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SCREENING PROCEDUE OF WOUND HEALING ANDANTIDIARRHEAL

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SCREENING OF WOUND HEALING Prensented by :- SNEHIL SINGH (P’CHEMISTRY) M.PHARMA (AIBS KANPUR) :

SCREENING OF WOUND HEALING Prensented by :- SNEHIL SINGH (P’CHEMISTRY) M.PHARMA (AIBS KANPUR)

Introduction :

Wound may be defined as loss or breaking of cellular and anatomic or functional continuity of living tissue. Introduction

Pharmacological screening :

Animal used : Sprague Dawley male rats Groups : Total Six groups Animal per group: EIGHT Pharmacological screening

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Animals After infliction of wound

Materials and methods:

Materials and methods Preparation of asiaticoside dosage forms Different concentrations of solution in sterile saline were prepared for in vivo studies. Methylcellulose disks were loaded with 20–80 mg asiaticoside for in vitro tests. A gum acacia suspension in water was used for oral administration . Models for wound healing activity In vivo- In vitro-

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STRUCTURE OF ASIATICOSIDE

In vivo models :

Normal animals :- Guinea pigs (male, 300– 325) were used in the study. Four cutaneous (full thickness, completely transdermal ) circular wounds of 8 mm diameter were made on the pre-shaved, sterile (wiped with 70% alcohol) dorsal surface of the animal with the help of a biopsy punch ( Acuderm , Louderole , USA). All surgical procedures were carried out under thiopentone sodium (25 mg:kg , i.p.) anaesthesia . Animals were allowed to recover and were housed individually in metallic cages containing autoclaved paper cuttings. They received food and water . In vivo models

Diabetic animals. :

Sprague Dawley male rats (150–180 g) were made diabetic by being given a single injection of streptozotocin (STZ) prepared in citrate buffer (0.1 M, pH 4.5) (50 mg:kg , i.p .) after overnight fasting. Blood was drawn from the orbital plexus 24 h after the injection and the glucose level was estimated using Glucometer (Ames, Bayer Diagnostic, India). Wounds were made on the rats showing elevated blood glucose (more than 250 mg:dl ) using the same method as described for the guinea pig. Diabetic animals.

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In the diabetic animals, 0.2 and 0.4% concentrations of asiaticoside were applied topically with the treatment schedule as in the case of guinea pigs. Blood glucose levels were estimated at the time of creation of the wounds as well as at excision of wounds to check whether the plant has got any antidiabetic activity per sec.

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Asiaticoside solution (20 ml:wound ) was applied topically in concentrations of 0.05%, 0.1%, 0.2% twice daily for 7 days. The control group received an equal amount of vehicle. In another series of experiments asiaticoside (0.5, 1.0 or 10 mg:kg ) was given orally for 7 days. Controls received the vehicle alone.

In vitro model:

Chick chorioallantoic membrane (CAM) model. This model was used to assess the angiogenic activity of asiaticoside ( Lobb et al., 1985). Nine day-old fertilised chick eggs were selected and a small window of 1.0 cm² made in the shell. A small hole was drilled at the air space and air was sucked out using a rubber bulb, as a result of which the membrane fell. In vitro model

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The window was opened and a sterile disk of methylcellulose loaded with different amounts of asiaticoside was placed in at the junction of two big vessels. The window was resealed by tape and the eggs were incubated at 37⁰C in a well-humidified chamber for 72 h. The eggs were then opened. New vessel formation was observed and compared with that in eggs containing disks without asiaticoside .

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Assessment of healing In each animal study eight animals group were taken. From each animal, two wounds were pooled to make one sample for hydroxyproline and one each was used for tensile strength and histology. Area of wound The surface area of healing (7 and 10th day) wound was measured by tracing the boundary of still open wound on semi-transparent paper and calculation of area was done by using a graph paper.

Tensile strength:

On the 7th day after creating the wound the animals were anaesthetised . Healing tissue along with normal skin at two ends was excised for tensile strength measurement using Tensile Testing Machine TKG-20 (from Fine Testing Machines, Miraz , India). Strips of 8 mm width and 20 mm length were cut out from the excised tissue in treated and control animals and were loaded between the upper and lower holder of the machine in such a way that the effective load bearing size was 88 mm with the wound remaining in the centre. The total breaking load is measured in Newtons and the tensile strength was calculated by the following equation: Tensile strength = Total breaking load x Cross-sectional area Tensile strength

Histopathological studies:

Wound tissue specimens from treated and untreated rats were collected in 10% buffered formalin after the usual processing 6 mm-thick sections were cut and stained with haematoxylin and eosin (McManus and Mowry , 1965). Sections were qualitatively assessed under the light microscope and graded in respect of congestion, oedema , infiltration of polymorphonuclear leukocytes and monocytes , necrosis, fibroblast proliferation, collagen formation, angiogenesis and epithelization . Histopathological studies

Collagen estimation:

Wound tissues were analysed for hydroxyproline content , which is a basic constituent of collagen . Tissues were dried in a hot air oven at 60–70 º C to constant weight and were hydrolysed in 6 N HCl at 130 º C for 4 h in sealed tubes. The hydrolysate was neutralised to pH 7.0 and was subjected to chloramine -T oxidation for 20 min . The reaction was terminated by addition of 0.4 M perchloric acid and colour was developed with the help of Ehrlich reagent at 60 º C ( Woessner , 1961) measured at 557 nm using a Pye Unicam spectrophotometer . Collagen estimation

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THANKS

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