Wound healing activity of Argyreia nervosa leaves extract.

BACKGROUND: Argyreia nervosa (Convolvulaceae) plant is an example of hallucinogenic plant. The antiseptic, anti-inflammatory, antispasmodic, antibacterial, antiviral, antifungal, anticonvulsant, nootropic, antifertility and aphrodisiac properties have already been reported for this plant. AIM: The aim of present work was to evaluate the wound healing property in normal and diabetic animals by oral and topical administration of ethanolic extract of leaves.
MATERIALS AND METHODS: Phytochemical investigations showed the presence of various biochemicals (alkaloids, flavonoids, carbohydrates, triterpenoids, proteins, saponins, steroids, tannins). A single injection of alloxan monohydrate (120 mg/kg, i.p.) prepared in citrate buffer (0.1 M, pH 4.5) was administered to produce diabetes in rats and mice, after overnight fasting. Excision wounds (sized 300 mm(2) and of 2 mm depth) were used for the study of rate of contraction of wound and epithelization. The means of wound area measurement between groups at different time intervalswere compared using one-way analysis of variance (ANOVA), followed by Dunnet's test.
RESULTS: Extracts of A. nervosa showed significant wound healing effect in normal (topically treated) and diabetic (both topically and orally treated) rats. In diabetic rats, the topically treated group showed more significant effect than the orally treated groups.
CONCLUSION: The present study demonstrates that A. nervosa leaves extract applied topically promotes healing of wounds more significantly as compared to oral application, in both normal rats and alloxan induced diabetic rats, where healing is otherwise delayed.

INTRODUCTION

Argyreia nervosa (family: Convolvulaceae) plant is also known as elephant creeper or woolly morning glory. This plant is found on river banks, edges of lakes and as undergrowth in semi-deciduous forests. Leaves of A. nervosa mainly contain β-sitosterol, 1-tricontanol and quercetin. Traditionally, the plant has been used therapeutically for its wide range of clinical effects such as antiviral, antibacterial, anti-fungal and anti-inflammatory properties. It has rejuvenating, age sustainer and spermatogenic activities as well.[1] The seeds contain the highest concentration of psychoactive compounds in the entire plant. In India, usually leaves and root parts of the plant are used as antiseptic and anti-inflammatory drugs.

In Unani system of medicine, its roots are known to possess aphrodisiac and diuretic properties, and have been used to treat gonorrhea; while its leaves are antiphlogestic, emollient, local stimulant, rubifacient and vesicant. Internally, the extract of A. nervosa leaves has been used to cure the boils and swellings. Externally, it is applied on eczema, etches, ringworm infections and skin diseases. Its seeds are used as hypotensive, spasmolytic and tonic.[2] 50% ethanolic extract of the seeds in a preliminary biological screening showed antispasmodic activity in the isolated guinea pig ileum[3] and antibacterial activity against Staphylococcus aureus. The alcoholic extract of the root exhibited statistically significant anti-inflammatory activity against granuloma technique in albino rats.[4] The seed-oil showed antifungal activity against Aspergillius flavus and Alternaria solani.[5] A paste of its tubers is applied externally in abscesses of stomach. A. nervosa Burm. has been reported for its anticonvulsant activity[6] and nootropic activity.[7]

Diabetic wound healing is an enigmatic and debilitating complication and poses a serious challenge in the clinical practice. The exact pathogenesis of the poor wound healing with the diabetic wound is not clearly understood, but evidence from studies involving both human and animal models reveal several abnormalities in the various phases of wound healing process.[8] Taking a clue from the antibacterial and anti-inflammatory activity of the plant leaves as reported in the traditional literature, the present study was planned to evaluate the wound healing property of its ethanolic extract, using normal and diabetic rats.

MATERIALS AND METHODS

Collection and identification

The plant leaves of A. nervosa were collected locally from Kapoor Chand Kulish Smriti Van, Jaipur, Rajasthan, and identified in the Botanical Survey of India/Arid Zone Circle, Jodhpur. The voucher specimen of the same was also deposited at the above-mentioned herbarium (specimen number JNU/JPR/PC/HG-1). Alloxan monohydrate was purchased from Spectrchem Pvt. Ltd. Company, Mumbai, India.

Selection of animals

Male Wistar rats weighing 150–200 g were used in the study, after obtaining the approval of the Institute's Animal Ethics Committee (approval code no. 005/2009/CPCSEA/JNU). Animals were fed on a standard pellet diet and water ad libitum and maintained at 24–28°C temperature and relative humidity (30% - 70%). Animals marked as fasted were deprived of food for 16 hours, but had free access to water.

Preparation of the extract

The freshly collected leaves were shade-dried and pulverized using a mechanical grinder. The powdered leaves were macerated with 90% ethanol for 3 days, with occasional shaking. The extract was subjected to preliminary phytochemical tests and percentage yield was calculated in the extract after drying.[9]

Phytochemical screening

Phytochemical screening was carried out to identify the presence of alkaloids, carbohydrate, glycoside, flavonoids, triterpenoids, protein, saponins, steroids, tannins, etc. in the ethanolic extract of A. nervosa.[10]

Vehicles

Alloxan was diluted in normal saline and injected intraperitoneally (i.p.). Plant extract and standard drug were suspended in 1% v/v Tween-80 and administered orally (p.o.) to animals with the help of oral feeder. 15% (w/w) extract-ointment was prepared with simple ointment formulation and was applied topically.

