INTRODUCTION:

Traditional herbal medicine and their preparations have been widely used for the thousands of years in developing and developed countries owing to its natural origin and lesser side effects or dissatisfaction with the results of synthetic drugs. The Acanthaceae (Acanths) derived from Acanthus are made up of 221 genera and 4000 species¹. Traditionally the most important part use in Acanthaceae is the leaves and they are used externally for wounds. Acanthaceae possess antifungal, cytotoxic, anti-inflammatory, anti-pyretic, anti-oxidant, insecticidal, hepatoprotective, immunomodulatory, Anti-platelet aggregation, anti-viral potential and many members of the family are used as medication for asthma². Dyschoriste is a genus of worldwide distribution in warm regions. Members of the genus are commonly known as snake herb.

Antipyretic, analgesic and anti-inflammatory activity were may be due to its bioactive principles lutolin and quercetin present in the extract and reveals that Dyschoriste littoralis could be used as potential drug for the treatment of pain, fever and inflammation. Dyschoriste littoralis Nees. are considered a very efficacious remedy for all sorts of coughs being administrated along with ginger. The leaves are used for rheumatism. The leaves were dried made into cigarettes and smoked in asthma and their juice is used treatment of diarrhea and dysentery³. Review of literature divulges that information on the wound healing analysis of Dyschoriste littoralis belonging to the family Acanthaceae is totally lacking. Hence, the objective of the present study is to investigate the wound healing effects of ethanol extract of D. littoralis on animal models.

MATERIALS AND METHODS:

MATERIALS:

The medicinal plant Dyschoriste littoralis was selected for the present studies. Mature and healthy plants were collected from different specific locations after the rainy and summer seasons. The specimens were authentified by Dr. M. Padma Sorna Subramanian, Research Officer (Scientist-II) in Botany, (CCRS, Govt. of India), Mettur Dam. Voucher specimens of the collections are deposited at the Herbarium of Medicinal Plants Garden, Mettur Dam, Tamil Nadu, India.

Experimental animals:

Male albino Wistar rats (150-180 g) used in the present study were procured from the small animals breeding station, Mannuthy, Kerala, India. They were housed in polypropylene cages (38 x 23 x 10 cm) with not more than six animals per cage and maintained under standard environmental conditions (14h dark /10h light cycles; temp 25±2°C; 35-60% humidity, air ventilation) and were fed with standard pellet diet (M/s. Hindustan Lever Ltd., Mumbai, India) and fresh water ad libitum.

The animals were acclimatized to the environment for two weeks prior to experiment use. Animals were fasted over night before the experimental schedule, but have free access for water ad libitum. The experiment was carried out according to the guidelines prescribed by Animal Welfare Board and with the prior approval of animal ethic committee.

Preparation of Ointment:

To 10 g of petroleum jelly 0.5 g of the ethanolic extract of the sample was added and stirred to produce the 5% low dose ointment. The 10% high dose ointment was prepared by stirring 10 g of Petroleum jelly with 1.0 g of the sample. This 5% and 10% ointment was used for topical application⁴.

Excision Wound model:

Wistar albino male rats (150-180 g) were divided into four groups of six animals each (n=6).

The experiment was designed as follows:

Group I : Excision wound (1.0 sq cm) induced rats

Group II : Induction + standard drug (Povidone iodine ointment for 21 days)

Group III : Induction + low dose (5%) for 21 days

Group IV : Induction + high dose (10%) for 21 days

Group I served as induced and Group II as standard which was topically applied with Povidone iodine ointment for 21 days after the wound excision. Groups III - IV were topically applied with 5% and 10% (w/w) ointments prepared using the sample extracts and petroleum jelly for 21 days. Wounds were created at the back of each animal of Groups I –IV. An area of about1.0 sq cm is marked out. The marked area is excised with sharp knife and scissors under ether anesthesia⁵. The length and breadth of the wounds were measured for 21 days using a vernier caliper. On days 0, 6, 12, 18 and 21 the wounds were photographed

RESULTS AND DISCUSSION:

In excision wound model the potency of wound healing activity of the D. littoralis ethanolic extract was found to be highly significant. Excision wound showed that there is almost complete healing on the 21^st post wounding day with ethanolic extract. The topical application of D. littoralis ointment increased the percentage of wound contraction and this indicates rapid epithelizaton. The administration of this extract D. littoralis accelerated the progression of wound healing by 21^st day i.e. (95.66%) compared with control (83.10%) in Table 1.

