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University Department of Pharmaceutical Sciences, Department of Pharmacology, Utkal University, Vani Vihar, Bhubaneswar, Odisha, India.
The current exploration of the research work was for screening the wound healing potential of hydro-alcoholic root extract of plant Clitoria ternatea Linn. Clitoria ternatea (Fabaceae), a most common medicinal plant in traditional Indian medicine. According to folk of medicine and experimental data raised an outlook on this plant for evaluating wound healing potential. Hydro-alcoholic root extract of Clitoria ternatea (HARCT) has been examined for its wound healing activity by utilizing excision wound model in Wistar albino rats following application of standard (Soframycin 5% ointment) and HARCT ointment topically. Animals have been categorized into 4 groups with 6 animals in every group. Group I treated with simple ointment base, Group II treated with standard Soframycin 5% w/w ointment, Group III and IV treated with HARCT 5% w/w and 10% w/w ointments respectively. Results obtained upon treatment with the plant extract groups shown activity close to standard drug activity, Soframycin in terms of wound contraction parameter, time taken for wound closure and period of epithelialization. HARCT 10% w/w ointment has produced better wound healing potential after comparing to 5% w/w HARCT ointment. The obtained results notified that Clitoria ternatea swifts the wound healing process by reducing wound’s surface area and enhancing hydroxyproline content.
Wounds are physical, chemical or thermal breaks in the normal continuity of skin or its underlying tissue leading to disruption of their anatomical and functional integrity [1]. An intricate natural process in which the skin or tissue repairs itself after injury, the wound healing, is governed by various phases namely; hemostasis, inflammation, proliferation, tissue remodeling or resolution and maturation [2]. These phases, in the complex and dynamic process, of wound healing involve a cascade of cellular and biochemical events mediated by various cytokines, growth factors and enzymes [3].Wounds are still a major problem in developing countries, often leading to serious complications [4] because their care is complex, time consuming, sometimes confusing, and almost always expensive [5]. Although, various therapeutic regimens involving antibiotics, analgesics and nonsteroidal anti-inflammatory drugs are available for the management of wound, majority of these therapies are not devoid of the numerous unwanted side effects [6].Clitoria ternatea Linn. is an enriched medicinal plant belonging to Fabaceae family. The plant generally grows in the humid, low land tropics, occurring naturally as well as in cultivated form [7]. It is popularly called as “Shankpushpi” in India as the flowers of this plant resemble a conch shell [8]. It is a common shrub. Different parts of this plant are used in Indian systems of medicine for various pharmacological actions like as Antihistaminic [9], Antimicrobial [10], Hepatoprotective [11], Alzheimer’s disease [12], Snake bite and scorpion sting [13], Immunomodulatory [14], Diuretic [15], Anti-ulcer [16], Antioxidant [17] and many more activities. In the present study, we designed a method to evaluate the wound healing potential of roots of C. ternatea using excision wound model in Wistar albino rats.
MATERIALS AND METHODS:
Collection of Plant Material and Plant Extraction: Fresh and mature plant roots of Clitoria ternatea Linn. has been collected in bulk quantities from the medicinal garden at U.D.P.S and Chandaka Forest, Bhubaneswar, Odisha, India. The plant specimen was authenticated by Dr. S. K. Dash, P.G. Dept. of Bio-sciences, Berhampur, Odisha, India. The voucher specimen of C. ternatea Linn. (Voucher No. Debananda-2017- 02) have been preserved in the institution herbarium of U.D.P.S, Bhubaneswar for future reference. The dried parts (root) were then grated to coarse powder by mechanical grinder and allowed to extract with hydro-alcohol (30:70) in Soxhlet apparatus. The crude extracts were evaporated to dryness under vacuum and placed it in vacuum desiccator for further drying. Preliminary phytochemical investigation was performed and on the eve of the phytochemicals of hydro-alcoholic root extracts of C. ternatea (HARCT) was selected for wound healing screening in albino rats. The herbal ointment was prepared by levigation method.
