Wound-Healing Effect of Aqueous Anthocephalus cadamba Leaf Extract....Prabhakar Kumar

1. Wound-Healing Effect of Aqueous Anthocephalus cadamba Leaf Extract in a
Diabetic Rat Model.
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Presented by-
Prabhakar Kumar
En. no- 67/MPB/SPS/2021
DPSRU, New Delhi
Supervisor
Dr. Mukesh Nandave
Associate Professor
Department of Pharmaceutical Biotechnology
DPSRU, New Delhi
Delhi Pharmaceutical Sciences and Research University, New Delhi-110017
Journal Club Presentation

2. Title: Accelerative Wound-Healing Effect of Aqueous
Anthocephalus Cadamba Leaf Extract in a Diabetic Rat
Model
Author’s Name: Shoket Ali, Sharmeen Ishteyaque, Foziya
Khan, Pragati Singh, Abhishek Soni and Madhav N.
Mugale*
Journal Name: The International Journal of lower
Extremity Wounds
Impact factor: 2.057
Publisher: SAGE
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3. Introduction
Cascade of biomolecular and cellular events like hemostasis, inflammation, cellular
migration and remodeling.
These processes replace dead debris and cellular layers and restore injured wound tissue to its
original state by formation of collagen connective tissue.
The pathogenic mechanism of delayed diabetic wound healing includes prolonged
inflammatory and oxidative states, deferred proliferation and remodeling phase.
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4. Aim & Objective
Current study was designed to understand the mechanism involved in
wound healing and therapeutic potential of Anthocephalus cadamba in
a diabetic rat model.
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5. Methodology
Schematic Presentation
Extract
Animal (Disease Induction)
Excision wound model
Analysis
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Histopathology Analysis
Biochemical
Western Blotting

6. Procurement of Animal
The female Sprague Dawley rats of 5 to 7 weeks and body weight 150 to 200 g were used. The
total number of animals (n=40) was divided into 4 groups (10 rats/group). Rats were housed in
polypropylene cages under relative humidity (RH 55 ±20%) and room temperature (22±3 °C) in a
12 h light/dark cycle.
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7. Induction of Diabetes
Diabetes was induced by single injection of STZ (Streptozotocin) at the dose of 60
mg/kg intra-peritoneally (dissolved in citrate buffer pH of 4.5; except for the
nondiabetic control group (I) rat).
After 4 to 5 days of STZ administration, the blood glucose level was measured.
Animals with fasting blood glucose levels >250 mg/dL were assumed to be diabetic
and used in the study.
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8. Excision Diabetic Wound Model
All the rats were anesthetized with ketamine (87 mg/kg) and xylazine (13 mg/kg) and their dorsal
(last thorax to first sacral vertebra) hair was shaved.
Wound field sketching was outlined by tracing a circle on butter paper with a marker. On the
dorsal midline, one full-thickness circular excision of 1.77 cm2 area was created (1.5 cm in
diameter).
 A surgically sterile deep skin wound was created in both diabetic rats and nondiabetic rats. All
wounds were cleaned daily with 0.9% sterile normal saline solution.
After cleaning, the extract was applied topically at 500 mg/kg evenly to cover the wounds.
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9. Experimental Design
The animals were randomly divided into 4 groups:-
Group I (NDC) (n = 10): normal control without diabetes (served as untreated negative
control).14
Group II (DC) (n = 10): diabetic control (wounds allowed to heal naturally without any
treatment).
Group III (D +KPLE) (n = 10): diabetic rats treated with the topical application of A cadamba
aqueous extract (500 mg/kg), once a day for 28 days.
Group IV (5% D +PIS) (n = 10): diabetic rats treated with topical application of marketed 5% PIS
solution/ marketed standard treatment group on wounds for 28 consecutive days.
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10. Statistical analysis was carried out and the results were expressed as mean ± SD.
All results were analyzed by one-way analysis of variance at a level of
significance P< .05, P < .01, P < .001, and 0.0001 (GraphPad Prism, Version
7.0).
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Results
(D +KPLE) showed a significant increase in the percentage of wound closure
(82%) at day 21 as compared to the diabetic control group (42%), nondiabetic
control group (I) (49%), and povidone–iodine
treatment group (75%) group (IV).

