IN-VITRO CYOTOTOXIC STUDIES OF CAESALPINIA BONDUCELLA F. USING AQUEOUS EXTRACT OF SEED POWDER AND CALLUS

IN-VITRO CYOTOTOXIC STUDIES OF CAESALPINIA BONDUCELLA F. USING AQUEOUS EXTRACT OF SEED POWDER AND CALLUS

Shirsat Rajani ¹ Kamerkar Urwashi ² and Rai Aruna ^*1

Department of Botany ¹, Smt. C.H.M. College (Affiliated to University of Mumbai), Ulhasnagar - 421003, Thane, Maharashtra.

Srujan Biotech ², Dombivali, Thane, Maharashtra

ABSTRACT:  In the traditional system of Indian medicine Caesalpinia bonducella Flem. is widely used for its medicinal properties. All parts are valuable as it contains various secondary metabolites. Seeds contain 20-30% of oil. On the basis of phyto-chemical analysis of the seed it is known to contain alkaloids, flavonoids, glycosides, saponins and furano di terpenes. The plant possesses antidiabetic, antioxidant, antitumor, antiproliferative and anti imflammatory activities. “Cytotoxicity” is the ability to kill the cells. In the present study experiments were carried out to check for the cytotoxic nature of seeds and callus using Daphnia cultures. The method used is simple, inexpensive and even a low toxin amounts are sufficient to perform the test. Cytotoxic nature was tested using aqueous extracts of seed powder as well as callus. From the experiments it was observed that extract from seed powder as well as callus expressed cytotoxic activities, however, seed powder extract was more effective compared to callus extract.

callus, cytotoxic, Daphnia

INTRODUCTION: Plants are a valuable source of natural products for maintaining human health, with more intensive studies for natural therapies. In developed countries, about 80% individuals rely on   traditional medicines which are derived from plants, for their primary health care needs ¹. There is a need to investigate such plants sources to better understand their pharmacological properties with safety and efficacy. Plant phytochemicals have been reported to prevent a number of diseases, including cancer, cardiovascular disease, infection and inflammation ².

Caesalpinia bonducella from family Leguminosae and sub family Caesalpinae is a prickly shrub widely distributed all over the world especially in India, Sri Lanka and Andaman and Nicobar Islands. In India it is specially found in tropical regions. The seed of the plant, which is extremely bitter in taste, commonly known as natphal to the rural people of Tripura.

C. bonducella is used in traditional medicine like Ayurveda, Siddha, Unani and Homoeopathy ^{3, 4}. All parts of the plant have medicinal properties. Seeds are used to treat colic, convulsions, leprosy and palsy. The oil from seed helps to soften the skin and removes the acne. Singh and Raghav ⁵ reported that, Caesalpinia bonducella. Contain all major phytochemicals such as amino acids, fatty acids, hydrocarbons, isoflavones, phenolics, phytosterols and steroidal saponin. The presence of antioxidative activity of Caesalpinia bonducella seeds has been reported ^{6, 7}. The evaluation of acute and sub-acute toxicity of methanolic extract of Caesalpinia bonducella in albino mice has been reported ⁸. The animal model selected for the present study is Daphnia which is one of the several small aquatic crustaceans commonly called “water fleas” because of their jerky swimming movements. The lifespan of a Daphnia does not exceed one year and depends on temperature, food availability and habitat. Cytotoxicity has been explained as those bioactive compounds which are toxic/fatal to living cells ⁹.

Some toxic chemicals, toxic venom of different animals and immune cell are the main cause of cytotoxicity.  Many cytotoxic compounds from natural products have been investigated for the discovery of the novel compound as an anti cancer drugs ². With this background present study was carried out to test for the cytotoxic properties of Caesalpinia bonducella seed powder and callus extracts against Daphnia.

MATERIALS AND METHODS:

Collection of plant material: The dried fruits of Caesalpinia bonducella were collected in the month of November from the farmyard, located at Parle village, Satara district. Fruits were peeled and the seeds were sundried and stored in plastic bags.

