INTRODUCTION:

Healing of Wound is a dynamic, complex, process. It consists of series of cellular and biochemical events that leads to re-establishment of anatomical continuity. Wounds heal without complications. But various factors may affect healing of wound. They may interfere with one of the phases of wound healing, which leads to impaired wound healing. In turn that is responsible for chronicity of wound eg. chronic anal fissure, diabetic foot wound, venous ulcers.. To prevent chronicity of wound and to improve wound healing, there is need of continued research on wound healing in human as well as animals.

Anal fissure is one of the painful, chronic, non-healing wound. It’s chronicity is due to spasm of anal sphincter and local ischemia.^1,2Commonly anal fissure is treated surgically by sphincterotomy to eliminate sphincter spasm. Glyceryl trinitrate(GTN) is used as non-surgical alternative treatment due to its spasmolytic action.^3,4

GTN and calcium channel blockers are also used to promote wound healing.^5,6,7,8. Diltiazem, a calcium channel blocker, and GTN are used in chronic anal fissure and compared for their therapeutic effect.⁹ The prohealing effect of GTN and Diltiazem are due to smooth muscle relaxation which results in mitigation of sphincter spasm and local ischemia.^10,11As wound healing is a complex phenomenon, there is a possibility that wound healing potential of GTN and Diltiazem may not be only due to smooth muscle relaxant action.

Therefore, the present study was undertaken to investigate, if GTN and Diltiazem have any wound healing property apart from their spasmolytic action.⁹ We also studied effect of ointment base of GTN and Gel base of Diltiazem to see whether wound healing is due to drug itself or potentiated by their bases.

Many ayurvedic preparations are used in the management of different types of wounds. ‘Ropan Tail’ is one of the ayurvedic preparation,, which is used traditionally to promote healing of clean wounds.

Therefore, the present study was undertaken to investigate, if GTN and Diltiazem have any wound healing property apart from their spasmolytic action and to determine wound healing potential of Ropan tail. To compare wound healing potential of GTN, Diltiazem and Ropan tail.We also studied the Ointment Base of GTN and Gel Base of Diltiazem to study whethere the wound healing effect of GTN and Diltiazem is potentiated by their bases.

AIM AND OBJECTIVES:

1) To determine the wound healing potential of Ropan tail.

2) To determine the wound healing potentials of Glyceryl trinitrate and Diltiazem.

3) To compare wound healing potentials of Glyceryl trinitrate, Diltiazem and Ropan Tail.

MATERIAL AND METHODS:

Study was carried out in 6 groups of wistar rats ( 6 rats per group) for comparison of wound healing at Dept of Pharmacology and Central Animal House, Bharati Vidyapeeth Deemed University, Medical College & Hospital Sangli.

Synopsis was approved by IAEC (Institutional Animal Ethical Committee). IAEC approval registration no – BVDUMC & H, Sangli CAH/ 2015/11 and it was conducted according to CPCSEA guidelines. Total 36 Wistar rats weighing 150-250 gm were taken. These were divided into six groups, six rats per group.

Chemicals / Drugs and surgical material used:

Glyceryl trinitrate ointment 0.2% (GTN) –Troikaa Pharmaceuticals Ltd.

Ropan tail – Shrikrishna Aushadhalaya

Diltiazem Gel -.Troikaa Pharmaceuticals Ltd.

Thiopental sodium, Spirit and Normal Saline, scissor, artery forcep ect.

Table No 1: Group of animals and Drug Treatment Schedule for excisional wound

Group No.	No. of A nimals	Drug Treatment (Topical Application)
1	6	Control (Normal Saline)
2	6	0.2% Glyceryl trinitrate ointment
3	6	GTN Ointment Base
4	6	Diltiazem 2% Gel
5	6	Diltiazem Gel Base
6	6	Ropan Tail

Methodology:

It was a double blinded study. Randomization was done by simple random method.

The animals bearing experimental wounds were divided and treated with Normal Saline, GTN, GTN Ointment Base,, Diltiazem, Diltiazem Gel base and Ropan Tail respectively.

Wounding Procedure:

The nape of neck of rat was shaved. All the rats were starved overnight with water ad libitum and on the next day, surgical intervention under general anaesthesia was done to create excisional wound.

