Efficacy of Annona squamosa L in the synthesis of glycosaminoglycans and collagen during wound repair in streptozotocin induced diabetic rats

Abstract

The aim of this work was to find out the effects of Annona squamosa on the formation of glycosaminoglycans and collagen during wound healing in normal and diabetic rats. Diabetes induced rats were segregated into 4 groups, each containing six animals. Groups I and III served as the normal and diabetic control while groups II and IV served as normal and diabetic treated. The animals were treated with 200 μL of Annona squamosa extract topically. The granulation tissues formed were removed on the 8th day and the amount of glycosaminoglycans (GAGs) and collagen formed was evaluated by sequential extraction and SDSPAGE, respectively. Histological evaluation was also carried out using Masson's trichrome stain. In vitro wound healing efficacy of A. squamosa in human dermal fibroblast culture (HDF) was also carried out. The fibroblasts treated with varying concentrations of A. squamosa were examined for proliferation and closure of the wound area and photographed. A. squamosa increased cellular proliferation in HDF culture. The granulation tissues of treated wounds showed increased levels of glycosaminoglycans (P < 0.05) and collagen which were also confirmed by histopathology. The results strongly substantiate the beneficial effects of A. squamosa on the formation of glycosaminoglycans and collagen during wound healing.

Figure 1

Efficacy of A. squamosa extract on the proliferation of HDF cells for 48 hrs. HDF cells were cultured with graded concentrations of A. squamosa extract (7.8 ppm–1000 ppm) and the number of viable cells was determined by MTT assay after 48 hrs. The results revealed that the A. squamosa extract increased the viable cells with increasing concentrations and there is no toxic or adverse effect at any concentration. All the data were expressed as mean ± SD (n = 6) and statistically significant (*P < 0.05) when compared to control culture.

Figure 2

In vitro wound healing of HDF cells of control culture and A. squamosa treated (supplemented with 7.8–125 ppm) cultures. Proliferation of HDF cells was observed for 2 days after wound creation. The double headed black arrow shows the margin of scratched area on day 0 as initial wound (IW), and double headed white arrow shows the incomplete healing (IH), whereas white arrow shows the complete healing (CH). A. squamosa treatment shows complete healing on day 2, which clearly indicates that the A. squamosa accelerates the rate of proliferation faster in treated culture than the control.

Figure 3

(a) Glycosaminoglycans of control and A. squamosa treated wound tissues on day 8 after wounding. Values are expressed as mean ± SD for six animals. *P < 0.05 is considered as significant compared with the control. (b) Percentage proportions of various glycosaminoglycans formed in control and A. squamosa treated wound tissues on day 8 after wounding. HA: hyaluronic acid; CS C + CS A: chondroitin sulphate A and C; DS: dermatan sulphate; KS: keratan sulphate; H + HS: heparin and heparin sulphate.

Figure 4

Normal (a) and interrupted (b) SDS-PAGE photograph showing the distribution of collagen. All lanes were loaded with equal concentration of collagen (25 μg). RTT: rat tail tendon; C: control; T: treated; DC: diabetic control; DT: diabetic treated.

Figure 5

Masson's trichrome staining for collagen in control and A. squamosa treated wound tissues from first and second week, respectively (magnification 20x). Control (a) shows collagen fibers with incomplete epithelialization whereas (b) treated tissue shows dense collagen deposition. In the second week, control (c) depicts thin collagen layer at the wound site and (d) treated tissue shows complete epithelialization with uniform collagen deposition. IE: incomplete epithelialization, CE: complete epithelialization, E: epithelialization, BV: blood vessels, and C: collagen. Scale bar: 50 μm.

Figure 6

Masson's trichrome staining for collagen in control and A. squamosa treated diabetic wound tissues from first, second, and third week, respectively (magnification 20x). Control (a) shows large number of proliferating blood vessels, whereas (b) treated tissue shows less collagen formation. In the second week, control (c) depicts adequate amount of blood vessels formation and collagen fibers and (d) treated tissue shows thick collagen fiber deposition under thin epithelial layer. In the third week, control (e) shows accumulation of loosely arranged collagen bundles at the wound site and (f) explores the completed epithelial layer with highly organized collagen deposition. IE: incomplete epithelialization, E: epithelialization, C: collagen, and BV: blood vessels. Scale bar: 50 μm.