The percent wound contraction by ethanolic extracts of D. littoralis from day 3 to day 21 has been shown in Plate 3 and 4. In D. littoralis a rapid closure of wound in standard and extract treated groups was observed between 6 to 9 days of post surgery as represented by a decrease in the wound area (Table: 2).

After day 9 of post surgery, the wound closure was gradual till the closure of the wound. The extract ointment treated groups showed significant wound healing from the third day onwards, which was comparable to that of the standard drug, i.e. povidone iodine ointment treated group of animals (Plate 1). The wound healing activity of induced drug was shown in Plate 2.

The wound closure time was almost similar, as well as the percentage of wound contraction was very close to standard with the extract ointment treated group.

Wounds are physical injuries that result in an opening or break of the skin. Proper healing of wounds is essential for the restoration of disrupted anatomical continuity and disturbed functional status of the skin⁶. Healing is a complex and intricate process initiated in response to an injury that restores the function and integrity of damaged tissues⁷.

Wound healing involves continuous cell–cell and cell–matrix interactions that allow the process to proceed in three overlapping phases viz. inflammation (0–3 days), cellular proliferation (3–12 days) and remodeling (3–6 months) ^8,9,10.

Healing requires the collaborative efforts of many different tissues and cell lineages¹¹.

It involves platelet aggregation and blood clotting, formation of fibrin, an inflammatory response to injury, alteration in the ground substances, angiogenesis and reepithelialization.

Plate 1: Wound healing activity of standard drug

Plate 2: Wound healing activity of induced drug

Plate 3: Wound healing activity of the ethanolic extract of D. littoralis at low dose

Plate 4: Wound healing activity of the ethanolic extract of D. littoralis at high dose

Table: 1 Percentage reduction of samples on Wound size

Day	Induced		Standard		Low Dose (5%)		High Dose (10%)
Day	Wound size (mm)	% Reduction	Wound size (mm)	% Reduction	Wound size (mm)	% Reduction	Wound size (mm)	% Reduction
0^th day	131.33±1.57	-	127.26±0.89	-	130.97±1.63	-	123.82±0.50	-
3^rd day	124.71±0.74	5.04	104.08±1.03	18.22	115.72±1.18	11.65	110.61±1.00	10.67
6^th day	113.01±1.33	13.94	89.80±1.05	29.44	114.19±0.83	12.81	103.92±0.56	16.07
9^th day	81.49±0.92	37.95	40.90±0.97	67.86	80.73±3.62	38.36	58.96±3.97	52.38
12^th day	72.73±0.68	44.62	21.00±0.78	83.50	62.09±3.36	52.60	48.55±3.58	60.79
15^th day	60.06±1.15	54.26	5.04±0.19	96.04	45.70±2.62	65.11	35.26±2.90	71.53
18^th day	50.24±0.58	61.74	0.04±0.09	99.68	28.18±2.79	78.49	22.28±2.12	82.01
21^st day	22.20±0.53	83.10	-	100	12.19±1.90	90.69	5.37±0.70	95.66

Table: 2 Wound healing activity of the ethanolic extract of D. littoralis (Excision model)

