Evaluation of Wound Healing Activity of Isolated Compound Quercetin and Alcoholic Extract of Leaves of Mussaenda frondosa Linn.

Patil Suhas A.¹*, Joshi V.G.² and Sambrekar S.N.³

¹Dept. of Pharmacognosy, Maratha Mandal’s College of Pharmacy, Belgaum, India.

²Dept. of Pharmaceutics. Government College of Pharmacy, Bengaluru, India

³Dept. of Pharmacology, Maratha Mandal’s College of Pharmacy, Belgaum, India.

ABSTRACT:

Since the time immemorial, our traditional system of medicine and folklore claiming that medicinal plants as a whole or their parts are being used in all types of diseases. Natural remedies from medicinal plants are considered to be effective and safe alternative treatment for wounds. Alcoholic extract of leaves of Mussaenda frondosa Linn. (Rubiaceae) and its isolated compound quercetin were evaluated for wound healing activity by using different types of wound healing models such as excision wound, incision wound and dead space wound. The results were obtained in terms of wound contraction, epithelialization time and tensile strength. All results were significant for different parameters in wound healing activity in quercetin and alcoholic extract treated animals compared with control groups. The isolated compound quercetin was confirmed by preliminary phytochemical investigation, IR, NMR and Mass spectroscopic methods.

KEYWORDS: Mussaenda frondosa Linn, wound healing activity, alcoholic extract, quercetin.

INTRODUCTION:

Wound can be defined as a cut or break in the continuity of any tissue which may arise due to physical, chemical or microbial agents¹. Wound a clinical entity contemporary to mankind is a common clinical problem till today. Wound healing or wound repair is an intricate process in which the skin or some other organ repairs after injury². Healing is essentially a survival mechanism and represents an attempt to maintain normal anatomical structure and function. All wound heal following a specific sequence of phases which may overlap^3,4. The healing depends on the type of tissue which has been damaged and the nature of tissue disruption. The treatment is aimed to shortening the time required for healing or minimizing the undesirable consequences.

The basic principles of optimal wound healing which include minimizing tissue damage, maximizing tissue perfusion and oxygenation, proper nutrition and moist wound healing environment have been recognized for many years⁵.A number of drugs ranging from simple analgesics to complex and expensive chemotherapeutic agents administered in the management of wound affect healing either positively or negatively.⁶Aspirin, indomethcin, cytotoxic agents and immunosuppressant have been proved to experimentally to affect healing negatively^.7-10.

Management of wound healing, particularly the under healing is complicated and expensive programme. Research on wound healing drugs is a developing area in modern biomedical sciences, Several drugs from plants have been screened scientifically for evaluation of their wound healing activity in different pharmacological models and patients, but the potential of most remains unexplored. Hence there is a dearth of safe, economic and effective prohealing agents for wound management programme, which can enhance healing as well as control infection.

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, wound,¹³; antimicrobial¹⁴This plant has been investigated by several workers. Diuretic activity¹⁵ Hepatoprotective activity¹⁶, activity on fever asthama and cough¹⁷ was reported in the leaf extract. Hypolipidemic effect of Methanolic extract of Mussaenda frondosa linn. Leaves in wistar rats was investigated.¹⁸

In the Ayurvedic system of medicine, herbal extracts but not purified compounds have been used from centuries because many constituents with more than one mechanism of action are considered to be beneficial but no scientific reports were available for its wound healing activity. Hence the present study is designed to fill up the lacunae in the literature for 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:

Around 1 Kg of fresh shade dried powdered 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. The yield was 7.4 g. The extract was stored in a cool place for further study.

Isolation of compound:^19,20

The dried samples were separately soxhlet extracted in 80% methanol (100 ml/gm dry weight) on a water bath for 24 hrs (Subramanian and Nagarajan, 1969). Each of the extracts was concentrated and reconcentrated in petroleum ether (40°- 60°C) (fraction-I), ethyl ether (fraction-II) and ethyl acetate (fraction-III) in succession. Each of the steps was repeated three times to ensure complete extraction in each case. Fraction I was rejected since it was rich in fatty substances whereas fraction II was analysed for the free flavonoids in each of the samples.

