European Journal of Medicinal Plants 7(2): 67-76, 2015, Article no.EJMP.2015.069 ISSN: 2231-0894
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Evaluation of Wound Healing Activity of Erythrophleum suaveolens (Guill. & Perr.) Brenan and Moringa oleifera Lam. On Infected Albino Rats O. C. Akanji1* and M. A. Sonibare2 1
Department of Plant Science and Biotechnology, Adekunle Ajasin University, Akungba Akoko, Nigeria. 2 Department of Pharmacognosy, University of Ibadan, Ibadan, Nigeria. Authors’ contributions
This work was carried out in collaboration between both authors. Author OCA was responsible for practically carrying out the laboratory experiments. Author MAS supervised the laboratory experiment. Both authors read and approved the final manuscript. Article Information DOI: 10.9734/EJMP/2015/15222 Editor(s): (1) Marcello Iriti, Department of Agricultural and Environmental Sciences, Milan State University, Italy. Reviewers: (1) Anonymous, Malaysia. (2) Anonymous, Thailand. (3) Shivananda Nayak, The University of the West Indies, Faculty of Medical Sciences, Department of Preclinical Sciences, Trinidad and Tobago. Complete Peer review History: http://www.sciencedomain.org/review-history.php?iid=1018&id=13&aid=8260
Original Research Article
Received 14th November 2014 th Accepted 17 December 2014 Published 25th February 2015
ABSTRACT Introduction: Erythrophleum suaveolens (Guill. & Perr.) Brenan (Caesalpiniaceae) and Moringa oleifera Lam. (Moringaceae) are important medicinal plants that have been reported to possess various medicinal properties including wound healing. In the present study methanolic extracts of Erythrophleum suaveolens and Moringa oleifera were evaluated for their wound healing activity using excision wound model in rats. Methods: Wistar rats (150-200 g) were placed into two groups of non-infected and infected groups. Wound was inflicted on the rats by excision wound model. Local infection was introduced into rat with Staphylococcus aureus and Pseudomonas aeruginosa using a 108 cell/ml inoculum. Gentamycin (8 mg/ml) and normal saline were used as positive and negative controls respectively. The rate of wound healing was assessed by contraction and period of epithelization. Results: Contraction rate on day 12 in the non- infected group were 61.0, 36.5 and 46.8% respectively for M. oleifera leaf, E. sauveolens leaf and stem bark; 93.1, 92.1 and 92.0% in the _____________________________________________________________________________________________________ *Corresponding author: E-mail:
[email protected];
Akanji and Sonibare; EJMP, 7(2): 67-76, 2015; Article no.EJMP.2015.069
Staphlococcus aureus infected group; 25.8, 28.0 and 30.1% in P. aeruginosa infected group. Complete epithelization in non-infected group was achieved in both plants on day 14, on day 16 in Staphylococcus aureus group and on day 18 in P. aeruginosa group. Conclusion: The results proved M. oleifera leaf extract as the most active in a dose dependent manner compared to other extracts. The present study provides justification for the use of Moringa oleifera and Erythrophleum suaveolens in herbal medicine for the treatment of normal and complicated wounds as well as skin diseases.
Keywords: Erythrophleum sauveolens; Moringa oleifera; excision wound; infection; epithelization; wound healing. medium-sized tree up to 25(–30) m tall, often branching low; bark finely fissured, scaly, grey; twigs glabrous. Leaves alternate, bipinnately compound with 2–4 pairs of pinnae; stipules minute, soon falling; petiole and rachis up to 35 cm long, petiole thickened at base; leaflets alternate, 7–14 per pinna, ovate to ovateelliptical, up to 9 cm×5.5 cm, base asymmetrical, apex obtusely acuminate. The bark decoction of the plant is used as an emetic, anti-inflammatory and analgesic. It is also used to dress wounds, to treat chickenpox and gangrenous sores [22]. The bark has been used in arrow poisons and to kill or scare away stubborn pests from cultivated farms [23,24]. The stem bark of Erythrophleum suaveolens has been reported to compose of alkaloids, saponins, cardiac glycosides and tannins [25]. The alkaloid content of bark was found to range from 0.3% to 1.5%. The alkaloid content was found to vary with the age of the tree: A water extract of the bark contained 0.5% and 0.9% respectively in 60 year- and 150 yearold trees. Moringa oleifera Lam. (Moringaceae) commonly called Radish tree [26], is a fast growing deciduous shrub or small tree up to 12 m tall and 30 cm in diameter with an umbrella shaped open crown (unless repeatedly coppiced). It is a softwood tree with timber of low quality. The bark is corky and gummy. Leaves are alternate, oddly bi- or tri-pinnate compound, triangular in outline and 20– 70 cm long.
