IJCRR - 4(2), January, 2012
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IN VITRO ANTICANCER ACTIVITY OF ZINGIBER OFFICINALE AND ALLIUM SATIVA TOWARDS MCF- 7
AND K562 CELL LINES
Author: K.S.V.Prabhu Ratnam, Ranga Suresh Sannidhi, Y. Raja Ratna Reddy, Jagadeeswara Reddy Kanala
Abstract:Phytochemicals present in the genus Allium and Ginger have potential pharmacological effects, such as antimicrobial, antithrombotic, antitumor, hypo lipidaemic and hypoglycemic activities. In the present study, we examined the effects of garlic and ginger oils on human leukemia (K 562) and Breast Cancer (MCF-7) cells. Incubation of K 562 and MCF-7 with garlic and ginger mixture (5.0 \?g/ml) caused a marked suppression of K562 and MCF-7 proliferations when compared with DMSO (used as positive control) separately. The combination of garlic and ginger oil results were more effective (p < 0.05, 0.01) than DMS
Keywords: Tryphan Blue Assay, MCF-7, K562, Zingiber officinale (Ginger) and Allium Sativa (Garlic).
The role of natural products as a source for remedies has been recognized since ancient times. Despite major scientific and technological progress in combinatorial chemistry, drugs derived from natural product still make an enormous contribution in drug discovery today. Experimental agents derived from natural products are offering us a great opportunity to evaluate not only totally new chemical classes of anticancer agents, but also novel and potentially relevant mechanisms of action (Adriana B da Rocha et al, 2001). Cancer arises due to the uncontrolled growth of cells and it is a multi-step disease incorporating physical, environmental, metabolic, chemical and genetic factors, which play a direct and/or indirect role in the induction and deterioration of cancers. Breast cancer and lymphoma are one of the commonest malignancies affecting population. Breast cancer is one of the main life-threatening diseases that a woman may have to face during her lifetime (Angelopoulos et al., 2004). The increasing incidence of breast neoplasia reported over the last a few decades has led to development of new anticancer drugs, drug combinations, and chemotherapy strategies by methodical and scientific exploration of enormous pool of synthetic, biological, and natural products (Mukherjee et al., 2001). There is a large amount of scientific evidence showing that consumption of fruits and vegetables lower the risk of cancer (Chen et al., 2004), and medicinal plants constitute the main source of new pharmaceuticals and healthcare products, including medications for ethnoveterinary medicine (Ivanova et al., 2005). (Xiujie Wang et al, 2006)
Allium Sativa (Garlic) is known to exhibit anticancer effects in prostate, pancreatic and breast cancers. (Yogeshwer Shukla et al (2007)) Similarly, Zingiber officinale (Ginger) is used against breast, ovarian, gastric and lung cancers and lymphomas. (Yogeshwer Shukla (2007)). This study was undertaken to explore the cytotoxic activity of the plant extracts Zingiber officinale and Allium sativa in human leukemia (K 562) and Breast Cancer (MCF- 7) cells.
MATERIALS AND METHODS K562 cells (Leukaemic cell line) were obtained from ATCC, USA (CCL-243™) and MCF-7 cells (Breast cancer cell line) were obtained from ATCC, USA (HTB- 22™). RPMI 1640 medium containing 10% fetal bovine was obtained from Difco, In vitro gen Corp, Canada. All solvents were of HPLC grade. Acetonitrile was purchased from Merck (Darmstadt, Germany), and ultra pure water was obtained in a Milli-Q system from Millipore (Bedford, MA, China). Allium Sativa and Zingiber officinale were purchased from Acharya NG Ranga Agricultural University, Tirupati, India. The RPMI medium was prepared using Serum (Sigma, USA) and antibiotics (100u/mL Penicillin and 100µg/mL streptomycin) in a humidified atmosphere of 5% C02 at 37 0 C .
