Jyotirmoy et al
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ESTIMATION OF THE ANTI- PROLIFERATIVE POTENTIAL OF COSTUS PICTUS ON MOLT- 4
HUMAN CANCER CELL LINE
1. Department of Pharmacology, Royal College of
Jyotirmoy Das Choudhury1*, Anup Kumar
Maurya2, Smriti Tripathi2, Reetu Ranga2,
Monica Kahrana3, Manohar Lal Samyal4.
Pharmacy & Health Sciences, Berhampur (Gm) -
760002, (Odisha), India.
2. Department of Pharmaceutical Sciences, Shridhar
University Pilani- 333031, (Rajasthan), India.
3. Banasthali University,
4. CMJ University, Shillong- 793003, (Meghalaya),
Introduction: The present study is aimed at evaluating the anti-proliferative and apoptotic of Costus pictus on
MOLT- 4 human cancer cell line, and also evaluating its safety to normal human lymphocytes.
Materials and Methods: Dried leaves of C. pictus plant were used for aqueous and alcohol extraction. Different
concentration of these were evaluated for their cytotoxicity by trypan blue dye exclusion method and 3-(4, 5-
dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay on the cancer cell line (Human acute
lymphoblastic leukemia) MOLT- 4 and normal cell line (Human peripheral lymphocytes). The apoptic potential was
analyzed by DNA fragmentation analysis of the treated cells.
Results: The ethnolic extract of C.pictus was found to be anti-proliferative and cytotoxic at lower concentration and
induced cell death in MOLT- 4 cells. The ethanolic extract at the same concentration had no cytotoxicity on normal
lymphocytes. Campared to the ethanolic extract, aqueous and methanol extracts were less effective.
Conclusion: the present investigation, for the first time, reveals the anticancer potential of the ethanolic extract of
C.pictus on MOLT- 4 cells. It is very likely that the result can be extrapolated to animal or human system. The
extracts can be used for further purification of the active component for future application.
Keywords: Costus pictus, Lymphocytes, MTT assay, Cytotoxicity
Medicinal plants are frequently used by traditional healers to treat various ailments and symptoms, including
diabetes and cancer. Cancer is the second leading cause of death after cardiovascular diseases in India.  Cancer
cells, which are already irreversibly developed, obtain the capability to escape apoptosis by a number of ways.
Jyotirmoy Das Choudhury
Department of Pharmacology,
Royal College of Pharmacy & Health Sciences,
Berhampur (Gm) - 760002, (Odisha), India.
Phone no:- +91
Email id:- firstname.lastname@example.org
INTERNATIONAL JOURNAL OF BIOPHARMACEUTICAL & TOXICOLOGICAL RESEARCH
RESEARCH ISSN: 2249 - 4871
Jyotirmoy et al
The role of anticancer agents is to trigger the apoptosis-signaling system in these cancer cells while disturbing
their proliferation.  Zingiberaceae plants are well known for their medicinal properties and many of these
species are effectively used in the treatment of disease. Curcuma longa L., for example, has proven anticancer
properties.  The rhizomes of these plants possess several biologic activities such as antioxidant, cytotoxic,
and antitumor properties.[5-7] Costus pictus, commonly known as ‘Spiral ginger’, ‘Step ladder’, or ‘Insulin plant’
is a member of Zingiberaceae family and is newly introduced plant in India, originated probably in Mexico. In
India, it is grown in gardens, especially in the state of Kerala; here, patients with diabetes consume the fresh raw
leaves. In vitro studies on methanol, aqueous ethyl acetate, and ethanol extract of C.pictus have revealed good
inhibitory effect on carbohydrate hydrolyzing enzymes like α-glucosidase and α-amylase. Toxicity studies and
antidiabetic activity of methanol extract of this plant have been reported previously. [9-10] Since there are no
earlier reports about testing the anticancer property of C.pictus, we tried to evaluate potential anticancer effect of
C.pictus ethanol, aqueous and methanol extracts on MOLT- 4 cell line and also their effects on human peripheral
MATERIALS AND METHODS
Selection of medicinal plant
Costus pictus (insulin plant) was collected in the month of August 2010, from IIIM Medicinal garden, Jammu,
India, and identified by Dr. S. N. Sharma, a senior scientist in the Botany Division of the Indian Institute of
Integrative Medicine (IIIM), Jammu, India. A voucher specimen was retained and deposited at the crude drug
repository of the herbarium of IIIM, Jammu. The leaves were shade dried, reduced to coarse powder and stored
in airtight container till further use.
