INTRODUCTION:

Cancer is one of the most leading causes of death in the world, more than 1.7 million new cases every year and to increase about 45% in 2030 including breast and colon cancer¹^,². The earlier cancer to be identified was associated with tumor suppressor genes mutation which resulted from inducing inflammation, and proteins that play important role in malignancy cancer³. In malignancy conditions, it spread to another organ as known metastasis or secondary cancer⁴. In addition, 66.7% of cancer deaths were registered with metastases as a contributing cause⁵. Hence, there is a strong to develop novel anticancer drugs for treatment and prevention⁶.

Plant-derived bioactive compound is crucial in the development of novel medications, including new cancer drugs. Approximately 60% of approved medications were approved to be of natural origin⁷. Medicinal plants are often regarded as one of the most important sources of naturally occurring bioactive chemicals. It has been estimated that 50% of the treatment products in America and Europe come from natural products or derivatives⁸. Some research shows that certain chemical compounds either sourced from nature or synthetic that have anti-inflammatory and antioxidant effects can be potential and developed as anticancer and chemo preventive agents against the process of carcinogenesis⁹.

Acmella ulliginosa is one of the herbaceous plants, it have great analgetic and anti-inflammatory effects¹⁰^,¹¹. A.Ulliginosa used to treat toothache traditionally. In addition, various biological activities have been found from the extract including anti-inflammation, antioxidant, antinociceptive, and anti-bacterial¹²^,¹³. The aim of research to evaluate toxicity and cytotoxic effect on T47D breast cancer cell line and WiDr colon cancer cell line.

MATERIALS AND METHODS:

Materials:

Acmella uliginosa, Ethanol 98%, n-Hexane, Artemia Salina Leach, WiDr colon cancer cell line and T47D breast cancer cell line obtain from ATCC, RPMI medium, penicillin-streptomicin, L-glutamin, Trypsine.

Methode:

Extraction:

A.ulliginosa is obtained from Serang, Banten, Indonesia. Fresh plants (stems, leaves, and flowers) were washed and dried. A.ulliginosa was powdered. Then, 400g of powder was extracted in ethanol and n-hexane (1:7) for 3 days and macerated 3 times. The ethanol and n-hexane extracts filtered and evaporated with a rotary evaporator.

Phytochemicals Screening:

a) Alkaloid test:

2 ml of extract was added Mayer and Dragendorf reagent through the wall of the test tube. The presence of a white precipitate indicates the presence of alkaloids.

b) Phenolic test:

The extract was dissolved in water, then the extract was added with a solution of lead acetate (10%). A white precipitate indicates the presence of phenolic compounds.

c) Steroid test:

Acetic anhydride was added to 0.5 mL crude extract of plant sample with H₂SO₄. The color change from violet to blue or green in sample indicates the presence of steroids.

d) Flavonoid test:

The Extract was dissolved in ethanol 96%, then 0.1 g magnesium and HCl was added. The yellow or violet coloration disappears on standing¹⁴.

Brine Shrimp Lethal Test (BSLT):

The toxicity test of extracts against brine shrimp larvae was carried out according to the method described by Meyer (1982). Ethanol and n-hexane extract of A.uliginosa dissolved in DMSO and distilled water at varying concentrations, then shrimp larvae aged 24 hours were added to each concentration. Then, incubated for 24 hours and observed for each treatment. The test was repeated three times and compared with the control. The LC 50 value was calculated based on the linear curve of percent mortality and concentration¹⁵^,¹⁶.

Cytotoxic Assay:

The cytotoxic test was carried out by observing the viability of T47D breast cancer cells and WiDr colon cancer cells using MTT assay. The confluent cell cultures were harvested and then distributed into 96-well microplate wells with the number of 1x104 cells/well. The cells are incubated to adapt before treatment. n-Hexane extract of A.uliginosa dissolved in DMSO solvent and then diluted using culture media. Cells were washed with PBS and then 100uL of the test solution was added to each well. Each test sample was made 3 times (triple). Cells were incubated for 24 hours in a CO2 incubator. After incubation, the test solution was discarded and 0.5mg/mL of MTT reagent was added as much as 100uL into each well. The reaction was stopped with a stopper reagent (10% SDS in 0.01 N HCN) which was added after 3 h of incubation with MTT. Cells were incubated for 24h at room temperature and protected. The results are read with an ELISA reader at a wavelength of 595nm¹⁷^,¹⁸^,¹⁹.

