INTRODUCTION:

Alpinia conchigera Griff. (Begali name: Khetranga) belonging to the family Zingiberaceae, or the Ginger family, is a family of flowering plants consisting of aromatic perennial herbs with creeping horizontal or tuberous rhizomes. Zingiberaceae is one of the largest families of the plant kingdom with 53 genera and over 1300 species¹. The taxonomic study of the family Zingiberaceae was first studied by Kai Larsen². who proposed the key to genera of Thai Zingiberaceae. Zingiberaceous plants are distributed throughout Bangladesh. But wide varieties of species are mainly found in hilly areas like in Chittagong and Sylhet. The following species are identified in Bangladesh. Zingiberaceous plants are distributed throughout Bangladesh. But wide varieties of species are mainly found in hilly areas like in Chittagong and Sylhet. The following species are identified in Bangladesh³. The rhizome of A. conchigera is used as a condiment and occasionally in folk medicine along the east coast to treat fungal infections. In some states of Peninsular Malaysia, the rhizomes are consumed as a post-partum medicine and the young shoots are prepared into a vegetable dish.The rhizomes of A. conchigera are used in Thai traditional medicine to relieve gastrointestinal disorders and in the preparation of Thai food dishes ^4,5.

It was reported that the phenyl prepanoid derivatives, chavicol acetate and eugenol acetate are present in the fruit of A.conchigera⁶,and have anti-inflammatory activity .The milky juice of the plant is used in ophthalmia, scabies and as an antiseptic agent⁷. On the other hand, 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 familyPlumbaginaceae consists of 10 genera and 280 species. The genus Plumbago includes 3 species, namelyPlumbago indica. L, Plumbago rosea. L, Plumbagocapensis. L, and Plumbago zeylanica .L, which are distributed in several parts of India⁸. 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-sting⁹. 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. For this reason, Alpinia conchigera Griff. and Plumbago indica L. (Plumbaginaceae) two Bangladeshi plants under the family of Zingiberaceae and Plumbaginaceae, respectively were selected and subjected for chemical, biological and pharmacological investigations to explore the antimicrobial, cytotoxicity, antidiarrhoeal, anti-motility, analgesic activity of methanol extracts of the above mentioned plant species.

EXPERIMENTAL:

Collection of Plant material

The plants selected for present work A. conchigera (Family: Zingiberaceae) and Plumbago indica L. (Family: Plumbaginaceae) were 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 75 gm of powdered plant material of Plumbago indica L. (Family: Plumbaginaceae) was taken in a clean, flat bottomed amber glass container and soaked in 350 ml 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. On the other hand, About 185 gm of powdered plant material of A. conchigera (Family: Zingiberaceae) was subjected with 1700ml of methanol in a Soxhlet Apparatus. The obtained extract was collected and made to evaporate the solvent bellow 50°c temperature. The residues were stored in a refrigerator until further studies.

Animals:

Young Swiss-albino mice of either sex, average weight 18-25 gm of either sex were employed in the experiment taking four in a group. The mice were purchased from the Animal Research Branch of the Bangladesh Council of Scientific and Industrial Research (BCSIR), Chittagong, Bangladesh. The mice were kept separately in wooded cages having dimension of (15 x 10 x 8) inch. Soft wood shavings were placed in the cages for housing of the mice. The room where the mice were housed was well ventilated for air and light. Husk and excreta were removed from the cages on every day. Fresh water and pellets of mice foods were given to the mice regularly. The mice were kept at least one week in the laboratory to get them adapted with the environment before being employed in any experiment.

