Wound Healing and Antioxidant Activity of Achyranthes aspera

Abstract

Achyranthes aspera Linn (Amaranthaceae), commonly known as apamarga, is a commonly available plant in India. This plant has been used in the treatment of cuts by the people living in Tamil Nadu. The ethanol and aqueous extracts of leaves of Achyranthes aspera were prepared and its wound healing and antioxidant activity were evaluated. The wound healing activity was studied using two wound models, excision wound model and incision wound model. The free radical scavenging activity of the extracts was also assessed using two methods, DPPH radical scavenging activity, and superoxide scavenging activity. The extracts responded significantly in both the wound models tested. Also, the plant exhibited good antioxidant effect by preventing the formation of free radicals in the two models studied. Comparatively, the aqueous extract was found to be more effective. These findings could justify the inclusion of this plant in the management of cuts by the local people.

Keywords::

• Achyranthes aspera
• wound healing
• nitrofurazone
• antioxidant

Introduction

Wounds are physical injuries that result in an opening or breaking of the skin. Proper healing of wounds is necessary for the restoration of disrupted anatomical continuity and disturbed functional status of the skin. It is a product of the integrated response of several cell types to injury. Wound healing is a complex multifactorial process that results in the contraction and closure of the wound and restoration of a functional barrier. Repair of injured tissues occurs as a sequence of events, which includes inflammation, proliferation, and migration of different cell types. The reactive oxygen species (ROS) are deleterious to wound healing process due to the harmful effects on cells and tissues. Free-radical-scavenging enzymes (FRSE) are a cytoprotective enzyme group that has an essential role in the reduction, deactivation, and removal of ROS as well as regulating the wound healing process. Also, this complex phenomenon involves self-generated autocoids and hormones working in a systematic synchrony leading to wound healing (Meenakshi et al., 2006).

Plants are rich source of phytochemicals, which can have wound healing and antioxidant properties. Several indigenous drugs have been described in folkloric Indian medicine for the management of cuts, bruises, burns, and wounds. Achyranthes aspera Linn (Amaranthaceae), commonly known as apamarga, is an herb that grows wild and abundantly in India; folk practitioners and local people of Karungal village, Kanyakumari district, Tamilnadu, India use the leaves of this plant for healing wounds.

Achyranthes aspera is reported to have various activities including hepatoprotective (Bafna & Mishra, 2004), cancer chemopreventive (Chakraborty et al., 2002), anti-inflammatory, anti-arthritic (Gokhale et al., 2002), thyroid-stimulating, anti-peroxidative (Tahiliani & Kar, 2000), stimulates reproductive functions (Sandhyakumary et al., 2002), abortifacient (Pakrashi, 1977), anti-leprotic (Ojha et al., 1966), antibacterial (Verma et al., 1997), immunomodulatory (Rao, 2002), and contraceptive (Wadhwa et al., 1986). Saponins of this plant are reported to have phosphorylase activity on the heart (Ram et al., 1971). Root extract is used in malarial fever, asthma, hypertension (Neeru & Sharma, 2006), and diabetes (Akhtar & Iqbal, 1991). Decoction of the whole plant has diuretic properties and is used in the treatment of pneumonia (Neeru & Sharma, 2006).

So far no scientific evidence was found in a literature survey regarding wound healing properties of Achyranthes aspera. So, the present study focused on wound healing activity of Achyranthes aspera leaves to justify its traditional use and antioxidant activity, and also studied the mechanism behind the wound healing activity.

Materials and Methods

Plant material

The leaves of Achyranthes aspera were collected from the fields surrounding Mandsaur, Madhya Pradesh (MP), India, in the month of August and identified by Dr. H.S. Chatree, Botanist, Department of Botany, Govt. P. G. College, Mandsaur. A voucher specimen (AA/004/2005/BRNCOP) has been deposited in the Department of Pharmacognosy for future reference.