Extraction using ethanol
The dried leaves of the plant (50 g) were crushed, soaked in 75 ml of ethanol (80% v/v) for 24 h and then
percolated (5 h, 30 drops/min). The extract obtained was concentrated by a rotary evaporator and dried in oven at
40°C. Then 20 mg of solid residue was dissolved to 100 ml with distil water. The resulting solution was filtered
through 0.22 micro filters. The dilution was continued so that final concentration of extract was 40, 80 and
Extraction using methanol
Dried leaf samples were ground using an electric blender. Two grams of ground material was soaked in 25 ml of
methanol for 24 h and was filtered using filter paper. The material was again mixed with 25 ml of fresh methanol
and filtered after 24 h. The process was repeated (25 ml x 3 times).The filtrate was then transferred to vials and
allowed to dry. Dry extract was resuspended in 2ml of DMSO. The concentration of extract was 1mg/ml.  The
dilution was continued so that final concentrations of extracts used were 40, 80, and 120µg/ml.
Dried plant (50 g) was ground and mixed with 1 L boiling distilled water for 1 h. The mixture was filtered twice
through a funnel by using suction pump. The extract was evaporated under reduced pressure till completely dry
in a lyophilizer. The extract (1 mg/ml) was prepared by dilution of the stock with sterile distilled water. 
Cell line selected
Human peripheral lymphocytes were isolated in the laboratory and MOLT- 4 cell line was procured from
National Center for Cell Sciences (NCCS) Pune, India. MOLT-4 (Human acute lymphoblastic leukemia) cells
were grown in complete DMEM medium (2mM L-glutamine, 100g/ml of streptomycin, and 100U/ml of
penicillin) supplemented with 10% fetal bovine serum and maintained in a 5% CO2 humidified incubator at
37°C. Cells were seeded at a density of 1X105cells/ml, except where otherwise indicated.
Isolation of lymphocytes
Lymphocytes were obtained from the blood of five healthy male and female individuals, of age about 20 years,
apparently free from infection by pathogenic agents, and had not been under any treatment for the last six
months. Hisep medium (HIMEDIA, India) was used for the isolation. Cells were suspended in complete RPMI
Jyotirmoy et al
1640 medium supplement with 10% fetal bovine serum (HIMEDIA, India), 5g/ml phytohemagglutinin (PHA),
and maintained at 37°C in a 5% CO2 humidified incubator. Lymphocytes were used as control cells to assess the
cytotoxicity of plant extracts.
Determination of cell concentration and viability by Trypan blue dye exclusion
At the end of treatment period, the cells were counted with the aid of hemocytometer and cells viability was
determined by trypan blue dye exclusion method. Trypan blue was prepared at a concentration of 0.4% in
phosphate buffered saline (PBS). 
MTT assay was performed to assess the cytotoxicity of the plant extracts. (MTT is a yellow dye, which is
reduced into purple formazan crystals by the activity of mitochondrial succinate dehydrogenase enzyme in viable
cells). Cells were cultured in 96- well microtiter plates. Cells were treated with varying concentration of plant
extracts for 24 h (9 wells for each concentration) and incubated. At the end of treatment period, to each well, 20
µl of MTT was added. After addition of MTT, the plates were incubated for 3 h in a dark chamber. Then, 100 µl
of DMSO was added to dissolve the formazan crystals. The absorbance was read at 540 nm using ELISA reader. 
Analysis of DNA fragmentation
In a medium containing 10% FBS, 1.0X105 cells were incubated for 24 h. After 24 h, cells were treated with
C.pictus leaf, (aqueous, methanol, and ethanol) at different concentrations. After 24 h, cells were collected by
trypsinization; cells from different wells of the same concentration were pooled and rinsed twice in cold PBS (PH
7.4). Genomic DNA was extracts from MOLT- 4 cells as described earlier. Briefly, cells were re-suspended
twice in a lysis buffer containing 1% Nonidet-P40, 20mM Ethylenediaminetetraacetic acid (EDTA), and 50 mM
Tris-HCl at PH 8. The cells were centrifuge at 1,600g for 10 min, recovered supernatant were combined and
incubated with 0.5 mg/ml RNase A (Bangalore Genei, India) at 37°C for 4 h. The DNA was precipitated by the
addition of 1/10 volume of 7.5 M ammonium acetate and two volumes of ethanol and analyzed by agarose gel
Morphology observation under inverted microscope
Morphological changes were observed under the 40X lens of an inverted microscope and photographs were taken.
All experiments were carried out in triplicates. The results were calculated as mean±standard error (SE) values.
Statistical significance was calculated using one-way analysis of variance (ANOVA) and Student’s test. A value
of P<0.05 was taken as statistically significant.
When different concentrations of C.pictus were added to normal lymphocytes, the aqueous extract induced
proliferation in the lymphocytes. The cell concentration and cell survival both increased, as the concentration of
the extract increased [Figure 1]. The ethanol extract had no effect on the lymphocytes. Methanol extract showed
slight cytotoxicity when compared to the other two extracts.
Figure 1: Effect of C. pictus on the percentage viability of normal lymphocytes as measured by MTT assay.
