INTRODUCTION:

Several studies have shown that herbs with various pharmacological activity and potencies which when combined in a certain ratio might generate more promising results than individual herbs. Hence it was felt to develop plant bioactive based polyherbal topical formulation for non-healing burn wound. It can prevent inflammation and microbial infection in a synergistic fashion, and expedite the skin tissue remodeling process by acting against inflammation and different bacteria that cause wound complication¹. Non healing burn wound are especially difficult for the medical system and geriatric patients deal with².

Leg ulcers and pressure ulcers (diabetic ulcer, venous ulcers, ischemic ulcers) are the most frequent non healing wounds in western countries, accounting for approximately 70-80 percent of all such wounds. Many of these wounds occur in the geriatric patients and become non healing or chronic, requiring the patients to live with them for the rest of their life or perhaps die from them. The underlying contributing issue specific to the aged patient are not given adequate attention. Those factors are pathologic situation (diabetic disease, ischemia ofleg) and physiologic (atherosclerosis, aging skin, immune state)³. Alsoburns wound injuries to tissue caused by excessive heat, radiation, electricity and corrosive substances, which degrade protein in the exposed cells⁴. In burn wound healing process include re-epithelialization, inflammation, neovascularization granulation and wound contraction. Furthermore, in the first 24 hours after burn, open wounds particularly prone to bacterial infection such as Staphylococcus aureus or Pseudomonas aeruginosa which are primary cause of delayed healing⁵. Therefore, topical antimicrobial therapy is most critical aspect of burn wound care.

Literature survey revealed that almost every part of neemtree, Azadirachta indica (Maliaceae), has been used as traditional medicine to treat a wide range of human ailment⁶. The main chemical constituent is Nimbidin which show potent antibacterial, antipyretic, spermicidal, diuretic, anti-inflammatory activity. Nimbolide, Mahmoodin, gallic acid and catechin show potent antibacterial activity also it shows wound healing activity^7,8. Myrica esculenta belongs to family myricaceae and commonly known as katphala. As per ayurvedic literature stem bark mainly contains chemical constituents like myricanol, myricanone, castalagin, gallicacid, quarcetin, catechine, epigallocatechin, triterpene diol and β-sitosterol. Also reported that the bioactive of Myrica esculenta bark exhibited significant pharmacological activities like antimicrobial, anti-inflammatory, anti-allergic, antibacterial, wound healing, anixolytic, radical scavenging and antioxidant^9,10. Melaleucaalternifolia also commonly called tea tree oil or melaleucaoilbelongs to family myrtaceae¹¹. Many report describing tea tree oil show broad spectrum of antibacterial (terpinen-4-ol, a well-studied antibacterial component in TTO)¹², anti-inflammatory and anti-oxidant all help to wound healing ability^13,14. Also showantifungal¹⁵, antiseptic¹⁵, anti-acne ¹⁶, disinfectant activities¹⁵. The aim of this study was to develop topical emulsifying polyherbal ointment formulation containing mixture of tea tree oil, neem, katphalain AN specific ratio to investigate and explore the antimicrobial activity and wound healing activity.

MATERIALS AND METHODS:

Materials:

Analytical grade chemicals were used for the study. The white Vaseline, white wax, ethanol purchased from Research Lab Fine Chem Industry, Mumbai. Stearic acid from Thomas Baker Chemicals Pvt, limited Mumbai. Propylene glycol was obtained from Analab Fine Chemicals, Mumbai. Triethanolamine from Molychem, Mumbai. Tea tree oil was purchased from local market of Pune, India. The solid media and broth used for microbial culture were procured from Sisco Research Laboratory Pvt. Limited, Mumbai, India.

Plant material: The leaves of Neem and bark of Katphala were collected from the local area of Pune, Maharashtra.

Methods:

Method of extraction:

The leaves of neem and the bark of katphala were washed with double distilled water and shade dried for 11-12 days at room temperature. About 100g coarsely powder of neem leaves and katphala bark were subjected separately to solvent extraction with 250ml of ethanol using Soxhlet extractor apparatus for 24hours. Both extracts were collected separately and solvent was evaporated under reduced pressure using the rotary evaporator and stored separately in airtight container at 2 to 8°Cuntil use^17,18.

Formulation of poly-herbal ointment:

The emulsifying ointment base was prepared by fusion method. In this method ingredient i.e. stearic acid, white wax and white Vaseline were weigh accurately and allowed to melt at 70°C in water bath. After melting, the remaining ingredient were added continuous by maintaining temperature at 70°C and allowed cool to room temperature¹⁹. In prepared emulsifying base 10% w/w plant extracts with ratio (1:1:1) were incorporated with continuous trituration using mortar and pestle. The prepared ointments filled into the container and stored at room temperature.

