy.

There are numerous natural herbal wound healing agents available which have been used since ancient times for the management of wounds¹⁰. Sesame oil is known to soothe minor burns and also accelerates the process of wound re-epithelialization by making contraction of wounds⁴. Coconut oil gets absorbed rapidly in the skin and maintains moisture. At the cellular level, it accelerates the metabolism and wound healing process¹¹. Naphthalene and its derivatives, at low concentrations, are used as a preservative due to their antibacterial activity¹². Turmeric has many properties like anti-inflammatory, antibiotic, immunomodulatory, wound healing etc. which can be utilized for therapeutics purpose^13,14. Rosewater, along with having aromatic properties possess antiseptic properties¹⁵. Sandalwood topical application enhances wound healing rate via keratinocytes stimulation^16,17. Myrobalan, containing tannins, has wound healing properties via its protein precipitating activity¹⁶. Medicated dhupan or known as Rala, is a resin obtained from Sal tree, known to contain tannins that have wound healing properties¹⁸.

In this study, we have formulated an Ayurvedic ointment using the above-discussed components after optimization and its standardization and safety parameters have been evaluated. We have also tested the Ayurvedic ointment for acute dermal toxicity following OECD 402 guidelines in order to ensure its safety and finally, its evaluation for its burn wound healing potential has been done on a scalding burn wound rat model.

MATERIALS AND METHODS:

Study Material:

Chemicals:

Hematoxylin, eosin, isopropyl alcohol, paraffin wax were purchased from Merck US. Leica instruments, Silver Nitrate Ointment (Silverex™) Batch no. RDX0083, Mfg. date: Feb., 2020, Exp. Jan, 2022, Leica histological processing machine, embedding and microtome were procured from Leica, Germany. Natural drugs and components were procured from local market and their natural habitats, which were free from pollution. These were authenticated from the competent authority. All the other chemicals, solvents and reagents used were of analytical grades and standard quality only.

Study Methods:

Formulation of Ayurvedic ointment:

The Ayurvedic ointment was made by using the fusion method with slight modifications¹⁹. Ral gum was heated in a beaker taken on a water bath to its melting point, into which coconut oil and sesame oil were added. In a mortar, the powder constituents were incorporated and grounded till fine powder was obtained. Heated base and oil were added to the mortar while trituration was done to get an ointment with a semi-solid consistency. In the last rose water was added with vigorous trituration upon cooling of the ointment in order to avoid loss of volatile contents of rose water. Composition of the Ayurvedic ointment formulation was described in Table 1.

Standardization of Ayurvedic ointment:

The well-authenticated Ayurvedic materials were chemically standardized through phytochemical and physicochemical standards as per Ayurvedic Pharmacopoeia of India (API) evaluation methods. The physicochemical analysis of sample extracts was performed to determine chemical parameters like pH value, total ash, acid-insoluble ash, loss on drying, water-soluble extractive, alcohol-soluble extractive, and TLC profiles. While safety studies incorporated various tests viz., analysis of heavy/toxic metals (Pb, As, Hg, and Cd), microbiological analysis, aflatoxins (B1, B2, G1, and G2) and pesticidal residue analysis²⁰.

Viscosity measurement:

The viscosity of the Ayuvedic ointment was determined using Brookfield Viscometer (LVDV-II, USA) at 25°C²¹. Briefly, a fine amount of the Ayuvedic ointment was kept on the sample holder of the viscometer. It was firstly equilibrated for 20s and the corresponding viscosity (cP) was measured using a rotating spindle (s-64) at RPM of 20.

HPTLC based characterization:

Sample preparation:

1gm each of the test sample was soaked in 10ml of hexane and 10ml of toluene for 24 hours (h) and the extract obtained was filtered using a fine filter paper. The filtrate was further concentrated to 10ml and taken for the TLC profiling.

Stationary Phase:

Precoated (support on aluminum sheets) silica gel plate. Specification of the plate used was TLC Silica Gel 60F₂₅₄, Mfg. by Merck, 26.09.2016, Batch No. 1.05554.0007.