Fraction III of each of the test samples was hydrolysed by refluxing with 7% H₂SO₄ (10 ml/gm residue) for 5 hours. The mixture was filtered and the filtrate extracted with ethyl acetate in a separating funnel. The ethyl acetate layer was washed with distilled water till neutrality and dried in vacuo. The residues were taken up in small volumes of ethanol separately and then subjected to various tests for quercetin.

PHARMACOLOGICAL ACTIVITY:

Experimental animals:²¹

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 study:²²

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 (Table-3) for further investigation.

WOUND HEALING ACTIVITY:

Healthy young albino rats of either sex weighing between 150 to 200 gms (8 to 12 weeks old) were selected divided into different groups comprising of six animals in each group. The animals were depilated at the desired site before wounding. They were housed individually with free access to food and water, the basal food intake and body weights to the nearest gram were noted. The animals were starved for 12 h prior to wounding. Under light ether anesthesia wounding was performed semi-asceptically. The first group served as control, second received alcoholic extract and forth group quercetin by oral route.

Wound models:

Albino rats of either sex weighing between 150 to 200 gms (8 to 12 weeks old) were used for assessing Wound healing activity.

Incision wound:²³

Under light ether anaesthesia on the depilated backs of the animal was two paravertebral straight incisions of 6 cm. were made through entire thickness of the skin, on either side of the vertebral column with the help of a sharp blade as described by Ehrlich and Hunt, care was taken to see that the incisions were at least 1 cm apart, using four zero silk thread and straight round bodies needle. Wounds were then mopped with cotton swabs soaked in 70% alcohol. The animals were caged individually. Removal of sutures was done on 8^th post wounding day. Tensile strength was determined on 10^th post wounding day by continuous, constant water flow technique of Lee.

Granuloma studies (Dead space wound):²⁴

Physical changes in the granuloma tissue were studied in this model. Under light ether anaesthesia, subcutaneous dead space wounds were inflicted in the region of the axilla and groin by making a pouch through a small nick in the skin. Granuloma formation was induced by implanting grass pith. Cylindrical grass piths measuring 2.5 cm in length and 0.3 cm, in diameter were introduced in to the pouch similarly. Each animal received 2 grass piths in different locations. The wounds were sutured and mopped with an alcoholic swab. Animals were placed in to their individual cages after recovery from anaesthesia. Excision of the granulomas from the surrounding tissue was performed on the 10^th post wounding day under light ether anaesthesia. Granuloma surrounding the grass piths were excised and slit open by longitudinal rectangular strips. The tensile strength of a piece measuring about 15 mm. in length and 8 mm. in width (obtained by trimming the rectangular strip of granuloma tissue) was determined on 10^th post wounding day by continuous, constant water flow technique of Lee. The granulation tissue so harvested was subjected to hydroxyproline estimation. Their weights were expressed as mg/100 gms body weight as suggested by Gieson and Meli.

Excision wound:

An excision wound was inflicted by cutting away 500mm² of a pre-determined area on the depilated back of the rat. Epithilization period was noted as the number of days after wounding required for the scar to fall off leaving no raw wound behind. Wound contraction rate was monitored by planimetric measurement of the wound on a graph paper on4th, 8th, 12^th and 16th post wounding days and there after daily until healing was complete. Reduction in wound area was expressed as percentage of the original wound size on zero days.

Histopathological studies:

A section of granuloma tissue was subjected to histopathological examination to determine the pattern of lay-down for collagen using Van Gieson and Meli stains.

STATISTICAL ANALYSIS:

All the results were analyzed by ANOVA followed by Dunnett’s test. The level of significance was set at p<0.05.

RESULTS:

The powdered crude drug was subjected to determination of extractive value, total ash, water soluble ash, acid insoluble ash, loss on drying, heavy metals and microorganisms. These values are useful in determining the quality and purity of crude drug. Further these values indicate the nature of the constituents present in a crude drug. The results of physiochemical characterization are presented in Table-1

Since the alcoholic extract shows higher concentration of flavonoid it was subjected for the Flavonoid estimation.²⁵The alcoholic extract showed presence of flavonoid, 190 mgs/g equivalent to Rutin.

