|Year : 2015 | Volume
| Issue : 2 | Page : 50-55
Evaluation of the wound-healing potential of Amaranthus viridis (Linn.) in experimentally induced diabetic rats
Himanshu Bhusan Sahoo1, Saroj Kumar Sahoo2, Kirtimaya Mishra3, Rakesh Sagar1
1 Department of Pharmacology and Experimental Biology, Vedica College of Pharmacy, Ram Krishna Dharmarth Foundation (RKDF) University, Bhopal, Madhya Pradesh, India
2 Department of Pharmaceutical Chemistry, Sri Sivani College of Pharmacy, Srikakulam, Andhra Pradesh, India
3 Department of Analytical Chemistry, Sri Sivani College of Pharmacy, Srikakulam, Andhra Pradesh, India
|Date of Submission||20-Nov-2014|
|Date of Acceptance||07-Jan-2015|
|Date of Web Publication||23-Mar-2015|
Himanshu Bhusan Sahoo
Department of Pharmacology and Experimental Biology, Vedica College of Pharmacy, Ram Krishna Dharmarth Foundation (RKDF) University, Bhopal, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: This study aimed to evaluate the wound-healing potential of the ethanolic extract of Amaranthus (A.) viridis leaves in diabetic rats. Materials and Methods: Diabetes was induced by the administration of a single injection of alloxan monohydrate (120 mg/kg, i.p.) prepared in citrate buffer to experimental animals. The wound-healing activity was evaluated by three methods: excision, incision, and dead space wound models. The diabetic animals were divided into five groups, as follows: Group I, which received alloxan treatment only (diabetic control); Group II, which received topical mupirocin ointment (2% w/w) (standard); Groups III, IV, and V, which were treated with ointments prepared from A. viridis extract of 2%, 5%, and 10% w/w, respectively, daily (test groups) till the wounds completely healed. Wound-healing parameters such as the rate of wound contraction, epithelization, tensile strength, and concentration of hydroxyproline content were measured and compared with diabetic control using one-way analysis of variance (ANOVA) followed by Dunnett's test. Results: Rats treated with the prepared ointments of A. viridis extract had shown a significant (P < 0.01) increase in the percentage of wound closure, tensile strength, and hydroxyproline content of the granulation tissue when compared with the diabetic control, in a dose-dependent pattern. Conclusion: These findings demonstrated that the ointments of A. viridis leaf extract effectively stimulate wound contraction and accelerate wound healing in diabetic rats.
Keywords: A. viridis , hydroxyproline, tensile strength, wound contraction, wound healing
|How to cite this article:|
Sahoo HB, Sahoo SK, Mishra K, Sagar R. Evaluation of the wound-healing potential of Amaranthus viridis (Linn.) in experimentally induced diabetic rats. Int J Nutr Pharmacol Neurol Dis 2015;5:50-5
|How to cite this URL:|
Sahoo HB, Sahoo SK, Mishra K, Sagar R. Evaluation of the wound-healing potential of Amaranthus viridis (Linn.) in experimentally induced diabetic rats. Int J Nutr Pharmacol Neurol Dis [serial online] 2015 [cited 2017 Oct 20];5:50-5. Available from: http://www.ijnpnd.com/text.asp?2015/5/2/50/153792
| Introduction|| |
Diabetic wounds are very slow and nonhealing wounds occurring due to various abnormalities of the connective tissue. These abnormalities are caused by reduced biosynthesis and accelerated degradation of the collagen content. The decrease of collagen content in skin leads to the impairment of wound healing in diabetes.  Poor wound healing in diabetes may be due to deficiency in cell proliferation, proneness to infection, decrease in cell survival, and reduction of rates of wound contraction.  Thus, wound healing is a complex phenomenon involving different series of biological events, which include coordination between the dermal and epidermal tissues, existence of fibroblasts and keratinocytes, and repair of the connective tissue. 
