View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector J Acupunct Meridian Stud 2015;8(3):134e141 Available online at www.sciencedirect.com Journal of Acupuncture and Meridian Studies journal homepage: www.jams-kpi.com - RESEARCH ARTICLE - Antitumor and Wound Healing Properties of Rubus ellipticus Smith. Blassan Plackal George, Thangaraj Parimelazhagan*, Yamini T. Kumar, Thankarajan Sajeesh Department of Botany, Bharathiar University, Coimbatore, Tamil Nadu, India Available online 6 November 2013 Received: Jun 17, 2013 Abstract Revised: Sep 9, 2013 The present investigation has been undertaken to study the antioxidant, antitumor, and Accepted: Sep 23, 2013 wound healing properties of Rubus ellipticus. The R. ellipticus leaves were extracted us- ing organic solvents in Soxhlet and were subjected to in vitro antioxidant assays. R. ellip- KEYWORDS ticus leaf methanol (RELM) extract, which showed higher in vitro antioxidant activity, antioxidant; was taken for the evaluation of in vivo antioxidant, antitumor, and wound healing prop- antitumor; erties. Acute oral and dermal toxicity studies showed the safety of RELM up to a dose of Rubus ellipticus; 2 g/kg. A significant wound healing property was observed in incision, excision, and wound healing Staphylococcus aureus-induced infected wound models in the treatment groups compared to the control group. A complete epithelialization period was noticed during the 13th day and the 19th day. A 250-mg/kg treatment was found to prolong the life span of mice with Ehrlich ascite carcinoma (EAC; 46.76%) and to reduce the volume of Dalton’s lymphoma ascite (DLA) solid tumors (2.56 cm3). The present study suggests that R. ellip- ticus is a valuable natural antioxidant and that it is immensely effective for treating skin diseases, wounds, and tumors. 1. Introduction proliferative, and remodeling phases [1]. The bioactive compound that promotes these events can be therapeuti- Wound healing is the body’s natural process of regenerating cally used to improve the wound healing process. Reactive in a wounded area. When an individual is wounded, a set oxygen species (ROS) play a vital role in wound healing. of biochemical events take place, i.e., inflammatory, Superoxide is rapidly converted to H2O2 by superoxide * Corresponding author. Department of Botany, Bharathiar University, Maruthamalai Main Road, Coimbatore 641 046, Tamil Nadu, India. E-mail: [email protected] (T. Parimelazhagan). pISSN 2005-2901 eISSN 2093-8152 http://dx.doi.org/10.1016/j.jams.2013.10.002 Copyright ª 2015, International Pharmacopuncture Institute. R. ellipticus Wound Healing & Antitumor Properties 135 dismutase (SOD). Release of H2O2 may promote formation experimental clearance (Kovai Medical Center Research of other oxidants that are more stabledthis suggests that and Educational Trust, KMCRET/Ph.D/03/2011). The acute the wound site is rich in oxidants [2]. Antioxidants there- toxicity studies were carried out by following Organization fore enhance wound healing by reducing the damage for Economic Co-operation and Development (OECD) caused by radicals. Hence, plants showing antioxidant guidelines [9]. Thirty Swiss albino male mice were divided properties could also have wound healing activity. The into five groups of six animals and treated orally with target of many research studies has been the discovery of different doses of R. ellipticus leaf methanol (RELM) natural and synthetic compounds that can be used in the extract dissolved in 0.1% carboxy methyl cellulose (CMC) for prevention and/or treatment of cancer. Several studies 30 days: Group I, normal; Group II, control treated with have shown that the extracts from different medicinal 0.1% CMC; Group III, 50 mg/kg body weight (b. wt); Group plants have great significance in reducing the risk of inci- IV, 100 mg/kg b. wt; and Group V, 250 mg/kg b. wt. dence and progression of tumors. At the end of the experiment, animals were sacrificed, The genus Rubus is very diverse. It includes over 750 the liver was excised and washed in ice-cold trisamino- species in 12 subgenera and is found in all continents except methane hydrochloride (TriseHCl) buffer (0.1 M, pH 7.4), Antarctica [3]. R. ellipticus is a weedy raspberry, well and cytosolic samples of liver homogenate were prepared established in disturbed wet forests with an elevation of by centrifugation at 9500 x g for 30 minutes at 4C. 548e1700 m, and thrives in sunny open pastures as well as The estimation of total protein was carried out by deep rain forests [4]. Previously reported as an in vitro Lowry’s method [10]. The following parameters were antioxidant, its traditional uses and pharmacological prop- assayed in liver to assess the oxidative stress: SOD activity erties, such as