Determination of Antidiarrhoeal Activity:

The Antidiarrhoeal Activity was determined according to the method described by Shoba and Thomas, 2001 ¹⁰follows for this study. The animals were all screened initially by giving 0.3 ml of castor oil and only those showing diarrhoea are selected for the final experiment. The animals were divided into control, positive control and test groups containing five mice in each group. Control group received vehicle (1% Tween-80 in water) at a dose of 10 ml/kg body weight orally. The positive control group receives loperamide at the dose of 3 mg/kg orally; test group received the ethanol extract at the doses of 250 and 500mg/kg body weight orally. Each animal was placed in an individual cage, the floor of which was lined with blotting paper. The floor lining was changed every hour. Diarrhoea is induced by oral administration of 0.3 ml castor oil to each mouse, 30 minutes after the above treatments¹¹. During an observation period of 240 min, the total number of faecal output and the number of diarrheic faeces excrete by the animals is recorded. A numerical score based on stool consistency is assigned as follows: normal stool =1 and watery stool = 2.

Castor Oil Induced Diarrhoea:

Upon oral administration, castor oil mixes with bile and pancreatic enzymes and liberates ricinoleic acid from the tryglyceride. A small amount of ricinoleic acid is absorbed from the gastrointestinal tract and metabolized like any other fatty acid but most remains in the intestine where it produces its antiabsorptive or secretory effect. The ricinoleic acid thus liberated readily forms ricinoleate salts with sodium and potassium in the lumen of the intestine. The ricinoleate salt formed as such behaves like a soap or surfactant within the gut and at the mucosal surface. The precise mechanism of how ricinoleate salts induce diarrhoea is not yet to be known. But most agreed view is that it stimulates the intestinal epithelial cell’s adenyl cyclase, release prostaglandins and especially prostaglandins of the E series along with serotonin (5-HT) have been termed as ‘diarrhoeagenic hormones’.

Statistical Analysis:

All the values of antidiarrheal, tests were expressed as mean + SEM (Standard error of the Mean). Statistical differences between the mean of the various groups were analyzed by using Students “t” test¹². Probability (p) value of 0.05 or 0.01 was considered as significant. All the graphical presentation and statistical calculations were preparing“Microsoft Excel-2000”.

RESULTS AND DISCUSSION:

The test was performed to observe the effect of extract on castor oil induced diarrhoea in mice. Traditional study revealed that the plant exerts an inhibitory effect on diarrhoea. This method demonstrates that the extract or compound which has the capacity to inhibit the recinolic acid synthesis or some other autacoids such as Prostaglandins (mainly PGEs), and serotonin (5-HT), which has proven role in the pathogenesis of diarrhoea, has the power to prevent diarrhoea. The above evidence is justified by the anti-diarrhoeal activity screening. Antidiarrhoeal episode was determined by evaluating the latency period

and diarrhoeal frequency by counting the faeces time and number of the test groups in comparison to the control and standard loperamide groups.In the castor oil induced diarrhoeal mice, the methanol extracts of the A. conchigera and P. indica at the doses of 250 and 500mg/kg reduced the total number of faeces (65 ± 0.79 and 45±1.369 and 63±2.109 and 43±1.204) (Table 2and4) as well as latency period (69.6 and 79.4 and 56.6 and 74.6) (Table 1and3) respectively in comparison to the control group and the result was statistically significant (Figure 1and2).

Table-1: Effect of MEAC on the latent period of castor oil induced Diarrhoeal episode in mice:

Test groups	No. of mice	BW (Gm)	LP (Min)	MLP (Min)	SD	SE	t-test (P-value)
Control	01	21	45	51.0	12.748	6.374	2.99 (<0.05)
	02	22	61
	03	23	42
	04	24	68
	05	24	39
Positive control (Loperamide 3mg/kg)	01	20	80	81.2	12.049	6.025	3.68 (<0.05)
	02	21	81
	03	22	101
	04	23	75
	05	23	69
*Methanol Extract of Alpinia conchigera* Griff.** (250mg/kg)	01	25	66	69.6	7.765	3.8826	2.706
	02	26	76
	03	22	60
	04	21	79
	05	23	67
Methanol Extract of Alpinia Conchigera Griff. (500mg/kg)	01	20	78	79.4	5.727	2.863	3.36
	02	23	82
	03	21	75
	04	25	88
	05	22	74

