Screening Methods for Wound-Healing Agents

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Chapter: Pharmacognosy and Phytochemistry : Biological Screening of Herbal Drugs

The extracts obtained from plants are usually made into different formulations, either as ointment or as lotion and applied to the skin wound. Sometimes it is used internally or even injected if required depending on the nature of the constituents. The models usually used for the evaluation of the wound-healing activity can be described as follows:


SCREENING METHODS FOR WOUND-HEALING AGENTS

 

 

The extracts obtained from plants are usually made into different formulations, either as ointment or as lotion and applied to the skin wound. Sometimes it is used internally or even injected if required depending on the nature of the constituents. The models usually used for the evaluation of the wound-healing activity can be described as follows:

 

Excision Wound Model

 

Four groups of animals containing ten in each group are to be anaesthetized by open mask method with anaesthetic ether. The rats are depilated on the back. One excision, wound is inflicted by cutting away 500 mm2 full thickness of skin of a predetermined area. Rats are left undressed to the open environment. Then the drug, that is, the refer-ence standard (0.2% w/w nitrofurazone ointment), simple ointment BP (control), and test drug ointment or different other forms are administered till the wound is completely healed. This model is used to monitor wound contraction and epithelialization time. Epithelialization time is noted as the number of days after wounding required for the scar to fall off leaving no raw wound behind. Wound contrac-tion is calculated as percent reduction in wound area. The progressive changes in wound area are monitored plani-metrically by tracing the wound margin on a graph paper every alternate day. To determine the changes in healing of wound measurement of wound, area on graph paper is expressed as unit (mm2). For histopathological exami-nation, tissues are collected from the completely healed wound when the scar is removed. A transverse section of tissue is prepared from each group of rat and stained with haematoxilin and eosin to reveal the tissue section clearly. Then the tissues are observed under microscope to study different histopathological phenomenon.

 

Incision Wound Model

 

Four groups of animals containing ten in each group are anaesthetized, and two paravertebral long incisions of 6 cm length are made through the skin and cutaneous muscles at a distance of about 1.5 cm from midline on each side of the depilated back of rat. Full aseptic measures are not taken and no local or systemic antimicrobials are used throughout the experiment. All the groups are treated in the same manner as mentioned in case of excision wound model. No ligature is to be used for stitching. After the incision is made, the parted skin is kept together and stitched with black silk by 0.5 cm apart. Surgical thread (No. 000) and curved needle (No. 11) are used for stitching. The continuous threads on both wound edges are tightened for good adoption of wound. The wound was left undressed. The ointment of extract, standard drug (nitrofurazone ointment), and simple ointment BP is applied to the wound twice daily or feeded daily until complete recovery, to the respective groups of animals.

 

Tensiometer

 

It consists of a 6 × 12-inch wooden board with one arm of 4-inch long, fixed on each side of the possible longest distance of the board. The board is placed at the edge of a table. A pulley with bearing is mounted on the top of one arm. An alligator clamp with 1-cm width is tied on the tip of another arm by a fishing line (20-lb test monofilament) in such a way that the clamp could reach the middle of the board. Another alligator clamp is tied on a longer fishing line with 1 litre polyethylene bottle on the other end.

 

Tensile strength of wound represents the promotion of wound healing. Usually wound-healing agents promote the gaining of tensile strength. Tensile strength (the force required to open the healing skin) was used to measure the amount of healing. The instrument used for this purpose is called as tensiometer, which is explained as above. This was designated on the same principle as the thread tested in textile industry. One day before performing the experi-ment (measurement of tensile strength) the sutures are removed from the stitched wounds of rats after recovery and tensile strength is measured as follows.

 

Determination of Tensile Strength

 

The sutures are removed on ninth day of wounding and the tensile strength is measured on 10th day. Extract ointments along with simple ointment (control) and nitrofurazone ointment (standard) are administered through out the period, twice daily for 9 days. On 10th day again the rats are anaesthetized, and each rat is placed on a stack of paper towels on the middle of the board. The amount of the towels could be adjusted in such a way so that the wound is on the same level of the tips of the arms. The clamps are then carefully clamped on the skin of the opposite sides of the wound at a distance of 0.5 cm away from the wound. The longer pieces of the fishing line are placed on the pulley and finally to polyethylene bottle, and the position of the board is adjusted so that the bottle receive a rapid and con-stant rate of water from a large reservoir, until the wound began to open. The amount of water in the polyethylene bag is weighed and considered as tensile strength of the wound. The mean determinations are made on both sides of the animals and are taken as the measures of the tensile strength of the wound. The tensile strengths of the extract and nitrofurazone ointment treated wounds are compared with control. Tensile strength increment indicates better wound-healing promotions of the applied drug.

 

 

Dead Space Wound

 

Three groups of animals containing ten in each group are anaesthetized by open mask method with anaesthetic ether. Dead space wounds are created by subcutaneous implantation of sterilized polypropylene tubes (2.5 × 5 cm). The test drug is administered at different doses based on the design of the experiment for a period of ten days. The granuloma tissues formed on the tubes are harvested on the 10th postwounding day. The buffer extract of the wet granuloma tissue is used for the determination of lysyl oxidase activity, protein content, and tensile strength. Part of granuloma tissue is dried, and the acid hydrolysate is used for the determination of hydroxyproline, hexosamine, and hexuronic acid.

 

The progresses of wound healing in excision and inci-sion wound method have to be studied The measurement of the tensile strength, that is, the effect of the extract and standard drug on the wound-healing process by incision wound method have to be studied. Results are expressed as mean ± SE and compared with the corresponding control (simple ointment) values; p-values are calculated by student’s t-test by comparing with control. Percentages of wound contractions are calculated with respect to the corresponding 0 day’s wound area (mm2).

 

The contractions of wound with all the drugs comparing with simple ointment (control) are measured. The epithe-lialization period of the wound area of the extract treated group are compared with standard drug treated group. Tensile strength of wounds of rats treated with standard drug (nitrofurazone ointment), in case of incision wound model is measured increment in tensile strength indicates better wound healing.

 

The histopathological examination of the tissues of the wound area treated with extract, standard drug is performed. In these studies, test drugs (herbals) with good activity showed rapid increase in tissue regeneration in skin wounds, more relative fibrosis. The skin adrenal structures like pilocebaceous glands, sweat glands, etc. are better presented in wounds treated with extract compared to standard drug treated animal wounds.

 

The changes in the biochemical parameters affecting wound healing in dead space wound model like, granuloma weight, lysyl oxidase activity, as well as protein content are to be measured which is usually increased with effective test drugs. The hydroxyproline, hexuronic acid, hexosamine level are measured which are increased considerably. The observed increase in tensile strength could be attributed to the increase in lysyl oxidase activity which is responsible for cross-linking and maturation of collagen. The reduction in granuloma weight is also due to better maturation of collagen, which invariably leads to shrinkage of granulation tissue. However in this case, the observed increase in tensile strength is not only due to increased cross linking via lysyl oxidase but also due possibly to interactions (noncovalent, electrostatic) with the ground substance as evidenced by a highly significant increase in the hexosamine content.

 

Wound healing involves different phases such as con-traction, epithelialization, granulation, collagenation, etc. The glycosidal mixture of extract of Centella asiatica has been reported to be responsible to enhance incised wounds healing (Rosen et al., 1967) and in stimulating collagen in human skin fibroblast cell (Vogel and DeSouza, 1980).

 

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