Acta Biologica Indica 2014, 3(1):510-514
Qualitative phytochemical screening and in vitro anthelmintic activity of Exacum bicolor Roxb., an endemic medicinal plant from Western Ghats in India Appaji M. Ashwini, Mala Majumdar Department of Biotechnology, Centre for Post Graduate Studies, Jain University, Bangalore, Karnataka, 560011, India, Email:
[email protected]
ABSTRACT Exacum bicolor Roxb. is a deciduous herbaceous plant which is endemic to peninsular India. Due to its bitterness in taste local people use as herbal remedy for diabetes and skin disorder and also used for stomachic, febrifuge and antifungal diseases. The present study is designed to evaluate the qualitative phytochemical analysis and anthelmintic activity of extracts of leaves of Exacum bicolor. Extraction of the plant materials were carried out sequentially using different solvents (hexane>chloroform>ethyl acetate>methanol>water). Pheretima postuma was used as test organism for anthelmintic activity. Different concentrations (5, 10, 15 mg/ml) of all the extracts were tested for activity. The time of paralysis and time of death were studied and the activity was compared with albendazole (10mg/ml) as reference standard. Qualitative analysis confirmed the presence of phenols, flavonoids, alkaloids, cardiac glycosides, saponin, steroids and terpenoids in detectable amounts. It was found that all the extracts showed dose-dependent anthelmintic activity but the hexane extract exhibited better inhibition of spontaneous motility (paralysis) and death of earthworms when compared to reference standard (P < 0.0001). The potent extract was partially purified by TLC and 6 bands were visualized where band no. 4 showed lesser time of paralysis (3.186 ±0.14 min) and time of death (5.324±0.11 min) at 10mg/ml when tested for anthelmintic activity. From the present study, it can be concluded that E. bicolor possesses potent anthelmintic activity. As per literature, this is the first scientific report on the anthelmintic properties of leaf extracts of Exacum bicolor. Keywords: Exacum bicolor, qualitative analysis, anthelmintic activity, albendazole, Pheretima postuma
INTRODUCTION Exacum bicolor Roxb., is a perennial herb belonging to Gentianaceae family and an endemic to peninsular India in Western Ghats. This species has been extensively used for curing human ailments like diabetes [1], skin disorders [2], inflammation, purifies blood in treatment of malaria [3] and tonic for stomachic [4]. The leaves contain apigenin, luteolin, vanillic, p-hydroxybenzoic, protocatechuic and p-coumaric acids. The members of this family are rich sources in xanthonoids, flavonoids, irridoids and terpenoids. The species of the family Gentianaceae are best known for their bitter taste, which can be related to their content of iridoids such as amarogentin, the bitterest compound known. Medicinal plants contain some bioactive substances such as phenols, tannins, alkaloids, carbohydrates, terpenoids, steroids and flavonoid [5,6] which are of great importance to the health of individuals and communities. Studies have shown that they play an important preventive role in the development of cancer, heart diseases and ageing related diseases [7]. Research Article, Acta Biologica Indica 2014, 3(1):510-514 © 2014 Association for the Advancement of Biodiversity Science pISSN 2319-1244, eISSN 2279-0160 510
Acta Biologica Indica 2014, 3(1):510-514
Helminthiasis is a disease caused by infestation with one or more intestinal parasitic worms. The worms usually reside in the gastrointestinal tract but it may pose threat to other organs also [8]. Helminth infections cause many acute and chronic diseases among human beings as well as cattle. The disease is highly prevalent in third world countries due to poor management practices [9]. Anthelmintics are drugs that expel parasitic worms from the body by either stunning or killing them and are therefore also called as vermifuges. The most commonly used anthelmintic drugs are albendazole, piperazine citrate etc [10]. Chemical control of helminthes coupled with improved management has been the important worm control strategy throughout the world. However, increasing problems of development of resistance in helminths [5] against anthelmintics have led to the proposal of screening medicinal plants for their anthelmintic activity as they are potent, safe and affordable. They are also known to provide a rich source of botanical anthelmintics [6]. A number of medicinal plants have been used to treat parasitic infections in man and animals [7,11,12]. As per literature, this is the first report on anthelmintic activity of Exacum bicolor.
MATERIALS AND METHODS Collection and authentication of plant material Exacum bicolor leaves were collected from Kumar Parvatha at Kukke Subramanya, Karnataka. The plant was authenticated by Regional Research Centre, Bangalore, India [SMPU/MADRI/BNG/2010-11/557]. The plant material was dried in shade for 15-20 days, coarsely powdered and stored at 4°C for further studies.
Preparation of extracts The plant materials were extracted using different nonpolar-polar solvents (hexane>chloroform>ethyl acetate>methanol>water) in Soxhlet apparatus [13]. About 20 g of the powdered plant material was subjected to sequential extraction using 250 ml each of hexane, chloroform, ethyl acetate, methanol, water solvents. The extracts were evaporated to dryness by rotary evaporator and % yields were calculated for each extract.
Qualitative analysis of phytochemical constituents The preliminary phytochemical analysis was carried out to screen the phytoconstituents present in the crude extracts using standard protocols [14]. Different solvent extracts were tested for the presence of phytoconstituents such as phenols, flavonoids, saponin, tannin, steroids, terpenoids, alkaloids, cardiac glycosides, quinones, phlobotannins, coumarins, anthraquinones and acids using standard biochemical procedures [15].
Anthelmintic activity The anthelmintic activity was performed according to the method of Ajaiyeoba et al. [16] on adult Indian earthworm, Pheretima posthuma as it has anatomical and physiological resemblance with the intestinal roundworm parasites of human beings [17]. The earthworms of 3-5 cm in length and 0.10.2 cm in width and they were washed with normal saline to remove all the faecal matter. Desired formulations was prepared by dissolving the different leaf extracts (hexane, chloroform, ethyl acetate, methanol, water) in distilled water and were investigated for their anthelmintic activity. Approximately equal size earthworms consisting of six in each group were released into 15ml of each extract (5 mg, 10mg and 15mg/ml). Albendazole (10mg/ml) and distilled water were used as
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Acta Biologica Indica 2014, 3(1):510-514
positive and negative control respectively. Six replicates were used for each concentration. Anthelmintic activity was judged at two different stages, the time taken to paralysis and death of individual worms. Time for paralysis was noted when no movement of any sort could be observed except when the worms were shaken vigorously. Death was concluded when the worms lost their motility followed with fading away of their body colour.
Partial purification by TLC The potent extract (hexane) was partially purified by TLC [18] using toluene: ethyl acetate: formic acid (5:1.5:0.5) as the solvent system. The bands were visualized under 365 nm after proper drying. The Rf values were noted for separated components. The individual band was scraped and after centrifugation, the supernatant was dried and tested for anthelmintic activity with two different concentrations (5mg, 10mg/ml).The time of paralysis and death of individual worm were observed and noted.
Statistical analysis Data were analyzed with two-way ANOVA using Bonferroni test (Graph Pad Prism 6). The results were provided as mean ± SEM (n=6). The value of p
