One of the serious causes of morbidity and mortality all over the world is thermal burn injuries, which results in a large amount of expenditure and costs in health care. The estimated annual burn incidence in India is approximately 6-7 million per year. The high incidence is attributed to illiteracy, poverty and low level safety.¹⁰

Traditionally Mussaenda frondosa Linn commonly called as Nagavalli reported to possess number of medicinal properties ^11,12. Traditionally leaves are used in the treatment of jaundice, asthma, hyperacidity, fever, ulcers, leprosy, diuretic, inflammation, wound,¹³and antimicrobial.¹⁴ Plant contains bitter principles, tannins¹⁵, flavonoids-hirsutissima,quercetin, rutin,hyperin,ferulic acid,sinnapic acid,ß-sistoserol glucoside,saponin,resins¹⁶, mucilage colouring matterand anthocyanins.^17,18

Finding more efficient agents with fewer side effects for treatment of burns has always been a concern for researchers. Hence the present study is designed to fill up the lacunae in the literature for burn wound healing activity with a view to provide scientific evidence on wound healing.

MATERIALS AND METHODS:

Plant material and preparation of Extracts

In the present study, the leaves of Mussaenda frondosa were collected from Jamboti forest Dist. Belgaum, Karnataka in the month of July. The plant Mussaenda frondosa was authenticated from the Scientist Mr.Harsha Hegade of ICMR (Regional Medical Research centre, Belgaum.) Accession No.RMRC-484. The leaves shade dried powdered and then passed through sieve No.40 to get uniform powder.

Preparation of alcoholic extract of Mussaenda frondosa Linn

leaves was subjected to hot continuous extraction (soxhlet) with alcohol for 48h in batches of 250 g each. The extract was filtered, cooled and solvent was recovered under reduced pressure at 40±5^oC by rotary flash evaporator.

Powder analysis.¹⁹

The powdered crude drug was subjected to determination of extractive value, total ash, water soluble ash, acid insoluble ash etc.

Preliminary phytochemical investigation²⁰

All the extracts were screened for the presence of various secondary metabolites like steroids, glycosides, saponins, resins, mucilage and flavonoids using official methods. (Table-1)

The Development of herbal formulation:

In the present study, it was thought worthwhile to formulate extracts in the form of cream. The formulation was done by considering the solubility of extracts either in water or tween 80; to make^O/_W type cream using 10 % ^w/_w of extract in the formulation.

PHARMACOLOGICAL ACTIVITY:

Experimental animals²¹

Healthy young albino rats of either sex weighing between 150 to 200 gms (8 to 12 weeks old) were used for assessing Wound healing and Swiss albino mice of either sex weighing between 18-22 gms for acute toxicity study to determine LD₅₀ of various extracts. Animals were procured from Venkateshwara Enterprises, Bangalore. The animals were randomly selected, marked to permit individual identification, divided into different groups comprising of six animals in each group and kept in polypropylene cages for 5 days prior to dosing at 23±1⁰C in 12:12 dark: light cycle with free accession to standard pellet feed (Amrut Sangli) and water ad libitum.This project was cleared by Institutional Animal Ethical Committee. (Resolution No 0l, dated 2I-12-2009).

Acute Oral Toxicity study²²

The acute oral toxicity study was carried out as per the guidelines set by Organization for Economic Co–operation and Development (OECD), received draft guidelines 423, received from Committee for the Purpose of Control and Supervision of Experiments on Animals ( CPCSEA) , Ministry of Social Justice and Empowerment, Government of India. Swiss albino mice of either sex weighing between 18-22 gms were fasted over night prior to the acute experimental procedure. The principle, which is based on a stepwise procedure with the use of a minimum number of animals per step. The LD₅₀ of different extracts were determined. The therapeutic dose was calculated as1/10^th of the lethal dose for further investigation.

Burn wound Model:

Adult albino rats of weight around 150-200 gm were divided into 4 groups. On the zero day, animals were anaesthetized with anesthetic ether and secured to operation table in its natural position. An impression was made on the dorsal thoracic central region 5 mm away from the ears by using a round seal of 300 mm² diameter as described by Bairy and Somayaji²³. During the experimental period the animals were housed individually and resuscitation was done with Ringer lactate (0.1 ml/100 mg) daily.

Burn wound were created by pouring hot molten wax at 80ºC into a metal cylinder placed on the back of the rat

On solidification of wax (8 min), the metal cylinder with wax adhered to skin was removed, which left distinctly demarked circular wounds of 300 mm2. After this each animal was placed in a separate cage for full recovery from anesthesia before being returned to holding rooms. No local or systemic chemotherapeutic agents were given Animal showing signs of infection were excluded from the study. Actual amount of heat delivered by molten wax to create burn wound was calculated by the following formula:

∆H/∆A

ΔH/ ΔA = Amount of heat delivered by molten wax to sq. mm. of exposed skin.