Mobile Phase:

Toluene: Ethyl acetate: 9: 1 (v/v)

Sample application:

Applied volume 4µL, 6µL and 8µL of hexane and toluene extracts as 6mm band and applied at 15mm from the base of the plate. The Plate size was 10x10.

Development:

It was developed up to 90mm in CAMAG twin trough chamber, Plate preconditioning (temp 25°C and relative average humidity was 42%).

Photography of Developed HPTLC Plate:

Plate observed at 254nm, 366nm and in the white light of derivatized plate in 20% sulphuric acid.

Experimental Animals:

Healthy male Wistar rats of weight (260±20g) were bred and maintained in the animal house facility of Central Ayurveda Research Institute, Kolkata, India (Regn. No.694/GO/RBi/S/02/CPCSEA) were used in the experiment. The experiments were performed by adopting guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Fisheries, Animal Husbandry and Dairying, Government of India and the experimental protocol was approved from Institutional Animal Ethics Committee (IAEC) of CARI, Kolkata, with ethical approval No. 39/P/S/IAEC/2018, dated 25^th August 2020. Before the initiation of the experiment, animals were caged individually with proper marking and acclimatized for a period of seven days to the experimental animal house. Acclimitization period helps stabilize the rats in the new environment and thus promotes the concept of animal welfare and experimental reproducibility. The temperature of the experimental animal room was maintained at 25⁰C, and relative humidity was maintained at 60-70 percent. The lighting sequence was kept at 12 hours light and 12 hours dark. Standard pelleted animal diet obtained from the National Institute of Nutrition, Hyderabad, India was fed to the animals ad libitum during the study period. Normal purified water was provided to the animals ad-libitum using plastic bottles with a steel nozzle.

Evaluation of Acute Dermal Toxicity

Acute dermal toxicity test has been performed following the OECD guidelines 402 given for the chemical testing. Before the test, the rats were selected randomly and assigned to the treatment and control groups. Fur was removed 24 h before the test from the dorsal area of the trunk of the test animals by shaving. In the acute dermal toxicity study, twelve adult female Wistar rats of 7-9 weeks of age were taken, consisting of six animals each in the control and test groups. About 10% of the body surface area was cleared for the application of the test substance. The test drug was applied to the dorsum area and taped with a porous gauze dressing, non-corrosive, and non-irritating tape throughout a 24 hours exposure period. Coconut oil was applied topically in the skin as a vehicle. The test drug was applied topically only once on the 1^stday of the study. After exposure period, the residual test substance was removed, where practicable using water. The animals were clinically checked once on the 1^stday of the study and were closely monitored for the duration of 24 hrs, with special attention given to them for the first 6 h. Later, they were observed daily for the next 14 days. Bodyweight, feed and water consumption were recorded on 0^th day, 7^th day (1^st week) and 14^th day (2^nd week) of the experiment. Close observations of animals were made, including changes in fur, eyes and mucous membrane, and also the circulatory, respiratory, autonomic and central nervous system and somatomotor activity and behaviour pattern. Particular attention were directed and given on changes in physical appearance, injury, pain, tremor, convulsion, salivation, diarrhoea, lethargy, sleep, coma and mortality.

Evaluation of burn wound healing activity:

