Abstract: In vitro studies were carried out to evaluate the cytotoxic potential of three selected forest herbaceous species: Tectaria singaporeana (TS), Blechnum orientale (BO) and Tacca integrifolia (TCI). Methanol/methylene chloride extracts of three plant parts viz. leaves, roots and stems were assessed for their cytotoxic potential against human breast cancer cells (MCF-7wt.). Screening of these extracts was done using the microculture, followed by tetrazolium assay after a period of 72 h. There were significant differences between different parts of plants and dilution levels in terms of cytotoxicity, with roots and concentration of 100 μg mL-1 showing the highest cell mortality of 19.58 and 36.59%, respectively. However, the leaves and the stems of all three plant species did not induce any cytotoxic activity on the cells. Overall, the most promising material (IC50 <100 μg mL-1) were the methanolic extracts from the roots of all three plants. Tectaria singaporeana showed the highest cytotoxic potential with an IC50 value of 28.57 ± 11.74 μg mL-1 followed by Blechnum orientale, 32.07 ± 7.85 μg mL-1 and Tacca integrifolia, 95.03 ± 17.49 μg mL-1. From this study, the extracts of these plants may prove to be useful in cancer treatment and prevention.
INTRODUCTION
Malaysia is a country blessed with a broad spectrum of diversity in flora and fauna. Past and recent ethno-botanical studies suggest that at least 20% of the estimated 12 000 total higher plant species possess medicinal or other therapeutic properties (Parris and Latiff, 1997). The abundance of medicinal plants serves as an ideal resource that can unleash new discoveries in the medical breakthrough. They are proven beneficial because they contain various phytochemicals which are natural molecules produced by plants for protection. These plants serve as an alternative to modern medicine for most local and tribal communities. Traditional use of herbal medicine include herbs, herbal material, herbal preparations and finished herbal products that contain active ingredients in various parts of plants.
In the pursuit of determining the cure for cancer, especially breast cancer which results in 40,000 deaths yearly, making it the second leading cause of death from cancer in women (Lester and Cotran, 1999), various plants have been tested and have led to several therapeutically available anticancer drugs derived from plants such as vinca alkaloids from the Catharanthus roseus plant (Wendell et al., 1993), damnacanthal from Morinda citrifolia (Hisawa et al., 1999), diterpene lactone andrographolide, neoandrographolide and three minor diterpene constituents from Andrographis paniculata (Chan et al., 1971; Balmain and Gonnolly, 1973). Phenolic compounds, like gallic acid, ethyl gallate and luteolin with antineoplastic activities against a panel of human cancer cell lines have been isolated from the stem bark and leaves of T. arjuna (Roxb.). It is estimated that of the 250,000 to 500,000 plant species available, only a small percentage has been properly studied in terms of their pharmacological properties (Rates, 2001). Cardellina et al. (1999) reported that cytotoxicity screening models are the preliminary methods for selection of active plant extracts against cancer.
Hence, in this study, three local herbs (Tectaria singaporeana, Blechnum orientale and Tacca integrifolia) which are commonly found in the lowland Dipterocarp forests of Malaysia as well as widely known for their medicinal properties through undocumented traditional preparations were screened for their cytotoxic potential on human breast cancer cells.
MATERIALS AND METHODS
Sample Collection
Three plant specimens Tectaria singaporeana, Blechnum orientale
and Tacca integrifolia were collected from the Air Hitam Forest
Reserve, a Lowland Dipterocarp Forest situated in Puchong, Selangor. The
plants were randomly selected in areas located in Compartment 15. The
plants, especially the roots were washed thoroughly. Plants were then
separated into three portions i.e., roots, leaves and stems. The dried
plants were cut or hammered into small pieces. The dried samples were
then pulverized with grinders and the powder was kept in air-tight plastic
bags.
Sample Extraction
Plant specimens that have been ground were soaked in a mixture of
methylene chloride and methanol at a ratio of 1:1 at room temperature.
The solvent was filtered using a Whatman filter paper No. 1 and the marc
was then soaked in methanol for 30 min. The solution was then filtered
and pooled together with methylene chloride-methanol extracts to increase
the content of organic solvent fraction. The solvent of the extraction
was removed using a rotor evaporator (IKA Rotary Evaporator, RV05-ST)
with a water bath temperature not exceeding 55 °C. The concentrate
was then transferred into Bijour bottles and the residual solvent was
removed. A final drying occurred by placing the concentrate extract in
an incubator. The final dried extract was weighed and stored under -20
°C.
Sample Preparations
Dried samples were measured and dissolved in DMSO (Dimethylsulphoxide)
with 4X penicillin/streptomycin to make a stock concentration of 100 μg
mL-1. The stock solution were diluted 10-folds serial to prepare
the extract concentration ranging from 0.1, 1, 10 and 100 μg mL-1.