M = Mass of molten wax.

T₁ = Initial Temperature.

T₂ = Room Temperature.

S = Specific heat.

The animals were then placed back into individual cages. The physical attribute of healing viz. (wound closure) contraction which mainly contributes for wound closure was studied by tracing the raw wound area on the polythene paper on wounding day followed by 4,6,12 and 16^th days after removing scab till complete epithelization occurred, the criterion for complete epithelization being fall of scab without any raw wound area. Wound area was measured by retracing the wound on a millimeter graph paper.

The degree of wound healing was calculated as percentage closure in wound area from original wound area using the formula,

Percentage closure= 1- AD/AO X 100

Where,

AO = wound area on day 0

AD= wound area on corresponding days

The mean and S.E. values of raw wound areas were calculated. The number of days for complete epithelization was noted.

Time of Epithelization:

The time of complete epithelization were studied in days. The results so obtained were subjected to students ‘t’ test.

Scar Area Measurement:

The scar area on complete epithelization was determined in both groups by tracing the scar. The results obtained were subjected to students ‘t’ test..

Collagen Content:^24,25

The regenerated tissues extracted from the open wounds as described by Uduppa were collected from the animals and sacrificed at 16^th post wounding day were divided into 2 equal parts.

The regenerated tissue collected from the excision wounds were cut into pieces. They were washed with 0.5M sodium acetate and then suspended in 10 parts w/v of 0.5M acetic acid and stirred intermittently for 48 hrs. The solution was centrifuged for 2 hrs (intermittently) in the micro-centrifuge and then sodium chloride (5% w/v) solution was added to precipitate the collagen.

The collagen so precipitated was filtered using a pre weighed Whatman Filter paper – No.1.

The weight of the collagen precipitate obtained was calculated by taking difference between the initial and the final weights of the filter paper.

The same procedure was followed for the animals of both the control and the test groups. The results so obtained were subjected to students‘t’ test for statistical evaluation.

Estimation of Lipid Peroxides:^26,27

Assay for lipid peroxides is carried out by Thiobarbituric acid reactive substances (TBARS) method, measured as plasma level of malondialdehyde. Briefly, a 0.5ml aliquot of plasma was shaken with 2.5ml of 20% trichloroacetic acid (TCA) in a 10ml centrifuge tube. To the mixture, 1ml of 0.67% thiobarbituric acid (TBA) was added, shaken and warmed for 30 minutes in a boiling water bath followed by rapid cooling. Then 4ml of n-butyl-alcohol was added and shaken. The mixture was centrifuged at 3000 rpm for 10 minutes. The resultant n-butyl-alcohol layer was taken into a separate tube and MDA content in the plasma was determined from the absorbancy at 535nm by spectrophotometrically. Malondialdehyde (MDA) is used as an external standard. Levels of peroxidation products were expressed as the amount of MDA per milliliter of plasma. MDA level estimation was carried out on 7^th and 14^th post wound healing day.

Statistical Analysis:

Data were expressed as mean ± SE and were analyzed for statistical significance by One Way Analysis of Variance (One Way – ANOVA), followed by Dunnet’s‘t’ test for multiple comparison. P<0.05 is considered as significant.

RESULTS:

The creams prepared by using AE10 and AQE10 of MF were used to study different burn wound models in rats.

Significant wound healing activity was observed in both the groups of animals treated with the AE10 and AQE10 of MF. The percentage of closure of wound was significant (p < 0.001) in the animals treated with AE10 it was 52.50 ± 1.19 and 86.61 ± 1.19 on day 12^th and 16^th days. In the AQE10 treated group it was 44.89 ± 1.94 and 62.63 ± 1.29 on day 12^th and 16^th respectively (p < 0.05) compare to control group.

The time required for complete epithelization of the excision wound is an important parameter to assess the wound healing process. It was also found that the Mean time taken for complete epithelization of the excision wound in both the herbal cream treated group was less than the control group. The data was shown in the Table-2.

The herbal cream of AE10 and AQE10 showed a scar area of 8.71 ± 0.18 mm² and 14.78 ± 0.58 mm²respectively as compared to scar area of control 17.15 ± 0.61 mm²).The results are shown in Table-2.

The collagen content was estimated from regenerated tissues for control as well as treated groups. There was a significant increase (P<0.001) in collagen content on 4^th, 8^th, 12^th and 16^th days in AE10 treated group compared to the control group. The increase in collagen content in AQE10 treated group was also significant (P<0.05) as compared to the control group Table-3.