Preparation of Ginger Extract Dried ginger was prepared from ginger slices through air-drying at room temperature (25ºC) until the weight of ginger was constant. Steamed ginger was obtained by steaming raw ginger at different temperatures at various time points (100 ºC for 1 h, or 120 ºC for 0.5, 1, 2, 4, and 6 h). Ginger was air dried at room temperature (25 ºC) until the weight was constant. For the HPLC analysis, the ginger slices were accurately weighed (5 g for fresh ginger, 0.5 g for dried ginger and steamed ginger) and suspended in 40.0 mL methanol, and were ultrasonically extracted twice for 30 min each time. The extract mixture was cooled to room temperature, and filtered. The residue on the filter paper was washed with methanol. The combined methanol extracts were evaporated to dryness under vacuum at 45 ºC. After reconstitution in methanol, the extract was transferred to a 10-mL volumetric flask and made up to the volume with methanol. The resultant solutions were centrifuged at 12,000 rpm for five min; the supernatants were transferred to an auto sampler vial for HPLC analysis. For the in vitro antiproliferation studies, fresh, dried gingers, or steamed ginger (120 ºC, 4 h) were extracted with methanol as described above. The extract was evaporated under vacuum, and dissolved with DMSO. (XiaoLan Cheng et al, 2011)
Preparation of Garlic Extract The outer layers of garlic cloves were removed and cloves were cut up into 0.5 cm thick pieces, and distributed on sterile filter-papers as monolayers. The process of drying was done using a thermostat with natural circulation of warm medium at temperature of 45 °C ± 2 °C for 72 h. Dried garlic was cooled on 20 °C and was grinded into powder in the laboratory mill. Aqueous and ethanolic extracts were prepared from garlic powders as follows: 1 g of garlic powder was dissolved in 5 ml of re distilled water and absolute ethanol, respectively. The suspension was set aside for 24 h at room temperature. The supernatants (extracts) were collected after centrifugation at 5000 rpm for 15 min. Aqueous extracts were sterilized using a 0.22 μm filter. (M. Colic et al, 2002) For application to the cells, these oils and extract were diluted in dimethyl sulfoxide (DMSO) and further diluted in RPMI medium. (V. Bhuvaneswari et al, 2004).
Cell treatment Zingiber officinale and Allium sativa were used at different concentrations (alone and in various combinations) to evaluate their antiproliferative effect. These compounds were prepared as 10mM stock solution in 100% DMSO and were stored in dark colored bottle at 4°C. The stocks were diluted to the required concentration immediately before use with 1% DMSO. The cells were exposed to drugs individually and in different combination for a period of 48 hr. Cells grown in media containing equivalent amount of DMSO without drug serves as control. (Sivakumar Ramamurthy et al, 2011).
Cell Viability Assay K562 and MCF-7 cells were cultured at a density of 6 X 104 and 5 X 104 /well into 24-well plates to reach the 80-90% confluence using RPMI medium. After 48 hrs incubation, the cells were collected from each well in eppendroff and centrifuged at 1500 rpm for 5 min to get the cell pellet. (Liane Ziliotto et al, 2009) The cells were counted with a hemocytometer (Model S-plus, Coulter Co., FL), and cell viability was evaluated in terms of exclusion of Tryphan Blue, which was determined by microscopic observation.
Tryphan Blue Exclusion Assay The Tryphan blue exclusion assay is based on fact that the chromophore is negatively charged and does not interact with the cell unless the membrane is damaged. Therefore, all the cells which exclude the dye are viable. The cell suspension was diluted with 0.4% Tryphan blue solution (1:1), mixed thoroughly and was allowed to stand at room temperature for 5 min. Hemocytometer was used for cell counting. When observed under the microscope, nonviable cells were stained blue, viable cells remain unstained. % Dead cell = N° of dead cells / (Sum of the live cells and dead cells) X 100 Finally, cells were seeded in 48 well plates at the concentration of 5000 cells/ml and incubated in 5% Co2 at 37° C for 24 hours. After incubation, the cells were treated with the test compounds in various concentrations 5.0, 10.0 and 20.0 µg/mL. Percent of Growth Inhibition was calculated using the formula: % of growth Inhibition = ((Control Cells/ml – Test Cells/ml)/Control Cells/ml)*100.
Statistical Methods The data were subjected to one-way analysis of variance (ANOVA) and the differences among samples were determined by Dunnett‘s pair-wise comparison test using the Systat 12.0 software. P-value of < 0.05 was regarded as significant.
We acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. We are also grateful to authors / editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.