Thirty male rats of 7-8 weeks of age, was included in the study and were divided into 5 groups containing six rats in each group (Table 2). Each animal were and kept separately in the labelled cages with equal accessability of food and water inside the cages. Animal number was fixed on the basis of its need for appropriate statistics and based on previous in-vivo experiments done in the lab. Burn was induced in the animals by scalding burn injury method, according to a pre-devised method described by^22,23. Grouping of animals were done by following the principle of randomization with the help of Microsoft excel 2016 RAND function, and divided into five groups containing six rats in each group. The first group was normal control group which were not induced burn or given any treatment, second group was acute burn group in which burn was induced and no treatment was given, third group was vehicle control group in which vehicle which was coconut oil was applied after the burn induction throughout the study period, fourth group was the standard treatment group in which Silverex™ ointment which is a standard marketed ointment was applied throughout the study period, and the fifth group was the test group, the animals of this group were applied with the Ayurvedic ointment throughout the study after burn induction. The treatments were done on daily basis throughout the study period at a fixed time and by the same expert technical person. For induction of burn, the animals were restrained, and a dimeter of 3cm of the area on the dorsal surface of the rats were shaved using razor blade to expose the skin. A fixed volume of boiling water was poured for a time period of 15 seconds on the exposed skin to induce full-thickness burn. After the induction of burn injury, 0.8ml of normal saline were administered intraperitoneally to the animals to prevent spinal shock. The animals in which similar depth of burn was not induced were not taken in the further experiment and were discarded from the experiment and euthanized. Clinical observations of the experimental animals were recorded daily throughout the experimental period. Weekly feed and water consumption of all rats were recorded throughout the study period. Body weights were recorded weekly. The wound contraction and period of re-epithelialization was monitored by a technical expert whoi was kept blind for the experiment groupings. The number of days taken for the eschar to fall off itself from the burn wound surface without leaving any raw wound behind was considered as period of re-epithelialization. Wound Contraction was noted by measuring the wound bed area on day 1^st, day 3^rd, day 7, day 14^th and day 21^st with the help of vernier calliper. Other gross changes were recorded throughout the study period.

Percentage of wound contraction = [(Initial wound size – specific day wound size) / initial wound size] X 100.

Histological and histochemical techniques:

On the 21st day, after the necropsy was performed, the skin tissue containing the wound area were fixed in 10% formalin for 20 days. The fixed tissues were taken for further processing, including dehydration, clearing, and embedding. The blocks of the tissues were embedded with paraffin wax and later finely sectioned using microtome into 3.5 µm thick sections. The tissue sections were taken on the glass slides were dewaxed in the incubator and hydrated, followed by hematoxylin and eosin staining. In the last, the prepared slides were dehydrated with alcohol and mounted with DPX reagent and taken for light microscopic examination and reporting which was done by a technical expert who was kept blind for the experiment²⁴.

Statistical analysis:

Data was analyzed by appropriate statistical tools. The number of observations (N) for each analysis were 6 for each test, group mean, and Standard Deviations (SD) were calculated. Data were checked for normality by the Shapiro-Wilk test, and to check homogeneity of data, Levene statistic was applied. Normal and homogenous data were analyzed by independent sample t-test / one-way ANOVA taking the group as a factor. Normal but heterogenous data was analyzed by applying Welch test. Not normally distributed data was analyzed by Kruskal-Wallis H test. Dunett (2-sided) /Games-Howell/ Mann-Whitney U Post Hoc test, wherever applicable, was used to compare the groups over the control arm. All the statistical tests in the obtained results were done at 5% level of significance.

RESULTS:

Standardization of Ayurvedic ointment:

Chemical Standardization:

Chemical Standardization of the Ayurvedic ointment was done as presented in Table 3. Analysis of Ayurvedic ointment for the safety parameters like heavy metals (lead, cadmium, arsenic, mercury), microbial contamination, pesticide residue analysis, and aflatoxins as given in Table 5. indicates that all the batches of the formulation are safe and complied as per the WHO and API guidelines. The HPTLC profile of this formulation (Table 4) indicates eight prominent bands at 0.12, 0.14, 0.24, 0.32, 0.49, 0.51, 0.81 and 0.95 UV 254 nm; six bands at 0.07, 0.10, 0.14, 0.22, 0.29 and 0.40 under UV 366 nm; and nine bands at 0.13, 0.24,0.29, 0.41, 0.47, 0.50, 0.58, 0.84 and 0.97 under white light after derivatization with the anisaldehyde-sulphuric acid reagent (ASR).

Viscosity measurement:

Optimum viscosity is the characteristic feature of any efficient ointment that assists the application of the ointment on the wound surface. Ointment viscosity of the Ayurvedic ointment were found to be 21805 cp which confirmed the desired consistency of Ayurvedic ointment for topical administration on burn wound model.