Animals: Wistar albino rats weighing 150-200 g were used for the present study. All animals were maintained in the animal house of Dadhichi College of Pharmacy, Cuttack, Odisha, under controlled conditions of temperature 25 ± 2? C, relative humidity 55-60 %, and 12-h light-dark cycles for experimental purposes. They are grouped into experimental and control groups, kept in polypropylene cages with sterile paddy husk as bedding and free access to standard pellets to feed and water ad libitum. All the studies conducted were approved by the Institutional Animal Ethical Committee (IAEC) of Dadhichi College of Pharmacy, Sundargram, Cuttack, Odisha (Approval No. -1200/PO/Re/S/08/CCSEA).
EXPERIMENTAL DESIGN:
All the rats had been anaesthetized utilizing light vapour inhalation of diethyl ether. The rats have been categorized into 4 main groups (n=6) as shown in Table 1. Animals have been bared before wounding at dorsal thoracic areas.
Table 1: Experimental design of excision wound model
|
Groups |
Treatment |
Dose (applied topically/day) |
|
I |
Wound control |
Simple ointment base |
|
II |
Positive control |
Standard Soframycin (5% w/w) ointment |
|
III |
HARCT 5% ointment |
HARCT (5% w/w) ointment |
|
IV |
HARCT 10% ointment |
HARCT (10% w/w) ointment |
Excision Wound Model: Excision wound was made on the depilated region of the skin. A standardized wound area was marked on the dorsal surface of the desensitized rats and skin in full thickness was ablated to obtain a wound area of about 500 mm2 diameters. Homeostasis was attained by blotting the ulcerated area with cotton swab dipped in normal saline. The entire wound was left open. The wound diameter was measured using calipers on alternate days and the epithelialization period recorded at the end of the study. Number of days required for falling of the scar without any residual raw wound gave the period of epithelialization. It was measured in days from wounding day (day zero) till the full epithelialization. The wound area was measured with a translucent paper and traced the epithelialization period. The wound area of each animal was measured on days 0, 3, 6, 9, 12, 16 and 20 after inflicting the wound [18].The following parameters i.e., wound size reduction, percentage of wound contraction, period of epithelialization, hydroxyproline contents were recorded on post wounding days.
Measurement of Wound Closure: The rate of wound closure has been analyzed by tracing the wound on 0, 3, 6, 9, 12, 16 and 20 post wounding days utilizing permanent marker and transparent paper and noticed wound regions were calculated utilizing 1 mm2 scale of graph paper. The wound contraction progress was evaluated by using the following formula;
% Wound contraction=Initial wound size-specific day wound sizeInitial wound area×100
Measurement of Period of Epithelialization: Wound falling with no raw scab behind has been considered as perfect epithelialization and the days in number needed for was noticed as period of epithelialization [19].
Measurement of Biochemical parameter: At the end of the study, on 20th day, biochemical parameter like hydroxyproline content was determined from excised granulation tissue [20]. Hydroxyproline, an amino acid, is the major component of collagen which provides strength and support to the tissues. Collagen is a major protein of the extracellular matrix and is the major component that ultimately contributes to wound strength. Breakdown of collagen liberates free hydroxyproline and its peptides. Measurement of the hydroxyproline could be used as an index for collagen turnover [21].
STATISTICAL ANALYSIS:
All data were expressed as mean ± SD (n=6). Statistical analysis was performed at p< 0.05 to p< 0.01 between the groups by one-way analysis of variance (one-way ANOVA) followed by Tukey’s multiple comparison test using Graph Pad prism software (version 10).
RESULTS:
The intricate cellular process of wound healing allows damaged tissue to return as nearly to its pre-damaged state as feasible. The type and degree of damage, the tissues overall health and its capacity for repair all influence the healing process. The standard Soframycin serves as a common benchmark for evaluating the medicinal efficacy in comparison with the crude medication and the control. Animals in the extract and conventional treatment groups showed a significant reduction in the epithelialization period and an increase in the pace of wound contraction.
