Abstract: Hibiscus sabdariffa L. (Malvaceae) is a natural plant containing a lot of pigments that was found to possess anti-oxidant activity. Therefore, the present study was aimed to evaluate the anticancer potential of Hibiscus sabdariffa (H. sabdariffa) leaves on Hep 3B. Different extracts of methanol, ethanol, ethyl acetate and chloroform were prepared and tested for their cytotoxic effect by MTT assay in a dose and time dependent manner. Among the different organic solvent extracts tested, methanolic extract showed a greater cytotoxic effect (i.e.,) IC 50 value of 50% reduction when compared to others. The time required to show 75% decrease in cell number was found to be 24 h.
INTRODUCTION
Cancer is a disease that begins in the cells of the body. In normal situations, the cells grow and divide as the body needs them. No more, no less. This orderly process is disturbed when new cells form that the body doesn't need and old cells do not die when they should. These extra cells lump together to form a growth or tumor.
The liver is a common site of metastases from a variety of organs such as lung, breast, colon and rectum. When liver metastases occur at the time of initial diagnosis of the primary tumor, they are described as synchronous. If detected after the initial diagnosis, they are described as metachronous. The liver is frequently involved since it receives blood from the abdominal organs via the portal vein. Malignant cells detach from the primary cancer, enter the bloodstream or lymphatic channels, travel to the liver and grow independently. Potentially, the environment of the liver is suitable to the growth of certain tumor cells. Once a tumor begins to grow in the liver, it receives its blood supply from the hepatic artery.
Management of cancer will vary according to the individual. Some of the possible treatments are surgery, liver transplantation, chemotherapy and radiation. In recent times focus on plant research has increased all over the world and a large body of evidence has collected to show immense potential of medicinal plants used in various traditional system. Various medicinal plants have been studied using modern scientific approaches. The results from these plants have revealed the potential of medicinal plants in the area of pharmacology (Dahanukar et al., 2000). Recent research has identified food components (phytochemicals) that may have important anticarcinogenic activities (Mazur and Adlercreutz, 2000). These phytochemicals can suppress the initiation or reverse the promotion stage in multistep carcinogenesis.
Hibiscus sabdariffa commonly known as red sorrel and roselle. Roselle belongs to Malvaceae family. It is an erect, mostly branched, annual shrub. Stems are reddish in color and up to 3.5 m tall. Leaves are dark green to red, alternate, glabrous, long petiolate, palmately divided into 3-7 lobes, with serrate margins. Flowers are red to yellow with a dark center containing short peduncles (Yadong et al., 2005).
Many parts of roselle including seeds, leaves, fruits and roots are used in various foods. The young leaves and tender stems of roselles are eaten raw in salads or cooked as greens alone or in combination with other vegetables and/or with meat. They are added to curries as seasoning. They have acid, rhubarb like flavor (Yadong et al., 2005). The leaves are antiscorbutic, emollient, refrigerant and sedative. The leaves are very mucilaginous and are used as an emollient and as a soothing cough remedy. They are used externally as a poultice on abscesses. The leaves and flowers are used internally as a tonic tea for digestive and kidney functions. Experimentally, an infusion decreases the viscosity of blood, reduces blood pressure and stimulates intestinal peristalsis. The plant is also reported to be antiseptic, aphrodisiac, demulcent purgative. It is used as folk remedy in treatment of abcesses, cancer, cough, debility, fever, heart ailments (Yadong et al., 2005).
In this present study, we have evaluated the in vitro anticancerous effect of H. sabdariffa leaves on hepatocellular carcinoma cell lines Hep 3B by MTT assay and it will be certainly helpful in chemoprevention and therapy.
MATERIALS AND METHODS
The present research was carried out in the Biotechnology Laboratory at Prince Shri Venkateshwara Arts and Science College, Chennai (India) during August 2006 and January 2007.
Plant Material
Plant samples of H. sabdariffa were collected in Chennai, Tamilnadu
(India). The leaves were dried under shade and used for further experimentation.
Extraction Procedure
Five gram of H. sabdariffa was weighed, ground and dissolved in
50 mL of methanol. Similarly 5 g of the leaves were dissolved in 50 mL of chloroform,
ethanol and ethylacetate. The solutions were stirred by placing them in a shaking
incubator at 500 rpm at 20°C for 20 h. Later the solutions were filtered
using filter papers and allowed to dry in the petridish separately for 3-4 days.
After evaporation of the solvent, the powdered extract remaining in the plates
was collected and weighed. About 0.1 g of the extract was dissolved in 5 mL
of respective solvent. The extracts were stored at 4°C for further use.
Preparation of Extracts
Dry extracts of plant leaves were dissolved in 5 mL of respective solvents
to give a desired stock solution of extracts (20 mg mL-1). All the
extracts were stored at 4°C till end of the experimentation. During the
experiment, stock solutions were diluted to obtain an original concentration
of 0.2 mg mL-1 (10 μL of stock solution made up to 1 mL with
Minimum Essential Medium (MEM).
Cell Culture Conditions
Hepatocellular carcinoma Hep 3B cell culture was obtained from National
Center for Cell Science, Pune, India and used in this study. The cell lines
were grown at 37°C at humidified 5% CO2 in Minimum Essential
Medium (Eagle) with 2 mM L-glutamine and Earle’s BSS adjusted to contain
1.5 g L-1 sodium bicarbonate, 0.1 mM non-essential amino acids and
1.0 mM sodium pyruvate, 90%, fetal calf serum, 10% (Jenkins, 1999).
