Cytotoxicity Activity of Several Medicinal Plants Grow in Mangrove Forest against Human’s Cervical (HELA), Breast (T47D), and Colorectal (WiDr) Cancer Cell Lines

Mangrove forests are Indonesia’s most extensive forest ecosystems, reaching 27% (16 million hectares) of the world’s total mangrove forests. The largest mangrove forest area in Indonesia is in West Kalimantan, with about 119,327 hectares. It consists of 75% of the mangrove species in Indonesia, one of which is the Polaria Tanjung Pagar mangrove forest located in Sungai Kunyit sub-district, Mempawah district. Mangrove forest vegetation provides many benefits for human life, as protection from wave abrasion, a source of food and medicine.

Medicinal plants are biologic natural resources, both cultivated and noncultivated. The use of plants as medicines in Indonesia is usually only based on practical experience passed down from generation to generation without supporting data that meet the requirements.^[1] Therefore, exploring information on medicinal plants’ chemical compound content and bioactivity through scientific research is very important.

Plant bioactivity is greatly influenced by the chemical compounds contained in it. The difference in the chemical compounds’ content shows the plant’s pharmacologic activity.^[2] Plants generally contain active compounds in the form of secondary metabolites such as alkaloids, flavonoids, steroids, triterpenoids, coumarin, and others. Bioactive compounds are almost always toxic at high doses. The cytotoxic test is a method development and is an in vitro level study to predict toxic compounds in cells, which is an absolute requirement for anticancer drugs. These cytotoxic test results can provide information about the amount or level of drug concentration that still allows the cells target, such as cancer cells to survive.^[3]

Cancer is an abnormal cell growth that is continuous and uncontrolled. This cancer can spread to surrounding tissues and can metastasize to cancer cells. Some malignant cancer cells include colorectal cancer, cervical cancer, and breast cancer. Colorectal cancer is an abnormal growth in the colon or rectum. Most colorectal cancers start as a polyp (a benign or noncancerous growth on the lining of the colon or rectum) that later develops into an abnormal (cancerous) growth.^[4] Almost 95% of cervical cancer in women is caused by the human papillomavirus (HPV).^[5] HPV infection is common in women of reproductive age. This infection can persist, progress to dysplasia or resolve completely. Meanwhile, breast cancer is a cancer that forms in breast tissue. Breast cancer occurs when cells in the breast tissue grow uncontrollably and take over the healthy and surrounding breast tissue. Breast cancer can form in the glands that produce milk (lobules) or in the ducts (ducts) that carry milk from the glands to the nipple.^[6] Cancer can also form in the fatty tissue or connective tissue within the breast. Although more common in women, breast cancer can also affect men.

The increase in cancer cases to this day is very rapid, including the three types of cancer described previously. Treatment is carried out intensively with surgery, chemotherapy, and radiotherapy, but these treatments are still ineffective for treating colorectal cancer. The failure that often occurs is main treatment with chemotherapy due to the low selectivity of anticancer drugs against normal cells and the resistance of cancer cells to chemotherapy agents, causing serious side effects in cancer patients. Thus, it is necessary to support natural treatment, such as efficacious plants as drugs that naturally contain secondary metabolites (flavonoid, phenolics, tannins, saponins, terpenes, steroids, and alkaloids).^[7] Groups of compounds that are abundant in nature and have been widely studied for their anticancer activity.

Based on our previous investigation, the mangrove forests of Polaria Tanjung Pagar, Sungai Kunyit subdistrict, Mempawah district showed the potential therapeutic as traditional but not yet scientifically justified. Thus, it is necessary to assess the potential of natural materials as anticancer as a fundamental basis for optimal natural resource utilization. This study aimed to analyze the secondary metabolite components of medicinal plants in the mangrove forests of Polaria Tanjung Pagar, Sungai Kunyit subdistrict, Mempawah district, West Kalimantan, and analyze their cytotoxicity against several cancer cells.