Wound Contraction: Topical application of hydro-alcoholic root extract of Clitoria ternatea (HARCT) 5% and 10% w/w ointments showed effect on the process of wound healing on the rats. The progress size reduction of wound area was shown in Table 2 and Figure 1 as well as the percentage of wound contraction induced by treatment of HARCT 5% and 10% w/w ointments, control and Soframycin 5% w/w ointment were shown in Table 3 and Figure 2. The HARCT 10% w/w ointment treated group shown noticeable wound size reduction from the six days of treatment and with highly significant difference were seen from 12th day onwards in comparison with the control group. The wound size reduction on 6th day have showed much reduced in both group II and group IV (312.8 and 323.8 mm2) as compared to control group I (410.6 mm2). However, on 12th day the group IV showed wound size reduction (101.4 mm2) more than the group II (105.8 mm2). The percentage wound closure on 6th day of original wound was showed higher in group II and group IV (38.6% and 36.8%) when compared to normal control group I (21.8%). The percentage wound closure on 12th day of original wound was showed higher in group IV (80.2%) when compared to group II (79.2%). In comparison with group I (58.9%) there was no significant percentage of wound healing showed in group III (60.6%). On the day 20th, group IV (0 mm2 and 100%) and group II (0 mm2 and 100%) have showed higher wound size reduction and percentage of wound closure in comparison to the group I and group III.
Table 2: Effect of hydro-alcoholic root extract of Clitoria ternatea (HARCT) ointment on wound size reduction (mm2) on different groups of animals in excision wound model
|
Post wound healing (Days) |
Group I |
Group II |
Group III |
Group IV |
|
0 |
513.2 ± 1.47 |
510.2 ± 1.49 |
511.8 ± 0.74 |
512.8 ± 0.74 |
|
3 |
401.2 ± 3.05 |
432.8 ± 2.75 |
482.2 ± 3.31 |
437.2 ± 1.72 |
|
6 |
410.6 ± 1.02 |
312.8 ± 2.28 |
419.4 ± 1.02 |
323.8 ± 2.93 |
|
9 |
384.6 ± 2.05 |
228.2 ± 2.42* |
385.6 ± 3.0 |
218.8 ± 1.72* |
|
12 |
210.8 ± 1.16 |
105.8 ± 2.05** |
201.6 ± 1.02 |
101.4 ± 1.35** |
|
16 |
98.2 ± 1.72 |
41.2 ± 1.37** |
96.2 ± 1.32 |
47.2 ± 2.48** |
|
20 |
40.6 ± 1.35 |
0 |
31.4 ± 1.29* |
0 |
Values are expressed as mean ± SD, n=6. Using Tukey’s multiple comparison test, the intergroup variation between various groups was conducted by graph pad Prism software & Data were analyzed by using one way analysis of variances (ANOVA). **p < 0.01, *p < 0.05 when compared to control group; one way ANOVA.
Figure 1: Wound size reduction (mm2) of HARCT ointment on different groups of animals in excision wound model
Table 3: Effect of hydro-alcoholic root extract of Clitoria ternatea (HARCT) ointment on percentage of wound contraction (%) on different groups of animals in excision wound model
|
Post wound healing (Days) |
Group I |
Group II |
Group III |
Group IV |
|
0 |
0 |
0 |
0 |
0 |
|
3 |
19.9 |
15.1 |
5.7 |
14.7 |
|
6 |
21.8 |
38.6 |
18.1 |
36.8 |
|
9 |
25.1 |
55.2 |
24.6 |
57.3 |
|
12 |
58.9 |
79.2* |
60.6 |
80.2* |
|
16 |
80.8 |
91.9 |
81.2 |
90.7 |
|
20 |
92.1 |
100** |
93.8 |
100** |
Values are expressed as mean ± SD, n=6. Using Tukey’s multiple comparison test, the intergroup variation between various groups was conducted by graph pad Prism software & Data were analyzed by using one way analysis of variances (ANOVA). **p < 0.01, *p < 0.05 when compared to control group; one way ANOVA.
Figure 2: Percentage of wound contraction (%) and period of epithelization of HARCT ointment on post wounding days
Epithelialization Period: The time required for complete epithelialization of the excision wound is an important parameter to assess the wound healing process. It has been noticed during the study that the group IV show approximately 15 days as comparable with the group II which showed approximately 13 days for the epithelialization and found no significant difference in group IV (10% w/w HARCT) with the standard group I as shown in Table 4 and Figure 2.
Table 4: Effect of hydro-alcoholic root extract of Clitoria ternatea (HARCT) ointment on period of epithelialization (no. of days)
|
Groups |
Period of epithelization (days) |
|
I |
26.67 ± 0.57 |
|
II |
13.57 ± 0.57* |
|
III |
24.33 ± 0.57 |
|
IV |
15.57 ± 1.00* |
Values are expressed as mean ± SD, n=6. Using Tukey’s multiple comparison test, the intergroup variation between various groups was conducted by graph pad Prism software & Data were analyzed by using one way analysis of variances (ANOVA). **p < 0.01, when compared to control group; one way ANOVA.