Amaranthus (A.) viridis (Linn.) (Amaranthaceae) is an edible plant that is grown in all regions of India. In India, tribal people have used it to treat snakebite and scorpion stings.  In the Nepalese traditional system of medicine, it has been used to reduce labor pain and to act as an antipyretic.  Other traditional uses include the following: Anti-inflammatory action on the urinary tract; treatment of ulcers, venereal diseases, respiratory problems, eye problems, and asthma; as a vermifuge, diuretic, antirheumatic, analgesic, antiemetic, laxative, and antileprotic; and for the improvement of appetite.  The plant was found to be a rich source of flavonoids such as quercetin and rutin. It has also been used for the treatment of acne and for skin cleansing.  A number of studies found that the whole plant possesses analgesic, antipyretic, anti-inflammatory, antidiabetic, antihyperlipidemic, hepatoprotective, cardioprotective, antiproliferative, antiviral, and antioxidant properties. ,,,,, The present study has been undertaken to examine the wound-healing activity of the leaf extract of A. viridis on experimentally induced wounds in diabetic rats.
| Materials and Methods|| |
Chemicals and reagents
Alloxan, glucose, gluco strips, mupirocin ointment (2% w/w), diethyl ether, ethanol, sterilized cotton, hydroxyproline, and Ehrlich's reagent were procured from SD Fine-Chem Pvt. Ltd. (Mumbai, Maharashtra, India). All other chemicals used were of analytical grade.
Extraction of plant material
The leaves of A. viridis [Figure 1] were collected locally in the month of December. The collected leaves were air-dried and coarsely powdered. The powdered sample of 150 g was weighed and extracted in 500 ml ethanol by the cold maceration method with occasional shaking for 48 h. After 48 h, the extract was filtered through Whatman filter paper and dried under vacuum. Then, the extract was placed in a tightly closed container and stored at 4°C in the refrigerator for further use. The percentage yield of the ethanolic extract of A. viridis leaves was found to be 23% w/w.
Preliminary phytochemical screening
Preliminary tests of the prepared extract were carried out to detect the presence of phytoconstituents such as glycosides (including flavonoids and saponins), alkaloids, carbohydrates, sterols, proteins, phenolic compounds, and reducing compounds.
Procurement of animals
Male Swiss albino rats (150-180 g) were obtained from our animal house after approval from our Institutional Animal Ethical Committee. The animals were stabilized for 1 week and maintained under standard housing conditions at room temperature (24-27°C) and relative humidity (60-65%) with a 12-h light-dark cycle. They were given a standard pellet diet and water ad libitum throughout the course of the study.
Preparation of ointment
The ointment formulations with different concentrations of A. viridis Linn. Extract (2% w/w, 5% w/w, and 10% w/w ointment) were prepared using the fusion method, where 2 g, 5 g, and 10 g of the extract were incorporated in 100 g of simple ointment base, respectively. The ointment was packed in wide-mouthed containers.
Primary skin irritation test
The ointment prepared from the A. viridis extract was applied topically over the preselected areas of all the animals. The prepared formulation up to 20% did not show skin irritation till 48 h of topical application, which suggests that the formulations may be considered safe.
Induction of diabetes in rats
Rats were made diabetic after overnight fasting by a single injection of alloxan monohydrate (120 mg/kg, i.p.) prepared in normal saline. Seventy-two hours after alloxan injection, blood samples were withdrawn from the vein of the rat's tail, and blood glucose levels were estimated by glucometer (Accu-Chek Active, Chennai, India). Animals with normal blood glucose levels (≥250 mg/dl) were selected for the study.
