Vidhyabharti College of Pharmacy, Camp Road, Amravati 444601, Maharashtra India.
Wound healing is a complex process involving interactions between the immune and biological systems, with diabetes mellitus (DM) often leading to delayed wound healing. Adenanthera pavonina L. a tropical tree known for its medicinal properties, was investigated for its wound healing potential in diabetic mice. Ethanolic extracts of A. pavonina leaves were prepared and incorporated into cream formulations at 10% and 20% concentrations. Diabetes was induced in mice using alloxan, and excision wounds were created. The wound healing effects of the extract creams were compared to a control group, diabetic group, and a standard treatment group using 1% Soframycin cream. Wound contraction rate, epithelialization period, and histological parameters were assessed. Phytochemical analysis revealed the presence of flavonoids, saponins, tannins, alkaloids, and other bioactive compounds in the leaf extract. The 20% extract cream showed significant wound contraction and faster epithelialization compared to the control and diabetic groups, with effects comparable to Soframycin cream. Histological examination indicated enhanced tissue regeneration and collagen deposition in the extract-treated groups. The wound healing activity of A. pavonina leaf extract in diabetic mice is attributed to the presence of bioactive compounds, which may promote collagen synthesis, cell proliferation, and angiogenesis while reducing inflammation. These findings support the traditional use of A. pavonina for wound treatment and suggest its potential as an alternative therapy for wound management in diabetic patients.
A wound is defined as an impairment of the skin's structural and functional integrity at the affected site. A wound is a breach in the integrity of epithelium and it may also be followed by a disturbance of the structure and function of the normal tissue beneath. Thus, maintaining the skin's normal structural and functional integrity requires healing as a survival mechanism Healing of wounds, a fundamental response to tissue injury occurs by a process of connective tissue repair. Wound healing is the body's normal reaction to tissue damage. Wound healing is by no means a simple process, since the vascular system, cytokines, mediators, and several cell types interact delicately. The complex and time-consuming process of wound healing involves interactions between the immune and biological systems. A fibrous scar is the end product of this process, the pre-dominant constituent of which is collagen. The goal of the initial cascade of platelet aggregation and blood vessel constriction is to stop bleeding. After this, a variety of inflammatory cells start to infiltrate, starting with neutrophils. Diabetes mellitus (DM) is a serious worldwide public health issue and a chronic illness. Globally, there were 451 million adults (18 to 80 years old) living with diabetes in 2017, and by 2045, that number is predicted to rise to 693 million. Because of their macrovascular and microvascular abnormalities, people with diabetes often experience delayed wound healing spending each year, with diabetic wounds accounting for a significant portion of this amount. The morbidity, mortality, and quality of life of patients are significantly impacted over an extended period of time by DWs, which are more persistent and complex. To investigate novel therapy treatments and make further advancements in the current treatment modalities. The scientific name of Adenanthera pavonina L. is derived from the Greek words aden, meaning gland, and anthera, meaning anther. Commonly known as Red Sandalwood, Coral Wood, or Bead Tree, Adenanthera pavonina is a remarkable tropical tree indigenous to Southeast Asia, now widely found in tropical and subtropical areas globally. It is especially noted for its vibrant red seeds, which are frequently used as beads in jewelry and traditional decorations. As a member of the Fabaceae (legume) family, Adenanthera pavonina is a rapidly growing deciduous tree that can attain heights of 6–15 meters. It features compound leaves and produces fragrant yellow flowers that eventually develop into curved pods containing its distinctive red seeds. Its numerous pharmacological properties, particularly those associated with its leaves, bark, and seeds, render it an important therapeutic plant, often referred to as "raktakombol." The red bead tree, classifiedwithin the subfamily Mimosaceae of the Leguminosae (Fabaceae) family, is a notable medicinal plant indigenous to the Indian subcontinent. This species is originally from tropical Asia and thrives in moist soil conditions Due to the Presence of flavonoids, alkaloids and Tannins. The present study was planned to Evaluate wound healing activity in diabetic mice in Experimental Animal.