Methods

Sample preparation

The main ingredient in this research was the leaves of several medicinal plants obtained from the mangrove forests of Polaria Tanjung Pagar, Sungai Kunyit subdistrict, Mempawah district, located at latitude coordinates 0°23’49.65612"N and longitude coordinate 108°56’46.38309"E. These included Bruguiera cylindrica, Aegiceras corniculatum, Acrostichum aureum, Avicennia alba, and Rhizophora mucronata.

The first step was identifying medicinal plants used by locals, conducted by intensive interviews compared to the references regarding their effectiveness as medicine. The unidentified species were collected for further identification in Biology Laboratory, Universitas Tanjungpura. This step, called ethnobotanical and ecologic studies of medicinal plants, was conducted in our previous study.^[8]

In this study, the extract was obtained by macerated in methanol as the solvent. After sorting and cleaning, the leaf samples were powdered and then entered the maceration stage for 3 × 24 hours. The filtrate was collected and evaporated until methanol extract was obtained.

Screening of secondary metabolites

Methanol extract from five medicinal plants in the mangrove forest was then screened for secondary metabolites through flavonoids, saponins, terpenes, steroids, alkaloids, phenolics, and tannins. Qualitative analysis of these secondary metabolites identified by thin-layer chromatography method at 366 nm ultraviolet.

Cytotoxicity test

Cytotoxicity test was carried out by testing medicinal plant leaf extracts from mangrove forests using the MTT assay method, referring to Masriani.^[9] This cytotoxic test was carried out on three types of cancer cell lines. The cell lines included HeLa cells (tumor cells found in human cervical tissue), T47D cells (tumor cells found in human breasts), and WiDr cells (tumor cells found in the large intestine). The three types of cancer cells were obtained from the Faculty of Public Health Medicine and Nursing, Department of Parasitology, Gadjah Mada University, Yogyakarta. All cancer cells grew in RPMI 1640 (Gibco,USA) medium containing 5% v/v fetal bovine serum (Caisson, USA) and 1% v/v fungizone (Gibco,USA) added with 1% w/v penicillin–streptomycin, then incubated at 37°C with an incubator containing 5% CO₂ gas.

The cytotoxic test used the MTT method of cell suspension in complete media; about 100 μL (density 10,000 cells/well) was inserted into plate 96. The plate was incubated for 24 hours in a 5% CO₂ incubator. When incubation ended, the media of each well was discarded, new media was added, and a 100-μL sample in each different well was obtained. The final sample content was obtained with a specific variation level (500, 250, 125, 31.25, 15.625). Furthermore, the plates were incubated in a 5% CO₂ incubator for 24 hours at 37°C. At the end of incubation, the medium in each well was discarded, washed with phosphate-buffered saline. The plates were incubated again for 4 hours at 37°C. Live cells would react with MTT (3-(4,5-dimethyl thiazol-2il)-2,5-diphenyl tetrazolium bromide) to form purple formazan. The colored formazan formation reaction was stopped with 10% sodium dodecyl sulfate (SDS) solution in 0.01 N HCl, then incubated overnight at room temperature. After the incubation period, the absorption was read with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 550 nm. The percentage of living cells was calculated through the absorbance data of cells. A log relationship curve of the concentration versus the percentage value of living cells was calculated, and the price was calculated.

Data analysis

Cytotoxicity of the samples in the study was expressed in IC₅₀ value. The toxicity values in each plant were compared against WiDr, HeLa, and T47D cell lines through analysis of variance. Duncan multiple range tests were performed to specify values for comparisons between their means.

Results and Discussion

The samples used in this study were leaves from each type of medicinal plant originating from the mangrove forests of Polaria Tanjung Pagar, Sungai Kunyit subdistrict, Mempawah district. Those medicinal plants included B. cylindrica, A. corniculatum, A. aureum, A. alba, and R. mucronata. The results of the secondary metabolite test on the methanol extract are presented in [Table 1].