In the experiment, the rats were divided into five groups (n = 6):
Group I: Diabetic control (alloxan treatment only)
Group II: Diabetic wounds were treated topically with a standard drug, i.e., mupirocin ointment (2% w/w)
Groups III, IV, and V: Diabetic wounds were treated topically with A. viridis extract ointments daily (2%, 5%, and 10% w/w, respectively) till the wounds completely healed
Excision wound model
The animals were anesthetized with slight vapor inhalation of diethyl ether, and the hairs were removed from the dorsal thoracic central region. Excision wounds of 300 mm 2 size and 2 mm depth were made by cutting out pieces of skin from the shaven area. The entire wound was left open. The animals were closely observed for any infection and those that showed any sign of infection were separated, excluded from study, and replaced. Then the ointments (2%, 5%, and 10% w/w) and standard drugs were applied everyday to the specific groups for 16 days. Wound areas were measured on days 1, 4, 8, 12, and 16 for all groups, using a transparency sheet and a permanent marker. Wound areas recorded on those days were then measured on graph paper. The day the scar fell off, after wounding without any residual raw wound, was considered as the day of epithelization. This model was used to monitor the rate of wound contraction and epithelization. Wound contraction was calculated as percent reduction in wound area (% of wound contraction = healed area/total area × 100). The falling of the scab from around the wound was taken as the end point of complete epithelization and the total days required for this were taken as the period of epithelization. From the healed wound, a specimen sample of tissue was isolated from each group of rats for histopathological examination. 
Incision wound model
Rats in each group were anesthetized and longitudinal paravertebral-long incisions (6 cm in length) were made through the skin and cutaneous muscles at a distance of about 1 cm from the midline on each side of the depilated back of the rat. Full aseptic measures were not taken and no local or systemic antimicrobials were used throughout the experiment. After the incision was made, the parted skin was held together and stitched with black silk thread at 0.5 cm intervals. The wound was left undressed. The ointments prepared from the extract (2%, 5%, and 10% w/w) and the standard ointment were administered once daily for 8 days. The sutures were removed on the eighth day postwounding, and tensile strength was measured with a tensiometer. 
Determination of tensile strength
The prepared ointments along with the standard and control were applied throughout the period, once daily for 8 days. The sutures were removed on the ninth day and the rats were again anesthetized. A small piece of the healed wound was cut out from each rat such that the healed incision wound lay exactly in the middle. Four small curved needles (no. 14) were pierced through the healed skin, two on either side. On one side two needles were tied to a rod and on the other side two needles were tied to a plastic bottle, which hung freely in the air (either side of the needle was placed equidistant from the healed incision wound). Then slowly water was added to a bottle until the wound began to open. The amount of water in the bottle was weighed and considered as an indirect measure of the tensile strength of the wound. The mean determinations of tensile strength on the two paravertebral incisions on both sides of the animals were taken as the measure of the mean tensile strength of the wound for an individual animal. The tensile strength of the extract-treated wounds was compared with control. The tensile strength increment indicated better wound-healing stimulation by the applied drug.
Dead space wound-healing activity
Dead space wounds were inflicted by implanting sterile cotton pellets (10 mg each) on ventral side of the groin and axilla of each rat. On the 10 th day after wounding, the granulation tissue formed on the implanted cotton pellets was carefully removed under anesthesia. After noting the weight of the granulation tissue, the tissue was dried at 60°C for 12 h and the dry granulation tissue weight was recorded. To the dried tissue, 5 ml 6N HCL was added and kept at 110°C for 24 h. The neutralized acid hydrolysate of the dry tissue was used for the determination of hydroxyproline. 
The data were analyzed as mean SD. The values obtained in control and tests were compared using a one-way ANOVA, followed by Dunnett's test. A P value of 0.01 was considered to be significant.
| Results|| |
The ethanolic leaf extract of A. viridis showed a positive result for glycosides (including flavonoids and saponins), proteins, steroids, and terpenes.
Evaluation of wound healing in excision and incision wound models
All the ointments prepared from A. viridis induced a significant (P < 0.01) decrease in wound contraction and period of epithelization when compared to diabetic control [Table 1]. All these treatments of A. viridis also showed a dose-dependent pattern of wound contraction. The standard drug-treated animals among the diabetic animals showed significantly greater wound closure when compared with the extract-treated animals [Figure 2]. After the 16 th day of treatment, the percentage of wound contraction for standard borea very close resemblance with that for a high dose of A. viridis. The tensile strength of the incision wound after 8 days was significantly (P < 0.01) increased by all treatments [Figure 3]. The high dose of the prepared ointment led to similar tensile strength as did standard treatment. Besides this, all the prepared formulations induced dose-dependent wound contraction.
