INTRODUCTION:

Plumbago indica (Begali name: Agnichita ) belonging to the family Plumbaginaceae , is a family of flowering plants, with a cosmopolitan distribution. The family is sometimes referred to as the leadwort family or the plumbago family. Most species in this family are perennial herbaceous plants, but a few grow as lianas or shrubs. The plants have perfect flowers and are pollinated by insects. They are found in many different climatic regions, from arctic to tropical conditions, but are particularly associated with salt-rich steppes, marshes, and sea coasts. Plumbago popularly known as chittiramulam, in Tamil and white leadwort in English. Plumbaginaceae is distributed as a weed throughout the tropical and subtropical countries of the world. The family Plumbaginaceae consists of 10 genera and 280 species. The genus Plumbago includes 3 species, namely Plumbago indica. L, Plumbago rosea. L, Plumbagocapensis. L, and Plumbago zeylanica .L, which are distributed in several parts of India ^[1]. Plumbago Indica root increases digestive power, promotes appetite and has long been marked as a powerful antiseptic. A liniment made from bruised root mixed with a few amount of bland oil is used in treating rheumatism, paralysis, leucoderma, enlarged glands and buboes and scorpion-sting ^[2].

Scraped root is inserted into the mouth of the womb to procure illegal abortion, a tincture of the root is used in secondary syphilis, leprosy, dyspepsia, hemorrhage, piles, flatulence, loss of appetite and other digestive complaints, and the milky juice of the plant is used in ophthalmia, scabies and as an antiseptic agent. In order to establish the above assertion about their validity, these medicinal plants must be subjected to extensive study in different research works.Historically, natural products have served as a source of chemotherapy agents.The brine shrimp lethality bioassay has been used routinely in the primary screening of the crude extracts to assess the toxicity towards brine shrimp. The bioassay has a good correlation with cytotoxic activity in some human solid tumors and with pesticidal activity ^[3]. This in vivo lethality test has been successively employed for providing a frontline screen that can be backed up by more specific and more sophisticated bioassays once the active compound has been isolated. A number of novel antitumors and pesticidal natural products have been isolared using this bioassay ^[4]. The object of this research work was to investigate whether the extract of Plumbago indica. L possess cytotoxic activity.

MATERIALS AND METHODS:

Collection of Plant material

The plants selected for present work Plumbago indica. L (Family: Plumbaginaceae ) and was collected from Naramuk, Rajsthali of Rangamati district. After collection, suitable herbarium sheet for each plant with some general information were prepared and send to Bangladesh Council of Scientific and Industrial Research (BCSIR), Baluchara, Chittagong for identification. They provided us the scientific name of the plants.

Extraction:

The collected plant (leaves and stems) was separated from undesirable materials or plants or plant parts and was shed-dried (35-50°c). The plant was ground into a coarse powder with the help of a suitable grinder. The powder was stored in an airtight container and kept in a cool, dark and dry place until extraction commenced. About 185 gm of powdered plant material of Plumbago indica L. (Family: Plumbaginaceae ) was taken in a clean, flat bottomed amber glass container and soaked in 1700ml of methanol The container with its contents was sealed and kept for a period of 10 days accompanied by continuous shaking. The whole mixture then underwent a coarse filtration by a piece of clean, white cotton materials. Then they were filtered by using Whatman filter paper number 1 and the solvent was made to evaporate under the room temperature. The obtained extract was collected .The residues were stored in a refrigerator until further studies.

Preparation of sample:

25 mg of dried methanol extract of was taken in a 80 ml beaker and 500μl DMSO was added to it, finally the volume (5ml) was adjusted by 4.5ml methanol. The concentration of this solution was 5μg/μl.

Hatching of Brine shrimp:

Sea water was taken in the small tank and shrimp eggs were added to the one side of the divided tank and the side was covered. The shrimps were allowed for 36 hrs to hatch and mature as nauplii. During this period constant oxygen supply and temperature (around 37°c) was maintained. The hatched shrimps were attracted to the lamp through the perforations in the dam and they were taken for bioassay.

Application of test sample to the test tube containing brine shrimp nauplii:

42 clean test tubes were taken and marked 10ml by a permanent marker. 21 were for the samples in seven different concentrations (three test tubes for each concentration) and 21 for control (three test tubes for each concentration). With the help of a Pasteur pipette 10 living shrimps were kept to each of the test tubes ^{[5] .} Then with the help of the micropipette specific volume (15, 30, 45, 60, 75, 90, 105μg/10ml) of samples were transferred from the stock solutions to the sample tubes. For control, the DMSO and methanol of specified volume were transferred to the control tubes. The concentration of DMSO should not be exceeded 10 μl/ml of brine as because above this concentration DMSO may become toxic to the nauplii.

