Evaluation of antioxidant and wound healing activity of the leaves of bridelia airyshawii spreng.
Yogesh M. Bagad2*, Mayur R. Bhurat2, Anil U. Tatiya1, Sanjay J. Surana1, Shashikant D. Barhate2
1Department of Pharmacognosy, R.C. Patel College of Pharmacy, Shirpur (MS) – 425405
2Shree Sureshdada Jain Institute of Pharmaceutical Education and Research, Jamner, Jalgaon (MS)-424206.
*Corresponding Author E-mail: email@example.com
In recent years, oxidative stress and free radicals have been implicated in impaired wound healing. The wound healing parameters were evaluated by using incision, and dead space wounds in extract-treated rats and controls. free radical are generated in side every human body these free radicals are beneficial if they are generated in small amount but if they are generated in huge amount they are harmful to our body. At low concentration free radicals can stimulate the proliferation of cells as well as the formation of connective tissue and new blood vessels. "Skin wounds are rich in free radicals. There was a longer-lasting free radical effect in the wounds that had been treated with acetone and aqueous extract of LBR. The wound healing activity of acetone and aqueous extracts of Bridelia airyshawii Spreng. (Euphorbiaceae) was evaluated on incision and dead space wound models on albino rats. The healing activity was more significant in acetone extract treated animals. In incision wound models the wound healing process was evidenced by increase in the tensile strength. While in dead space wound model, the weight of the granuloma and tensile strength were increased indicating the increase in collagenation. The results were also compared with control and standard drug.
Bridelia airyshawii Spreng. is a deciduous tree which belongs to the family “Euphorbiaceae” and popularly known as Asana. (Kirtikar and Basu, 1935). Bridelia airyshawii is found to be distributed throughout the hotter parts of India, Ceylon and Burma. It is found throughout India up to an altitude of 1,000 m except in the very dry regions. In India it is widely distributed in Maharashtra and Karnataka state. The genus Bridelia is a shrub or climbers, comprising about 60 Spp. distributed from tropical Africa and Madgascar, easteard through the Indo- Malayan region of Southern China to tropical Africa and the Pacific Islands (Anon, 1958). The plant is pungent, bitter, heating; useful in Vata, lumbago, hemiplegia. (Kirtikar and Basu, 1935) The various extracts of the plant possesses anti-inflammatory (Mehare, et. al., 2003), and Hypoglycemic activity (Tatiya et. al., 2011).
There is no systematic studies have been carried out on the antioxidant and wound healing potential of leaves of this species. The present paper is reporting the antioxidant effect of different extracts and wound healing potential of the acetone and aqueous extracts of the leaves of Bridelia airyshawii on incision and dead space wound models on experimental rats.
MATERIAL AND METHODS:
The fresh leaves of B. airyshawii Spreng. was collected in the month of August (2006) from Nagziri (Toranmal) of Nandurbar District (MS), India. It was identified, confirmed and authenticated by Dr. D. A. Patil, Dept. of Botany, Dr. P. R. Ghogrey College, Dhulia (MS. The material were shade dried and powdered mechanically. In successive solvent extraction, dried material is extracted with different solvents of increasing polarity (Hideaki Otsuka et. Al., 2005)
Preparation of Plant Extract:
500 g of powdered Leaves of Bridelia airyshawii was extracted with methanol in several batches by using Soxhlet extractor. Methanolic extract obtained by continuous hot extraction was used for successive extraction and fractionation purpose.
The Methanolic extract thus obtained was successively extracted with pet ether 60-800C then Ethyl acetate and finally solvent remain is hydro alcoholic extract. These extracts were concentrated separately under reduced pressure. The acetone extract were prepared separately by cold maceration method by simply dissolved powder in the (70:30) acetone (70ml acetone and 30ml water). All extracts were dried in vacuum dryer and stored in desiccators care must be taken that no solvent is remain in the final extracts.
Acute Toxicity Studies:
Healthy young albino Westar rats of either sex weighing between 150-200 gm were procured from Yash Farm, Pune and The rats were housed in cages and maintained under standard conditions (12 hrs light and dark cycles, at 25±27°C and 35-60% humidity). Standard palletized feed and water were provided ad libitum. The study was approved by Institutional Animal Ethical Committee (IAEC) of R. C. Patel College of Pharmacy, Shirpur, India, registered under CPCSEA. (RCPCOP, IAEC/ 2006-07/07).
The animals were fasted overnight and divided into 6 groups (n=6). The Acetone and Aqueous extracts of B. airyshawii. were administered to each of the groups in aliquot doses (100, 150, 300, 500, 1000, 3000, 5000 mg/kg).the animals were continuously observed for mortality and behavioral responses for 48 h.