Acute Toxicity Study:

Clinical Signs and Mortality:

All rats were apparently normal, and no clinical signs and mortality were observed in any animal throughout the study. No abnormality in skin and fur, eyes and mucous membranes were detected. The respiratory and behavioural patterns were normal. There was no tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Body Weight:

The data of body weights of all rats were analysed statistically, and no significant difference was found between the control and treated group on 0^thday, 1^stday, 7^th day and 14^th day. However, there was a significant increase in the intra-group body weight in both control and treated groups, on day 7^th and day 14^th compared to the 0^thday and 1^stday (Table 6).

Feed Consumption:

No significant difference in feed consumption was found between the control and treated group at 1^st week and 2^nd week. However, a significant increase in feed consumption of the control group was observed on the 2^nd week as compared to the 1^st week (Table 7).

Water Consumption:

No significant difference in water consumption was found between the control and treated group at 1^st week and 2^nd week (Table 8).

Evaluation of Burn wound healing potential:

Absolute body weight:

The data of absolute body weight of all rats were analysed statistically and no significant difference was found between normal control and any other group on 0^thday (initial day). On 1^stweek significant decrease in absolute body weight of negative control group and test drug treated group as compared to normal control group were observed. On 2^nd week and 3^rd week, negative control group showed a significant decrease in absolute body weight as compared to normal control group. However, vehicle control, positive control and test drug-treated groups did not show significant difference in absolute body weight as compared to the normal control group in 1^st, 2^nd and 3^rd week (Table 9).

Relative body weight:

There was no significant difference in relative body weights of vehicle control, positive control and test drug-treated groups as compared to the normal control group on 1^st, 2^nd and 3^rd week. However, significant decrease in relative body weight of negative control group was observed on 1^st and 2^nd week as compared to a normal control group (Table 10).

Feed Consumption:

Significant increase in feed consumption of test group animals on 1^st week and significant decrease in feed consumption of negative control group on 2^nd and 3^rd week were observed as compared to normal control group (Table 11).

Water Consumption:

Significant decrease in water consumption of negative control, vehicle control and positive control group animals were observed on 3^rd week as compared to normal control group (Table 12).

Percentage burn wound contraction:

Significant increase in percentage burn wound contraction of vehicle control (Coconut oil), positive control (Silverex™) and test drug-treated group animals were observed on day 3, day 7, day 14 and on day 21 as compared to negative control (acute burn Injury) group (Table 13). The burn wound contraction in the test and positive control groups were significantly more as compared to vehicle control and negative control group.

Period of epithelization:

Days required for epithialization of both positive control (Silverex™) group and test drug treated group were significantly lower than negative control (acute burn injury) group (Table 14). The burn wound contraction in the test and positive control groups were significantly more as compared to vehicle control and negative control group (Figure 1).

Histopathological evaluation:

Histology of skin sections after H & E (Hematoxylin & Eosin) staining showed normal epidermis, dermis with mature fibrous tissue showing abundant collagen in bundles (arrows) in the normal control group in shown in Figure 2a. In case of the disease group, as shown in the Figure 2b, 2c and Figure 2d, the epidermal layer is not visible due to necrosis. The dermis shows signs of immature granulation reflecting inflammation as shown in the image with a highlighted circular ring. It also shows a necrotic area at the epidermis along with fibrin, and also the remnants of PMN (Polymorphonuclear leukocytes) can be found.

As shown in the Figure 2e, which is section from the vehicle treatment group, partial healing of epidermis under the scab (arrow) with mature granulation tissue can be observed. Figure 2f, is the image obtained from skin section of standard treatment group (Silverex™) which shows epidermal regeneration with mature granulation tissue in the dermis. Skin section obtained from the Ayurvedic ointment treatment group shows the epidermal regeneration along with mature granulation tissue in the dermis. The hair follicles regeneration in the section is also seen.

DISCUSSION:

Burn wound is of the common problem which is encountered by most of the people at least once in their lifetime. Their treatment and management mainly focuses on wound healing and protection of the wound from infections. In this study, we have aimed to develop a formulation which can have faster-wound healing thus, chances of infections can be eliminated.

Smooth spreading and optimum viscosity are the characteristic features of any efficient ointment that assists the application of the ointment on the wound surface. The obtained spreadability and viscosity were as per the compliance by the API and were appropriate for getting used in the burn wound studies, it was consistent with the previous study done by²¹.