1. INTRODUCTION Wound healing is an important biological process involving tissue repair and regeneration. Wound is an inescapable event of life and can be described as a break in continuity of soft tissue like skin mucous, tissue surfaces etc. caused by physical, chemical or microbial agents [1]. It is regarded as healed if there is a restoration of the wounded or inflamed tissue to normal condition [2]. Healing is a complex and intricate process initiated in response to an injury that restores the function and integrity of damaged tissues. Wound healing involves continuous cell–cell and cell–matrix interactions that allow the process to proceed in three overlapping phases viz. inflammation (0–3 days), cellular proliferation (3– 12 days) and remodeling (3–6 months) [3-7]. The basic principle of optimal wound healing is to minimize tissue damage and provide adequate tissue perfusion and oxygenation, proper nutrition and moist wound healing environment to restore the anatomical continuity and function of the affected part [8]. In spite of tremendous advances in the pharmaceutical drug industry, the availability of drugs capable of stimulating the process of wound repair is still limited [9]. Moreover, the management of chronic wounds is another major problem due to the high cost of therapy and the presence of unwanted side effects [10,11].
Among myriad of plants, Moringa oleifera, is one of the best known and most distributed species of Moringaceae family [27]. M. oleifera is considered one of the world’s most useful trees, as almost every part of the tree can be used for food, or has some other beneficial property. It is an exceptionally nutritious vegetable tree with a variety of potential uses. This plant has been well documented for its medicinal importance for a long time. The Moringa oleifera stem bark, root bark, fruit, flowers, leaves, seeds and gum are widely used in India folk medicine. Medicinal application reported for M. oleifera includes uses
Many plants have been evaluated for wound healing properties by various workers. Medicinal plants that have been studied recently for their wound healing properties include: Althernathera sessilis, Morinda citrifolia, Lycopodium srratum, Sesamum indicum, Catharanthus roseus, Cecropia peltata, Euphorbia hirta, Ginkgo biloba, Flabellaria paniculata, Ageratum conyzoides, Anthocleista djalonensis, Napoleona imperalis, Ocimum gratissimum, Psidium guajava [12-21]. Erythrophleum suaveolens (Guill. & Perr.) Brenan (Caesalpiniaceae) is a widely distributed 68
Akanji and Sonibare; EJMP, 7(2): 67-76, 2015; Article no.EJMP.2015.069
as an antiseptic, in treating rheumatism, venomous bites, and other activities such as antimicrobial, anti-spasmodic, anti-ulcer, hepathoprotective, anti-tumour and anti-cancer [28-34].
carrying out the study from the Institution’s ethics committee. They were used for a month and fed with standard commercial pellets and clean drinking water.
2.5 Wound Models and Infection with Micro Organisms
The present study is therefore on the wound healing effect of the Moringa oleifera leaves, Erythrophleum suaveolens leaves and stem-bark on wounds inoculated with Staphylococcus aureus and Pseudomonas aeruginosa.
