Cytotoxic Activity of Ethyl-para-methoxycinnamate from Kaempferia galanga L . on A 549 Lung Cancer and B 16 Melanoma Cancer Cells H )

Received: 6th December 2020 Revised: 27 February 2021 Accepted: 28th February 2021 Online: 28th February 2021 Kaempferia galanga L. belongs to the family of Zingiberaceae, an endangered medicinal plant with pharmacology activities. Ethyl-p-methoxycinnamate (EPMC) is an essential phytoconstituent of K. galanga rhizomes. Several studies have reported that EPMC has anticancer activities in several cancer cells, including CL-6 gallbladder cancer cells, HepG2liver cancer cells, MCF-7 breast cancer cells, and Raji lymphoma cancer cells. However, studies on A549 lung cancer and B16 melanoma cancer cells have not been reported. This study aimed to determine the anticancer activity of EPMC against A549 lung cancer and B16 melanoma cancer cells. EPMC was obtained by extraction using n-hexane, then recrystallized with chloroform. The isolate was then analyzed by thin-layer chromatography (TLC), and the structure was characterized by Fourier Transform Infrared (FTIR) and Nuclear Magnetic Resonance (NMR) spectroscopy. Cytotoxic activity was determined under Presto Blue assay. Based on the result, EPMC from K. galanga showed the cytotoxic effect on B16 cells with an IC5o value of 97.09 pg/mL, whereas EPMC showed no significant cytotoxic effect on A549 with an IC50 value of 1407.75 pg/mL. It was concluded that EPMC has potential cytotoxic on B16 melanoma cancer cells, but it showed inactive activity against A549 lung cancer cells. Further molecular mechanism underlying EPMC cytotoxic activity needs to be conducted.


Introduction
Cancer is one of the leading causes of death in the world. For several decades, lung cancer has been the most common cancer globally. In 2018, the incidence was 12.3 % of all cancer diagnose [1]. In Indonesia, lung cancer has the highest incidence in men, with 19.4 per 100,000 population with an average death rate of 10.9 per 100,000 population [2]. Similarly, the worldwide incidence of melanoma, a malignant skin cancer deriving from melanocytes, has increased more rapidly than other cancers [ 3 ]. The prognosis for patients with malignant melanoma is bleak, with an average survival time of 6-9 months. Melanoma is responsible for 80% of skin cancer patient mortalities [ 4 ].
There are several ways to treat cancer, including surgery, radiation therapy, chemotherapy, and a combination of those treatments. To date, the chemotherapeutic agent is the most common treatment [ 4 ]. However, chemotherapy drugs cause side effects such as hair loss, bone marrow suppression, drug resistance, gastrointestinal lesions, neurological dysfunction, and cardiac toxicity [ 5 ]. Besides, chemotherapy often causes failure due to the low selectivity of anticancer drugs [6]. Natural compounds represent attractive new drug leads. A recent report revealed that -50 % of all small molecule therapeutics are based on natural products or their derivatives [ 7 ]. Alkaloids such as vincristine, vinblastine, and taxol are examples of anticancer drugs that have long been used, and their molecular mechanisms are known [8]. Thus, exploring natural product isolates for cancer treatment needs to be increased to develop anticancer drugs.
Pharmacological activities of K. galanga rhizomes are mainly due to secondary metabolites of different nature [ 17 ]. Ethyl-p-methoxycinnamate (EPMC) is the essential compound reported as a bioactive secondary metabolite of K. galanga rhizome [ 14 ].
The potential of EPMC from K. galanga rhizome against anticancer activity needs to be explored further. Several previous studies reported that EPMC has anticancer activity against CL-6 bile duct cancer cells [18], HepG2 liver cancer cells [ 19 ], MCF-7 breast cancer cells [20], and Raji's lymphoma cancer cells [10]. However, the anticancer activity of EPMC on lung and melanoma cancer cells has not been reported yet. This study aimed to isolate EPMC from K. galanga rhizomes and investigated its potential for anticancer activity against A 549 lung cancer and B16 melanoma cancer cells. A 549 cells are adenocarcinomic human alveolar basal epithelial cells in the lungs responsible for the diffusion of substances such as water and electrolytes in the alveoli. These cells are commonly used as model cells to research respiratory diseases such as lung cancer [12]. B16 cells are derived from epithelial mice that produce melanin. Melanin has many living systems functions, and its synthesis alterations occur in many disease states [21]. Although melanin's primary function is to protect skin from UV-induced damage, melanin pigment can also regulate epidermal homeostasis and thus can affect melanoma behavior [22].