In SSD and AE10 treated animals showed significantly increased levels of Hydroxyproline content (p < 0.001) as compared with the control group of animals. The data was shown in the Table-4.

In 7^th and 14^th post-wounding day of Burn wound model, the TBARS (MDA) levels decreased in AE10 treated group (33.58 ± 0.94mm and 20.13 ± 0.67 mm respectively), where as it was less significant decrease (p<0.05) in AQE10 treated group (23.35 ±0.62 mm and 12.72 ± 0.71mm respectively) as compared to control group (Table no.5 and 6).

The AE10 showed parallel significant effect to that of animals treated with SSD. Histopathological studies accordingly revealed markedly increased collagen content, prominent granulation tissue and fibrous tissue in AE10 treated groups compared to control group.

DISCUSSION:

Burn and wound healing is a process by which damaged tissue is restored as closely as possible to its normal state and wound contraction is the process of shrinkage of the area of the wound. It is mainly dependent upon the type and extent of damage, the general state of health and the ability of the tissue to repair

In excision wound model significant wound healing was observed in the animals treated with SSD and AE10 cream of MF. Significant decrease in epithelization increase in wound contraction rate was observed in these groups of animals. While in AQE10 treated animals and control animals rate of contraction and epithelization was moderate and slow respectively.

Scar replaces the damaged tissue, Scar formation is a biological process by which an excision wound shrinks. Whereas the term contracture specifically implies to loss of normal stretching as a result of excessive scar formation. Scar undergoes certain changes indicating continuation of healing process.

Lipid peroxidation is an important process in several types of injuries like burns, infected wounds, skin ulcers, etc. because of cell destruction and inflammation during wound and burns, the reactive oxygen species are released and are behave as toxic “second messengers”.²⁸

There are several reports that the plants containing antioxidant constituents exhibited significant wound healing activity on various wound models, especially burn wounds.²⁹

ROS react with cells initiating chain reactions that result in tissue damage causing inflammation, spasm, pain, and disease. One of the important consequence of excessive production of ROS is lipid peroxidation.³⁰

Aging and disease result in diminished cell production of protective compounds leading to increased damage to cell membranes; inevitably, damage membranes which diminishes cellular ability to repair damaged tissue.³⁰

Free radical generation has been implicated in the inflammation of wound area. Estimation of lipid peroxides has been done.²⁸

A drug which inhibits lipid peroxidation is believed to increase the viability of cells by improving the circulation, preventing cell damage, promoting DNA synthesis, etc. and in turn promoting the wound healing.³¹

There are reports which suggested that administration of growth hormone prevents the lipid peroxidation in lung and liver tissues and also reduced the oxidant stress by increasing the endogenous antioxidant level after burn injury.In the present study, there is a decrease in TBARS level in SSD and AE10 treated group compared to control group, and also simultaneous increase in the wound healing property, which could be due to increase in the growth hormone level and also due to reduction in the oxidative stress by increase in the endogenous antioxidants level.

There are reports which suggested that administration of growth hormone prevents the lipid peroxidation in lung and liver tissues and also reduced the oxidative stress by increasing the endogenous antioxidant level after burn injury.³² In the present study, there is a decrease in TBARS level in alcoholic extract ointment treated group compared to control group, and also simultaneous increase in the wound healing property, which could be due to increase in the growth hormone level and also due to reduction in the oxidant stress by increase in the endogenous antioxidants level.

Histological evaluation was carried out for the treated and untreated samples. There was a marked infiltration of the inflammatory cells, increased blood vessel formation and enhanced proliferation of cells as a result of treatment with SSD and AE10 of MF. There was full thickness re-epithelization, in which epidermis was thin and well organized, comparable to the normal adjacent skin which was not involved in the wound generation and healing process. The granular layer was well formed and one cell in thickness.All the animals when treated with SSD and AE10 showed comparable results when compared with control. There was a full thickness epidermal regeneration which covered completely the wound area. The epidermis was thick and disorganized, especially when compared with the adjacent normal skin. In all, complete epithelization, vasculirisation and hair follicles formation were observed in treated animals. Early dermal and epidermal regeneration in treated mice also confirmed that the extract had a positive effect towards cellular proliferation, granular tissue formation and epithelization.

The AE of MF revealed presence of secondary metabolites like steroids, glycosides, saponins, resins, mucilage and flavonoids. Flavonoids are known to reduce lipid peroxidation not only by preventing or slowing cell necrosis but also by improving vascularity.Hence any drug that inhibits lipid peroxidation is believed to increase the viability of collagen fibrils by increasing the circulation, strength of collagen fibres, preventing the cell damage and by promoting the DNA synthesis.³³

Flavonoids³⁴ are also known to promote the wound healing process mainly due to their antimicrobial property which is responsible for wound contraction and increased rate of epithelialisation.