Wound healing activity (normal and diabetic rats)

Rats were made diabetic by a single injection of alloxan monohydrate (120 mg/kg, i.p.) prepared in citrate buffer (0.1 M, pH 4.5), after overnight fasting. Blood was drawn from the tail vein 24 hours after the injection and the glucose level was estimated using glucometer (Johnson and Johnson, Mumbai, India). Wounds were made on the rats showing elevated blood glucose level (>250 mg/dl). Blood glucose levels were estimated at the time of the creation of the wounds.

Excision wound creation

Excision wounds were used for the study of rate of contraction of wound and epithelization. All wounds were of full-thickness type, extending up to the adipose tissue. Animals were anesthetized with slight vapor inhalation of diethyl ether and the back side of each rat was shaved. Excision wounds sized 300 mm2 and of 2 mm depth were created along the markings using toothed forceps, a surgical blade and pointed scissors. Animals were closely observed for any infection, and those which showed any sign of infection were separated, excluded from the study and replaced. The treatment was done both topically and orally. The extract was applied in a dose of 100 mg/kg/day for 16 days. Wound areas were measured on days 1, 4, 8 and 16 for all groups, using a transparency sheet and a permanent marker. The recorded wound areas were measured on a graph paper.[11-15]

Sub-grouping of animals in normal and diabetic rat groups

Group I: Control group, i.e. rats treated neither with extract nor with standard

Group II: Rats treated topically with standard drug ointment, i.e. mupirocin ointment (2% w/w)

Group III: Rats treated topically with extract-ointment (15% w/w) of A. nervosa leaves

Group IV: Rats treated orally with ethanolic extract (200 mg/kg, p.o.) of A. nervosa leaves

Statistical analysis

The means of wound area measurement and percentage closure of wounds between groups at different time intervals were compared using one-way analysis of variance (ANOVA), followed by Dunnet's test. Data were analyzed using the Graph Pad Software (5.0 – demo version) and P value of <0.05 was considered to be significant.

Percentage wound closure

Percentage wound closure was calculated by using following formula:

RESULTS

The percentage yield of the alcoholic extract of A. nervosa leaves was found to be 30% w/w. Qualitative test showed the presence of alkaloids, carbohydrates, flavonoids, triterpenoids, proteins, saponins, steroids and tannins. An acute toxicity study on female rats showed no mortality and unusual effects at a dose of 2000 mg/kg, during a time period of 14 days. This helped to predict its non-toxicity and safety.

Wound healing in normal rats

In normal animals, the animals treated topically with the extract (group III) showed significantly more (P < 0.05) wound healing as compared to control animals (group I) on the 8th and 16th days. The standard drug treated animals (group II) showed significant (P < 0.05) wound healing on the 4th, 8th and 16th days, compared to the animals of the control group. Effect of the extract given orally (group IV) was nonsignificant compared to the control animals [Table 1].

Table 1

Combined wound area of normal rats (in mm2)

The wound closure was 92.96% with the extract ointment (group III) and 94.94% with 2% mupirocin (group II) compared to 80.27% in the control group (group I) on the 16th day of treatment, with a statistically significant difference. Wounds of animals treated orally with the extract did not show any significant closure at all phases [Table 2].

Table 2

Percentage wound closure in normal rats

Wound healing in diabetic rats

In diabetic animals, the extract treated groups III and IV (topically and orally, respectively) showed significant (P < 0.01) wound healing as compared to control animals on the 16th day, which was comparable to standard mupirocin (group II) treated animals [Table 3].

Table 3

Combined wound area of diabetic rats (in mm2)

The animals treated with ethanolic extract topically (group III) showed more wound closure (76.20%) than orally treated (group IV, 74.36%) animals, with a statistically significant difference compared to normal on all days [Table 4].

Table 4

Percentage wound closure in diabetic rats

DISCUSSION

Wound healing is characterized by three stages, viz., inflammation, proliferation, and remodeling. The proliferative phase typically demonstrates angiogenesis, collagen deposition, granulation tissue formation, epithelialization and wound contraction. In angiogenesis, new blood vessels grow from endothelial cells. In fibroplasia and granulation tissue formation, fibroblasts grow and form a new provisional extracellular matrix by excreting collagen and fibronectin. In epithelialization, epithelial cells crawl across the wound bed to cover it. Fibronectin, the major glycoprotein secreted by fibroblasts, has important functions of chemo-attraction for macrophages, fibroblasts and endothelial cells, promoting re-epithelialization and acting as a transduction agent in wound contraction. Wound contraction occurs by myofibroblasts, which establish a grip on the wound edges, bringing them in apposition.

The present study concluded that the topical application of ethanol extract of A. nervosa leaves plays a major role in wound healing in normal and diabetic animals. It was also found that the ethanolic extract of A. nervosa is more effective topically compared to oral preparation. The present study also demonstrates that A. nervosa leaf extract applied topically promotes healing of wound in alloxan-induced diabetic rats, where healing is otherwise delayed. These preliminary results can further be used a basis for a full-fledged study to evaluate the role of extract of A. nervosa in diabetic animal models, so as to elucidate its role in the treatment of diabetic foot.