INTRODUCTION:

Numerous medicinal therapies treat their patients with herbal medicines for its extraordinary influence, though relatively little knowledge about their mode of action is available. Ashoka is one of the most legendary and sacred trees of India which has been utilized from ancient times till date[1-3].

Ashoka tree, universally known by its binomial Latin name Saraca asoca (Roxb.) or Saraca indica belonging to the Caesalpiniaceae family[4], is found throughout India, especially in Kerala, West Bengal, regions of southern India and in the Himalayas up to an altitude of 750 m. It is a small, spreading evergreen tree of 7-10 m height whose bark is dark brown or almost grey with a warty surface. Its leaves are parpinnate, 15-20 cm long and the leaflets are 6-12 cm, oblong and rigidly subcoriaceous[5] while the flowers are fragrant and polygamous apetalous, yellowish orange turning to scarlet[6].

Stem bark of S. asoca is reported to contain glycosides, flavonoids, tannins and saponins[4,7].

It is used as a spasmogenic, oxytocic, uterotonic, antibacterial and antidysentric agent[5,8]. It has also been reported to possess antiprogestational and antioestrogenic activity against menorrhagia[4].

The reference standard drug used in this experiment is piperazine citrate. It causes hyperpolarization of muscle by its GABA agonistic action opening Cl^- channels that causes relaxation and depresses responsiveness to contractile action of acetylcholine thereby flaccid paralysis occurs. The worms recover if placed in a piperazine free medium [9]

An extensive search of the literature reveals no proper studies on the pharmacological activity of the stem bark of this plant. Thus, the present investigation aims towards the determination of preliminary phytochemical screening and assessment of the anthelmintic efficacy of extracts of S. asoca stem bark.

Helminth infections are among the commonest infections in man, affecting a large proportion of the world's population. In developing countries they pose a major threat to public health and contribute to the prevalence of malnutrition, anemia, eosinophilia, and pneumonia. Helminthiasis is rarely fatal, but is a major cause of morbidity [10].

MATERIALS AND METHODS:

Plant material:

The bark of the plant Saraca indica were collected from rural area of Jaunpur UP during November 2012. The sample was authenticated by Department of Pharmacognosy KHBS Pharmacy College Jaunpur. The shade dried barks were powdered and stored in a desiccators until evaporation.

Worm collection and authentication:

The anthelmintic activity was evaluated on adult Indian earthworm Pheretima posthuma (Annelida). It resembles anatomically and physiologically with the intestinal round worm parasite of human being. Indian earthworms were obtained from vermiculture area and were identified at KHBS Pharmacy College Jaunpur.

Chemicals:

Piperazine citrate was obtained from local medical shop. Ethanol [RANKEM], methanol [RANKEM], dimethylsulfoxide (DMSO) is purchased from SD-Fine Chem. limited, India.

Preparation of extract:

The coarse powdered bark was stored in a desiccator. The powdered bark was extracted by both Maceration and Soxhlet methods.

1) Maceration method:

The powdered bark (200gm) of Saraca indica was extracted using the maceration method. The powdered bark was macerated in 60ml of 95% ethanol for 3 days at room temperature. The resulting extract was filtered through filter paper (Whatman No.1). The residue was further extracted using the same procedure. The filtrates obtained were combined and then evaporated to dryness. We also followed the above-mentioned method of extraction using methanol instead of ethanol.

2) Soxhlet method:

The powdered bark (500gm) of Saraca indica were successively extracted using solvents in order of increasing polarity, viz. ethanol and methanol. After extraction, each time the marc was dried and later extracted with the next solvent. Both the extracts were dried by distilling the solvents in a rotary vacuum evaporator [11]. Both the extracts were dissolved in dimethylsulfoxide (DMSO) [12].

Preliminary phytochemical Investigation:

The preliminary phytochemical investigation of prepared plant extract was performed and presence of, tannins, alkaloids, saponin anthraquinone glycoside, steroids, flavonoids, phenolics and terpenoids in prepared plant extract was confirmed. The result of phytochemical extraction was as shown in Table – 1.

Phytochemical analysis of the crude extract revealed the presence of tannins along with other chemical constituents contained within them. Tannins have been reported to produce anthelmintic activities [13,14], as they can bind to free proteins in the gastrointestinal tract of host animal[15] or glycoprotein on the cuticle of the parasite and thereby cause deaths[16]. The wormicidal activity of the alcoholic extracts against earthworms suggests that it is effective against parasitic infections of humans [17]. It would be interesting to identify the active principle responsible for the anthelmintic activity and to study its further pharmacological actions.