Table-1: Physiochemical characterization of Mussaenda frondosa

Sl. No.	Parameters	As per IP/BP Standards	Results
1	Loss on Drying	NMT 10% w/w	7.41
2	Total Ash	-	10.34
3	Acid insoluble Ash	-	4.13
4	Water soluble Ash	-	1.44
6	Total Microbial count	NMT 10,000	Confirms
7	Heavy metals
	Mercury	NMT 10 ppm	Confirms
	Lead	NMT 10 ppm	Confirms
	Lithium	NMT 10 ppm	Confirms
	Cadmium	NMT 10 ppm	Confirms

The phytochemical tests revealed that the leaves of Mussaenda frondosa Linn possess presence of various secondary metabolites like steroids, glycosides, saponins, resins, mucilage and Flavonoid in the alcoholic extract as shown in table-2

Table-2: Phytochemical Screening of Mussaenda frondosa Linn.

Extracts	Ster oid	Flav onoid	Glyc osides	Sapo nin	Resin	Muci lage
Alcohol	++	+++	+	++	+	+

+++ = High concentration, ++ = medium concentration, + = low concentration, - = absent.

The LD₅₀ was found to be more than 2000 mg/kg of b.w. in acute toxicity testing. The therapeutic dose was calculated as1/10^th of the lethal dose (Table-3) for further investigation. The results of oral toxicity studies of various extracts is shown in table-3

Table-3: Results of Acute Toxicity studies

Sl. No.

Extracts/

Isolated compound

LD₅₀(mg /kgb.w.)

ED₅₀ (mg/ kg b.w.)

Alcohol

2000

200

Quercetin

300

The isolated compound showed positive tests for flavonoid (Ayurvedic pharmacopoeia of India 1990) and for quercetin (Indian Herbal Pharmacopoeia 2002). IR, Mass and NMR spectral analysis confirmed the chemical structure of the compound.

¹H NMR spectrum (400 MHz, DMSO-d₆), δ, ppm (J, Hz): 6.17 (1H, s, Phenyl-C₇ proton); 6.39 (1H, s, Phenyl-C₉ proton); 6.88 (1H, m, Phenyl-C₁₅ proton); 7.54 (1H, m, Phenyl-C₁₆ proton); 7.66 (1H, s, Phenyl-C₁₂ proton); 9.36 (3H, b, of-OH proton); 10.72 (1H, b, of-OH proton); 12.48 (1H, s, of-OH proton); MS spectrum, m/z: 303 [M+1]⁺.

2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one

The results of the excision wound model are given in table-4.In an excision significant wound healing activity was observed in both the groups of animals treated with alcoholic extract and quercetin respectively. The percentage of wound closure was significant p<0.001)in the animals treated with quercetin 66.22 ± 2.13 on day 12^th and 93.32 ± 1.95 on day 16^th respectively. While in control animals it was only36.20 ± 1.66 and 63.95 ± 3.34 respectively. The time required for complete epithelization of excision wound is an important parameter to assess the wound healing process. It was found that mean time taken for complete epithelization was less in quercetin treated group than animal treated with alcoholic extract. The quercetin and alcoholic extract treated groups showed a scar area of 06.80 ± 0.38 and 7.83 ± 0.52mm² respectively compared to scar area of control 15.22 ± 1.41 mm².

The results of incision wound model are given in table-5.In an incision wound study, the quercetin and alcoholic extract treated groups showed significant breaking strength (378.30 ± 2.20 and 372. 25 ± 2.29 respectively) compared to control (335.20 ± 2.05).