Values are expressed as mean±S.E.M. (n=9). *P<0.05 (in comparison with control group)
Jyotirmoy et al
When cell count was taken by hemocytometry, it was observed that [Figure 2] the ethanol extract had no
significant effect on the peripheral blood lymphocytes, where as the methanol extract was slightly cytotoxic and
the aqueous extract had increased the cell count significantly.
Figure 2: Effect of C. pictus on the cell concentration of normal lymphocytes measured by Trypan blue
dye exclusion method. Values are expressed as mean±S.E.M. (n=4). *P<0.05 (in comparison with control
Figure 3: Effect of C. pictus on the percentage viability of MOLT-4 human cancer cells as measured by
MTT assay. Values are expressed as mean±S.E.M. (n=9). *P<0.05 (in comparison with control group)
C.pictus aqueous extract administered to MOLT- 4 cells at 40 and 80 µg/ml concentration showed slight
cytotoxicity, through it was not significant. The ethanol extract was highly cytotoxic; reducing the cell viability
to less than 50% at 120 µg/ml concentrations (IC50) value was found to be 120 µg/ml from the graph.
Figure 4: Effect of C. pictus on the cell concentration of MOLT-4 human cancer cells measured by trypan
blue dye exclusion method. Values are expressed as mean±S.E.M. (n=4). *P<0.05 (in comparison with
Jyotirmoy et al
As the concentration of the extract increased, cell viability decreased. The methanol extract was cytotoxic at 80
and 120 µg/ml concentration. By trypan blue dye exclusion method, the cell concentration and cell viability were
determined with the help of a hemocytometer [Figure 4].
Figure 5: DNA fragmentation assay of HT-1080 cells treated with different concentrations of C. pictus
ethanol extract for 24 h. Lane C- control, Lane 3- 40 μg/ml, Lane 2- 80 μg/ml, and Lane 1- 120 μg/ml of C.
pictus ethanol extract.
The ethanol extract (120 µg/ml) had reduced the cell concentration to half of that of the control. The ethanol
extract was safe to normal human lymphocytes but has shown cytotoxicity to the cancer cell line.
DNA fragmentation was tested by agarose gel electrophoresis. [Figure 5] indicates a significant increase in inter-
nucleosomal DNA fragmentation of MOLT-4 cells. When the DNA isolate from C.pictus-treated cells was
subjected to agarose gel electrophoresis, a DNA ladder characteristic of apoptotic DNA was observed in the cells
treated with different concentration of the ethanol extract.
The morphology of MOLT-4 cells was observed under the inverted microscope, and it was obvious that the
control cells were firmly attached to the substrate [Figure 6] and C.pictus (80 µg/ml) - treated cells started
becoming round and were detached [Figure 7] from the substrate. The ethanol extract had induced apoptosis in
the fibrosarcoma cells.
Jyotirmoy et al
Accumulating evidence suggests that many dietary factors may be used alone or in combition with traditional
chemotherapeutic agents to prevent or treat cancer. The main disadvantage of using natural or dietary
compounds as anticancer remedy is that they seem to have low toxicity and show very few adverse side effects.
Ginger (Zingiber officinale Roscoe), is widely used all over the world as a spice and condiment in daily cooking.
It has been shown to have anticancer and antioxidant effects. The antidiabetic activity of the aqueous, ethanol
ethyl acetate and methanol extract of C.pictus has been reported earlier. [8-10] Effect of C.pictus on glucose uptake
by L6 myotube cell line ((Skeletal muscle) has also been reported.  However, to best of our knowledge, this is
the first report on the first report on the cytotoxic activity of C.pictus on any human cancer cell line.
In conclusion, the result of the present work show that the ethanol extract of C.pictus activated the apoptotic
pathway in (Human acute lymphoblastic leukemia cell line) MOLT- 4 cells. The ability of the extract to trigger
and execute apoptosis in cancer cells is unclear but the MTT assay suggests a mitochondrial involvement.
Anticancer activity of the ethanol extract of C.pictus has not been reported in literature. In our present in vitro
study through MTT assay, Trypan blue dye-exclusion method, and DNA fragmentation analysis, we confirm the
pro-apoptotic and anticancer potential of C.pictus ethanol extract. The IC50 value was found to be 120µg/ml.
Additionally; it was revealed that the key bioactive compound was in the ethanol fraction, showing its promising
anticancer drugs used in cancer therapy are toxic and have adverse side effects. Thus, studies pinpointing
confirmed efficacy of a particular fraction or compound among several found in crude extract are important for
therpeacutic purpose. Although the efficacy of C.pictus ethanol extract has presently been tested against an in
vitro cancer cell line, it is very likely that the result can be extrapolated to animal or human systems. However, to
determine this, more experiments should be carried out on in vivo animal models, which will hopefully be taken
up in the next phase of our program.
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