About 500 mm² full thickness, circular, skin was excised with scalpel blade on the nape of the neck by using method of Morton and Malone.¹²

Drug Schedule:

36 rats were wounded, 6 rats in each group received Normal Saline, GTN, GTN Ointment Base, Diltiazem, Diltiazem Gel Base and Ropan Tail respectively. Day of wounding was considered as zero day. Drug application was done locally once daily from next day of wounding. Topical application was continued till wound completely healed as shown by total epithelization of the wound.

Monitoring of healing:

Excisional wounds:

Two parameters viz; contraction of wound and epithelization period were monitored. Wound was observed daily upto 5^th day and from 6^th day recording of wound size was done by planimetry and then on mm²paper every 4^th day. The degree of wound healing was calculated as percentage of wound contraction of the original wound area using the following formula-

Percentage of wound contraction = A0 -Ad/ A0 х 100

Where A0 = wound area on zero day and

Ad = wound area on corresponding day.

The mean percentage of wound contraction and standard error of mean were calculated in control(Normal Saline), GTN, GTN Ointment Base, Diltiazem, Diltiazem Gel Base and Ropan Tail treated groups. The time required for epithelization was assessed in terms of days required for total fall of eschar with no trace of wound and full covering by glistening young epithelium.

Statistical Analysis:

Statistical analysis was carried out using one way ANOVA (Analysis of varience) for significance between groups. Data was expressed as mean ± SEM. For statistical analysis Instat softwere was used.

The level of significance between individual group was detected using unpaired ‘t’ test.

For all tests, effects with a probability of P< 0.05 were considered to be significant. For statistical analysis SPSS softwere was used.

After completion of epithelization, animals were followed up by standard procedures as outlined by CPCSEA guidelines and rehabilitated.

Observations And Results:

Table No.2- Percentage of wound contraction.

Group	No of Animals	Drugs	Mean Percentage Wound Contraction ±.S.E.M.
Group	No of Animals	Drugs	6^th day.	10^th day	14^th day
1	6	Control(Normal Saline)	55.33± 6.04	74.00± 1.63	90.33± 1.82
2	6	GTN	70.67± 2.04 ^*	81.00± 1.98 *	94..6± 1.34
3	6	GTN Ointment Base	67.33± 8.04	72.33± 7.42	90.33± 1.50
4	6	Diltiazem	69.33± 1.69	82.33± 2.85 *	94.00± 0.52
5	6	Diltiazem Gel Base	46.24± 8.76	66.73± 7.03	90.33± 1.82
6	6	Ropan Tail	75.33± 4.1 *	91.33± 2.04* #@	100± 0.00

Data was expressed as Mean ± S.E.M. p < 0.05 is significant *= p < 0.05 When compared with Control # = p < 0.05 compared with GTN @ = p < 0.05 compared with Diltiazem

There was statistically significant difference in means of percentage wound contraction of Ropan Tail when compared with Controrl (NS) indicating Ropan Tail has increased percentage wound contraction than Control on 6^th day.

There was statistically significant difference in means of percentage of wound contraction of drug treated groups – GTN, Diltiazem and Ropan Tail when compared with Control, indicating GTN, Diltiazem and Ropan Tail have enhanced percentage of wound contraction than Control on 10^th day.

Ropan tail has shown statistically significant difference in means of percentage of wound contraction when compared with GTN, Diltiazem on 10^th day indicating Ropan Tail has increased percentage wound contraction GTN Ointment base, Diltiazem Gel Base did not show statistically significant difference in percentage of wound contraction compared to Control, GTN, Diltiazem on 6^th, 10^th and 14^th day.

Period Of Epithelization In Days:

Group No.	No of Animals	Drugs	Mean Period of Epithelization
1.	6	Control	19.00±0.45
2.	6	GTN	17.83± 0.31
3.	6	GTN Ointment Base	19.00±0.45
4.	6	Diltiazem	17.50±0.56
5.	6	Diltiazem Gel Base	16.00± 00
6.	6	Ropan Tail	12.50± 0.22 * # @

Data was expressed as Mean ± S.E.M. p < 0.05 is significant * = p < 0.05 compared with Control # = p < 0.05 compared with GTN @ P < 0.05 compared with Diltiazem

There was no statistically significant difference in means of period of epithelization with drug treated groups – GTN, Diltazem, GTN Ointment base, Diltiazem Gel base when compared to control indicating similar effect.