anti-inflammatory, analgesic, and antipyretic was measured by the nitro blue tetrazolium (NBT) reduc- activities [5], encouraged us to carry out this study. The tion method of McCord and Fridovich [11]. Catalase (CAT) Rubus species is well known for its pharmacological proper- activity was estimated by the method of Aebi [12] by ties; a survey of the literature revealed that the antioxidant, measuring the rate of decomposition of hydrogen peroxide antitumor, and wound healing potential of this plant has not at 240 nm. Glutathione (GSH) activity was assayed by the been fully evaluated. Keeping this in mind, the present method of Moron [13]; assay of glutathione peroxidase investigation was undertaken to study the antioxidant, (GPX) was carried out by the method of Hafeman [14] based antitumor, and wound healing activities of R. ellipticus leaf on the degradation of H2O2 in the presence of GSH. and to put forward a scope to develop an effective drug for the treatment of wounds, bacterial infections, and tumors. 2.4. Wound healing activity 2. Materials and methods Wistar male rats (150e250 g) were used to assess the wound healing property (Rats, mice: Small animals breeding sta- 2.1. Plant material and extract preparation tion, College of Veterinary and Animal Sciences, Mannuthy, Trissur, India; Cell lines: Amala Cancer Research Centre, Trissur, India). The experimental protocol was subjected to Fresh R. ellipticus plant was collected from Marayoor Shola the scrutiny of the institutional animal ethical committee forest, Kerala, India and was identified by the Botanical for experimental clearance. Acute dermal toxicity was Survey of India (Tamil Nadu, India; voucher specimen No. checked as per OECD guidelines 404 [15]. The most BSI/SRC/5/23/2010-11/Tech.1659). The powdered leaf was commonly used trituration method as mentioned in British extracted successively in Soxhlet (Borosil Glass Works Pharmacopeia [12] was followed for extract ointment Limited 1101, Crescenzo, G-Block, Opp. MCA club, Bandra preparation (1% and 2%). The animals were divided into Kurla Complex, Mumbai 400051, India) using organic sol- groups (8 animals per group) as follows: vents. The chemicals such as DPPH, Nitric oxide, Superox- Excision model: Group I, control; Group II, control ide, Tris HCl, SOD, Catalase, GSH and GPx were purchased treated with simple ointment base; Group III, Betadine 5% from Sigma Aldrich Chemicals (Bommansandar Jigani Link (w/w); Group IV, RELM 1% (w/w); and Group V, RELM 2% Road Industrial Area, Bangalore 560100, Karnataka, India). (w/w). Infected model: Group I, control; Group II, control 2.2. Determination of in vitro antioxidant activity treated with simple ointment base; Group III, Betadine 5% (w/w); Group IV, RELM 1% (w/w); Group V, RELM 2% (w/w); The antioxidant activities of the leaf extracts were deter- and Group VI, Neomycin 5% (w/w). mined in terms of hydrogen donating or radical scavenging Incision model: Group I, control; Group III, Betadine 5% ability using the stable radical 2,2-diphenyl-1-picrylhy- (w/w); Group IV, RELM 1% (w/w); and Group V, RELM 2% drazyl (DPPH), according to the method of Blois [6]. Nitric (w/w). oxide [7] and superoxide [8] radical scavenging activities of the R. ellipticus leaf extracts were also assayed to confirm 2.4.1. Excision and infected wound models its antioxidant potential. An excision wound of 1.5 cm in diameter and 0.2 cm depth was created on the shaved dorsal side. In the infected 2.3. Determination of in vivo antioxidant activity model, 0.1 mL of saline containing 106 colony forming units (CFU)/mL of Staphylococcus aureus suspension was swab- All of the animal experimental protocol was subjected to bed on the wounded area. The healing property was eval- scrutiny by the institutional animal ethics committee for uated by wound contraction percentage and closure time. 136 B.P. George et al. Table 1 Free radical scavenging activities of RELM. Sample DPPH radical scavenging Nitric oxide radical scavenging Superoxide radical scavenging activity (IC50 mg/mL) (%inhibition) (100 mg/mL) (%inhibition) (100 mg/mL) Petroleum ether 182.48 Æ 3.43c 26.54 Æ 2.74c 59.23 Æ 0.83c Chloroform 270.27 Æ 2.44d 34.09 Æ 3.34c 45.34 Æ 1.45c Acetone 7.05 Æ 2.02a 70.37 Æ 2.36b 65.71 Æ 2.07b Methanol 6.96 Æ 2.32a 71.08 Æ 3.34b 66.08 Æ 2.11b Hot water 7.39 Æ 1.23a 20.01 Æ 3.68d 63.44 Æ 0.54b BHT 13.18 Æ 1.43b 91.02 Æ 1.77a 94.20 Æ 2.12a BHA 4.88 Æ 1.45a d 94.70 Æ 1.78a Rutin 5.81 Æ 2.34a 93.24 Æ 1.96a d Quercetin 4.12 Æ 1.67a dd Values are mean of triplicate determinations (n Z 3) Æ standard deviation (SD). BHA Z butylated hydroxyanisole; BHT Z butylated hydroxytoluene; DPPH Z 2,2-diphenyl-1-picrylhydrazyl; IC50 Z an inhibitory con- centration 50; RELM Z Rubus ellipticus leaf methanol.