Anthelmintic activity:

The suspension of both the extracts, obtained from the maceration and the soxhlet methods, was prepared in DMSO to obtain 10, 30 and 60mg/ml concentrations. Solutions of similar concentrations of the standard anthelmintic drug like Piperazine citrate (as positive control) were also prepared in distilled water. For our study DMSO and distilled water were used as negative controls.

2ml. of each concentration of both methanolic and ethanolic fractions and Piperazine citrate were diluted to 10ml separately with normal saline and poured into Petridishes. Nine groups of approximately equal size of earthworms, consisting of six in number in each group, were released into each Petridish. The anthelmintic activity was evaluated by adopting the standard method [18]. Adult Indian earthworms Pheritima posthuma were selected for the study because of their anatomical and physiological resemblance with the intestinal round worm parasite of human being [19-20].

Table 1 - Preliminary Phytochemical Evaluation:

Sr.No.	Phytochemical Test	Maceration method		Soxhlet method
Sr.No.	Phytochemical Test	Ethanolic extract	Methanolic extract	Ethanolic extract	Methanolic extract
1	Tannins	-	+	-	+
2	Alkaloids	-	+	-	+
3	Saponin	+	+	+	+
4	Anthraquinone glycoside	+	+	+	+
5	Steroids	+	-	+	-
6	Flavonoids	+	+	+	+
7	Protiens	-	-	-	-
8	Terpenoids	-	+	-	+

Table-2: Anthelmintic activity of methanolic and ethanolic extracts (by Maceration method) of Saraca indica bark

Treatment group	Concentrations (mg/ml)	Time taken (min.)
Treatment group	Concentrations (mg/ml)	Paralysis	Death
Methanolic extract	10	2.0±0.08	3.33±0.10
	30	1.83±0.04	2.91±0.04
	60	1.5±0.10	2.75±0.10
Ethanolic extract	10	2.41±0.9	3.08±0.07
	30	1.5±0.10	2.5±0.08
	60	1.16±0.10	1.83±0.09
Piperazine citrate	10	25.33±0.23	43.5±0.34
	30	20.5±0.17	31.66±0.23
	60	13.33±0.14	27.83±0.20

Values are mean ± SD, n = 6, * P<0.001 was significant.

Table-3: Anthelmintic activity of methanolic and ethanolic extracts (by Soxhlet method) of Saraca indica bark

Treatment group	Concentrations (mg/ml)	Time taken (min.)
Treatment group	Concentrations (mg/ml)	Paralysis	Death
Methanolic Extract	10	6.5±0.30	11.83±0.23
	30	5.5±0.26	10.66±0.04
	60	3.5±0.19	7.58±0.09
Ethanolic Extract	10	10.83±0.03	16.08±0.23
	30	8.83±0.15	13.16±0.17
	60	8.33±0.09	11.16±0.23
Piperazine citrate	10	25.33±0.23	43.5±0.34
	30	20.5±0.17	31.66±0.23
	60	13.33±0.14	27.83±0.20

Values are mean ± SD, n = 6, * P<0.001 was significant.

Statistical analysis:

The results were analyzed for statistical significance using one way ANOVA followed by student t-test. The P value (<0.001) was considered significant.

RESULTS AND DISCUSSION:

In case of the maceration method, the ethanolic extract showed more potent anthelmintic activity than the methanolic extract (Table -2). On the other hand, the methanolic extract, obtained from the soxhlet method of extraction, indicated that it had better anthelmintic activity than the ethanolic extract (Table-3). Overall the anthelmintic activity revealed the concentration- dependent nature of the extracts. In cases of both the methods of extraction, we had found that the methanolic as well as the ethanolic extracts were more potent than the positive control as far as the anthelmintic property was concerned. We did not find any anthelmintic activity of DMSO and distilled water against earthworms.

Although the leaves of the plant has got anthelmintic activity (Indian Medicinal Plants, 1996), we found that even the bark of Saraca indica possessed very potent anthelmintic activity. From this study it may be concluded that, in addition to leaves, other parts of plant should be explored thoroughly (using several extracts) to know its exact anthelmintic activity.

CONCLUSION:

From the above study it may be concluded that stem bark extracts of Saraca indica Roxb. exhibited significant anthelmintic activity (In vitro study against Pheretima posthuma ) as evidenced by decreased paralyzing time and death time. The results thus support the use of Saraca indica Roxb. as an anthelmintic agent.

ACKNOWLEDGEMENTS:

Authors thank Dr. Anand Kumar Singh Head of Chemistry Department, Mariyahu PG College Mariyahu Jaunpur UP for providing financial support for the entire project work.

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Received on 09.11.2013 Modified on 02.12.2013

Asian J. Research Chem. 7(2): February 2014; Page 141-143