In-vitro callus induction:  Seeds were soaked in water for 6 h, testa was removed and the embryos were washed with liquid detergent for 15 min. Seed embryos were treated with 1% Bacillocide and 1% Bavistin, for 10 min each. Alternate washing was provided with sterilized distilled water.  Finally the seed embryos were treated with 0.1% HgCl₂ for 5 min with continuous shaking. The embryos were washed with the sterilized distilled water, thrice to get rid of traces of HgCl₂. The embryos were cut into small pieces and were placed to the culture tubes containing Murashige and Skoog’s¹⁰ media supplemented with   3mgL^-1 2, 4-Dichloro phenoxy acetic acid.   The callus thus obtained was sub cultures repeatedly to get more biomass.

Preparation of Extracts: The dried seeds were ground to form a fine powder. The extracts were prepared using cold infusion method. Five g of powder was soaked in 50 ml of distilled water for 48 h.  Same method was applied to get the extract from the callus. Five gram of callus was homogenized in mortar and pestle using distilled water, volume was made to 50 ml.

Preparation of Daphnia Culture: The dried eggs of Daphnia were hatched in glass beaker (500 ml) containing fresh water with constant aeration and illumination. After 24 h the newly hatched Daphnia were collected using pipettes. Ten nauplii were transferred to different vials having varying concentrations of extracts. The nauplii were counted macroscopically in the stem of pipette against a light background. The vials were maintained under illumination at room temperature 25°C to 28°C. Survivors were counted with the aid of 3x magnifying glass at specific time intervals. .

RESULTS:

From Table 1, it is evident that Caesalpinia bonducella seed powder extract has cytotoxic effect on Daphnia. Even the lowest concentration is found to be toxic to Daphnia as after 8 h 50% of the nauplii were found to be immotile. As the concentration of extract increased, cytotoxicity was also found to be increasing, with the highest concentration of 100% there are 50% of the nauplii affected just in 3 h. Form Table 2, it is clear that callus extract does not show the same severity as that of seed powder extract. Lower concentrations of extract do not affect the motility of Daphnia even up to 8 hr, however, highest concentration (100%) does affect the motility of the organisms

TABLE 1: CYTOTOXIC EFFECT OF CAESALPINIA BONDUCELLA SEED POWDER EXTRACT ON DAPHNIA

(IM: Immotile, M: Motile)

TABLE 2: CYTOTOXIC EFFECT OF CAESALPINIA BONDUCELLA CALLUS EXTRACT ON DAPHNIA

(IM: Immotile, M: Motile)

DISCUSSIONS: Caesalpinia bonducella seed powder extract is found to be cytotoxic on Daphnia, as after 24 h 100% of the nauplii were immotile whereas callus extract showed 90% immotility in them. Daphnia assay is a primary assay conducted to detect cytotoxic property of plant extract.   Khalighi-Sigaroodi et al. on the basis of their experiments reported the cytotoxic nature of leguminous plants ¹¹. Caesalpinia gilliesii expressed potent lethality activity against brine shrimp. Prema et al. ¹² reported the potent cytotoxic activity of the combined extracts of Cissus quadrangularis and Aegle marmelos against the Colon cancer- HT-29 cell lines. The phytochemical constituents such as flavanoids and terpenoids are the major components which are responsible for the potential cytotoxic activity.

CONCLUSION: On the basis of above findings it is clear that seed powder extract affects the motility of Daphnia cultures adversely. Callus extract also shows adverse effect, but only when used at higher concentrations.

ACKNOWLEDGEMENT: We are thankful to Principal, Smt. C.H.M. College, Ulhasnagar and Srujan Biotech, Dombivali for providing the necessary facilities to carry out the experiments.

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    How to cite this article:

    Rajani S, Urwashi K and Aruna R: In-Vitro Cyototoxic Studies of Caesalpinia Bonducella F. Using Aqueous Extract of Seed Powder and Callus. Int J Pharm Sci Res 2015; 6(3): 1181-83.doi: 10.13040/IJPSR.0975-8232.6(3).1181-83.

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