1. INTRODUCTION:

Skin, being the largest organ, protects the body from the external aggressors. Injury to the epidermal layer of skin leads to disruption of integrity of the skin and ultimately results in formation of wound. A number of factors are responsible for the formation of wound such as mechanical abrasions, insect or animal bites, trauma etc. ¹. Invasion of various pathogenic microorganisms at the injured tissue results in severe infection. Wound infection implies the multiplication of microorganisms resulting in a prolonged (and excessive) inflammatory response, a delay in collagen synthesis, retarded epithelialisation and finally in tissue damage². Worldwide it is estimated that at least six million people suffer from chronic wound infection every year³.

Wound healing is a natural phenomenon by which body restores the cellular and functional integrity of tissue⁴.

Appropriate wound healing is necessary to regain the functional and anatomical status of the damaged tissue that got disturbed due to wound. Complex biochemical events involved in the wound healing process are divided into three phases: inflammatory, proliferative and remodelling phase⁵. Various endogenous and exogenous factors such as microbial infection on wound, poor blood supply to the wound area and nutritional deficiency such as vitamin C and zinc results in delay of healing of wound⁶. On the basis of mechanism and duration of healing, wound can be classified as acute and chronic⁷. Acute wound heals in timely manner by the events involved in the healing of wound while tissue affected by chronic wound gets healed after a prolonged period of time. Cellular functioning and regulation are altered in chronic wound infections due to excessive production of matrix molecules like fibrinogen and fibrin. These matrix molecules results in scavenging of various growth factors and other molecules which play an essential role in healing of wound; as a result, infection in the chronic wound is more severe condition than acute wound. Other than this, wound can also be classified on the basis of layer of skin that gets damaged: superficial wound, partial thickness wound and full thickness wound⁸.

Currently a wide range of topical antibiotics, antifungal and antiseptic drugs are used for the treatment of wound infections. A number of cleansing solutions and gels are also used for rinsing and cleaning of wound such as sterile normal saline, sterile water, povidone-iodine, poly-hexane biguanide and sodium hypochlorite⁹. Marketed antimicrobial drugs used for the treatment of wound infections are associated with various side effects like itching, redness of skin and blister formation. Resistant cases with the use of topical antibiotics and antifungal are also rising nowadays¹⁰. In addition to conventional approaches, new effective techniques such as tissue growth engineering, recombinant growth factors and silver dressings are also used, nowadays, for the treatment of wound infections. But all these approaches are expensive and recommended for developed countries only¹¹.

Medicinal plants such as Neem, Aloe vera and Tulsi are rich source of alkaloids and flavonoids; and are reported to enhance the wound healing process. Combination of medicinal plants in one formulation often gives more promising pharmacological and therapeutic effects in comparison to formulation containing single medicinal plant. Formulations composed of several medicinal plants extract is known as polyherbal formulation (PHF). Due to their effective medicinal and therapeutic properties, such formulations are being used all over the world. The need of the hour is to evaluate the mechanism of action and efficacy of these PHF by using various scientific methods¹². The present study is proposed to evaluate the wound healing potential of a Septaheal, a PHF containing Till oil, Coconut oil, Karanj Beej oil, Jafi, Patol patra, Glycyrrhiza Glabra, Daru Haridra, Saariva satiava, and Terminalia chebula.

2. MATERIAL AND METHODS:

2.1. Formulation:

Polyherbal formulation (Septaheal) was received as a gift sample from Shree Dhanvantri herbals, Jalandhar, Punjab, India.

2.2. Animals:

Male Wister albino rats of weight between 150-250gm were used in this study. Animals were kept under standard laboratory conditions maintained at 25±2℃ at 12h day: night circle and were given free access to food chow and water ad libitum. The ethical clearance for experimental study was provided by Institutional Animal Ethics Committee, Lovely Professional University (NCP/IAEC/CL/247/2017-18) and the tests were performed as per guidelines given by Committee for the Purpose of Control and Supervision of Experiments on Animals. Excision wound, incision wound and burn wound models were used to evaluate the wound healing potential of the PHF as per the following study design:

3. Evaluation of wound healing potential:

3.1. Excision wound model:

Animals were anesthetized by diethyl ether and hairs were removed from the dorsal thoracic region. Then a cutaneous circular wound of 3mm diameter was inflicted on the pre-shaved area of rat skin with a sterile surgical blade. Wound area was measured on day 1, followed by measurement on every 4th day till 12^th day. Percentage wound contraction was calculated regularly as per formula given in equation (1). Time for complete epithelization of tissue was also measured¹³.