Picture 1. Acmella uliginosa

RESULT:

Extraction of Acmella uliginosa:

Extraction is separation process of metabolites from plants using polar and non-polar solvents²⁰. In this study, dried A.uliginosa simplicia was extracted using two solvents with different polarities, ethanol and n-Hexane. The ethanol solvent aims to extract polar to semi-polar compounds in A.uliginosa. whereas, n-hexan solvent aims to extract non-polar compounds. Figure 1 show the yields of A.uliginosa ethanol and n-Hexane extract was 2.4% and 1.53% respectively. The difference extract yields in the two solvents is related to the polarity of the compounds contained in A.uliginosa.

Figure 1. Yield Extract of Acmella uliginosa

Phytochemical’s screening:

The next stage of this research is phytochemical screening which aims to determine secondary metabolite contained in A. uliginosa²¹. The results of phytochemical screening in table 1 show that the A. uliginosa contain several secondary metabolite components such as phenolics, steroids, alkaloids, and flavonoids. Phytochemical compounds contained in the ethanolic extract of A. uliginosa such as alkaloids and flavonoids, while in the n-Hexane extract such as phenolics, steroids, alkaloids and flavonoids. In the steroid test, the addition of acetic anhydride and sulfuric acid showed a colour change and ring formation which indicated the presence of steroid compounds in the n-hexane extract. Testing for alkaloids was carried out using two reagents, Dragendorf and Mayer. The addition of Dragendorf reagent in the ethanol and n-hexane extracts showed a red precipitant, it indicated the presence of alkaloid compounds contained in the two extracts. Flavonoid testing shows the presence of colour fluorescence when observed in a UV lamp which indicates that there are flavonoid compounds in these compounds.

Table 1. Phytochemical Screning of Acmella uliginosa extract

	Ethanol extract	n-Hexane Extract
Fenolic	─	─
Steroid	─	+
Alkaloid (Dragendorf)	+	+
Alkaloid (Mayer)	+	─
Flavonoid	++	+

Brine Shrimp Lethality Test (BSLT):

Brine Shrimp Lethality Test (BSLT) is a simple bioassay to determining the toxicity of a bioactive chemical²². Artemia Salina is a species of brine shrimp could be used as toxicity test subjects because sensitivity to toxic compounds in the environment. In addition, BSLT not only to see the toxic effect of a compound, but this test could be used as preliminary testing for cytotoxic or anti-cancer compound screening¹⁶.

Table 2: Toxicity of Acmella uliginosa Against Brine Shrimp Assay

Extract	Concentration	Log Concentration	% Mortality
Ethanol	800 ppm	2.90	100
	400 ppm	2.60	73.33
	200 ppm	2.30	46.67
	100 ppm	2.00	33.33
	50 ppm	1.70	16.67
n-Hexane	250 ppm	2.40	100
	125 ppm	2.10	76.67
	62,5 ppm	1.80	46.67
	31,25 ppm	1.49	23.33
	15,62 ppm	1.19	10

Figure 2. Corellation between concentration and percent mortality of Artemia salina. (a) Ethanol extract, (b) n-Hexan Extract

Figure 2 shows correlation between log concentration and percent mortality of Artemia salina, it shows that the lethal concentration 50 (LC 50) of the ethanol and n-Hexane extract was 174.8751mg/L and 60.06807mg/L, respectively. According to Meyer et al. (1989) that plant or animal extracts are classified as cytotoxic if the LC50 value is <1000ppm. The extract is considered very toxic if it has an LC50 value of < 30ppm and is toxic if it has an LC50 value of 30-1000ppm and is considered non-toxic if the LC50 > 1000ppm²³. Therefore, n-hexane and ethanol extract of A.uliginosa with an LC50 value of 30-1000 categorized as toxic compounds. The Cytotoxic ability of A.uliginosa correlation with secondary metabolite in ethanol and n-Hexane extract. Based on these data, it can be concluded that A.uliginosa extract have ability as cytotoxic agent. Furthermore n-hexane extract has a better toxicity than ethanol extract.