These drug concentrations were then used for the cytotoxic analysis.
Cell Culture
Human Breast Cancer (HBC) cells, MCF-7wt., provided by the Cancer
Research and Drug Discovery Laboratory, Faculty of Medicine and Health
Sciences, Universiti Putra Malaysia, were grown in RPMI 1640 standard
culture medium supplemented with 10% Heat Inactivated Fetal Calf Serum
(HIFCS), 2 mM L-glutamine with 100 IU mL-1 penicillin and 100
μg mL-1 streptomycin in 25 and 75 cm2 tissue
culture flask under conventional culture conditions, such as 37 °C,
5%CO2, 95% air and 100% relative humidity. Adherent cancer
cells approaching 80% confluency were harvested with trypsin-EDTA to detach
the cells from the culture flask. The cells were then collected in a fresh
medium and were sub-cultured at densities of 1.0 -5.0x105 cells/10
m into 25 cm2 flask every 3-4 days as being adopted by Stanslas
(1998).
Cytotoxic Assay
The log phase cells with viability of 95% were mildly trypsinized
(trypsin 0.5 mg mL-1; EDTA 0.2 μg mL-1) to
detach the cell from the culture flask. Cells were then collected in fresh
medium. A syringe (21G) was used to prepare a suspension of single cells
in order to determine the cell number. Appropriate number of cells in
180 μL of medium was transferred to a 96-well flat bottom tissue
culture plate. The cells were allowed to attach for about 24 h before
the drug was introduced. The extract concentrations were obtained by a
serial dilution of the 100 mg mL-1 of stock solutions in the
culture medium, thus decreasing the concentrations to 1 mg mL-1,
100, 10 and 1 μg mL-1. A volume of 20 μL from these
concentrations of extracts (1 μg mL-1-1 mg mL-1)
was added into each well to yield a final concentration ranging from 0.1-100
μg mL-1. The final mixture used for treating the cell
contained not more than 1% of the solvent, the same for control wells
as well. The plates were then incubated at 5% CO2, 37 °C,
under high humidity for four days and the viability of the cells in the
plates were determined using the Microculture Tetrazolium (MTT) assay.
Fifty microliter of MTT (2 mg mL-1) were added to each well,
giving a final concentration of 0.4 mg mL-1. The plates were
incubated for 3-4 h for the living cells to convert the soluble MTT salt
into insoluble purple formazon. The medium was then aspirated from the
wells as completely as possible without disturbing the formazon crystals
and cells on the plastic surface (Twentyman and Luscombe, 1987). Hundred
microliter of DMSO were added to dissolve the purple formazon. The plate
was then analyzed using the SOFTProMax spectrophotometer.
Data Analysis
The absorbance of the formazon solution at 550 nm was determined on
the spectrophotometer. IC50 values (dose of drug that produces
a 50% reduction in the absorbance compared to the control) were determined
from the dose-response cytotoxic curves. Data obtained were analyzed using
the 2-Way Analysis of Variance (ANOVA) and Duncan`s Mean Separation Tests
using the percentage of cell viability and IC50 values as parameters
RESULTS AND DISCUSSION
Cytotoxic Potential of Plant Extracts
Medicinal plants have proven to be an invaluable source of drugs for
the advancement and efficacy of modern medicine. Indeed, despite all the
advances in biotechnology and synthetic chemistry, plants are still an
indispensable source of medicinal preparations, both preventive and curative.
Accordingly, several therapeutic properties of medicinal plants are known
in obstetrics and gynaecology (Abo et al., 2000), in respiratory
disorders (Neto et al., 2002), in skin disorders (Graf, 2000) in
cardiac cases (Ankli et al., 2002) and many more. In addition,
several studies have demonstrated that medicinal plants could bring to
the identification of antitumor compounds as recently suggested by Fumoleau
et al. (1995) and Mantle et al. (2000).
In the pursuit of breast cancer treatment and prevention, methanolic extracts of three different types of plants were tested on the MCF-7wt. breast cancer cell lines at various concentrations ranging from 0.1-100 μg mL-1. The extracts were obtained from three different parts of the individual plants namely the leaves, stems and roots. MCF-7wt. cells containing oestrogen receptor (ER+) were used as a model for studying the events associated with response to chemotherapy of ER+ breast cancer cells (Perry et al., 1995). These cells are able to exhibit fast responses such as inhibition effects and therefore serve as a suitable model.