MATERIALS AND METHODS
Animal and Housing condition
The experiment is performed on albino mice (weighing 25-30gm), which are obtained from the animal house of Department of Pharmacology. Vidyabharati college of pharmacy, Amravati. ( 1504/PO/RE/S/11/CPCSEA). All the animals are acclimatized to the animal house prior to use. They are kept in cages in animal house with n 12 h light: 12 h dark cycle. Animals are fed on pellets and top water ad libitum. The care and handling of mice were in accordance with the internationally accepted standard guidelines for use of animals (CPCSEA).Permission und approval animal studies were obtained from the Institutional Animal Ethics Committee (IAEC) of Vidyabharati college of Pharmacy, Amravati. SG) Amravati University.
Plant Collection Identification and Extract Preparation
Collection and Authentication of plant
The plant of Adenanthera pavonina has been collected from local area of Akot Dist. Akola, Maharashtra, India. The Plant were identified and authenticated by Ms L.D. Khalid mam, Department of Botany, Vidyabhrati Mahavidyalaya, Amravati.
Extraction Process
The heartwood of Adenanthera Pavonina was processed by washing with clean water, air-drying, pulverizing, and sieving through a 0.3 mm sieve. tool consists of several parts including a heat round bottom flask, Soxhlet extractor, and condenser. The solid coarsely powdered were placed in thimble and placed in an extractor. The bottom end of the extractor was connected to a round bottom flask containing a solvent (Ethanol 1000ml was chosen as the solvent), and was connected to a reflux Condenser. Process was repeated until the all the material extracted from the solid leaves powder. The percentage yield of the extract was calculated and the extract was then subjected to different phytochemical tests.
Drugs And Chemicals
A) Inducing Agent: Alloxan Monohydrate (120mg/kg) was used to induced diabetes on mice. CASNumber: 2244-11-3 Was purchased from Mass Dye Chem, Pvt. LTD, Pune, India
B) Standard Drug: 1%Soframycin cream was purchased by Shiv medical, Amravati.
C) Test Drug: Adenanthera pavonina plant were obtain from local areas
Phytochemical Screening
Qualitative Phytochemical investigation
Qualitative chemical tests were conducted in order to identify the various phytoconstituents Present in Adenanthera pavonina.
Preparation Of Formulation
Table 01: Formula for the preparation of Cream
|
Sr no |
Ingredients |
Quantity |
|
1 |
Cetyl alcohol |
10gm |
|
2 |
Soft white paraffine wax |
10gm |
|
3 |
Liquid paraffin |
29ml |
|
4 |
Water |
50ml |
|
5 |
EEAP |
1gm |
Induction Of Diabetes
After mice were adapted for 1 week, induction of diabetes was prepared by injecting alloxan (120 mg/kg) intraperitoneally after overnight (12 hrs) of fasting. Immediately after alloxan induction, mice were given food necessarily. Alloxan was applied in an isotonic saline solution and the In the first 48 hours after alloxan induction, mice were given 10% glucose orally to avoid hypoglycemia and to prevent mice death. On day 7 after alloxan induction, mice blood glucose levels were measured by glucometer . Mice with blood glucose >150mg/dL were confirmed as diabetic mice.
Grouping And Dosing of Animal
Healthy white albino mice (25-30g) and 6-8 weeks old age are used. five groups of mice is used each containing six mice were used. Animal in Group I will treated with Simple cream Base as control, Group II will treated as inducing agent i.e Alloxan (120mg/kg) i.p and with simple Cream base, Group III as Standard treated with 1% Soframycin cream, Group IV and V will be treated with 10%and 20% extract cream respectively. All the experiments are conducted in accordance with the internationally accepted guideline for Laboratory animal use and care
Excision Wound Model: -
On wounding day, animals were anesthetized by using antiesthetic ether. After wound area preparation with 70% alcohol, the dorsal fur of the animals was shaved with shaving machine and the anticipated area of the wound to be created was outlined on the back of the animals on the dorsal thoracic region 1 cm away from vertebral column on the anesthetized mouse. Full thickness circular excision wounds sized about 300 mm² were created along the markings using toothed forceps, scalpel, and scissors. Hemostasis was achieved by blotting the wound with cotton swab soaked in normal saline. The entire wound was left open. The mice were divided into five groups (6 mice per group) randomly and each mouse was placed in a separated cage. The treatment was done once daily topically in all the cases .The wounding day was considered as day 0. The standard drug, extract and simple cream base will applied topically for 21 days once in day.
