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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.
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 .
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 (35-50°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 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 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 [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
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 (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.
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.
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