It is essential for the detection of contaminants of formulation and useful for finding the authenticity and quality of materials used in the formulation of Ayurvedic ointment. quantitative tests method, i.e., physicochemical analysis, is an essential part of standardization to set the pharmacopoeial standards of Ayurvedic ointment. Almost similar results of all the three batches for these parameters indicate that all the three batches of Ayurvedic ointment are found in close proximity. The result of the safety parameter of Ayurvedic ointment showed that all the batches are safe as per the WHO and API guidelines. Moreover, the outcomes of aflatoxins, microbial load, and pesticidal residue analysis help to draw an inference that Ayurvedic ointment with its ingredients are free from chemicals, toxins and safe for consumption^25,26.

The acute dermal toxicity results showed that there was no treatment-related mortality in any of the groups after the application of Ayurvedic ointment in either male or female rats kept under observation for 14 days. One of the important markers of gross toxicity is the loss of body weight in rats after treatment of toxic substance. Drastic toxicity is related to interference with the absorption of nutrients will be reflected in bodyweight reduction. The body weight in the group treated with Ayurvdic ointment had not brought any significant difference when compared with the control group. Hence, it can be inferred that the formulation has no tendency to produce drastic tissue destruction or any interference with the absorption of the nutrients. Food and water consumption among treated and control groups. Thus, we can say that the Ayurvedic ointment is biocompatible and non-immunogenic.

After burn wound induction, there has been no significant change in the body weight and feed intake behavior of the rats which shows that the rats are showing normal physiological behavior. Absolute body weight of the rat signifies the normal well being of the rats and it can be used to show the normal physiological growth of rats as function of time²⁷.

The significantly higher wound contraction in the Ayurvedic ointment group indicates the healing potential of the formulation for burn wound^10,28. Period of re-epithelialization after the injury is the time period after which the burn wound would start healing. It is characterized by granulation process in the wound mediated by macrophages and fibroblasts.These are responsible for the recovery of the lost extracellular matrix supported by neovascularization along with the involvement of important cytokines. A shorter period of re-epithelization ensures faster burn wound healing¹⁰.

Histological evaluation of skin tissues shows wound healing in the three groups namely vehicle group, standard treatment and Ayurvedic ointment treatment groups. Coconut oil treatment gives a partial healing, where as both standard drug and Ayurvedic ointment treatment goups show epidermal tissue granulation. The hair follicle growth in the Ayurvedic ointment treatment groups shows an edge against the standard drug treatment as it signifies a faster rate of healing²⁹. Results obtained from the present experiment suggest that treatment of burn wound with Ayurvedic ointment provides an accelerated wound healing. It was effective in reducing burn healing which was comparable with that of standard drug Silverex™. However, further studies can be done to explore the active physto-constituents involved and the mechanism of action of the formulation through detailed molecular studies. It can be a potential ointment for burn wound treatment, and in future, it can be considered for clinical studies to validate its safety and efficacy in humans.

Table 1: Showing composition of Ayurvedic ointment formulation

Each 200gm of Ayurvedic ointment contains
Name	Part use	Quantity
Sesame oil	Oil	20ml
Coconut oil	Nut oil	80 ml
Naphthalene	Powder	0.001gm
Turmeric powder	Powder	1gm
Sandal	Powder	1gm
Myrobalan	Powder	1gm
Ral gum (Dhup)	Powder	20gm
Rosewater	Liquid	q.s. to 200gm

Table 2: Showing Group description

Group I (n=6)	Group II (n=6)	Group III (n=6)	Group IV (n=6)	Group V (n=6)
Normal Control group	Acute Burn injury group	Vehicle Control group	Standard group	Test group
Animals of this group just made to undergo shaving procedure on back of the animals and then will be kept undisturbed throughout the study period.	Animals of this group were given scalding burn Injury and then were kept undisturbed throughout the study period.	Acute Burn injury group + Coconut oil as vehicle without medicament were applied topically for 21 days.	Acute Burn injury group + Silver Nitrate Ointment (Silverex™) were applied topically for 21 days.	Acute Burn injury group + Ayurvedic ointment were applied topically for 21 days.