Measurement Of Wound Contraction Rate:
The wound closure rate was assessed by tracing the wound on days 0,3,7,10,14,21days using transparent paper and a permanent marker. The wound areas recorded were measured using 1 mm² scale of graph paper. Changes in wound area were evaluated, giving an indication of the rate of wound contraction and epithelialization period. The evaluated surface area was used to calculate the percentage of wound contraction, taking initial size of the wound as 100% as shown below:
Percentage Wound closure - Ao-Ad/Ao x 100
Where,
Ao = Wound area on zero day
Ad = wound area on corresponding days like 3,7,10,14, 21 day .
Epithelization Period Measurement
Falling of scab leaving no raw wound behind was taken as end point of complete epithelialization and the days required for this were taken as period of epithelialization.
Statistical Analysis
The results are expressed as mean standard error of mean (SEM) for each group. The statistical significance was analysed using one-way analysis of variance (ANOVA) followed by Tukey multiple comparision test was employed and P value <0.01 was considered statistically significant
RESULT
Table: 1 Phytochemical Analysis of ethanolic extract of Adenanthera Pavonina
|
Sr. No |
Chemical Constituents |
Results |
|
1 |
Carbohydrate |
+ |
|
2 |
Glycosides |
+ |
|
3 |
Alkaloids |
+ |
|
4 |
Phytosterols |
- |
|
5 |
Saponins |
+ |
|
6 |
Phenols |
+ |
|
7 |
Tannins |
+ |
|
8 |
Flavonoids |
+ |
|
9 |
Protein and Amino acids |
+ |
Where (+) indicates Present& (- ) indicates Absent
Phytochemical testing carried out to find out the secondary metabolite because secondary metabolic possess biological activity. Phytochemical studies of Adenanthera pavonina performed for the presence of, Glycosides, Alkaloids, Saponins, Tannins, Flavonoids, Protein.
Wound Contraction Rate
Table No 2: Effect of topical application of the EEAP on wound contraction of excision wound model in mice
|
Groups |
Day0 |
Day7 |
Day14 |
Day21 |
|
Control |
0.0 ±0.0 |
24.8 ±0.6 |
58.3±0.8 |
93.8 ± 0.6 |
|
Diabetic |
0.0 ±0.0 |
11.5 ±0.4 |
35.1 ±0.6 |
71.1 ± 0.6 |
|
1%Soframycin |
0.0 ±0.0 |
32.5 ±1.0 |
65.0± 1.0* |
97.5 ± 1.0* |
|
10%EEAP |
0.0 ±0.0 |
16.5 ±0.4 |
44.0 ±0.5* |
82.0 ± 0.5* |
|
20%EEAP |
0.0 ±0.0 |
19.00 ±1.4 |
48.0 ± 1.4* |
86.5 ± 1.0* |
Values are expressed as mean ± SEM ( n = 6 animals in each group) and analyzed by Two- way ANOVA followed by Tukey multiple comparision test compared with simple cream base (control group) *p<0.0001 and compared and Diabetic group *p< 0.0001. Similar percentages of wound contraction were observed in animals treated with the 20%EEAP cream from the 10th to 21th day. The 20% extract cream revealed better observable effect compared to the soframycin cream. The animals treated with 10%EEAP extract cream showed significant wound contraction from 14th day onward as compared to control group (p< 0.0001) Significant wound contraction was also observe Soframycin treated group from 3rd day onward as compared to extract.However, there was no significant difference in wound healing activity between Soframycin cream and 20% EEAP, Furthermore, complete wound closure was observed in 20% (w/w) EEAP extract and 1%soframycin cream treated groups within 10 to 21 days, respectively.