CONCLUSION:

The use of leaves of MF in folk medicine for the treatment of wounds has been justified by this work, as it showed remarkable burn wound healing property. These findings clearly justifies for the inclusion of leaves of Mussaenda frondosa in the management of burn wound. The wound healing activity of this plant may be due to presence of flavonoid .The present study confirms the use of Mussaenda frondosa leaves for Burn wound treatment.

Photomicrograph of granuloma tissue in control

Photomicrograph of granuloma tissue in cream AE10

Table-2: Effect of herbal creams (AE10 andAQE10) OF Mussaenda frondosa on burn wound model

Group	Topical application	Mean ± SEM of % of wound closure				Epethelization (Days)	Scar area (mm²)
Group	Topical application	4^th Day	8^thDay	12^thDay	16^thDay	Epethelization (Days)	Scar area (mm²)
I	Control	11.97 ±0.80	18.32 ± 0.80	31.95 ± 1.54	48.09 ± 1.26	26.32 ± 0.93	17.15± 0.61
II	SSD	20.05± 0.66***	32.50±0.72***	58.60±1.39***	91.43±0.61***	17.40 ±0.38***	7.05±0.19***
III	AE10	19.40±0.85***	29.68 ±0.79***	52.50 ±1.19***	86.61±1.19***	18.13 ±0.44***	8.71 ±0.18***
IV	AQE10	15.79±1.00*	23.18 ±1.02*	44.89 ±1.94*	62.63 ±1.29*	23.03 ±0.65*	14.78±0.58*

Note: Data analysed by ANOVA followed by Dunnett’s test. ***=p < 0.001,*= p < 0.05.

Table-3: Effect of herbal creams (AE10 andAQE10) of Mussaenda frondosa on collagen content

GROUP	Topical Application	Collagen Content
GROUP	Topical Application	4^th Day	8^thDay	12^thDay	16^thDay
I	Control	10.33±0.51	15.10±0.73	20.03±0.38	30.32±2.01
II	SSD	23.78±0.62***	33.03±0.78***	38.47±0.52***	50.60±2.19***
III	AE10	21.57±0.44***	30.33±0.88***	35.88 ±0.66***	47.68±2.20***
IV	AQE10	12.85±1.46*	20.12±0.50*	23.27±0.46*	36.85±1.62*

Note: Data analysed by ANOVA followed by Dunnett’s test. ***=p < 0.001,*= p < 0.05

Table-4: Effect of herbal creams (AE10 andAQE10) Mussaenda frondosa extracts on Hydroxyproline content

Animal	Control	Standard	AE10	AQE10
1	6.4	8.6	8.2	7.1
2	6.1	8.4	8.1	6.3
3	5.9	7.8	7.4	6.8
4	6.3	9.2	7.6	6.8
5	6.4	8.4	8.2	7.0
6	6.7	8.2	8.0	7.2
MEAN	6.30	8.43	8.08	6.86
SEM	0.11	0.18	0.16	0.13
P-value	-	<0.001	<0.001	<0.05

Note: Data analysed by ANOVA followed by Dunnett’s test.

***=p < 0.001,*= p < 0.05

Table-5: Effect of herbal creams (AE10 and AQE10) on TBAR- 7^th day

Animal	Control	AE10	AQE10
1	22.1	35.2	23.3
2	24.5	30.1	25.0
3	21.6	36.0	22.5
4	19.8	35.4	20.8
5	23.4	32.1	24.1
6	22.0	32.7	24.4
MEAN	22.23	33.58	23.35
SEM	0.65	0.94	0.62
P-value	-	<0.001	<0.05

Note: Data analysed by ANOVA followed by Dunnett’s test.

***=p < 0.001,*= p < 0.05

Table-6: Effect of herbal creams (AE10 andAQE10) on TBAR- 14^th day

Animal	Control	AE10	AQE10
1	10.5	22.3	12.1
2	12.8	17.4	14.0
3	14.0	20.0	15.6
4	9.8	19.5	11.1
5	10.6	21.2	11.3
6	11.1	20.4	12.2
MEAN	11.47	20.13	12.72
SEM	0.66	0.67	0.71
P-value	-	<0.001	<0.05

Note: Data analysed by ANOVA followed by Dunnett’s test.

***=p < 0.001,*= p < 0.05

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Received on 16.02.2012

Modified on 01.03.2012

Accepted on 18.03.2012

Research J. Pharmacology and Pharmacodynamics. 4(3): May-June, 2012, 163-168

Extracts	Steroid	Flavonoid	Glycosides	Saponin	Resin	Mucilage
Alcohol	++	+++	+	++	+	+
Aqueous	-	++	+	+	-	+