Healed area

% wound contraction = –––––––––––––––––––– × 100

Total wound area

(1)

Healed area = Original wound area – Present wound area

3.2. Incision wound model:

After anesthesing the animals, 1.5cm long incision was made at the dorsal portion of rat skin with the help of sharp scalpel. After complete homeostasis, the wound was stitched with the help of black silk surgical thread (number 000) and a curved needle (number 11). After stitching, the wound was kept undressed for 10 days. On the 10^th day, all rats were anesthetized and sutures were removed and tensile strength of cured wound skin was measured¹⁴.

3.3. Burn wound model:

A cylindrical stainless steel rod of 1cm diameter and 100 g weight was used for the infliction of burns. The rod was immersed in a flask of boiling water and heated up to 100℃. Wound was induced on the dorsal thoracic region of animal with the rod that was 1cm away from the vertebral and 5 cm away from the ear. Wound area was measured on first day followed by measurement on every 2^nd day to till 20^nd day and % wound contraction was measured as per formula given in equation 1. Epithelization period of wound was also calculated out ¹⁵. Skin specimens from treated rats were collected in 10% buffered formalin and analysed for keratinisation and scar formation.

3.4. Histopathological evaluation:

On the terminal day, histopathological analysis of the tissue was done to evaluate the extent of wound healing.

3.5. Statistical analysis:

Statistical analysis was performed using the Sigma stat software. Data were expressed as mean ± standard error of mean (SEM) or standard deviation (SD). Statistical variations were determined using one-way analysis of variance (ANOVA). Any value of p<0.05 was considered to be significant, p<0.01 very significant and p<0.001 as extremely significant.

4. RESULTS AND DISCUSSION:

4.1. Incision Wound model:

The wound healing potential of PHF was evaluated by the measurement of tensile strength which indicates the resistance to break under tension. The incision wound model reside on the formation of the granuloma tissue i.e. increase in the synthesis of collagen which provides more stability to the intra and intermolecular crosslinks. The parametric data were expressed as mean+SEM. The control group rats showed tensile strength of 4.173+0.281 on 10^thday while rats of treatment and standard group exhibit tensile strength as 9.3+0.074 and 7.86+0.034 respectively. The excellent wound healing ability of the PHF is proved by means of two-fold rise in the tensile strength in animals of the treated group when compared to the control group.

Table 2: Effect of Polyherbal formulation on tensile strength in incision wound model

Group	Drug	Tensile strength (gm)
Group I	Control	4.173 ± 0.281
Group II	Povidone iodine	7.86 ± 0.0348***
Group III	PHF	9.33 ± 0.74***

Values are expressed as mean ± SEM. (n=6), Data were analysed by one-way ANOVA followed by Dunnett’s test. *P<0.05 vs control; **P<0.01 vs control, ***P<0.001 vs control

4.2. Excision wound model:

A significant increase in percentage wound contraction was observed in animals treated with PHF as compared to that of control group. Percentage increase in contraction of wounded tissue observed was high in animals of the standard group than the treatment group on 2^ndand 4^thday but on 8^th to 16^th days, it was high in animals of treatment group. Period of re-epithelization was found to be 16 days in animals treated with PHF which was almost same as that of standard group (Figure 1). Figure 2 depicts the digital images of wounds of standard and treatment groups on different days.

Figure 1: % increase in wound contraction observed on post-wounding days in excision wound model

Figure 2: Digital images of wounds of standard and treatment groups on different days in excision wound model. A-E: Images of standard group; F-J: Images of Polyherbal formulation treated group. Images of day 1 (A,F), 4 (B,G), 8 (C,H), 12 (D,I) and 20 (E,J).

4.3. Determination of the hydroxyproline and hexosamine content:

Hexosamine and hydroxyproline are important constituents of the extracellular matrix and major markers to detect the wound healing activity. A significant increase in levels of hydroxyproline and hexosamine in PHF treated group as control group on 10^th day indicated the efficacy of formulation in tissue injury. The absolute values of hydroxyproline and hexosamine in all the groups have been mentioned in table 4.