Table 1: Formulation of polyherbal ointment

Sr. No	Ingredient	Quantity
1	Azadirachta indica (Leaves extract)	3.3%
2	Myricaesculenta (Bark extract)	3.3 %
3	Tea tree oil	3.3 %
4	Stearic acid	15.1%
5	White vaseline	1.9 %
6	White wax	8.2 %
7	Triethanolamine	1 %
8	Propylene glycol	8.1%
9	Purified water	q.s to 100 %

Evaluation of formulated ointment:

Appearance:

Organoleptic properties like nature, odor, color, consistency, phase separation and homogeneity of the formulation was examined by visual examination.

pH:

The pH of ointment was determined by usinga pre-calibrated digital pH meter (Auto Digital pH-meter LB-901). One gram of ointment dissolved in 100 ml distilled water and set aside for 2hours. pH of sample solution was measured in triplicate and the average value was taken¹⁹.

Spreadability:

The Spreadability of the formulation was determined using apparatus suggested by Multimer which was used for the study with some modification²⁰. The apparatus consists of a wooden block with a pulley attached to one end. A ground glass plate was fixed on the block. About three grams of polyherbal ointment placed on the ground plate, and the ointment was sandwiched between these plates and another glass plate with the same dimensions as the fixed ground plate. One kg of weight was placed on top of these two plates for five minutes to remove air and ensure that the ointment was evenly distributed across the plates. Scrapped off the excess ointment around the edges. The spread capacity expressed in terms of time in second taken by two glass slide slip off from the ointment, when sandwiched in between two slides under the direction of certain load. The less time it takes to separate two slides, the better the ointment’s spreadability. Spreadability was calculated using the formula

S=ML/T ²¹

Where,

S= Spreadability

M= Weight tied to the upper slide

L= Length of glass slides.

T= Time taken to separate the slides.

Viscosity:

The viscosity of produced ointment was determined using a Brookfield Viscometer (Brookfield DV II+ Pro) and viscosity was measured in centipoise^22,19.

Extrudability:

It is the common and simplest method which is generally used to obtain the force required to expel the substances from the tube. The approach employed in the study for evaluating ointment formulation for extrudability was based on the amount of ointment in % and after the single application of pressure the ointment from the tube get extrude. More the amount of ointment extruded better the extrudability¹⁹. The ointment was placed in the collapsible tube container. The extrudability was measured in amount of ointment needed to extrude 0.5cm ribbon of ointment in 10 seconds²¹.

Antimicrobial activity:

Antimicrobial activity study of polyherbal ointment was conducted in Microbiology laboratory, Modern College of Pharmacy, Nigdi, Pune. Antimicrobial screening of ointment formulation against gram positive strains Bacillussubtilis, Staphylococcus aureus and gram negative strain Pseudomonas aeruginosa by agar well diffusion method. Antibacterial Povidone iodine ointment (10% w/w) was used as a standard. Selected bacterial strains were initially cultured in nutrient agar medium under shaking condition for 24hours at 37°C and then 100ul of the test inoculums were spread evenly on nutrient agar plates by using sterilized glass spreader and allow to dry at 37°C in the incubator (Bio-Technics India, BTI-25). Then the required wells were punched by using sterile 6mm cork borer in seeded nutrient agar medium plates. Standard ointment and prepared polyherbal ointment 0.2gm was added immediately in to the wells and then plates kept at 10-11°C as a period of pre diffusion for 30 minutes. After get normalized to room temperature the plates were incubated at 37°C for 24hours. After completing incubation period, the antimicrobial activity was determined by measuring zone of inhibition(i.e. observed for bacterial growth inhibition, which was indicated by a clear zone surrounding the wells) by using antibiotic zone reader and expressed in terms of average diameter of zone of inhibition in millimeters^23,24.

Wound healing activity:

Experimental animals and grouping:

The 16-male Wistar albino rats were procured from National institute of biosciences, Pune, Maharashtra. Wistar rat of weighing between 160-260gm. They were kept under controlled conditions of temperature (24°C± 2°) and humidity (about 50-75%), with a 12-hour light/dark cycle, with ad. Libitum access to standard food and water²⁵. Protocol was approved by IAEC, Modern college of pharmacy, Nigdi, Pune. (MCP/IAEC/007/2019)

Burn induction and treatment:

Reported method was used to induce burn wound in the rats²⁶. In brief, the anesthesia in rats was induced using diethyl ether. The skin on back was shaved and the burn wound was induced (250 mm²)²⁷and all the post wound care was taken as per the ethical guidelines. Then, the animals were divided into 3-groups: 1. Control group (No treatment) 2. Experimental ointment (10% w/w polyherbal ointment with herbal extract), 3. Standard ointment (10% w/w povidone iodine ointment USP). After 2-hours of burn wound induction, the thin layer of 1gm polyherbal test ointment and standard ointment was applied to the burn area once a day. Wound area was measured for contraction every fourth day of ointment application under careful observation.