Table 3. Showing chemical standardization results for Ayurvedic ointment.

Sl. No.	Test Parameter	Result (% w/w)
1	LOD at 105^oC	62.33
2	Ash value	0.125
3	Acid insoluble ash	Nil
4	Alcohol soluble extractive value	6.75
5	Water soluble extractive value	0.15
6	pH	4. 78

Table 4: HPTLC profile of the Ayurvedic ointment

UV 254 nm		UV 366 nm		White light/ Derivatized plate

Track: 1 to 3 are Hexane extracts (4 µL, 6 µL and 8 µL respectively); Track :4 to 6 are Toluene extracts (4 µL, 6 µL and 8 µL respectively)
R_f Values for Track 6 (Toluene extract 8µL) at UV 254 nm	R_f Values for Track 6 (Toluene extract 8µL) at UV 366 nm		R_f Values for Track 6 (Toluene extract 8µL) in White light/ derivatized plate
0.12, 0.14, 0.24, 0.32, 0.49, 0.51, 0.81 and 0.95	0.07, 0.10, 0.14, 0.22, 0.29 and 0.40		0.13, 0.24,0.29, 0.41, 0.47, 0.50, 0.58, 0.84 and 0.97

Table.5: Analysis of Ayurvedic ointment:

Test Parameters	Units	Results	Limits	LOQ	Method of Analysis
Elemental Analysis- Lead	mg/kg	Not detected	NMT 10	0.05	API
Elemental Analysis- Mercury	mg/kg	Not detected	NMT 1	0.05	API
Elemental Analysis- Arsenic	mg/kg	Not detected	NMT 3	0.05	API
Elemental Analysis- Cadmium	mg/kg	Not detected	NMT 0.3	0.05	API
Aflatoxin B1	ppb	BDL	NMT 2	1.0	API
Aflatoxin B1,B2,G1,G2	ppb	BDL	NMT 5	1.0	API
Micobioloical Parameters Total fungal count	cfu/gm	<10	NMT 10^3	NA	API
Total viable aeobic count	cfu/gm	<10	NMT 10^5	NA	API
E.coli	Per gm	Absent	Absent	NA	API
Salmonella	Per gm	Absent	Absent	NA	API
P.auriginosa	Per gm	Absent	Absent	NA	API
S.aureus	Per gm	Absent	Absent	NA	API
Pesticide residue- Alachlor	mg/kg	BDL	NMT 0.02	0.01	API
Pesticide residue-Aldrin and dieldrin	mg/kg	BDL	NMT 0.05	0.01	API
Aziphos methyl	mg/kg	BDL	NMT 1	0.01	API
Bromopopylate	mg/kg	BDL	NMT 0.3	0.01	API
Chlordane	mg/kg	BDL	NMT 0.05	0.01	API
chlorfenvinphos	mg/kg	BDL	NMT 0.5	0.01	API
chlorpyrifos	mg/kg	BDL	NMT 0.2	0.01	API
Chlorpyrifos methyl	mg/kg	BDL	NMT 0.3	0.01	API
Cypemethrin and its isomers	mg/kg	BDL	NMT 1	0.01	API
DDT	mg/kg	BDL	NMT 1	0.01	API
Deltamethrin	mg/kg	BDL	NMT 0.5	0.01	API
Diazinon	mg/kg	BDL	NMT 0.5	0.01	API
Dichlorvos	mg/kg	BDL	NMT 1	0.01	API
Dithiocarbmates	mg/kg	BDL	NMT 2	0.01	API
Endosulfan	mg/kg	BDL	NMT 3	0.01	API
Endrin	mg/kg	BDL	NMT 0.05	0.01	API
Ethion	mg/kg	BDL	NMT 2.0	0.01	API
Fenitothion	mg/kg	BDL	NMT 0.5	0.01	API
Fenvelaarte	mg/kg	BDL	NMT 1.5	0.01	API
Fonofos	mg/kg	BDL	NMT 0.05	0.01	API
Heptachlor	mg/kg	BDL	NMT 0.05	0.01	API
Hexachlorbenzene	mg/kg	BDL	NMT 1.0	0.01	API
Hexachlorcyclohexane isomers	mg/kg	BDL	NMT 0.3	0.01	API
Lindane	mg/kg	BDL	NMT 0.6	0.01	API
Malathion	mg/kg	BDL	NMT 1.0	0.01	API
Methidathion	mg/kg	BDL	NMT 0.2	0.01	API
Parathion	mg/kg	BDL	NMT 0.5	0.01	API
Parathion methyl	mg/kg	BDL	NMT 0.2	0.01	API
Permethrin	mg/kg	BDL	NMT 1.0	0.01	API
Phosalone	mg/kg	BDL	NMT 0.1	0.01	API
Piperonyl Butoxide	mg/kg	BDL	NMT 3.0	0.01	API
Pirimos methyl	mg/kg	BDL	NMT 4.0	0.01	API
Pyrethrin	mg/kg	BDL	NMT 3.0	0.01	API
Quintozene	mg/kg	BDL	NMT 1.0	0.01	API
Total acidity	NA	complied	NA	NA	NA
Spreadability	NA	complied	NA	NA	NA