MEAC= Methanol Extract of Alpinia conchigera Griff. , BW = Body weight, Gm = Gram, LP = Latent period, MLP = Mean of latent period, SD = Standard deviation, SE = Standard error, Min = Minute ,

Figure 1: Graphical representations of antidiarrhoeal activityof the methanol extract of MEAC and MEPI compared with positive control and control

Table- 2 : Effect of MEAC on castor oil (0.3 ml each mouse at 240 min.)-induced diarrhoea in mice

Test groups	No. of mice	MBW (gm)	No. of faeces (240 min)	MD (240 min)	TNF (240min)	SD	SEM	t-test (P-value)
Control	01	22.8	13	13.8	69±1.597	3.194	1.597	2.95 (<0.05)
	02		16
	03		18
	04		12
	05		10
Positive control	01	21.8	04	6.4	32±1.151	2.302	1.151	3.50(<0.05)
	02		07
	03		10
	04		05
	05		06
*Methanol Extract of Alpinia conchigera* Griff.** (250mg/kg)	01	23.4	14	13	65 ± 0.79	1.58	0.79	2.83 (<0.05)
	02		12
	03		15
	04		11
	05		13
*Methanol Extract of Alpinia conchigera* Griff.** (500mg/kg)	01	22.2	9	9	45±1.369	2.739	1.369	3.265 (<0.05)
	02		13
	03		7
	04		6
	05		10

MEAC= Methanol Extract of Alpinia conchigera Griff. , MBW = Mean body weight, MD = Mean defecation, TNF = Total number of faeces (Total values ± SEM)

Figure 2: Graphical representation of antidiarrhoeal activity of the methanol extracts of MEAC and MEPI compared with positive control and control

Table-3: Effect of MEPI on the latent period of castor oil induced Diarrhoeal episode in mice

Test groups	No. of mice	BW (Gm)	LP (Min)	MLP(Min)	SD	SE	t-test (P-value)
Control	01	21	45	51	12.748	6.374	2.99 (<0.05)
	02	22	61
	03	23	42
	04	24	68
	05	24	39
Positive control (Loperamide 3mg/kg)	01	20	80	81.2	12.049	6.025	3.68 (>0.05)
	02	21	81
	03	22	101
	04	23	75
	05	23	69
Methanol Extract of Plumbago Indica (250mg/kg)	01	20	70	56.6	9.289779	4.64488	3.2254
	02	22	47
	03	24	51
	04	21	62
	05	19	53
Methanol Extract of Plumbago Indica (500mg/kg)	01	23	76	74.6	10.5023	5.2511	3.70 (<0.05)
	02	24	69
	03	26	71
	04	20	92
	05	21	65

MEPI = Methanol Extract of Plumbago Indica , BW = Body weight, Gm = Gram, LP = Latent period, MLP = Mean of latent period, SD = Standard deviation, SE = Standard error, Min = Minute

Table-4: Effect of MEPI on castor oil (0.3 ml each mouse at 240 min.)-induced diarrhoea in mice

Test groups	No. of mice	MBW (gm)	No. of faeces (240 min)	MD (240 min)	TNF (240min)	SD	SEM	t-test (P-value)
Control	01	22.8	13	13.8	69±1.597	3.194	1.597	2.95 (<0.05)
	02		16
	03		18
	04		12
	05		10
Positive control	01	21.8	04	6.4	32±1.151	2.302	1.151	3.50 (<0.05)
	02		07
	03		10
	04		05
	05		06
Methanol Extract of Plumbago Indica (250mg/kg)	01	21.2	19	12.6	63±2.109	4.219005	2.109502	3.39
	02		14
	03		8
	04		12
	05		10
Methanol Extract of Plumbago Indica (500mg/kg)	01	22.8	12	8.6	43±1.204	2.4083	1.204	3.16 (<0.05)
	02		8
	03		7
	04		10
	05		6

MEPI = Methanol Extract of Plumbago Indica , BW = Mean body weight, MD = Mean defecation, TNF = Total number of faeces (Total values ± SEM)