Evaluation of Antioxidant Activity:
1. Determination Free radical scavenging activity by DPPH method:
Different concentrations of test and standard drug solutions were prepared. To this 5 ml of methanolic solution of DPPH was added, shaken well and mixture was incubated at 37°C for 30 min. A blank was prepared in the similar way and the absorbance was measured at 517nm. Scavenging activity was expressed as the percentage inhibition calculated using the following formula (fig. No. 1) (Yen, et. al., 1997). (Blois, et. al., 1958)
2. Determination of Nitric oxide radicals scavenging activity:
Different concentrations of sample solutions were prepared in 100ml volumetric flasks. To this 0.1489 g of sodium nitroprusside (5mM) was added and kept for incubation. At different time intervals 5.6 ml was taken, 0.2 ml of Griess reagent A was added, and kept for incubation at 30° C for 10 min. After incubation 0.2 mL of Griess reagent B was added and kept for incubation at 30° C for 20 min. After incubation, absorbance was measured at 542 nm against blank. Concentration of NO was calculated from standard calibration curve. (fig. No. 2) (Ilavarasan, et. al., 2005)
Fig. N0. 1 % Inhibition Vs concentration plot of successive extracts of leaves of Bridelia airyshawii by DPPH radical method.
Fig No. 2. % of Inhibition Vs concentration plot of successive extracts of leaves of Bridelia airyshawii by Nitric oxide assay.
3. Determination of Anti-lipid peroxidation effect:
0.5mL of homogenate was taken and to it 1mL of 0.15M KCL and 0.5ml of test drugs at different concentrations (10, 20, 40, 60, 80 µg/ml) were added. Lipid peroxidation was initiated by adding 100µl of 1mM ferric chloride. The reaction was stopped by adding 2ml of ice cold 0.25N HCL containing 15% trichloroacetic acid, 0.38% thiobarbituric acid, and 0.2 ml of 0.05% butylated hydroxyl toluene. These reaction mixtures were heated for 60min at 80ºC then cooled and centrifuged at 6900 rpm for 15min.The absorbance of supernatant was measured at 532nm against blank, which contained all reagents except liver homogenate and drug. Same experiments were performed to determine the normal (without drug and FeCl3) and induced (without drug) lipid peroxidation level in the tissue. The percentage of anti-lipid per oxidation effect (%ALP) was calculated by the following formula (fig. No. 3) (Feldman, et. al., 1999).
Fig No. 3. % of ALP Vs concentration plot of successive extracts of Leaves of Bridelia airyshawii by In-vitro Lipid peroxidation method.
Evaluation of Wound Healing Activity:
Drug formulations / Preparation of Ointment:
The ointment in concentration of 5% (W/W) and 10% (W/W) of the Acetone (70%) and aqueous leaves extract were prepared in lipophillic base. For oral administration, suspensions of 25mg/ml of the acetone (70%) and aqueous extracts were prepared in 2% gum tragacanth.
1) Resutured incisional wound model:
The rats were anaesthetized with anesthetic ether and animals were secured to operation table in its natural position. Two Para vertebral straight incisions of 6 cm each was made through the entire thickness of the skin on either side of the vertebral column with the help of a sharp blade as described by Ehrlich and Hunt. No local or systemic chemotherapeutic agents were given. Care was taken to see that the incisions were at least 1cm lateral to the vertebral column. After complete homeostasis, the wounds were closed by means of interrupted sutures placed at equidistant points about 1cm apart, using black silk thread and curve needle (No. 11). Wounds were then mopped with cotton swabs soaked in 0.9% NaCl and Animals were caged individually.On the 8th post wounding day, the sutures were removed and skin breaking strength was measured on 10th post wounding day by continuous constant water flow technique as described by Lee . (fig. No. 4) (Ehrlich, H.P., 1968)
2) Dead space wound model:
Under the ether anesthesia, the dead Space wound were created by implanting 2 polypropylene tubes (0.5 X 2.5 cm each) one on either side in the lumber region on the dorsal surface of the rat. The sutured wounds were mopped with an alcoholic swab and animals were placed into their individual cages.
The animals were divided in to five groups. The group 1 received normal saline which was considered as control and groups 2, 3 received aqueous leaves extract and 4and 5 received acetone extract in the dose of 250 mg/kg, 500 mg/kg and mg/kg orally of leaves extracts which was reconstituted in distilled water, respectively.
Fig No. 4. Skin Breaking Strength Measurement in Incisional Wound Model
Animals received drugs from 0 day to 9th post wounding day. On 10th post wounding day, the granulation tissue harvested on each implanted tube was carefully dissected out along with the tube and employed for determination of breaking strength. and granuloma excised from dead space wounds were dried overnight 60°C so as to obtain constant dry weight. Their weights were expressed as mg/100gm body weight. (fig. No. 5) (Udupa S.L., 1999, Lodhi, S. et.al, 2006, Shetty, S. B., 2006, Bhaskar H N.-2005)
The data were subjected to One way ANOVA followed by Bonferroni test and the values of P< 0.05 were con-sidered statistically significant.