Subculturing of Monolayer Cell Culture
Using a sterile pipette, the old medium was removed and discarded. Five
milliliter of the trypsin versene glucose solution was added to the flask and
left it for few min so as to detach the cells from the flask. Then 5 mL of growth
medium was added to the cell suspension. The suspended cells were collected
in a 10 mL glass centrifuge tube and centrifuged at 1000 rpm for 10 min. The
pellet obtained was collected, diluted appropriately and the correct amount
of cells were added to each culture vessel. Then fresh medium was added to the
culture vessel. The flasks were placed in the CO2 incubator at 37°C
with 5% CO2 concentration (Jenkins, 1999).
Cell Counting and Cell Viability
Trypan Blue Exclusion Assay
0.5 mL cell suspension was mixed with 1mL of trypan blue by vortexing. Then,
20 μL of the sample was withdrawn and carefully loaded into a clean hemocytometer.
The percentage viability was calculated (Chang et al., 2005)
MTT Based Cytotoxicity Testing
Concentration Dependent Dose
The subconfluent monolayer culture was trypsinized and collected in growth
medium. The suspension was centrifuged to pellet the cells and resuspended in
the growth medium and counted. The cells were diluted to 2.5-50x103
cell mL-1. Two hundred microliter of the cell suspension was added
into each well of the tube leaving a tube as blank. Two hundred microliter of
growth medium was added to all the wells including the blank control medium.
Serial dilutions of the cytotoxic drug (various extracts) were prepared for
15 concentrations. Then 200 μL of each drug concentration was added to
the tubes excluding the blank. The tubes were then incubated at 37°C for
24 h. The contents in the tubes were transferred to the microtitre plates. At,
the end of the drug exposure period, the medium was removed and fresh medium
was added to the tubes. To it 50 μL of MTT was added to all the tubes.
The tubes were incubated for 4 h at 37°C for 4 h. Medium and MTT from the
wells were removed and the formazon crystals were dissolved by adding 200 μL
0.04 N HCl - isopropanol to all the tubes. The absorbance was taken immediately
at 570 nm since the product was unstable. The results were tabulated (Table
1) and the IC50 concentration was determined, that was required
to reduce the absorbance to half that of the control.
| Table 1: | The absorbance at 570 nm by the cells after treatment with various extracts of H. sabdariffa by concentration dependent manner |
Time Dependent Dose
The above mentioned procedure was followed but the drug exposure time was
varied i.e., the time of drug exposure was maintained as 0, 6, 12, 24 and 48
h (Chang et al., 2005).
RESULTS AND DISCUSSION
In the search for new anticancer drugs, the most common screening methods employ cytotoxicity tests against panel of cancer cell lines. These are High Throughput Screening (HTS) assays, revealing compounds with the highest cytotoxic activity. Although anticancer drugs are designed to kill cells such activity should be selective towards tumor cells (Popiolkiewicz et al., 2005). The use of natural products as anticancer agents has a long history that began with folk medicine and through the years has been incorporated into traditional and allopathic medicine (Costa-Lotufo et al., 2005).
Cell Counting and Viability
The cells were maintained at a density of 3.4x10 cells mL-1 after
subculturing and remained 86.66% viable.
MTT Based Cytotoxicity
Concentration Dependent Dose
Different concentrations of the extract were added to the cells and absorbance
was taken at 570 nm and values in Table 1 were observed. It
indicated that methanolic extract could inhibit the cells even at a lower concentration
whereas other solvents did not show such effect at the particular concentration.
The IC50 value for the methanolic extract was found to be 0.494x10-4
that showed cytotoxic effect on Hep3B cells to 50% reduction.
Hibiscus anthocyanins extracted from calyx of H. sabdariffa possess anti carcinogenic and antimutagenic activity (Tseng et al., 2000). Cancerous cell lines derived from different origins were used like Hep G2, NIH3T3, Caco-2, Hep3B, HL-60 and AGS. It was found that H. sabdariffa inhibited the growth of these cancerous cells through apoptosis. H. sabdariffa exhibited the strongest cytotoxicity potency towards Human Leukemia HL-60 and Hep 3B the next most sensitive. Present study was highly correlating with the aforesaid authors study but on the purified anthocyanins from the calyx on H.L 60 and Hep 3B cell lines. Christian et al. (2006) also evaluated the methanolic extract of Hibiscus sabdariffa flower had significantly higher activity than the other solvent extracts. This result agreed with our findings, the methanolic extract had higher cytotoxic activities than other solvent extracts.
Time dependent dose
The concentration of the drug was maintained as 11.856x10-4 and
the time of exposure was varied and the absorbance was observed as in Fig.
1. The time required to show 75% decrease in cell number was found to be
24 h. Chang et al. (2005) found that Hibiscus sabdariffa anthocyanin
treatment markedly induced apoptosis in HL-60 cells in a dose and time dependent
manner. This result was comparable with that of present findings.
The present investigation suggests that the H. sabdariffa has potent
anticancer activity against Hep 3B cell lines. Methanol was predicted to be
the suitable solvent for the extraction of anticancer (Hepatocellular carcinoma)
compounds from H. sabdariffa leaves. Further studies are needed for the
identification of mechanism(s) of inhibition of cancerous cells. As leaves of
H. sabdariffa are considered as dietary material by most of the Indians,
this could reduce the chemotherapeutic side effects in the patients by suggesting
herbal therapy.
| Fig. 1: | The absorbance at 570 nm by the cells after treatment with various extracts of H. sabdariffa by time dependent manner |