Table 1 Secondary metabolites of medicinal plants from mangrove forests of Polaria Tanjung Pagar, Sungai Kunyit subdistrict, Mempawah district Click here to view

Based on the secondary metabolite analysis results, all the leaves samples of mangrove medicinal plants contained flavonoids, saponins, terpenes, steroids, phenolics, and tannins but negative alkaloid compounds. Secondary metabolites from natural ingredients are influenced by inherent (genetic) and external (environmental) factors. The active ingredient content cannot be guaranteed to remain constant.^[10] The same plant species, but different places to grow, compounds in these plant species will differ due to several factors. These factors can be divided into two, external factors [nutrients, water, temperature, altitude, plants that grow around them (allelopathy), sunlight] and internal factors (plants themselves: pests infections).^{[11],[12],[13],[14][15]}

Cytotoxicity

This test was conducted to determine the cytotoxic potential of medicinal plant leaf extracts in the mangrove forests of Polaria Tanjung Pagar against three human cancer cells, namely HeLa, T47D, and WiDr cell lines in vitro. The method used in this cytotoxic test was the MTT method (3-(4,5-dimethyl thiazol-2il)-2,5-diphenyl tetrazolium bromide). The basis for measuring the MTT method is the measurement of the formazan crystals formed. Formazan crystals are purple crystals insoluble in water but soluble in 10% soluble SDS. The formation of formazan crystals results from the reaction between the MTT salt and the tetrazolium succinate reductase system found in living cells’ mitochondria.^[3] Hence, the living cancer cells will form more formazan crystals than dead cancer cells.

Prayong et al. classified substances as cytotoxic and selective into five categories: active (IC₅₀ < 100 μg/mL) and high selectivity (IS ≥ 3); moderate active (100 μg/mL < IC₅₀ < 1000 μg/mL) and high selectivity (IS ≥ 3); moderate active (100 μg/mL < IC₅₀ > 1000 μg/mL) but low selectivity (IS < 3); toxic only for normal cells, and nontoxic (IC₅₀ > 1000 μg/mL).^[16] The toxicity level of methanol extracts from several medicinal plants in mangrove forests against HeLa cervical cancer cells, T47D breast cancer cells, and WiDr colorectal cancer cells are presented in [Table 2].

Table 2 Cytotoxicity of methanolic extracts ofmedicinal plants from Polaria Tanjung Pagar mangrove forests, Mendalok village, Sungai Kunyit subdistrict, Mempawah district against HeLa cervical cancer cells, T47D breast cancer cells, and WiDr colon cancer cells Click here to view

The cytotoxicity testing of medicinal plant methanol extracts in mangrove forests of Polaria Tanjung Pagar, Mendalok village, Sungai Kunyit subdistrict, Mempawah district is summarized in [Table 2]. It was found that several types of medicinal plants had an active cytotoxic activity as their IC₅₀ value of >100 μg/mL. The plants were A. corniculatum and A. alba, which showed cytotoxicity against HeLa cervical cancer cells in IC₅₀ of 49.40 ± 5.85 and 74.75 ± 26.97 μg/mL, respectively. In T47D breast cancer cells, mangrove forest plants A. corniculatum and A. alba also showed active cytotoxics in IC₅₀ of 78.12 ± 11.38 and 50.76 ± 9.92 μg/mL, respectively. On the contrary, for WiDr colorectal cancer cells, methanol extract from A. corniculatum, A. aureum, A. alba, and R. mucronata showed the most active cytotoxicity in IC₅₀ of 45.60 ± 7.35, 89.19 ± 4.22, 73.25 ± 17.63, 57.49 ± 17.63, and 57.49 ± 11.70 μg/mL, respectively. Statistically, the cytotoxic value of the same plant against different cell lines was significantly different (P < 0.001) [[Table 2]].

