Study of wound healing activity of Tectona grandis Linn. leaf extract on rats.

AIMS: The aim of the study is to determine the wound healing activity of Tectona grandis (TG) Linn. leaf extract on rats.
MATERIALS AND METHODS: Healthy albino rats (150-200 g) of either sex were taken for excision and incision wound model. Animals were divided into four groups of six animals in each group. For Group simple ointment served as control. The Groups 2 and 3 had 5 and 10% ointment of TG leaf extract and Group 4 soframycin ointment served as standard. In excision wound percentage of wound contraction was assessed, whereas in incision wound tensile strength was assessed. Statistical analysis was performed by one-way analysis of variance followed by t-test.
RESULTS: In excision wound model, 5% ointment of TG leaf extract showed a reduction in wound area 8(th) day onwards. Reduction in wound area was very significant (P < 0.01) as compared to control. Whereas 10% ointment of TG leaf extract and standard showed a reduction in wound area fourth day onwards, which was highly significant (P < 0.001) as compared to control. In incision wound model, animals treated with 5% ointment of TG leaf extract showed significant (P < 0.05) increase in tensile strength as compare to control. However, animals treated with 10% ointment of TG leaf extract showed very significant (P < 0.001) increase in tensile strength as compare with control. However, animals treated with soframycin showed highly significant (P < 0.001) increase in tensile strength as compare with control.
CONCLUSIONS: TG leaf extract showed significant wound healing activity.

INTRODUCTION

Natural products are a source of synthetic and traditional herbal healing activities or antimicrobial and other related properties medicine.[1] Some medicinal plants have been employed in folk medicine for wound care.[2345678] Some of these plants either possess pro-woundhealing activities or antimicrobial and other related properties which are beneficial in overall wound care. Tectona grandis (TG) Linn. Family Verbenaceae also known as teak in English, Śāka in Sanskrit. Literature reveals that almost all parts of the plant possess various medicinal properties, including leaves which are used for hemostatic, depurative, anti-inflammatory, vulnery, leprosy, skin diseases, puritus, stomatitis, indolent ulcers, hemorrhages, hemoptysis, vitiated conditions of pitta.[9101112131415]

MATERIALS AND METHODS

Materials

Animals

The present study was conducted in healthy adult Wistar rats of either sex weighing 150-250 g. They were maintained under standard laboratory conditions (12 h light and dark cycle and temperature of 27°C ± 2°C), with access to food and water.

Drugs and instruments

Pentobarbitone (Loba Cheme IndoAustranal Co, Mumbai)

Soframycin (Aventis Pharma Limited, Goa)

Simple ointment soft and hard paraffin, lanolin

Percolator Borosil (Alka Scientific Co. Nagpur)

Silk (3-0) (Centenial Surgical Suture Limited, Thane, Mumbai).

Plant material

Collection of the plant materials

The frontal leaves of TG were collected from nearby area and authenticated by the Local Botanist of Science College. Frontal leaves were shade dried and powdered in the Department of Pharmacology.

Preparation of extract

The powder of leaves was macerated for 24 h in 70% ethanol. The hydro-alcoholic extracts were obtained by percolation using 70% ethanol as a solvent. Percolated solution was again shade dried and extract was obtained. The yield of extract was around 25%.

Preparation of drug formulation

TG leaf extract was mixed with simple ointment as mentioned below:

Preparation of ointment- Simple ointment (100 g IP):

Soft paraffin - 85 g

Hard paraffin - 10 g

Fat - 5 g.

5% ointment:

Simple ointment - 95

TG leaf extract -5.

10% ointment:

Simple ointment - 90g

TG leaf extract -10 g.

Ethical clearance

Ethical clearance was taken from Institutional Animal Ethics Committee.

Methods

Groups for excision and incision wound model

The albino Wistar rats of either sex were divided into four groups, six animals in each group (total 24 animals).

Group 1: Control-simple ointment locally once daily

Group 2: TG leaf extract (5% ointment) locally once daily

Group 3: TG leaf extract (10% ointment) locally once daily

Group 4: Standard drug soframycin ointment locally once daily.