There were significant differences in the mean values for the various plant parts and dilutions used (Table 1). However, there were no significant differences in the mean values for the plant types when tested against cell viability.
| Table 1: | ANOVA summary: Analysis of variance for 3 factors (species of plant, parts of plants, dilution levels) and their interactions |
| *Significance level at p<0.05, ns: non-significant | |
| Fig. 1: | Mean separation test for parts of plants. Different letter(s) indicate significant differences at p<0.05 |
The roots displayed the highest cell mortality at 19.58% compared to the leaves and stems as shown in Fig. 1. As for the various drug dilutions that were tested against the cell line, the results show that the highest significance is at 100 μg mL-1 with a cell mortality of 36.59%. Generally, the mechanism of cell death induced after treatment is dependent on the drug, its concentration and the particular cell line used for the study. This was also suggested by Junaidah (1997), where observations were made between 10 and 30 μg mL-1 of drug concentrations on cell lines. The results indicated that a higher drug concentration caused much severe damages to the cell line than at a lower concentration.
The parameters measured were according to the National Cancer Institute (NCI) guidelines in which IC50 < 100 μg mL-1 is considered active. The IC50 value shows the inhibition concentration at which only 50% of the cells are viable. The criteria of cytotoxic activity for the crude extracts, as established by the American National Cancer Institute (NCI) is an IC50<30μg mL-1 in the preliminary assay (Suffness and Pezzuto, 1990). According to Quah (1995), crude materials that require a drug concentration greater than 30 μg mL-1 to exhibit cytotoxicity were not considered as cytotoxic agents. Therefore, among all the three plants tested based on their roots, leaves and stems, the roots of Tectaria singaporeana, TS displayed the most cytotoxic potential with an IC50 value of 28.57 μg mL-1 as shown in Table 2.
As for Tectaria singaporeana, the results obtained suggest that the root of this fern is a cytotoxic agent since it is most effective against the breast cancer cell as compared to the other parts of the plants. The percentage of cell viability obtained from the root extracts were 16.62% which were far lower than those of the leaves (88.46%) and stems (90.68%). However, no studies were done on this particular fern species regarding their medicinal value or cytotoxicity against cancer cells. It is difficult to compare the findings from this study to other previous studies in terms of IC50 and cell viability due to the lack of studies conducted.
| Table 2: | IC50 values (μg mL-1) of roots of TCI, BO and TS in MCF-7wt. breast cancer cell lines. Values are mean of 3 separate determinations and errors represent the SD values |
In our screening of Blechnum orientale, the roots of the plant showed the lowest percentage of cell viability that is 31.47% as compared to the leaves and stems of the plant, 81.44 and 89.05%, respectively. The roots also displayed an IC50 value of 32.065 μg mL-1 where changes significantly occurred from 10 to 100 μg mL-1 of drug concentrations. Earlier studies have shown that Blechnum orientale and Blechnum spicant contain presumed caffeic acid derived lignans, e.g., the 8-2-linked (-) blechnic and (-) brainic acids (Wada et al., 1992; Davin et al., 2003). Based on existing chemotaxonomic data, lignans are present in primitive plants, such as the fern Blechnum orientale (Wada et al., 1992) in which these lignans take the role of phytoestrogens and thus are currently being studied for possible use in cancer prevention, particularly breast cancer.
As for Tacca integrifolia, significant changes occurred between 10 to 100 μg mL-1 concentrations for the various parts. The methanol/methylene chloride extracts of TCI leaves and stems showed an average cell viability percentage of 52.36 and 80.78%, respectively. However, the root extracts displayed the lowest cell viability percentage of 46.48% with an IC50 value of 95.03 μg mL-1. No studies have been reported for the cytotoxic effects of this plant, therefore comparisons of this study were made based on research findings made by Tinley et al. (2003) in which a new class of microtubule-stabilizing agents, the taccalonolides were identified. Taccalonolide A was first isolated from Tacca plantaginea by Chen et al. (1987) and Taccalonolide E was isolated in 1991 (Shen et al., 1991). In this study, the roots of Tacca chantrieri also contained the active constituents, taccalonolides E and A, which are complex, highly oxygenated steroids. These drugs caused the formation of abnormal mitotic spindles, thus inhibiting the mitotic progression and causing a G2-M arrest in cancer cells. It is possible that taccalonolides could account for one of the active compounds responsible for the observed cytotoxic activity on the cancer cells.
CONCLUSIONS
From this study, we can conclude that different parts of the plants such as the roots, leaves and stems produce different effects on the MCF-7wt. cell cancer lines. Only the roots of Tectaria singaporeana showed potential cytotoxicity effects on MCF-7wt. cell cancer lines with the lowest IC50 value of 28.57 μg mL-1. Further investigation of the active extracts and isolated compounds in animal models for the therapeutic efficacy and toxicity would provide evidence to determine whether these medicinal plants could be beneficial for drug discovery.