Table 6: Bodyweight (g) of Wistar rats in acute dermal toxicity study of Ayurvedic ointment

Group	Day 0^th (Mean ± SD)	Day 1^st (Mean ± SD)	Day 7^th (Mean ± SD)	Day 14^th (Mean ± SD)	P value
Control group	203.16 ± 2.23^Aa	206.83 ± 2.22^Aa	216.16 ± 1.94^Ba	224.0±3.52^Ca	<0.001
Treated group	203.5 ± 2.16^Aa	207.0 ± 2.75^Aa	216.66 ± 3.72^Ba	224.5±4.08^Ca	<0.001
P value	0.788	0.892	0.673	0.838

‘a, b’ denote statistical significance means for columns according to paired sample ‘t’ test (two tailed) at 5% level of significance (95% confidence interval) A, B, C denote statistical significance means for rows according to one way ANOVA by SPSS 16.0; values not sharing common superscript differ significantly in Bonferroni post hoc test at p< 0.05 level and at 95% confidence interval.

Table 7: Weekly Feed consumption (gm per day/100gm b.wt.) of Wistar rats in acute dermal toxicity study of Ayurvedic ointment

Group	1^st Week (Mean ± SD)	2^nd Week (Mean ± SD)	P value
Control group	3.72±0.32^Aa	3.86±0.29^Ba	0.017
Treated group	3.94±0.15^Aa	4.10±0.15^Aa	0.079
P value	0.077	0.076

‘a, b’ denote statistical significance means for columns and ‘A, B’ denote statistical significance means for rows according to paired sample ‘t’ test (two tailed) at 5% level of significance (95% confidence interval).

Table 8: Weekly water consumption (ml per day/100gm b.wt.) of Wistar rats in acute dermal toxicity study of Ayurvedic ointment

Group	1^st Week (Mean ± SD)	2^nd Week (Mean ± SD)	P value
Control group	8.36±0.96^A,a	8.71±0.67^A,a	0.319
Treated group	9.01±0.88^A,a	9.02±0.82^A,a	0.955
P value	0.272	0.583

‘a, b’ denote statistical significance means for columns and ‘A, B’ denote statistical significance means for rows according to paired sample ‘t’ test (two-tailed) at 5% level of significance (95% confidence interval).

Table 9: Mean absolute body weight (g) of male Wistar rats in Evaluation of Burn wound healing potential

Group	0^th day	1^st week	2^nd week	3^rd week
I-Normal control	206.16±5.49	216.66±5.50	226.16±4.49	238.16±5.07
II-Negative control	202.67±2.34^ns	197.66±2.25^*	211.66±5.16^*	229.66±5.53^*
III- Vehicle control	202.66±2.66^ns	213.66±3.14^ns	223.66±2.73^ns	233.66±3.14^ns
IV-Positive control	202.16±1.94^ns	216.33±3.61^ns	225.0±3.34^ns	236.16±4.30^ns
V-Test group	202.0±2.45^ns	210.5±2.16^ns	222.16±3.18^ns	233.33±1.96^ns
p value	0.183	<0.001	<0.001	0.022

Statistical analysis was done by using one way ANOVA followed by Dunnet (2-sided) post hoc test. ^nsNo significant difference compared with the control group (p>0.05), ^*significant difference compared with the control group (p<0.05).