Method for evaluating wound healing activity

Excision wound model

The animals were anaesthetized by using pentobarbitone (30 mg/kg i.p). An impression was made on the dorsal thoracic region 1 cm away from the vertebral column and 5 cm away from ear on the anaesthetized rat. The particular skin area was shaved 1 day prior to the experiment. The skin of impressed area was excised to the full thickness to obtain a wound area of about 500 mm2. Hemostasis was achieved by blotting the wound with a cotton swab soaked in normal saline. The wound contraction was studied by tracing the raw wound area on the subsequent days 1, 4, 8, 12, 16, 18 and 21 on graph paper. Scar area and time for complete epithelization were also measured. The percentage of wound contraction was recorded.[1617]

Incision wound model

The animals were anaesthetized by using pentobarbitone (30 mg/kg i.p). Para vertebral straight incision of 6 cm length was made through the entire thickness of the skin, on either side of the vertebral column with the help of a sharp scalpel. After complete hemostasis, the wound was closed by means of interrupted sutures placed at equidistant points about 1 cm apart. Animals were treated once a day with drugs (TG ointment and soframycin) from 0 day to 9 post-wounding day. The wound breaking strength was estimated on 10 day by continuous, constant water flow technique as describe by Lee. The breaking strength was expressed as minimum weight of water necessary to bring about gaping of area.[181920]

Statistical analysis

All the results were expressed as mean ± standard deviation. The differences between experimental groups were compared by one-way analysis of variance followed by t-test. The results were considered to be statistically significant when *P < 0.05 - significant, **P < 0.01 - very significant, ***P < 0.001 - Highly significant as compare with control.

RESULTS

In excision wound model, 5% ointment of TG leaf extract showed a reduction in wound area on day 4, which was not significant but day 8 onwards reduction in wound area was very significant (**P < 0.01) as compared with control. However 10% ointment of TG leaf extract and standard showed a reduction in wound area on day 4 onwards, which was highly significant (***P < 0.001) as compared to control [Table 1].

Table 1

Wound healing activity of Tectona grandis leaf extract by excision wound model on rats

In incision wound model, animals treated with 5% ointment of TG leaf extract showed significant (P < 0.05) increase in tensile strength as compare with control. However, animals treated with 10% ointment of TG leaf extract showed very significant (**P < 0.01) increase in tensile strength as compare to control. However, animals treated with soframycin showed highly significant (***P < 0.001) increase in tensile strength as compared to control [Table 2].

Table 2

Wound healing activity of Tectona grandis leaf extract by incision wound model on rats

DISCUSSION

According to Naira and Karvekar frontal leaves of TG contains phenol and flavonoids 26 μg/g and 15.07 μg/g respectively.[21]

Majumdar suggests that the antioxidant property of the TG leaves, due to the presence of high amounts of tannin may also be responsible to the prohealing action of the extract.[22]

Literature survey has revealed that tannins promote wound healing activity through several mechanisms that include chelation of free radicals; antioxidant, antimicrobial and astringent property.[23] Phenolic acids have been reported to possess anti-inflammatory, analgesic, anti-oxidant and wound healing properties.[24]

Lipid peroxidation results in cellular membrane damage, which leads to swelling and cell death. The free radicals attract the different inflammatory mediators that are responsible for the general inflammatory response and tissue damage. During the injury, there is an increase in the consumption of the endogenous antioxidants that bring about a decrease the amount of antioxidants. Flavonoids may contribute an additive effect to the endogenous antioxidants and to inhibit the eicosanoid biosynthesis therefore decreasing the formation of the inflammatory metabolites, which is responsible for its anti-inflammatory property. Hence, by virtue of their free radical scavenging, antioxidant and anti-inflammatory properties, flavonoids may help in healing of wounds.[25]

In excision wound model, 5% ointment of TG leaf extract showed significant (**P < 0.01) reduction in wound area day 8 onwards as compared to control. Whereas, 10% ointment of TG leaf extract and standard drug soframycin showed a reduction in wound area day 4 onwards, which was highly significant (***P < 0.001) as compared to control [Table 1].

In incision wound model, animals treated with 5% ointment, 10% ointment of TG leaf extract and standard drug (soframycin) showed significant (*P < 0.05, **P < 0.01, ***P < 0.001) increase in tensile strength as compare with control [Table 2].

Thus, it can be suggested that phytochemical constituents present in TG leaves may be responsible for its wound healing activity. Hence, this present research supports traditional claims of the plant in wound healing. Thus, it may be concluded that TG leaf extract have the potential to satisfy all requirements of an ideal dressing material. In that it provides an environment at the surface of the wound in which healing can take place at the maximum rate. However, further study is needed to provide a rationale for the use of TG leaves preparations in the traditional system of medicine to promote wound healing.

Furthermore, it can be concluded that the TG leaf extract has a beneficial effect as an injury healing promoter.