Table 4: Effect of polyherbal formulation on levels of hydroxyproline and hexosamine

Group	Drug	Hydroxyproline content (mg/g)	Hexosamine content (mg/g)
Group I	Control	12.4±1.8	20.3±62.1
Group II	Povidone iodine	36.8±1.2**	35± 4.5***
Group III	PHF	41.2±1.7***	42.4±1.5***

Values are expressed as mean ± SEM. (n=6). Data were analysed by one-way ANOVA followed by Dunnett’s test. *P<0.05 vs control; **P<0.01 vs control, ***P<0.001 vs control

4.4. Exudate analysis:

Large amount of exudate rich in blood was observed in animals of all the groups on 1^st day. Types of exudates were compared in animals of the control group, standard group and treated group respectively on different days. The watery pink colour discharge was observed in both standard and treatment group till 8^th day while in control group, it was observed till 18^th day. Complete drying of the injured tissue was observed on 14^thday in animals of the treatment group which was much early in comparison to standard group (on 16^th day) and control group (20^th day).

4.5. Burn wound model:

A significant increase in % contraction of injured tissue was observed in animals of the treatment group as compared to control group animals (Fig. 3). Period of re-epithelization was found to be 14 days in animals treated with PHF, while it was 18 days in standard group.

Figure 3: % increase in wound contraction observed on post-wounding days in burn wound model.

4.6. Histopathological evaluation:

Results of histopathological study provide evidence of wound healing as the generation of epidermal skin and collagen fibres were observed in rats of treatment and standard groups. However, formation of new epidermal skin and deposition of collagen tissue was better in treatment group than the standard group. In control group, new generation of epidermal skin was not observed; slight deposition of connective tissue was observed on 21^thday. Hence, the results of histopathology is in accordance to other parameters observed.

Figure 4: Histopathological analysis of tissue in burn wound model.

A: Standard; B: Treatment; C: Control.

5. DISCUSSION:

A wound is defined as the disruption of protective epidermal layer of skin. Physiological and biochemical processes involved in healing of injured tissue can distinctively be divided into three phases- inflammation, granulation and remodelling phase. Appropriate wound healing depends upon the severity of skin damage, immunological status of patient and self-ability of tissue to repair the damage¹⁶. Contraction of wounds occurs by the movement of myofibroblasts from the healthy skin to damage tissue that occur throughout the wound healing process and results in the formation of healed scar tissue. Natural agents potentiate collagen deposition, decrease the microbial load on wounded tissue, fasten the re-epithelization, and decrease the oxidative stress produced by cytotoxic free radicals¹⁷.

Treatment with PHF significantly potentiated the wound healing process due to its angiogenic and mitogenic potential. The newly generated tissue synthesizes collagen which modulates the homeostasis and re-epithelization at the latter phase of wound healing process. Biochemical analysis provided the evidence of marked increase in levels of hydroxyproline and hexosamine content in animals treated with PHF in comparison to standard group which is reflection of increase in the synthesis of collagen. Mean tensile strength was also high in animals treated with PHF than the animals of the standard group and significantly high than the control group. In the excision wound models, high % rate of contraction in the injured tissue was observed in animals treated with PHF. Mean time for the complete re-epithelization in the treatment group was almost comparable to the standard group and significantly less than that of control group. Amount of exudates and nature of exudates observed on various days also provide marked evidence about the wound healing potential of PHF. Pronounced effect of PHF on wound healing may be attributed to the anti-oxidant and anti-microbial activities of herbal drugs present in it.

6. CONCLUSION:

The present study confirms significant wound healing activity of the PHF (Septaheal). High % wound contraction, rise in the level of hexosamine and hydroxyproline, increased tensile strength, mild to moderate antimicrobial and antioxidant activities demonstrate the potent wound healing of activity of PHF. The exact active principals and mechanisms still remains to be investigated.

7.ACKNOWLEDGMENT:

Authors are thankful to second International Conference of Pharmacy, held at School of Pharmaceutical Sciences, Lovely Professional University on September 13-14, 2019 to fund the publication of this manuscript.