The results of dead space wound model are given in table-6.The tensile strengths of granuloma tissue were determined by the water flow technique of Lee. The quercetin and alcoholic extract treated group showed highly significant increase in tensile strength (500.30±3.19 and 475.30±2.73 resp.) compared to control (424.40 ± 2.30).The quercetin and alcoholic extract treated groups also showed significant increase in the dry weight of granuloma tissue (42.57 ± 1.63 and 41.48 ± 1.60 respectively) compared to control (32.23 ± 1.57). The The quercetin and alcoholic extract also showed significant increase in the hydroxyproline content (10.20 ± 0.47 and 9.93± 0.44 resp.) as compared to control (7.35 ± 0.11).

Histopathological studies of granulation tissue of alcoholic extract treated animals showed significant increase in the collagen deposition with macrophages, tissue edema and more fibroblasts compared to control. (Fig.1, 2 and 3).

Control

Quercetin

Alcoholic Extract

Table-5: Influence of different extracts on Incision wound model

Group	Dose (oral) mg/kg b.w.	Wound breaking strength(g)
Control	1% CMC	335.2 ± 2.05
Quercetin	30	378.3 ±2.20***
Alcoholic extract	200	372.25 ± 2.29***

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

Table-6: Influence of different extracts on Dead space wound model

Group	Dose (oral) mg/kg b.w.	Wound breaking strength(g)	Granuloma tissue dry weight (mg)	Hydroxyproline
Control	1% CMC	424.40 ± 2.30	32.23 ± 1.57	7.35 ± 0.11
Quercetin	30	500.30± 3.19***	42.57 ± 1.63***	10.20 ± 0.47***
Alcoholic extract	200	475.30± 2.73***	41.48 ± 1.60***	9.93 ± 0.44***

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

DISCUSSION:

In the present ionvestigation quercetin was isolated from the alcoholic extract of the leaves of Mussaenda frondosa. The crude alcoholic extract and the isolated compound quercetin were concomitantly tested for wound healing activity.

Granulation, collagen, maturation and scar formation are some of the phases of wound healing The various wound healing models have been chosen in our study because use of single model is inadequate and no reference standard exist that can collectively represent the various phases of wound healing. The wound breaking strength is determined by the rate of collagen synthesis so by the maturation process where there is covalent binding of collagen fibers through inter and intra molecular cross linking. The significant increase in breaking strength, hydroxyproline concentration and dry weight of granulation tissue by this we can assume that quercetin and alcoholic extract of Mussaenda frondosa have increased collagen and have altered the maturation process by affecting cross linking of collagen or quality of collagen fibrils. Increase in dry granulation tissue weight indicates presence of higher protein content.

The measurement of hydroxyproline could be used as an index for collagen turnover. In present study there is significant increase in hydroxyproline content the animals treated with quercetin and alcoholic extract indicating rapid collagen turnover.

The alcoholic extract of Mussaenda frondosa 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.²⁶

Flavonoids²⁷ 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.

Between the three tested groups the wound healing activity of the constituent quercetin was comparatively more. In the alcoholic extract the lesser activity may be due to the presence of other chemical constituents that hindered the activity of quercetin

CONCLUSION:

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

To conclude the quercetin and alcoholic extract of Mussaenda frondosa Linn. exibited significant wound healing activity in excision, incision and dead space wound model. Hence the present findings provide scientific evidence that Mussaenda frondosa Linn as potent wound healer.

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Received on 10.08.2011

Accepted on 30.09.2011

Research Journal of Pharmacognosy and Phytochemistry. 3(6): Nov. - Dec. 2011, 266-271

Group	Mean ± SEM of % of wound closure on				Epithilization Time (Days)	Scar Area (mm²)
Group	4^th day	8^th day	12^th day	16^th day	Epithilization Time (Days)	Scar Area (mm²)
Control	12.20± 1.00	22.87± 1.29	36.20± 1.66	63.95± 3.34	25.52± 1.49	5.22± 1.41
Quercetin	21.13± 1.00***	39.53± 2.90***	66.22± 2.13***	93.32± 1.95***	12.70± 0.34***	6.80± 0.38***
Alcoholic extract	20.20 ± 1.05***	37.93± 2.81***	62.95± 3.15***	90.43± 2.11***	13.88± 0.36***	7.83± 0.52***