Preparation of extracts

Shade-dried, coarsely powdered leaves (500 g) were defatted with petroleum ether (60–80°C) for 72 h. The defatted drug was Soxhlet-extracted with ethanol and distilled water successively for 72 h each. The obtained extracts were evaporated in vacuum to give residues, and percentage yield of the extracts were found to be 10.1% and 5.5%, respectively. To identify the type of constituents present in the extracts, qualitative chemical tests were performed (Khandelwal, 2005). The extracts were used in the form of ointment. Plant extract ointments (10% w/w) were prepared by mixing the extracts separately in yellow soft paraffin (Muthusamy et al., 2006).

Animals

Healthy Wistar rats of either sex (150–200 g) with no prior drug treatment were used for the present studies. The animals were fed a commercial pellet diet (Hindustan Lever, Bangalore, India) and given water ad libitum. The animals were acclimatized to laboratory hygienic conditions for 10 days before starting the experiment. The animal study was performed in the Division of Pharmacology, B R Nahata College of Pharmacy, Mandsaur with due permission from the Institutional Animal Ethics Committee (registration number 23/M.Ph/06/IAEC/BRNCOP).

For incision and excision wound models, animals of either sex were divided into four groups in each model consisting of five animals in each group: group I – ointment base; group II – 0.2% nitrofurazone ointment; group III-aqueous extract; group IV – ethanol extract. The extracts and the standard ointment were used twice daily to treat the different groups of animals.

Wound healing activity

Excision wound model

Animals were kept under light ether anesthesia throughout the surgical procedures. An impression of 500 mm² as described by Nagappa and Binu (2001) was made after leaving at least 5 mm space from the ears. The skin of the impressed area was excised carefully to the complete thickness and a wound of 500 mm² was produced. Hemostasis was achieved by application of normal saline solution. Ointment base, nitrofurazone ointment, and extracts in ointment form were applied topically, until the wound was completely healed. The physical attributes of wound healing, i.e., wound closure (contraction), epithelization and scar features were recorded. The wound contraction was studied by tracing the raw wound area on transparent paper on day 0, 8 and 16 subsequently until complete epithelization has occurred. The criterion for complete epithelization was fixed as a formation of the scar with absence of raw wound area. The wound area was measured planimetrically by the help of mm² scale graph paper. The degree of wound healing was calculated as percentage wound closure area with the original area using the formula as below: where Ao = wound area on day zero, Ad = wound area on corresponding days.

Incision wound model

The incision wound model was studied as described by Ehrlich and Hunt (1969). Under light ether anesthesia the animals were secured to an operation table in its natural position. Two paravertebral straight incisions of 6 cm each were made on either side of the vertebral column with the use of a scalpel blade. Incisions were made at least 1 cm apart to the vertebral column. The wounds were closed with sutures at equidistant points of 1 cm apart by silk thread of zero grade with the help of a straight round-bodied needle.

Wounds were cleaned with 70% alcohol soaked cotton swabs. The animals were treated with the ointment base, nitrofurazone ointment, and extracts in ointment form topically. The sutures were removed after 8 days. The tensile strength of the wound was determined on both sides by a continuous constant water supply technique (Singh et al., 2006).

In the continuous constant water supply technique, the anesthetized animals were secured to the operating table. All the forceps were firmly fixed on lines facing each other. The forceps on one side was hooked to a metal rod to keep it in position while the other forceps connected to a polythene reservoir by a string run over a pulley. The water was allowed to flow at a constant rate into a polythene reservoir and the pulling force necessary to disrupt the wound was gradually built under controlled conditions.

The flow of water was regulated with the help of an occlusion clamp on polythene tubing, which was connected to the reservoir and was raised to a suitable height. As soon as the gaping of the wound was formed, the water flow was cut off. The pulling force of the wound was immediately released by lifting up the polythene reservoir to avoid further opening of wound. The volume of water accumulated in reservoir was measured and was converted to corresponding weight by considering the density of water as 1. The tensile strength was expressed as the minimum weight of water necessary to bring out the gaping of the wound.

Statistical analysis

Data are expressed in mean ± Standard Error Mean (SEM). The difference among means was analyzed by one-way Analysis of Variance (ANOVA), p values less than 0.05 were considered as significant.