Fig No. 5. Tensile Strength Measurement of Granuloma in Dead space wound model.
Fig No. 6. Dry Weight Granuloma in Dead Space Wound Model
RESULTS AND DISCUSSIONS:
DPPH is relatively stable nitrogen centered free radical that easily accepts an electron or hydrogen radical to become a stable diamagnetic molecule. DPPH radicals react with suitable reducing agents as a result of which the electrons become paired off forming the corresponding hydrazine. The solution therefore loses colour stoichometrically depending on the number of electrons taken up (Mangathayaru et al., 2007).
Successive extracts were tested for the antioxidant activity in various concentrations ranging from 10 – 80 μg/ml by DPPH method, Nitric oxide radicals scavenging and Anti-lipid peroxidation effect. The results show the effect of these extracts to scavenge free radicals. It was observed that free radicals were scavenged by the extracts in a concentration dependent manner. The maximum percentage inhibition of DPPH for methanol, ether, ethylacetate, and aqueous extracts was 62.06 %, 44.44 %, 50.63 %, and 55.39 % respectively at 80 μg concentration. Standard drug ascorbic acid showed 75.39 % inhibition of the DPPH radical at 80 μg concentration(fig. No. 1). The maximum percentage inhibition of Nitric oxide radicals scavenging activity for methanol, ether, ethylacetate, and aqueous extracts was 66.03 %, 41.37 %, 53.27 %, and 62.58 % respectively at 80 μg concentration(fig. No. 2). The maximum percentage inhibition of Anti-lipid peroxidation effect for methanol, ether, ethylacetate, and aqueous extracts was 67.34 %, 43.67 %, 55.30 %, and 56.32 % respectively at 80 μg concentration(fig. No. 3).
The overall results shows that the acetone extract of leaves of Bridelia airyshawii possesses significant wound healing activity as compared with aqueous extract of the same plant and with control. The result from phytochemical screening shows that the acetone and aqueous extract of leaves mainly contains tannins and polyphenols. There was reported that tannins and different polyphenolics from the plant having wound healing activity. The wound healing process involves various phases viz. granulation, wound contraction, collagenation, collagen maturation and scar formation. The two different models have been used in our study to assess the effect of various extract of leaves of Bridelia airyshawii. In incisional wound model the animals treated with acetone extract showed increase in the tensile strength on 10th post wounding day. The tensile strength of the animals treated with acetone and aqueous extracts and controls were depicted in (fig. No. 4). similar type of work has been reported on Trigonella foenum gracecum, Malva sylvestris, Calendula officinalis, and Fucus vesiculosus. The effect of oral administration of suspensions of the leaves extracts on the dead space wound models were also assessed by the increase in its weight granuloma and by increase in tensile strength. The increase in tensile strength of the granuloma tissue indicates enhanced collagen maturation by increased cross-linking of collagen fibers. While, an increase in the granuloma weight indicates the presence of higher protein content. Among these treated animals the response was shown to be the best in acetone extract treated animals than aqueous one.(fig. No. 5 and 6)
In the acute toxicity test, oral administration of maximum 5 g/kg of aqueous extracts of the leaves of the plant Bridelia airyshawii Spreng. produced neither mortality nor changes in behavior or any other physiological activities. So, the LD50 of aqueous leaves extract was more than 5000 mg/kg.
The acetone and aqueous extracts Bridelia airyshawii. spreng were shows positive test for tannins. It also shows positive test for carbohydrate. It was observed by determination that leaves contains (18-20%) of tannins. The acetone extract of Bridelia airyshawii. Spreng shows significant wound healing activity in both models such as incisional wound model, and dead space wound model.
In incisional wound model, the plant extract ointment of both acetone and aqueous prepared in lipophillic base (5% and 10%) treated animals demonstrated significant (P<0.01) increase in skin breaking strength as compared to the control group but there was standard treated animals shows better skin breaking strength.
In dead space wound model, there was no significant increase in tensile strength, and dry weight of granulation tissue at the dose of 250 mg/kg but significant increase was observed at the doses of 500 mg/kg as compared to the control.
Acetone and aqueous extract shows significant wound healing property comparable to that of standard group. The external application of these extracts on the wound prevented the microbes to invade through the wound, resulting in protection of wound against the infections of the various microorganisms. At the same time, external application of the extracts entrapped the free radicals liberated from the wound surrounding cells, which are having inherent machinery to protect the cells from the microbes. Therefore, it can be assumed that the Synergistic effect of both anti microbial and anti oxidant activity accelerated the wound-healing process.
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Received on 12.01.2013 Modified on 20.01.2013
Accepted on 04.02.2013 © RJPT All right reserved