Hydoxyproline Content: The hydroxyproline contents were measured in terms of μg/mg and it was increased significantly as dose dependent manner in group IV (0.69 μg/mg) as comparison to the group II (0.74 μg/mg) and group III (0.62 μg/mg) as shown in Table 5 and Figure 3. So, the 10% HARCT ointment showed the remarkable increased hydroxyproline content level but comparatively less than the standard group.
Table 5: Effect of hydro-alcoholic root extract of Clitoria ternatea (HARCT) ointment on biochemical parameters
|
Groups/Treatment |
Hydroxyproline (μg/mg tissue) |
|
Group-I (control) |
0.60±0.024 |
|
Group-II (Standard) |
0.74±0.049** |
|
Group-III (HARCT 5% w/w) |
0.62±0.049* |
|
Group-IV (HARCT 10% w/w) |
0.69±0.024** |
Values are expressed as mean ± SD, n=6. Using Tukey’s multiple comparison test, the intergroup variation between various groups was conducted by graph pad Prism software & Data were analyzed by using one way analysis of variances (ANOVA). **p < 0.01, *p < 0.05 when compared to control group; one way ANOVA.
Figure 3: Effect of HARCT ointment on hydroxyproline content of excised wound in rats
DISCUSSION:
Wound is a major problem of health in relation to morbidity and mortality. It involved in wound healing are epithelialization, contraction and connective tissue deposition. It depends on regulated biosynthesis and deposition of new collagens and their subsequent maturation [22].
In the tissue repair process, inflammatory cells which synthesize extracellular matrices including collagen and keratinocytes for reepithelialization of wound tissues [23].It is a complex process of restoring cellular structures and tissue layers in damaged tissue together to its normal state and commencing in the fibroblastic stage where the area of the wound undergoes shrinkage [24]. It comprises of different phases such as contraction, granulation, epithelization and collagenation [25, 26].
Studies were revealed that flavonoids are also known to promote the wound healing process mainly due to their astringent and antimicrobial properties which appear to be responsible for the wound healing and increased rate of epithelialization [27]. Similar types of wound-healing activity were reported on Vernonia arborea [28] and Pentas lanceolata [29].Collagen is a major protein of the extracellular matrix and is the major component that ultimately contributes to wound strength [30]. Breakdown of collagen liberates free hydroxyproline and its peptides. Measurement of the hydroxyproline could be used as an index for collagen turnover [31]. Hydroxyproline, an amino acid, is the major component of collagen which provides strength and support to the tissues. The liberation of hydroxyproline may be due to collagen breakdown. Hence its measurement can be used as an index for collagen turnover as well as a biochemical marker for tissue collagen [32].
CONCLUSION
In this research work, hydro-alcoholic root extract of Clitoria ternatea plant was examined for its wound healing activity in excision wound model in rats. The plant part material proved its significant potentials against wound healing activity. The results were also comparable to those of a standard drug, Soframycin, in terms of wound size reduction, percentage of wound contraction, epithelialization periods and improvement of hydroxyproline content during the experimental period in excision wounding in Wistar albino rats. So, this plant part may contain the phytoconstituents or chemicals those potentially act on the biological systems and prove the wound healing potentials by various mechanism on the affected wound sites.
ETHICAL MATTER:
Authors would like to thank to Dadhichi College of Pharmacy, Sundargram, Cuttack, Odisha for providing the facility of experimental animal work and IAEC approval.
AUTHORS CONTRIBUTIONS
All the authors have contributed equally from conceptualization to quality control of the document.
CONFLICT OF INTEREST:
Author has no conflict of interest regarding submission of manuscript for publication.
REFERENCES
Debananda Champatisingh, Ranjit Mohapatra, Evaluation Of In-Vivo Wound Healing Activity of The Hydro-Alcoholic Root Extract of Clitoria Ternatea (Linn.) Ointment In an Excision Wound Model on Wistar Albino Rats, Int. J. of Pharm. Sci., 2026, Vol 4, Issue 2, 2024-2033. https://doi.org/10.5281/zenodo.18628288
10.5281/zenodo.18628288