Measurement of wound contraction:

The wound diameter was measured a different time interval such as on day 0, 4, 8, 12, 16 with the help of Vernier caliper, wound contraction was measured every 4^th day until the wounds were completely healed²⁵. Percentage healing of the wound area was calculated by using following formula:

First day wound area – Specific day wound area

% Wound contraction = ------------------------------- × 100

First day wound area

Epithelialization period:

Number of days required for falling of eschar without any residual raw wound was recorded as the period of epithelialization⁴.

Histopathological analysis:

On the 16th day following wounding, a specimen sample was taken from each group of rats for histological evaluation. The fixative solution was replenished every 2 days until the tissues stiffened, and skin specimens were immediately fixed in 10% (v/v) neutral-buffered formalin. Each specimen was fixed in paraffin and thin slices (3m) were cut and stained with Masson's trichrome (to detect collagen fibers) and hematoxylin and eosin (H&E) (for general morphological observations). Using a mild to intense scale (+ to +++), slides were inspected qualitatively under a light microscope for collagen formation, fibroblast proliferation, angiogenesis, epithelization, and granulation tissue creation^28,29,30.

Accelerated Stability study:

The developed ointment formulations were subjected to stability study as per the International Conference on Harmonization (ICH) guidelines. The formulated ointment was filled in the collapsible tubes and stored at different temperatures and humidity conditions, namely, 25°C±2°C/60%±5% RH, 30°C±2°C/65%±5% RH, and 40°C±2°C /75%±5% RH for a period of 3 months and studied for appearance, pH, viscosity, and spreadability ^17,18.

RESULT AND DISCUSSION:

Evaluation of polyherbal ointment:

The present study was done to prepare and evaluate polyherbal ointment formulation. The herbal extracts were obtained from soxhlet extraction method, with no adverse effects on the chemical contents or their activity. The ointment preparation was done by using fusion method so that consistent mixing of the herbal extract with ointment base which was stable throughout storage. pH, viscosity, spreadability, extrudability and other physiochemical properties were studied which show satisfactory result (Table 2).

Table 2: Physiochemical evaluation of formulated ointment

Sr. No.	Observation parameter	Observation
1	Nature	Semisolid
2	Odor	Characteristic
3	Color	Light brown
4	Consistency	Smooth (free from lumps)
5	Phase separation	No
6	Homogeneity	Homogeneous
7	pH	6.7±0.1
8	Spreadability (sec)	10±0.2
9	Viscosity (centipoise)	12450±0.3
10	Extrudability (g)	0.27±0.01
11	Accelerated Stability study (4°C, 25°C and 37°C)	Stable

Antimicrobial activity:

The result of the antimicrobial activity of the prepared ointment and standard povidone ointment against the test organism are shown in table 3 And figure 1 respectively. From result obtained it was observed that 10% w/w polyherbal ointment preparation showed more zone of inhibition, better activity against selected strain than standard povidone ointment.

(a) (b)

(c) (d)

(e) (f)

Figure 1: Antimicrobial activity of polyherbal ointment formulation against (a) Pseudomonas aeruginosa, (b) Staphylococcus aureus, (c) Bacillus subtilis and standard povidone iodine ointment against (d) Staphylococcus aureus, (e) Bacillus subtilis, (f) Pseudomonas aeruginosa.

Table 3: Zone of inhibition shown by polyherbal ointment formulation on bacterial strain.

Micro-organism	Zone of inhibition (mm)
Micro-organism	Formulation	Standard
Bacillus subtilis	21.5±0.1	14.1±0.3
Staphylococcus aureus	23.3±0.2	12.7±0.4
Pseudomonas aeruginosa	19.7±0.1	15.4±0.3

In vivo burn wound healing activity:

Wound contraction:

In control group, the wound area on day-1 was found to be 248±2.0mm². On day-4, the wound area was found to be 219±1.01mm², and the percent wound contraction on day-4 was found to be 11.69±2.2%. On day-8, the wound area was found to be 177±1.3mm², and the percentage wound contraction was found to be 28.62±2.4%. On day-12, the area of wound was measured as 131±2.1mm², and the percentage wound contraction was found to be 47.17±3.2%. On day-16, the wound area in was found to be 110±1.1mm², and the percent wound contraction was found to be 55.64±3.1%.

In polyherbal ointment treated group, the wound area on day-1 was found to be 250±1.0mm². On day-4, the wound area was found to be 192±2.0mm², and the percentage wound contraction on day-4 was found to be 23.2±1.2%. On day-8, the wound area was found to be 135±1.2mm² and the percentage wound contraction was found to be 46.0±2.3%. On day-12, the area of wound was measured as 86±0.11mm², and the percentage wound contraction was found to be 65.6±4.2%. On day-16, the wound area was found to be 0.10±1.0mm², and the percentage wound contraction was found to be 99.96±1.0%.

