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RESEARCH ARTICLE

Cytotoxic Activity of Methanolic Extract of Alpinia conchigera Griff (Family: Zingiberaceae)

 

Dibyajyoti Saha*, Swati Paul

Department of Pharmacy, BGC Trust University Bangladesh, Chittagong.

*Corresponding Author E-mail: saha.dibyajyoti@gmail.com

 

 

ABSTRACT:

Investigation with crude methanolic extract of Alpinia conchigera Griff was carried out to evaluate its possible cytotoxic activity. Pharmacological history of this plant promoted us to check the possible cytotoxic activity. By using the brine shrimp lethality bioassay method, the LC50 and LC90 value of Alpinia conchigera Griff were assayed. In this study, DMSO was used as solvent. The extract evidence cytotoxic activity against brine shrimp nauplii and calculated LC50 and LC90 value was 6.1mg/ml and 12.2 mg/ml respectively.

 

KEY WORDS: Alpinia conchigera Griff, Cytotoxicity, LC50, Brine shrimp lethality bioassay, LC90.


 

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 species[1]. The taxonomic study of the family Zingiberaceae was first studied by Kai Larsen [2]. 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 Bangladesh [3]. 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.conchigera,[6] and have anti-inflammatory activity .

 

 

Received on 16.04.2012 Accepted on 08.05.2012

Asian Pharma Press All Right Reserved

Asian J. Pharm. Res. 2(2): April-June 2012; Page 86-88

 

 

The milky juice of the plant is used in ophthalmia, scabies and as an antiseptic agent [7]. 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 [8] . 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 antitumor and pesticidal natural products have been isolated using this bioassay [9]. The object of this research work was to investigate whether the extract of Alpinia conchigera Griff possess cytotoxic activity.

 

MATERIALS AND METHODS:

Collection of Plant material

The plants selected for present work A. conchigera (Family: Zingiberaceae) 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 (35-50c). 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 A. conchigera (Family: Zingiberaceae) was 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 37c) 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 [10]. 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 90 values were calculated.

 

RESULTS AND DISCUSSION:

In brine shrimp lethality bioassay using brine shrimp nauplii, Methanolic Extract of Alpinia conchigera Griff showed positive result in comparison with the control thats 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 (LC50, the concentration at which 50% mortality of brine shrimp nauplii occurred) were determined and LC90 values were also determined to check the toxic level of the extract. The crude extract of Alpinia conchigera Griff showed significant cytotoxic activity against brine shrimp nauplii and LC50 value was 6.1mg/ml (Table-1 and Figure-1) .The 90% mortality rate(LC90) was also calculated to get the therapeutic index and the value was 12.2 mg/ml (Table-1 and Figure-1).A s negative control DMSO was used to validate the test method.

 


 

Table-1: Brine shrimp lethality bioassay of MEAC

Test groups

Conc. (mg/ml)

Log (Conc.)

No. of alive shrimp

Mean alive

% mortality

LC50 (mg/ml)

LC90 (mg/ml)

t 1

t2

T3

 

 

 

MEAC

20

1.30

8

9

8

8.33

16.7

 

 

 

 

6.1

 

 

 

 

12.2

40

1.60

6

7

5

6

40

60

1.77

4

4

5

4.33

56.7

80

1.90

3

3

5

3.66

63.4

100

2

3

2

1

2

80

120

2.07

2

2

1

1.66

83.4

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 Alpinia conchigera Griff


Figure- 1: Determination of LC50 and LC90 Methanolic Extract of Alpinia conchigera Griff

 

In brine shrimp lethality bioassay, the methanolic Extract of Alpinia conchigera Griff showed LC50 at 6.1mg/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 (10).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 [11].

 

CONCLUSION:

From this study, it can be concluded that Alpinia conchigera Griff 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. Kai Larsen, K. 1980. Annotated key to the genera of Zingiberaceae of Thailand. Nat. Hist. Bull.Siam Soc. 28: 151-169.

2. E.W.C. Chan, Y.Y.Lim, S.K.Ling, S.P. Tan, K.K. Lim and M.G.H. Khoo Caffeoylquinic acids from leaves of Etlingera species (Zingiberaceae). LWT - Food Science and Technology, 2009 June, Volume 42, Issue 5, Pages 1026-1030.

3. Ghani A., (1998). Medicinal Plants of Bangladesh with Chemical Constituents and Uses. 2nd edition.pp.4-19 Asiatic Society of Bangladesh, Dhaka.

4. Baby Sabulal, Mathew Dan, Anil John J, Rajani Kurup, Nediyamparambu Sukumaran Pradeep, Renju Krishna Valsamma and Varughese George, Caryophyllene-rich rhizome oil of Zingiber nimmonii from South India: Chemical characterization and antimicrobial activity. Phytochemistry, 2006 November, Volume 67, Issue 22, Pages 2469-2473.

5. K. C. Wong, K. S. Ong, C. L. Lim. Compositon of the essential oil of rhizomes of Kaempferia galanga L. Flavour and Fragrance Journal, 2006, Volume 7, Pages 263-266.

6. Pino.J.A, Marbot.R, Rosado.A, Batista.A. Chemical composition of the essential oils of Zingiber officinale ( Roscoe L). from Cuba. Journal Essential Oils Research, 2004. Volume 16, Pages 186-188.

7. M.A. Sukari, N.W. Mohd Sharif, A.L.C. Yap, S.W. Tang, B.K. Neoh, M. Rahmani, G.C.L. Ee, Y.H. Taufiq-Yap and U.K. Yusof. Chemical constituents variations of essential oils from rhizomes of four zingiberaceae species. The Malaysian Journal of Analytical Sciences, 2008, Vol 12, No 3: 638 644.

8. 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).

9. 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.

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

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