Matured albino wistar rats of both sexes weighing 150 to 200 g were randomly divided into two groups: A non-infection group representing control conditions of 20 animals was divided into five sub-groups of 4 each. Moringa oleifera; leaves (100 mg/ml), Erythrophleum suaveolens; stem-bark (100 mg/ml), Erythrophleum suaveolens; leaves (100 mg/ml) were applied on wound of three subgroups of animals while Gentamycin (8 mg/ml) was applied on the wounds of the fourth subgroup and the fifth sub-group received equal quantity of normal saline and infection groups of 40 animals representing experimental conditions. They were housed 2 by 2 in cages. Under the general anaesthesia from intraperitoneally administered ethyl carbamate, a 2 cmx2 cm skin excision was made (Fig. 1) on a pre-shaved, sterilized left dorsolateral flank of the rat. The resulting area of skin wound was measured using a sterile transparent white plastic which had been previously sterilized with methylated spirit. Haemostasis was achieved by blotting the wound with cotton swab soaked in normal saline. 8 In the infection groups, 0.5 ml of a 10 bacterial per inoculum, prepared by the Mcfarland # ½ standard was introduced gently into the wound using a tuberculin syringe and fine needle [35]. There were two infection groups based on the organism used – Staphylococcus aureus and Pseudomonas aeruginosa. Swabs were taken on day 5 to confirm the presence of the expected organisms in the pus [36,37].
2. METHODS 2.1 Plant Material The plant materials used for this study were leaves of Moringa oleifera, leaves and stem bark of Erythrophleum sauveolens. The plant materials were collected at Ido Local Goververnment, Oyo State on ESNY farmland and were authenticated by Dr. (Mrs) O. A. Ugbogu of Forest Herbarium, Ibadan (FHI). The voucher specimens were deposited at FHI in Ibadan as FHI 108885 and FHI 108886 for M. oleifera and E. suaveolens respectively.
2.2 Plant Preparation and Extraction The specimens were oven-dried at 45°C. The dried samples were powdered with electric mill and kept in air-tight containers until used for analysis. Powdered sample (500 g) was macerated with 70% ethanol for five days. The filtrate for each extraction was combined, and evaporated to dryness with the use of rotary evaporator. Dilutions of each dried crude extract were prepared to give final test concentrations of 100 mg/ml used for wound healing screening.
2.3 Micro-organisms
The first group of animals in the non-infection group was divided into five sub-groups. Moringa oleifera; leaves (100 mg/ml), Erythrophleum suaveolens; stem-bark (100 mg/ml), Erythrophleum suaveolens; leaves (100 mg/ml) were applied on wound of three sub-groups of animals while Gentamycin (8 mg/ml) was applied on the wounds of the fourth sub- group as positive control and the fifth sub-group received equal quantity of normal saline as negative control. The wounds were then packed with sterile gauze soaked in the agent and then covered with adhesive Zinc oxide plaster. Wound dressing was done at 2 daily intervals until complete wound epithelization (Fig. 2) had taken
The laboratory stock cultures of Staphylococcus aureus NCTC 6571 and Pseudomonas aeruginosa NCTC 6750 were collected from the Department of Pharmaceutical Microbiology University of Ibadan, Ibadan.
2.4 Animals A total of sixty rats of both sexes weighing between 150-200 grams were used for the study. They were obtained from the Animal house, Department of Physiology in the University of Ibadan after obtaining ethical approval for 69
Akanji and Sonibare; EJMP, 7(2): 67-76, 2015;; Article no.EJMP.2015.069 no.
place. The wound area was measured on day 5, 7, 12, 14, 18 and 21. Similar procedure was adopted ted for the infection groups only that the administration of drugs did not commence until the infection was established.
wound closure sure on the same day. Figs. 3 3-5 show the analysis of percentage wound contraction for the test drugs. In the infection group of S. aureus aureus, the M. oleifera leaves, E. suaveolens leaves and E. suaveolens stem-bark bark were much more active than the control. Likewise, M. oleifera leaves, E. suaveolens leaves and E. suaveolens stem-bark proved more potent compared to the reference drug on dayy 12 (Fig. 4) with 93.1% 93.1%, 92.1% and 92.0% % respectively as against 83.5 83.5% of the reference drug. Also in P. aeruginosa inoculated group, the crude extracts demonstrated high potency compared to control (Fig. 5). In this group, both crude extracts and reference drug achieved complete epithelization pithelization on day 18 i.e. 100% % as compared to control. The crude extracts of M. oleifera leaves demonstrated highest potency against the non-infected infected and infected wounds compared to other extracts.
2.6 Statistical Analysis Data obtained were expressed as mean ± SE and analyzed by Microsoft SPSS. Values of p