Group	1^st week	2^nd week	3^rd week
I-Normal control	5.09±0.62	9.72±1.66	15.54±1.91
II-Negative control	-2.46±0.82^*	4.43±2.00^*	13.32±2.61^ns
III- Vehicle control	5.43±0.83^ns	10.37±1.47^ns	15.31±2.09^ns
IV-Positive control	7.01±2.18^ns	11.30±2.03^ns	16.82±2.35^ns
V-Test group	4.21±0.89^ns	9.98±0.62^ns	15.52±1.62^ns
p value	<0.001	<0.001	0.117

Group	1^st week	2^nd week	3^rd week
I-Normal control	6.35±0.56	6.22±0.51	6.07±0.43
II-Negative control	7.04±0.52^ns	4.52±0.34^*	4.81±0.16^*
III- Vehicle control	6.58±0.46^ns	6.38±0.43^ns	6.28±0.41^ns
IV-Positive control	6.72±0.46^ns	6.58±0.45^ns	6.39±0.43^ns
V-Test group	7.05±2.16^ns	6.82±0.14^ns	6.49±0.22^ns
p value	0.062	<0.001	0.003

Group	1^st week	2^nd week	3^rd week
I-Normal control	12.86±0.73	13.06±0.65	14.10±0.58
II-Negative control	13.37±1.40^ns	10.47±1.72^ns	11.50± 0.84^*
III- Vehicle control	12.04±1.62^ns	12.13±0.69^ns	12.12± 0.46^*
IV-Positive control	12.88±1.48^ns	12.59±0.94^ns	12.70± 0.49^ns
V-Test group	14.36±1.15^ns	13.71±0.51^ns	13.37± 0.66^ns
p value	0.069	0.003	<0.001

Group	Day 1	Day 3	Day 7	Day 14	Day 21
I-Normal control	-	-	-	-	-
II-Negative control	0	1.11± 1.72	4.44± 1.72	7.22± 2.50	8.89± 2.71
III- Vehicle control	0	7.77± 3.44^*	12.77±3.27^*	18.33±1.82^*	30.55±2.50^*
IV-Positive control	0	17.77±2.72^*	22.22±3.44^*	39.44±`4.9^*	62.22±5.44^*
V-Test group	0	11.11±3.44^*	16.38±2.67^*	23.88±3.89^*	50.0± 2.97^*
p value		<0.001	<0.001	<0.001	<0.001

MATERIALS AND METHODS:

Study Material:

Chemicals:

Study Methods:

Experimental Animals:

Evaluation of Acute Dermal Toxicity

Evaluation of burn wound healing activity:

Histological and histochemical techniques:

Statistical analysis:

RESULTS:

Clinical Signs and Mortality:

Body Weight:

Feed Consumption:

Water Consumption:

Evaluation of Burn wound healing potential:

Absolute body weight:

Relative body weight:

Feed Consumption:

Water Consumption:

Percentage burn wound contraction:

Period of epithelization:

DISCUSSION:

Table 2: Showing Group description

Table 9: Mean absolute body weight (g) of male Wistar rats in Evaluation of Burn wound healing potential

Table 10: Relative body weight (%) of male Wistar rats in Evaluation of Burn wound healing potential

Table 11: Mean feed consumption (gm/100gm body weight/day) of male Wistar rats in Evaluation of Burn wound healing potential

Table 12: Mean water consumption (ml/100g body weight/day) of male Wistar rats in Evaluation of Burn wound healing potential

Group	Period of epithelization in days (Mean±SD; N=6)
I-Normal control	-
II-Negative control	19.0±0.89
III- Vehicle control	18.66±0.81^ns
IV-Positive control	14.83±0.75*
V-Test group	16.0±0.89*
p value	<0.001