MTT Assay:

Control cell of WiDr cell line

WiDr cell line treatmen 500 ppm

Control cell of T47D cell

T47D cell line treatmen 500 ppm

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay is colorimetric test for analyzing cell metabolic activity¹⁸^,²⁴. Determine the cytotoxic effect of compounds on a cell which is measured based on the value of Inhibition Concentration (IC50). The IC 50 is the concentration of the extract that inhibit of 50% cell growth of the cell population²⁵^,²⁶. In this study, the cytotoxic effect of n-Hexane extract of A.uliginosa was observed on T47D breast cancer cells and WiDr colon cancer cells.

(a)

(b)

Figure 3. Corellation between concentration and percent cell mortality, (a) WiDr (b) T47D

Determination of the IC 50 was obtained from the linear regression of extract concentration on cell viability. The IC 50 values of AU n-hexane extract were 1235.84 µg/ml and 628.48 µg/ml against T47D and WiDr cancer cells, respectively.

DISCUSSION:

In this study, preliminary testing with the BSLT method showed the LC50 value of the n-hexane extract was better than the ethanol extract, this was related to the secondary metabolites contained in the extract. In this study, phytochemical screening (Table 1) showed that the n-hexane extract contained several compounds such as flavonoids, alkaloids, flavonoids and steroids. According to Maimulyati et al (2018), the major secondary metabolites contained in the n-hexane extract of A.uliginosa include N-Isobutyl-2(E),6(Z),8(E)-decatrienamide (Spilantol), (9Z)- 9,17-Octadecadienal, Neophytadiene and 5-(4-nitrophenyl)-1,3,4-oxadiazole-2(5H)-one¹⁴^,²⁷. The compound N-Isobutyl-2(E),6(Z),8(E)-decatrienamide (Spilantol) has the highest percentage compared to other compounds¹⁴, so the toxic effect of n-hexane extract may be related to these compounds.

Cytotoxic analysis on WiDr dan T47D cell line show n-hexane extract of A.uliginosa better inhibit on WiDr cell line than T47D cell line. The characteristics of WiDr cells are Cox-2 overexpression and p53 mutation²⁸. Cox-2 is a protein that plays an important role in the inflammatory process²⁹. According to Hanahan and Weinberg (2011) inflammatory are one of the markers and hallmark in the development of cancer cells, inflammation plays a role in such as increasing cell proliferation, stromal activation and triggering DNA damage³⁰. In addition, Paul et al (2016) explain the n-hexane fraction of A.uliginosa showed anti-inflammatory activity against inflammation-induced wistar rats¹⁰. Therefore, cytotoxic activity of A.uliginosa correlation with anti-inflammation activity. The above correlation needs to be explored further.

In this preliminary study, it can be concluded that etanol and n-Hexan extract of A.uliginosa have potention to be develop as anticancer agent, specifically for colon cancer. The results of this study provide important information for further research on the isolation, and characterization of cytotoxic compounds from the n-hexane extract of A.uliginosa.

CONCLUSION:

This reseach demonstrated that extract of A.uliginosa have anticancer effect. The results indicated that n-hexane extract of AU may be useful as an alternative to produce the anticancer used in pharmaceutical. However, further research is needed to determine the anticancer compounds contained by A.uliginosa.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank the research grant of Kementerian Riset dan Pendidikan Tinggi (Kemenristek) BRIN through the research scheme of Penelitian Dosen Pemula (PDP) year 2020-2021 with contract number of 065/SP2H/LT/DPRM/2021 for funding this research.

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Received on 11.11.2021 Modified on 17.04.2022

Research J. Pharm. and Tech 2023; 16(2):640-644.

DOI: 10.52711/0974-360X.2023.00109