There was statistically highly significant difference in mean of period of epithelization of drug treated group – Ropan Tail when compared with Control, GTN, Diltiazem indicating Ropan Tail has reduced period of epithelization

DISCUSSION:

GTN is used to treat chronic anal fissure. It acts by release of nitric oxide (NO).NO increases concentration of Guanylyl cyclase. It increases cyclic GMP(cGMP) and brings out vasodilatation^13,14. GTN promotes wound healing by increasing blood supply and there by nourishment to the wound area. In addition, GTN activates cGMP, an intracellular intermediate, that inhibits the calcium activity.Calcium is important in modulation of cellular proliferation, modification as well as maturation of keratinocytes and fibriblasts¹⁵

GTN increases angiogenesis. Angiogenesis in wound bed is important to maintain newly formed granulation as it increases wound contraction through myofibroblast modification¹⁵

By releasing NO, GTN plays an impotant role in inflammatory process.As cytokines are important for initiation of inflammatory process, NO modulates inflammation induced oedema and it inhibits inflammatory cell infiltration into granuloma^.15. It is vital for the activity of pro-angiogenic cytokines. It is also important in the Vascular Endothelial Growth Factor (VEGF) dependent and independent angiogenesis. GTN application stimulates the production of organized collagen fibres and dampens inflammatory response by reducing number of polymorphonuclear cells.¹⁵

Keratinocyte proliferation and wound re-epithelization is NO dependent, which is mediated by VEGF. It plays an important role in collagen synthesis which is important in the proliferative phase of wound healing, as collagen gives strength to the wound^13,14. In a previous study it has been observed that NO has promoted re- epithelization in healing of wounds¹⁶. In our study GTN has enhanced the percentage of wound contraction when compared to control. So, it has wound healing property apart from its spasmolytic action.

Diltiazem is a calcium channel blocker. It belongs to dihydropyridine group¹⁷. The calcium channel blockers brings out relaxation by decreasing intracellular availability of Calcium.^17,18,Calcium dose dependently inhibits keratinocyte chemotaxis as well as adhesion. This effect was reversed by calcium channel blockers¹⁹.

At the wound site, calcium concentration changes consistently with biological events of healing process. But excessive calcium concentration in wound environment may reduce proliferation and chemotactic responses and that will lead to delayed wound healing²⁰

Calcium is having important role in normal wound healing process. Metabolism of calcium in cells is important in regulating extracellular matrix production. Calcium channel blockers may inhibit smooth muscle cell proliferation which is stimulated by Fibroblast Growth Factor and Platelet Derived Growth Factor. Metalloprotinase expression and collagenolytic activity in smooth muscle cells can be inhibited by Diltiazem¹⁹ In present study we observed that Diltiazem has enhanced percentage of wound contraction when compared to control.

Ropan Tail acts as a debriding agent by removing slough and necrotic tissue. It reduces pain, burning sensation and itching. It also decreases discharge, redness and enhances epithelization. The ingredients of Ropan Tail have many pharmacological effects such as wound healing, analgesic, local anti-inflammatory and anti-infective.^21,22,23,24,

Among three drugs, Ropan tail demonstrated significant increase in wound contraction throughout observation period of 6^th day and 10^th day after wounding and there was complete wound closure on 12^th day.

The results of the present study showed that GTN, Diltiazem and Ropan Tail promoted wound healing, though the period of epithelisation was different as compared to control. Ointment Base and Gel base havenot any effect on wound contraction or period of epithelization indicating, wound healing effect of GTN and Diltiazem isnot potentiated by their bases.

Ropan Tail has not only enhanced the percentage of wound contraction, it has also significantly decreased time required for epithelization as compared to control.

CONCLUSION:

To conclude GTN and Diltiazem have wound healing property apart from spasmolytic action. Ropan tail has shown better wound healing compared to GTN and Diltiazem.It indicaes better wound healing potential.

CONFLICT OF INTREST:

No Conflict of intrest.

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Received on 22.10.2019 Modified on 08.12.2019

Research J. Pharm. and Tech 2020; 13(9):4395-4398.

DOI: 10.5958/0974-360X.2020.00777.5