Epithelization Period
Table 3: Effect of topical application of EEAP on period of epithelization (in days) of excision wound model in mice
|
Treatment groups |
Period of epithelization |
|
Control |
18.6 ± 0.6 |
|
Diabetic |
24.8 ± 0.3 |
|
1%Soframycin |
15.0 ± 0.3* |
|
10% EEAP |
21.8 ± 0.3* |
|
20% EEAP |
19.8 ± 0.3* |
Values are expressed as mean ± SEM (n = 6) one-way ANOVA followed by Dunetts multiple comparison test compared to diabetic (*p< 0.0001)
Fig No 1: Effect of topical application of EEAP on period of epithelization (in days) of excision wound model in mice
The 1% Soframycin group treated group showed faster rate of epithelialization (p <0.001) compared to control group and (p < 0.0001) compared to10%EEAP. Similarly 20% EEAP showed significant (P < 0.04) difference of epithelialization period as compared to simple cream. The 20%EEAP also showed significant(P < 0.001) difference of epithelialization period as compared to simple cream treated group. The Diabetic group has the longest epithelization period, confirming impaired wound healing due to diabetes. 1%Soframycin shows the shortest period, indicating highly effective healing. EEAP exhibits a dose-dependent response that is 20% EEAP shows better healing than 10% EEAP. Both are significantly better than the Diabetic group.
DISCUSSION
Wound healing, a common clinical entity contemporary to human beings has been recognized to be influenced by several factors such as nutrients like Vitamin-C, zine and hormones like growth hormone, insulin etc. Products of traditionally used plants such as Bryophyllum pinnatum and Moringa oleifera has also been reported to promote wound healing in experimental animals probably in virtue of their antimicrobial activity. Because of it is well known that infection interferes with healing. Antimicrobials are suspected to suppress infection and promote healing process in infected wounds. Management of healing wounds is a complicated and expensive program and research on drugs that increase wound healing is a developing area in modern biomedical sciences. Several drugs obtained from plant sources are known to increase the healing of different types of wounds. Though some of these drugs have been screened scientifically for evaluation of their wound healing activity in different pharmacological models and patients the potential of many of traditionally used herbal agents remain unexplored. In the field of wound healing, there are several unknowns; this includes the wound itself. These agents available to traditional systems of medicine should not disappear from use before they can be fully accessed. There are number of plants which a used traditionally and by the tribal people which have not been evaluated. In the present investigation, preliminary phytochemical analysis of ethanolic leaves extract revealed the presence of flavanoids, saponins, tannins, lipids, proteins, alkaloids, terpenoids. The faster wound healing is due to presence of flavonoids because it reduces lipid peroxidation not only by preventing or slowing onset of cell necrosis but also improving vascularity, thus increases collagen strength and preventing cell damage by promoting the DNA synthesis. Tannins, flavonoids, triterpenoids and sesquiterpenes are also known to promote the wound healing mainly due to their astringent and antimicrobial properties which increases the rate of epithelialization. In excision wound healing model, the leaves extract of Adenanthera pavonina. showed statistically significant wound area contraction compared to the control. The 20%EEAP treated group revealed faster wound area contraction from day 4 to day 21, whereas the 10%EEAP treated group showed statistically significant wound area contraction starting from the 8th day onwards. The higher wound contraction rate of the extract cream may be due to either its dose-dependent anti-inflammatory effect or induction of macrophage cell proliferation. The leaf of Adenanthera pavonina was traditionally claimed to be used for inflammation and analgesic activity. Therefore, in the present study we scientifically explored these traditional claims. The results of this study on wound healing activity revealed that the leaves extract significantly increases wound healing effects with both 20% and 10% EEAP cream treated groups in the excision wound models. This can be supported by the fact that the greater the reduction in the rate of wound contraction is, the better the efficacy of medication is and the wound will close at faster rate if the medication is more efficient. Furthermore, the period of epithelialization was significantly reduced from 19.18 days to 12, 11.41, and 14.20 days for Soframycin cream, 10% and 20%EEAP cream treated groups, respectively. The shorter period of epithelialization and faster wound area