Cytotoxic is the primary testing process for the utilization of plant extracts in medicine. This drug is expected to have selective toxicity, meaning that it destroys disease cells without damaging normal tissue cells.^[17] Cytotoxic assays are used to predict the presence of new cytotoxic drugs from natural ingredients. The cytotoxic test is an in vitro test using cell culture to detect antineoplastic (cytostatic) activity. The basis of cytotoxic test is that the biologic activity determination system. It will produce a dose–response curve and response criteria that show a linear correlation to the number of cells. The end of the cytotoxic test can provide information on drug concentrations that still allow cells to survive.^[3] The use of cytotoxic tests on a cell is one way of determining in vitro to obtain cytotoxic drugs. This system is a qualitative test by determining cell death. The cytotoxic test is developed to predict toxic compounds in cells, an absolute requirement for use as a medicinal substance.

The method of calculating the number of cells used in this study was the MTT method. The MTT method is an indirect cell count. This method is relatively fast, sensitive, accurate, and quantitative for measuring large samples. The results can predict the cytotoxic properties of a material.^[3] The MTT method principle is based on the metabolic reduction of the tetrazolium salt of MTT (3-(4,5-dimethyltiazol-2-il)2,5-diphenyltetrazolium bromide). The colorless activity of mitochondrial enzymes in living cells form purple formazan salts, which can be quantified colorimetrically. The colorimetric test estimates the number of cells directly by measuring the amount of sensitivity index of cellular protein, which is linearly related to cell density.^[18] The addition of a stopper reagent (detergent in nature) will dissolve the formazan crystals. The absorbance of the formation can be measured using an ELISA reader Doyle and Griffiths at a wavelength of 500 to 600 nm.^[3] The intensity of the purple color formed is proportional to the number of living cells. If the purple color intensity is high, it means that the number of living cells will increase.

In this study, cytotoxic properties were determined on three types of cancer cells. Cervical cancer cells are cervical epithelial carcinoma triggered by HPV infection.^[19] One type of breast cancer cell is T47D cells isolated from ductal tumor tissue in the human breast. These cancer cells are different from human breast cancer cells Michigan Cancer Foundation-7 (MCF-7).^[20] The apoptotic process of T47D breast cells involves both caspase 7 and caspase 3, whereas MCF-7 does not show caspase 3. Several colorectal cancer cells are usually observed, including HT-29 cells, WiDr cell lines, and CaCo₂. ^[21,22] The WiDr cell line is a cancer cell isolated from the colon of a woman with colorectal cancer.

The presence of secondary metabolites probably influences the cytotoxic activity of medicinal plants growing in mangrove forests. Secondary metabolites are identified as a form of plant defense to form antibacterial, antifungal, and anticancer properties by interacting with target molecules in cells, including nucleic acids, proteins, or protein membranes.^[23] Besides, phenolics, alkaloids, and terpenoids have been shown to interfere with the activation of ATP-binding cassette transporters so that they have anticancer properties.^[24]

The observation showed that the plants in the mangrove forest, mainly A. corniculatum and A. alba, had very active toxic properties against the three types of cancer cell lines. Apart from the secondary metabolites identified in this study, previous studies found that the twigs and stems of A. corniculatum also contain a 2-O-acetyl-5-O-methylembelin component. This component shows cytotoxicity to HeLa cell lines.^[25] Another study on A. alba showed that the leaf extract of this plant showed more active cytotoxicity against HeLa cells than MCF-7 human breast cancer cells and was suspected of the activity of phenolic compounds.^[26] Our study showed a potential basis for developing the medicinal therapeutic of cancer from mangrove forest’s natural resource.

Conclusion

Of the five types of mangrove plants observed (B. cylindrica, A. corniculatum, A. aureum, A. alba, and R. mucronata), all contain compounds containing flavonoids, saponins, terpenes, steroids, phenolics, and tannins, but the alkaloid compounds were absent.

Mangrove plants with an active cytotoxic activity against HeLa cancer cells were A. corniculatum and A. alba. Mangrove plants that showed the most active cytotoxic activity against T47D cancer cells were A. corniculatum and A. alba. Meanwhile, the most active cytotoxic activity against WiDr cancer cells was A. corniculatum, A. aureum, A. alba, and R. mucronata.