Antioxidant activity

The antioxidant activity of the ethanol and aqueous extracts was carried out using two methods, DPPH radical scavenging activity, and superoxide scavenging activity.

DPPH scavenging activity

DPPH (α, α -diphenyl β -picrylhydrazyl) scavenging activity was measured by the spectrophotometric method. A stock solution of DPPH (1.5 mg/ml in ethanol) was prepared, and 75 μ l in 3 ml of ethanol gave an initial absorbance of 0.9. Decrease in the absorbance in presence of sample extracts at different concentration (100–500 μ g/ml) was noted after 15 min. IC₅₀ was calculated from percentage inhibition (Sheeja et al., 2006).

Superoxide scavenging activity

Superoxide scavenging activity was determined by the NBT reduction method of McCord and Fridovich. The assay was based on the capacity of the sample to inhibit blue formazan formation by scavenging the superoxide radical generated in riboflavin-light-NBT system. The reaction mixture contained EDTA, riboflavin, nitro blue tetrazolium (NBT), various concentration of extract and phosphate buffer (pH 7.8) in a final volume of 3 ml. The tubes were uniformly illuminated with an incandescent lamp for 15 min and absorbance was measured at 590 nm before and after illumination. The percentage inhibition of superoxide generation was measured by comparing the absorbance values of control and those of the test compound (Sheeja et al., 2006). Ascorbic acid was used as positive control.

Statistics

The decolorization was plotted against the sample extract concentration, and a linear regression curve was established in order to calculate the IC₅₀ (μ g/ml) being the amount of sample necessary to decrease by 50% the absorbance of radicals.

Results

Phytochemical screening of the tested extracts showed the presence of alkaloids, carbohydrates, tannins, proteins, saponins and flavonoids in both the extracts.

The results of the excision wound model are presented in Table 1. The extracts of the leaves of A. aspera studied for wound healing activity showed significant wound healing activity when compared to control. Percentage closure of original wound area was calculated at different time intervals. The measurement on day 8 showed that the percentage closure of the original excision wound area was found to be 56.99 (standard), 57.28 (aqueous extract), and 49.09 (ethanol extract). The tested extracts significantly (P < 0.001) promoted wound closure compared to control and standard. On day 16 the extent of percentage wound closure was 97.58 (standard), 98.18 (aqueous extract), and 88.62 (ethanol extract).

Table 1.  Effect of extracts of Achyranthes aspera on excision wound model.

	Remaining of original excision wound area (mm^2)
Treatments	Day 0	Day 8	Day 16	Epithelization time (d)
Control	484.00 ± 14.80	311.00 ± 14.97 (35.75)	151.40 ± 8.10 (68.64)	24.30 ± 1.54
Nitrofurazone	480.30 ± 11.33	219.80 ± 14.55** (56.99)	11.40 ± 0.48** (97.58)	18.50 ± 0.51*
Aqueous extract	479.00 ± 11.59	204.50 ± 10.96** (57.28)	8.00 ± 0.84** (98.18)	20.00 ± 0.63*
Ethanol extract	488.30 ± 13.49	247.90 ± 13.59** (49.09)	54.80 ± 3.14** (88.62)	22.00 ± 0.82

Values are expressed as mean ± SEM for five observations. * P < 0.01, **P < 0.001 versus control; values in parenthesis are percentage closure of original excision wound area.

The most important and commonly used parameter to evaluate wound healing is tensile strength or wound breaking strength of granulated tissue. The results of the incision wound model are given in Table 2. The mean tensile strength of granulated tissue in control animals was less when compared to the extracts and standard ointment treated groups. These observations of the incision wound model confirm the prohealing effect of II, III and IV groups as observed in the excision wound model (Table 2). Comparatively, aqueous extract was found to be more effective than ethanol extract.

Table 2.  Effect of extracts of Achyranthes aspera on incision wound model.