RESULTS AND DISCUSSION
The present study was undertaken to investigate the potential anticancer activity of the Zingiber officinale and Allium sativa in two different Cell lines. The In vitro assay resulted that Zingiber officinale and Allium sativa were able to induce significant inhibition of cell growth by applying them in combination when compared to separate and single agent treatment in K-562 (leukemia) and MCF-7 (breast cancer) cell lines by Tryphan blue assay. Rational drug development in cancer therapy appears to concentrate on the discovery of effective pharmaceutical agents that can intervene in diverge signaling pathways. (John Stone et al., 2002) The result obtained from the study revealed that the anticancer activity of drugs was higher in combination therapy when compared with the individual drug treatment at lower concentrations. Zingiber officinale rhizome is typically consumed as a fresh paste, dried powder, tablets, slices preserved in syrup, candy (crystallized Zingiber officinale) or for flavoring tea. Zingiber officinale has been found to be anti carcinogenic via multiple pathways. Although chemo preventive activities of Zingiber officinale have been examined (Koshimizu et al., 1988; Katiyar et al., 1996), very little information is available in the literature with regard to the effects of individual constituents of Zingiber officinale on experimental carcinogenesis. Its use in inflammatory conditions was consistent with anti-inflammatory activities of its components In vitro (Kiuchi et al., 1982; Mascolo et al., 1989). Two studies suggested that these compounds suppress the proliferation of human cancer cells through the induction of apoptosis (Lee and Surh, 1998; Lee et al., 1998) and were found to exert inhibitory effects on the viability of human HL-60 (promyelocytic leukemia) cells (Lee and Surh, 1998). Moreover, results of the recent study showed that the cell viability was decreased by 35% and 89% after 5 hrs of treatment with 20 and 100 µM -paradol, respectively (Lee and Surh, 1998). In another study, it was observed that - paradol induced apoptosis in JB6 cells at low concentrations (up to 25 µM), but apparent necrotic cell death was resulted at concentrations greater than 50 µM (Huang et al., 1996). For the present in vitro antiproliferation studies, fresh, dried gingers, or steamed ginger (120ºC, 4 h) were extracted with methanol. For application to the cells extract were diluted in dimethyl sulfoxide (DMSO) and further diluted in RPMI medium. The data revealed that Zingiber officinale was effective (p < 0.05) at 20.0 µg/mL compared with the DMSO in both the MCF-7 and K562 cells. (Figures 1 and 3) Recent studies have been stated that Allium sativa inhibits stomach, colorectal and prostate cancer. Garlic and onion have also shown to possess anti allergic, anti bacterial and anti inflammatory property. (Shilpa Srivastava et al, 2010). Allium sativa powder/extract has shown antiproliferative activity in HL-60, K562, MCF-7 cell lines and it inhibits anchoragedependent growth of cells. Incubation of HL-60 with Allium sativa or onion oil (20 mg/mL) caused a marked suppression of HL-60 proliferation; the suppression was almost identical with those obtained by alltrans-retinoic acid (ATRA) or dimethyl sulfoxide (DMSO). (Taiichiro Seki et al, 2000). In our present study Allium sativa was tested at different concentration as similar to Zingiber officinale. It was found that the Allium sativa was more effective (p < 0.05) at 20.0 µg/mL towards MCF-7 cells while it was more effective (p < 0.05) at 10.0 µg/mL towards K562 cells (Figures 1 and 3). The IC50 value of ginger and garlic extracts grown in RPMI for MCF-7 and K 562Cells were 10.0 and 20.0 µg/mL. The combination of Allium sativa and Zingiber officinale resulted in a potential anticancer effect at the low concentration level of 5.0 µg/mL (p < 0.05, 0.01) (Figures 1 and3) whereas the percent inhibition is higher (p < 0.05) at 10.0 µg/mL for Allium sativa and Zingiber officinale individually and in combination (Figures 2 and4). Morphological assessment of MCF-7 Cell cultures revealed that there were no morphological differences in the cultures treated with Curcumin, Allium sativa and Zingiber officinale as single agents as well as when treated with combinations. The mode of synergistic interaction between these compounds, however, was not well investigated.
Cancer is one of the extensive diseases in humans and there is substantial scientific and commercial attention in continuing discovery of new anticancer agents from natural product sources. Currently, about 50% of drugs used in clinical trials for anticancer activity were isolated from natural sources such as herbs and spices or any other related to them. (Newman and Cragg, 2007) The results showed strong inhibitory activity of extracts on human breast cancer (MCF-7) and leukemia cells (K562). According to the report of the American National Cancer Institute (NCI), the criterion of anticancer activity of the crude extracts of herbs is: IC50 ≤ 30 µg/mL (Itharat et al, 2004). In conclusion, our In vitro results on the anticancer activity of Zingiber officinale and Allium sativa showed that the beneficial effects in the breast cancer and leukemia cells in combination therapy. Therefore, more focused clinical studies are necessary to establish whether these combinations can be exploited to reach cancer blocking or remedial effects in human body.
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