|Figure 2: Effects of A. viridis treatment on excision wound-healing model|
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|Figure 3: Effects of A. viridis treatment on percentage of wound contraction, tensile strength, and dry tissue weight in diabetic rats. Values were represented as mean ± S.D of six animals in each group, **P < 0.01 as comparedto diabetic control|
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|Table 1: Effects of A. viridis treatment on excision wound mode l in diabetic rats|
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Evaluation of dry tissue weight and hydroxyproline content in the dead space wound model
The breaking strength of 10-days-old granulation tissue was significantly enhanced by all the treatments. The dry tissue weight and hydroxyproline content were significantly increased (P < 0.01) by all the treatments when compared to diabetic control [Figure 3] and [Figure 4].
|Figure 4: Effects of A. viridis treatment on hydroxyproline content (ìg/g of tissue) in mice. Values were represented as mean ± SD of six animals in each group, **P < 0.01 as compared to diabetic control|
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| Discussion|| |
Diabetes is associated with delays in wound healing, which may be due to the elevation of blood glucose levels, denaturation of proteins and cellular components, overproduction of oxidative free radicals, and alterations in connective tissue metabolism, that is, loss of collagen.  Collagen is an extracellular protein in the granulation tissue of a healing wound and acts as a homeostatic agent as well as an initiator of epithelization by providing strength and integrity to a tissue matrix. These alterations of collagen may be due to decreased levels of synthesis or enhanced catabolism of newly synthesized collagen, or both.  Collagenation, epithelization, wound contraction, and fibrosis are very crucial phases of wound healing, which run concurrently but independent of each other and are also regulated by the body as part of cellular defense mechanisms.  The topical application of prepared ointments of A. viridis improved the breaking strength, wound contraction, and period of epithelization in the excision and incision models of experimental wounds. The faster wound contraction rate may be due to the stimulation of interleukin or induction of more rapid maturation of granulation tissue. A significant increase in collagen content due to the enhanced migration of fibroblasts and epithelial cells to the wound site was observed during the wound-healing process in the treated group. A close observation of granulation tissue sections revealed that tissue regeneration was much quicker in the treated group compared to diabetic control.
In diabetes, poor wound healing is caused due to impaired blood flow and oxygen release from increased blood sugar, decreased collagen and fibronectin synthesis from protein malnutrition, impaired local immune defenses, and decreased anabolic activity with decreased insulin and growth hormones.  These defects of wound healing are due to the hyperglycosylation of cellular fibronectin, which affects the neutrophilic functions, that is, migration and phagocytic and antibacterial activity.  Growth hormones promote cell collagen formation and fibroblast proliferation from the granulation tissue. , The increase in dry granulation tissue weight in the treated groups indicated high protein content. Collagen is composed of the amino acid hydroxyproline, which has been used as a biochemical marker for tissue collagen.  In the dead space wound model, the increase of dry tissue weight and hydroxyproline content was observed in each treatment derived from A. viridis, which suggests greater deposition of collagen. The formation and maturation of collagen may be due to the presence of flavonoids and steroids in A. viridis, which are responsible for free radical-scavenging activities and help to promote the most important phase of wound healing. Hence, on the basis of the observed results, the faster wound-healing activity of A. viridis in diabetic animals may be due to the presence of phytochemicals and their effect on components of wound healing.
| Conclusion|| |
The present study demonstrates that A. viridis extract applied topically promotes the healing of wounds, with enhanced rates of collagen turnover and wound contraction in diabetic-induced rats. These preliminary results further suggest that A. viridis facilitates healing by increasing the rate and extent of wound closure and hydroxyproline content in wounds subject to delayed healing. However, further phytochemical studies are needed to isolate the active compound(s) responsible for these pharmacological activities and will be helpful in projecting this plant as a therapeutic target for healing wounds and treating various diseases.
| Acknowledgments|| |
The authors are very grateful to Dr. M. L. Kori (Director), Vedica College of Pharmacy, Bhopal for his valuable guidance and for providing lab facilities throughout the research work.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]