Treatment	Incision wound breaking strength (g)
Control	121.20 ± 9.67
Nitrofurazone	435.30 ± 18.17*
Aqueous extract	428.60 ± 19.34*
Ethanol extract	317.60 ± 19.17*

Values are expressed as mean ± SEM for five observations.

* P < 0.001 versus control.

Since there is a definite role of free radicals in the pathogenesis of wounds, the antioxidant activity was also studied using two models. The study showed that the extracts of the leaves of A. aspera could inhibit the oxygen free radicals as seen from scavenging of super oxide and DPPH radicals, and therefore possesses antioxidant activity (Table 3). The antioxidant activity of the aqueous extract was better than that of the ethanol extract. Both the extracts showed less activity than the standard ascorbic acid.

Table 3.  Antioxidant activity of extracts of A. aspera by DPPH and NBT methods.

	IC50(μ g/ml)
Extracts	DPPH	NBT
Ethanol	556.07	490.66
Aqueous	444.69	392.20
Ascorbic acid	122.22	102.44

Discussion

Wound healing is a complex process, characterized by homeostasis, re-epithelization, granulation, tissue formation, and remodeling of the extracellular matrix. Although healing process takes place by itself and does not require much help, various risk factors such as infection and delay in healing has brought attention to promote this process (Shanmuga et al., 2002).

Our present study examined A. aspera leaves for wound healing activity. Topical application of aqueous and ethanol extracts of leaves at the wound site in an excision wound healing model produced significant (P < 0.001) wound healing activity. The healing of the excision wounds can be monitored by recording wound area changes (closure rate) at conveniently fixed intervals of time. It was also monitored by recording complete epithelization. Epithelization can be directly measured in terms of days for complete closure of wounds. In addition, studies with an animal model showed an enhanced rate of wound contraction and a drastic reduction in healing time, which might be due to enhanced epithelization (Shanmuga et al., 2002). In an incision model, tensile strength was measured indirectly to assess collagen content and maturation. Increase in the protein and collagen content is responsible for the enhanced migration of fibroblast cells, epithelial cells, and synthesis of extracellular matrix, including collagen during the healing process in treated rats (Muthusamy et al., 2006). The results indicate that aqueous and ethanol extracts of A. aspera significantly promoted (P < 0.001) tensile strength as compared to that of control.

Tannins have been used in dermatology because of their strong astringent property, which positively effects wound healing. Tannins also promote capillary vasoconstriction, which decrease vascular permeability and cause a local anti-inflammatory effect (Lopes et al., 2005). Phytochemical screening revealed the presence of tannins in both extracts and this may be one component that contributes to the plant's wound healing effect.

Further antioxidant activity contributes favorably to the cicatrization of wounds, because the production of reactive oxygen species during the process of tissue injury aggravates the disorders in the tissue (Lopes et al., 2005). Free radicals can damage cell structures including membrane lipids, proteins, enzymes, and nucleic acids. So a scavenging effect might be one of the most important components of wound healing. Aqueous and ethanol extracts of the leaves showed free radical scavenging activity in a dose-dependent manner. The antioxidant enzymes (superoxide dismutase and catalase) are known to quench radicals and, thus, prevent the damage of cells caused by free radicals (Singh et al., 2006). The results indicate that the plant may possess potent antioxidant activity by inhibiting lipid peroxidation and increase SOD and catalase activity (Table 3).

Flavonoids are well known for their antioxidant potential. Moreover, phytoconstituents like flavonoids are also responsible for wound healing potential (Mukherjee et al., 2000). In our present study, a preliminary phytochemical investigation revealed that aqueous and ethanol extracts of leaves contain tannins, flavonoids, glycosides, and alkaloids. Thus, the enhanced wound healing may be due to the free radical scavenging action and immune-enhancing property (Vasudeva et al., 2006) of the plant.

The results of the present study conclude that aqueous and ethanol extracts of Achyranthes aspera have significant wound healing and antioxidant activities. The results of this study seem to confirm the traditional use of A. aspera for the treatment of wounds. The extracts of these plants can further be developed into phytomedicines for the management of septic wounds.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.