contraction could be due to the ability of A.pavonina leaf extract to enhance collagen synthesis. induction of cell proliferation, anti-inflammatory, antimicrobial activities of bioactive constituents. The present work showed interesting healing action of the extract that was evidenced by the inhibition of vascularity and inflammation in all treated groups. However, use of the extract at doses of 20%EEAP was suitable since they produced changes. the tissue scar was not analysed for the extent of dermis and epidermis formation and regeneration. A semiquantitative method was carrried out to assess the following histological processes and structures: reepithelization, inflammatory cells, and new collagen. The histological examination showed that the original tissues regeneration was much greater the skin wound treated with extracts and Soframycin cream treated group without any oedema, congestion or inflammatory changes. The histological studies of the granulation tissue of the control group of animals showed more aggregation of macrophages with lesser collagen fibres. More relative fibrosis was observed in the soframycin and 10%EEAP treated groups with comparing to control group. In acute diabetes, the inability of wounds to heal is related to aberrations of the wound healing process. Acute diabetes can be responsible for deficiencies in defense cells (low density of neutrophils) and in repair tissue failures. In our study, we observed an impaired wound repair in diabetic treated mice associated with a prolonged inflammatory phase and reduced collagen production. Collagen is an essential building block of the skin that promotes wound healing. Thus, when wound repair is defective, stimulating the synthesis of the collagen would be beneficial for promoting wound healing. These results suggest that topical application of the cream based on A.pavonina extract may stimulate the deposition of collagen and thereby promoted the wound healing. Moreover, the Adenanthera pavonina cream seems to enhance wound healing in diabetics by promoting the collagen and growth factors. Angiogenesis increases the delivery of oxy gen and other nutrients that are necessary for local collagen synthesis.
CONCLUSION
In this study, in Excision models, the different phases of wound repair, wound contraction, epithelialization, and tensile strength, were enhanced by the ethanolic extract cream of the leaves of Adenanthera pavonina as compared to the simple cream base treated group. Applying EEAP to diabetic wounds has been shown to increase collagen and neovascularization while decreasing pro- inflammatory CKs, suggesting the wounds may heal more quickly than they would otherwise These results collectively demonstrate that the 80%EEAP possesses wound healing activity and this justifies the use of the leaves of Adenanthera pavonina for treatment of wounds as claimed in the folklore literature. Also in diabetes there is delayed in wound healing but the 20% extract cream may possess ability to heal wound faster in diabetic patients. The wound healing activity of the plant could be due to the presence of alkaloids, flavonoids, steroids, proteins, glycosides and polyphenolic compounds. Hence the present findings extend support for the traditional claims of the plant for the treatment of wound. From the histological examination we concluded that the original tissue regeneration was much greater in the skin wound treated with extracts and Soframycin cream treated group when compared with control and diabetic group. In the present study we found that ethanolic leaf extract of Adenanthera pavonina may promoted the wound healing activity significantly in diabetic patients. High rate of wound contraction, decrease in period for epithelialization, high skin breaking strength and granulation strength, increase in dry granulation tissue weight and increased Collage nation in histopathological section were observed in animals treated with ethanolic leaf extract when compared to control and induced group. Hence the present findings extend support for the traditional claims of the plant for the treatment of wound and helps in faster wound healing in diabetic patients. Future work needs to be done in direction to elucidate the molecular mechanism of EEAP leaves for the wound healing. It could to be the next better, safer& cheaper herbal alternate in management of wound healing activity in diabetic patients and also modulation of herbal drug with highest inflammatory activity to potentiate the activity.
REFERENCES
Roshni Gawande*, Dr. Anjali Wankhade, Dr. Vivek Paithankar, Wound Healing Potential of Adenanthera Pavonina On Alloxan Induced Diabetic Wound in Mice Model, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 6, 196-205. https://doi.org/10.5281/zenodo.15574846
10.5281/zenodo.15574846