In standard povidoneiodine ointment treated group, the wound area on day-1 was found to be 251±1.0mm². On day-4, the wound area was found to be 183±2.1mm², and the percentage wound contraction on day-4 was found to be 27.0±2.5. On day-8, the wound area was found to be 119±1.2mm² and the percentage wound contraction was found to be 52.58±2.6%. On day-12, the area of wound was measured as 69±2.1mm², and the percentage wound contraction was found to be 72.50±1.2%. On day-16, the wound area in was found to be 0.99±1.0mm², and the percentage wound contraction was found to be 99.60±1.1%.

The observations revealed that the wounds in group-2 (10% w/w polyherbal ointment) formulation and with group 3 (10% w/w standard povidone iodine ointment) healed considerably faster during the 16-days study when compared with control (left untreated) group. Test ointment treatment group showed maximum wound contraction as evidenced by the wound contraction observations. Maximum wound closure rate percent was observed in 10% w/w polyherbal ointment treated group from 250± 0mm² on (day-1) to 0.10±0.0mm² on (day-16) and in the standard 10 % w/w povidone iodine ointment treated group, the wound contraction was observed from 249±0 mm² (day-1) to 0.99±0.87mm² (day-16) respectively. Control group rats (left untreated) revealed very slow wound contraction rate with 110± 0.18mm2 on the 16^th day. Thus it can be said that wound contraction rate of 10% w/w polyherbal ointment showed greater wound contraction when compared with control group.

Epithelialization period:

Average number of days required for rats treated with 10% w/w polyhedral ointment to shed eschar without leaving any residual raw wound (epithelialization period) was the shortest i.e. 14.01±0.12 day followed by standard povidone iodine ointment i.e 15.99±0.11 day. Control (left untreated) rats had the longest epithelialization durationi.e 23.2±0.14 day.

Histopathological examination results:

Figure 3 shows the histological characteristics of the healed wounds on day-16 after the induction of wound. The povidone ointment-treated group showed more tissue regeneration, as evidenced by full epithelization, much higher collagen deposition, and the appearance of granulation tissues. The tissue from the control group that got only a basic ointment, on the other hand, showed disordered fibroblasts, reduced collagen fiber deposition, and angiogenesis. Less scar formation, increased fibroblast proliferation, newly created blood capillaries (angiogenesis), and re-epithelialization were observed in wounds treated with herbal ointment. The table 4 below shows the scoring of the distinctive histological findings of the experimental animals' healed wounds.

Accelerated stability study:

The preparedpolyherbal ointment formulations were subjected to stability study as per International Council on Harmonization guidelines. Throughout the stability studies, the appearance of formulations was clear and no significant variation in appearance, pH, spreadability and viscosity for optimized formulation for the period of 3 months.

CONCLUSION:

The present investigation develops and evaluate a topical polyherbal ointment for antimicrobial and wound healing activity using burn wound model, which included the observation of different physical, histological activities. The healing effects appeared due to promoting faster collagen deposition, the development of other connective tissue constituents, and antibacterial activity.

ACKNOWLEDGEMENTS:

The authors would like to thank PES Modern College of Pharmacy, Nigdi, Pune, Maharashtra. for supporting this research.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 07.06.2022 Modified on 19.11.2022

Research J. Pharm. and Tech 2023; 16(10):4610-4616.

DOI: 10.52711/0974-360X.2023.00751

Day 1	Control	Formulation	Standard
Day 1
Day 4
Day 8
Day 12
Day 16

Sr. no.	Observations	Before stability study	After stability study at different temperature and humidity conditions 25°C±2°C/60%±5% RH, 30°C±2°C/65%±5% RH, and 40°C±2°C /75%±5% RH
Sr. no.	Observations	Before stability study	One months	Two months	Three months
1.	pH	6.7±0.1	6.69±0.11	6.72±0.2	6.61±0.1
2.	Spreadability (sec)	10±0.2	9.9±0.34	9.7±0.1	10.1±0.2
3.	Viscosity (centipoises)	12450±0.3	12340±0.1	12410±0.4	12480±0.3
4.	Extrudability (gm)	0.27±0.01	0.28±0.1	0.26±0.03	0.27±0.5


(a) Simple ointment base	(b) Standard Treatment Povidone iodine ointment (10%)	(C) Polyerbal ointment formulation (10%)

Treatments	Collagen formation	Fibroblast proliferation	Angiogenesis	Granulation tissue	Re-epithelization
Simple Ointment	+	+	++	+	+
Herbal Formulation (10% w/w)	+++	+++	++	+++	+++
Standard Povidone Ointment(10% w/w)	+++	+++	+++	+++	+++