Preparation of control group:

Control group was added in cytotoxic activity to validate the test method and result obtained due to the cytotoxic activity of the test agent. In this case, only 30 μl of DMSO was added to premarked glass vials containing 5 ml of simulated sea water and 10 shrimp nauplii to use as control groups. No extract was added to prepare control solution. If the brine shrimp in these vials show a rapid mortality rate, then the test was considered as invalid as the nauplii died due to some reason other than the cytotoxicity of the compound.

Counting of nauplii:

After 24 hours, the vials were inspected using a magnifying glass and the number of survived nauplii in each vial were counted and the LC₅₀ and LC ₉₀values were calculated.

RESULTS AND DISCUSSION:

In brine shrimp lethality bioassay using brine shrimp nauplii, Methanolic Extract of Plumbago indica L.showed positive result in comparison with the control that’s why it can be assumed that the extract is pharmacologically active.By plotting the log of concentration (log C) versus (%) mortality for all test samples showed an approximate linear correlation.From the graph, the median lethal concentration (LC_50,the concentration at which 50%mortality ofbrine shrimp nauplii occured) were determined and LC₉₀values were also determined to check the toxic level of the extract. The crude extract of Plumbago indica L. showed significant cytotoxic activity against brine shrimp nauplii and LC₅₀ value was 5.0mg/ml (Table-1 and Figure-1) .The 90% mortality rate(LC₉₀) was also calculated to get the therapeutic index and the value was 12 mg/ml (Table-1 and Figure-1).A s negative control DMSO was used to validate the test method.

In brine shrimp lethality bioassay, the methanolic Extract of Plumbago indica L. showed LC₅₀at 5.0mg/ml, which revealed that the extract is pharmacologically active. Both the LC₅₀ and LC₉₀ showed significant cytotoxic activity against brine shrimp nauplii and it can be considered for compound isolation in order to detect future anti-tumor compounds .Moreover, the significant lethality of the crude plant extract(as LC ₅₀ value less than 100 ppm or mg/ml) to brine shrimp is indicative of the presence of potent cytotoxic and probably insecticidal compounds which warrants further investigation. This bioassay has a good correlation with the human solid tumor cell lines ^[6]

Figure- 1: Determination of LC₅₀ and LC₉₀ Methanolic Extract of Plumbago indica L.

Table-1: Brine shrimp lethality bioassay of MEPI

Test groups	Conc (mg/ml)	Log(Conc.)	No. of alive shrimp			Mean alive	% mortality	LC₅₀(mg/ml)	LC₉₀ (mg/ml)
Test groups	Conc (mg/ml)	Log(Conc.)	t 1	t2	T3	Mean alive	% mortality	LC₅₀(mg/ml)	LC₉₀ (mg/ml)
MEPI	20	1.30	7	7	8	7.33	26.66	5.0	12.0
	40	1.60	6	5	6	5.66	43.33
	60	1.78	4	3	4	3.66	63.33
	80	1.90	3	4	3	3.33	66.66
	100	2	2	2	2	2	80
	120	2.07	1	2	0	1	90
	140	2.14	0	0	0	0	100
control	20	1.30	10	10	10	10	0
	40	1.60	10	10	10	10	0
	60	1.77	10	10	10	10	0
	80	1.90	10	10	10	10	0
	100	2	10	10	10	10	0
	120	2.07	9	10	9	9.33	6.7
	140	2.14	9	8	8	8.33	16.7

TC = Test Column, LC = Lethal Concentration, MEAC=Methanolic Extract of Plumbago indica L

CONCLUSION:

From this study, it can be concluded that Plumbago indica L. can be investigated as a source of anti-tumor agent. This is only a preliminary study and to make final comment the drug should thoroughly investigated phytochemically and pharmacologically to explore their medicinal and pharmaceutical potentialities.

REFERENCES:

1. Van Der Vijver Lm. Distribution of plumbagin in the Plumbaginaceae. Phytochemistry 1974, Vol 11, Pages 3247–3248.

2. Bala Rathinasabapathi, Walid M. Fouad, and Celia A. Sigua . β-Alanine Betaine Synthesis in the Plumbaginaceae. Purification and Characterization of a Trifunctional, S-Adenosyl-l-Methionine-Dependent N-Methyltransferase from Limonium latifolium Leaves. Plant Physiol. 2001, Vol 126(3), Pages 1241–1249.

3. Jerry L Mclaughlin, Lingling L, Rogers and Jon E Anderson. The use of biological assays to evaluate botanicals. Drug Information Journal, vol:32,pp:513-524(1998).

4. Meyer, B.N., N.R. Ferrigni, J.E. Putnam, L.B. Jacobsen, D.E. Nichols and J.L. McLaughlin, 1982. A convenient general bioassay for active plant constituents. Planta Medica, 45: 31-34.

5. McLughilin JL, Rogers LL. (1991), The use of biological assays to evaluate botanicals. Drug Information J. 32:513-524.

6. Anderson, J E., C.M. Goetz , JL McLughilin (1991), Phytochem. Anal.2:107-11.

Received on 16.04.2012 Accepted on 21.05.2012

Asian J. Pharm. Tech. 2(2): April-June 2012; Page 59-61