Day	Wound measurement (mm)
	low Dose (5%)		High Dose (10%)		Induced		Standard
	Length	Breadth	Length	Breadth	Length	Breadth	Length	Breadth
0^th day	11.44±0.10	11.46±0.12	11.17±0.04	11.09±0.03	11.47±0.09	11.44±0.07	Length	Breadth
3^rd day	10.70±0.09	10.82±0.07	10.53±0.07	10.50±0.08	11.18±0.06	11.16±0.05	11.29±0.06	11.27±0.04
6^th day	10.65±0.04	10.73±0.04	10.19±0.02	10.20±0.04	10.56±0.10	10.7±0.06	10.28±0.04	10.13±0.06
9^th day	8.86±0.20	9.09±0.21	7.84±0.25	7.48±0.28	9.12±0.09	8.93±0.03	9.48±0.08	9.48±0.05
12^th day	7.75±0.21	7.99±0.23	7.08±0.25	6.81±0.27	8.62±0.08	8.44±0.05	6.24±0.09	6.55±0.09
15^th day	6.75±0.21	6.75±0.20	5.91±0.25	5.92±0.24	7.82±0.09	7.68±0.11	4.46±0.12	4.71±0.07
18^th day	5.30±0.32	5.25±0.23	4.70±0.25	4.69±0.21	7.03±0.06	7.15±0.08	2.31±0.08	2.18±0.04
21^st day	3.46±0.28	3.42±0.28	2.39±0.19	2.20±0.16	4.70±0.09	4.73±0.09	0.65±0.10	0.6±0.09

The animals that did not receive the plant extract treatment, the wounds appear to be hard and crusty with undermined margins and generally unclean with bioflim glaze on the surface. In contrast the animals treated with plant extract were clean, and showed bright red healthy granulation tissue. The wound treated with Povidone iodine ointment showed the healthy granulation tissue (Plate: 1). Treated excision wounds showed an increased rate of wound contraction, leading to faster healing as confirmed by the increased healed area when compared to the control group. Tensile strength was measured to confirm the wound healing activity claimed for this plant. An increase in tensile strength of treated wounds may be due to an increase in collagen concentration and stabilization of the fibres facilitating wound healing.

Wound healing involves various phases which include granulation, collagenation, collagen maturation and scar maturation¹². Many plant extracts and medicinal herbs have shown potent antioxidant activity. Tannins the main components of many plant extracts, act as free radical scavengers^{13, 14}. Research into the role of antioxidants from plant extracts in wound healing has been published widely¹⁵.

Phytochemical work reveals that ethanolic extract of selected plants contains high amount of tannins, implied that tannin is one of the active compounds which may be responsible for the antioxidant activity. So in this study scavenging effect might be one of the most important components of wound healing which may be responsible to support wound healing property. Thus the enhanced wound healing may be due to the free radical scavenging action of the plant as well as enhanced antioxidant enzyme level in granuloma tissues.

Flavonoids have been documented which is believed to be one of the most important components of wound healing. The enhanced wound healing may be due to free radical scavenging action and the antibacterial property of the phytoconstituents present in it which either due to their individual or additive effect fastens the process of wound healing¹⁶. This could be the reason for wound healing activity of D. littoralis.

In conclusion, the observations and results obtained in this study indicated that the selected plant extracts significantly stimulated wound contraction. The application of ethanolic extract of selected plant was found to improve the different phases of wound repair, including collagen synthesis and maturation, wound contraction, and epithelialization. As all the selected plant ethanolic extract possesses an antifungal property and is traditionally used in folk medicine, the present findings may provide scientific rationale for the use of these plants to promote healing of infected wounds. The present study showed remarkable wound healing activity and it may be suggested for treating various types of wounds in human beings. Further studies with purified constituents are needed to understand the complete mechanism of wound healing activity.

ACKNOWLEDGEMENT:

The Authors are very grateful and thankful to Dr. M. Padma Sorna Subramanian, Research Officer (Scientist-II) in Botany, (CCRS, Govt. of India), Mettur Dam. for his constant help.

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Received on 30.07.2017 Modified on 16.08.2017

Res. J. Pharmacology & Pharmacodynamics.2016; 8(3): 181-185.

DOI: 10.5958/2321-5836.2017.00031.3