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Groups Day 1 Day 7 Day 14 Day 21 Day 28
NDC Gropus 1.77±0 1.41±0.13 0.88±0.3 0.58±0.4 Scar
DC group II 1.77±0 1.37±0.08 0.95±0.16 0.64±0.28 0.2±0.4
D+KPLE group IIII 1.77±0 1.21±0.05 0.36±0.09*** 0.071 ±0.08*** Scar
5% D+PIS group IV 1.77±0 1.33±0.08 0.28±0.1*** 0.15±0.05** Scar
DC, diabetic control; NDC, nondiabetic control; D+KPLE, diabetic+Kadam plant leaf extract; D+PIS, diabetic+povidone–iodine sol.

12. Results:- Body Weight and Hydroxyproline
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Figure 1. Change in body weight of different groups. *P
< .05,
**P < .01, ***P < .001 (significant when compared to the
nondiabetic control group).
Figure 2. Hydroxyproline content in the wound tissue. *P
< .05, **P < .01, ***P < .001 (significant when compared
to the nondiabetic control group).

13. Results:- Feed Intake and Blood glucose
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Figure 4. Blood glucose levels of different groups. *P <
.05, **P < .01, ***P < .001 (significant when compared
to the nondiabetic control group).
Figure 3. Feed intake (gram) in different treatment
groups (no statistical differences were found between
groups).

14. Results:- Vascular endothelial growth factor
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Figure 5. Quantification of vascular endothelial growth factor (VEGF) in different groups evaluated by normalizing
against β-actin.
*P < .05, **P < .01, ***P < .001 (significant when compared to the nondiabetic control group).

15. Results:- Histopathology
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Figure 6. Rat: wound area histopathology at 21 and 28 post wounding days in different groups (scale bar=10×,
hematoxylin & eosin [H&E] staining).

16. Results:-
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Figure 7. Rat: wound area Masson’s trichrome staining in different groups (scale bar=10×, hematoxylin & eosin
[H&E] staining).

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Conclusion
The present study demonstrates that the topical application of aqueous leaf extract:
 Kadam plant indicates novel healing properties of wounds in diabetic rats.
 The healing effects seemed to be due to an increased amount of hydroxyproline, fibroblasts, granulation tissue,
total protein, and collagen deposition in the wound.
 Therefore, the findings of the current study showed the extract promotes healing of diabetic wound in rats and
provides a scientific perspective for the use of A cadamba aqueous leaf extract as an alternative to synthetic
drugs with expensive costs and adverse effects.
 However, to the best of our knowledge, this is probably the first study that evaluates the wound-healing effect of
A cadamba leaf extract in diabetic wounds.

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References
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methanolic extract in streptozotocin-induced diabetic rats. J Wound Care. 2019;28(Sup10):S26-S36.
2. Ponrasu T, Suguna L. Efficacy of Annona squamosa L in the synthesis of glycosaminoglycans and collagen during wound
repair in streptozotocin induced diabetic rats. Biomed Res Int. 2014;2014:1-10. doi:10.1155/2014/124352
3. TanWS, Arulselvan P, Ng SF, Taib CN, SarianMN, Fakurazi S. Improvement of diabetic wound healing by topical application
of vicenin-2 hydrocolloid film on Sprague Dawley rats. BMC Compl Alternative Med. 2019;19(1):1-6.
4. Harsha L, Brundha MP. Role of collagen in wound healing, Drug Invent Today. 2020;13(1):55-57.
5. Barrett EJ, Liu Z, Khamais M, et al. Diabetic microvascular disease: an endocrine society scientific statement. J Clin
Endocrinol Metab. 2017;102(12):4343-4410.

19. Thank You 19