8. CONFLICT OF INTEREST:

Nil.

9. REFERENCES:

1. Shevchenko RV et al. A novel skin substitute biomaterial to treat full-thickness wounds in a burns emergency care. In Biodefence, Edited by Mikhalovsky S and Khajibaev A. Springer Dordrecht. 2011; pp. 247-255.

2. Rondas AA et al. Prevalence of chronic wounds and structural quality indicators of chronic wound care in Dutch nursing homes. International Wound Journal. 2015; 12(6): 630-635.

3. Kumar K et al. Ethnotherapeutics of Bryophyte plagiochasmaappendiculatum among the Gaddi tribes of Kangra valley, Himachal Pradesh, India. Pharmaceutical Biology. 2000; 38(5): 353-356.

4. Lazarus GS et al. Definitions and guidelines for assessment of wounds and evaluation of healing. Archives of Dermatology. 1994; 130(4): 489-493.

5. Thomas CH. Checklist for factors affecting wound healing. Advances in Skin and Wound Care. 2011; 24(4): 192.

6. Bowler PG. Wound microbiology and associated approaches to wound management. Clinical Microbiology Reviews. 2001; 14(2): 244-269.

7. Leach MJ. Making sense of the venous leg ulcer debate: a literature review. Journal of Wound Care. 2004; 13(2): 52-56.

8. Dabiri G et al. Choosing a wound dressing based on common wound characteristics. Advances in Wound Care. 2016; 5(1): 32-41.

9. Lipsky BA and Hoey C. Topical antimicrobial therapy for treating chronic wounds. Clinical Infectious Diseases. 2009; 49(10): 1541-1549.

10. Church D et al. Burn wound infections. Clinical Microbiology Reviews. 2006; 19(2): 403-434.

11. Thakur R et al. Practices in wound healing studies of plants. Evidence-Based Complementary and Alternative Medicine. 2011; 2011: 438056.

12. Aslam MS et al. Antioxidant and wound healing activity of polyherbal fractions of Clinacanthus nutans and Elephantopus scaber. Evidence-based Complementary and Alternative Medicine. 2016; 2016: 4685246.

13. Hasan A et al. Bovine reticulum derived extracellular matrix (b-REM) for reconstruction of full thickness skin wounds in rats. Wound Medicine. 2016; 12: 19-31.

14. Garg VK and Paliwal SK. Wound-healing activity of ethanolic and aqueous extracts of Ficus benghalensis. Journal of Advanced Pharmaceutical Technology & Research. 2011; 2(2): 110-114.

15. Cai EZ et al. Creation of consistent burn wounds: a rat model. Archives of Plastic Surgery. 2014; 41(4): 317-324.

16. Velnar T et al. The wound healing process: an overview of the cellular and molecular mechanisms. Journal of International Medical Research. 2009; 37(5) 1528-1542.

17. Boyapati Land Wang HL. The role of stress in periodontal disease and wound healing. Periodontology. 2000; 44(1): 195-210.

Received on 26.11.2019 Modified on 29.03.2020

Research J. Pharm. and Tech. 2021; 14(4):2195-2199.

DOI: 10.52711/0974-360X.2021.00389

Group	Control (n=6)	Standard (n=6)	PHF (n=6)
Incision Wound	Untreated or Diseased group	50 mg of povidone iodine ointment applied twice in a day	1 mL of sufficient PHF applied twice in a day topically to cover wound area
Excision Wound	Untreated or Diseased group	50 mg of povidone-iodine ointment applied twice in a day	1 mL of sufficient PHF applied twice in a day topically to cover the wound area
Burn Wound	Untreated or Diseased group	50 mg of Silver nitrate ointment applied twice in a day	1 mL of sufficient PHF applied twice in a day topically to cover the wound area

Groups	Drug	Wound area (mm²)
Groups	Drug	Day 0	Day 4	Day 8	Day 12
Group I	Control	2.97±0.32	2.82±0.2	2.41±0.89	1.68±0.49
Group II	Povidone iodine	2.95±0.15	2.46±0.27***	1.97±0.36***	1.15±2.21***
Group III	PHF	2.97±0.34	2.55±0.69***	1.83±0.89***	0.85±2.45***