Methodology
The rhizome part of K. galanga was obtained from Sukawening, Garut, West Java . The  n -hexane, ethyl acetate, chloroform.

Isolation and Characterization of ethyl-pmethoxycinnamate
The rhizome of K. galanga was washed with water, then dried in an oven at 35°C . After drying, the sample was ground with a mechanical blender. K. galanga rhizome dry powder (820 g) was then extracted using 2 L of n-hexane solvent by maceration method for 3 x 24 hours at 25°C . The macerated extract was then filtered using a filter funnel and then concentrated using a vacuum rotary evaporator at 45°C . The concentrated nhexane extract was purified by recrystallization. An impure EPMC crystal was mixed with hot chloroform as a solvent to form a saturated solution to perform recrystallization. The resulting crystals were tested for purity using Thin Layer Chromatography (TLC), Fourier Transform Infra-Red (FTIR) spectroscopy, and Nuclear Magnetic Resonance (NMR).

Cell Culture
These two-cancer cells, A 549 , and B16 cells were obtained from the Laboratorium Sentral of Universitas Padjadjaran. The cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were inoculated at a density of lxio 3 cells in a 6-well plate and were maintained at 37°C in a humidified atmosphere containing 95 % air and 5 % CO2.

. Cytotoxic Test with Presto Blue Assay Method
EPMC compound was tested for their cytotoxic activity against A 549 and B16 cells using the Presto Blue assay method. A number of lxio 4  hours. Cisplatin was used as a positive control. At the end of incubation, the media and extract were discarded and then added with 100 pL of PrestoBlue with a ratio of 1: 9. Cells were incubated again for 1-2 hours until a color change was seen in a 5 % C02 incubator at 37°C . Living cells will reduce Presto Blue from the blue compound resazurin to the red compound resorufin. Absorption was measured at 570 nm and 600 nm (reference wavelength) using a multimode reader [ 23 ].

. Ethyl-p-methoxy cinnamate isolation
EPMC is found as a major ester and easily isolated compound from the rhizome of K. galanga [ 24 ]. Purification of n-hexane extracts of the rhizome of K.
galanga produced a white crystal of EPMC ( 76.9 g). In case, the percentage of yield was 49.99 %. EPMC isolate was then confirmed for purity using Thin Layer Chromatography (TLC). The stationary phase in TLC was in the form of a GF 254 silica gel plate, while the mobile phase used was n-hexane and ethyl acetate with a ratio

. FTIR Characterization
The Infra-red spectra of the isolated compound showed a very intensely broad peak at 3450 cm 1 for 0-H bond vibrations, and moderately intense peaks at 1510 and 1600 cm 1 were observed for the aromatic part of C = C vibration. The unsaturated C-H vibration was observed at 2900 cm 1 . This is supported by the presence of absorption at wavenumbers 1367 cm 1  To ensure the appropriate obtained structure of EPMC, we compared our original spectrum values with other spectra that had been reported. The comparison between original and reference spectrum values can be seen in Table 1 and Table 2.    2 showed that the NMR data for isolated EPMC had similarities with the NMR reference. It can be seen that the spectrum value of isolated EPMC approached to spectrum value of the reference. In order that we conclude that the structure of isolated EPMC was the same as the reference.

. Cytotoxicity Activities of A 549 Lung Cancer and B16 Melanoma Cancer Cells
To examine whether EPMC has anticancer activity, the cytotoxic activity of EPMC against A 549 lung cancer and B16 melanoma cancer cells was performed. The analysis was done using the Presto Blue assay method described in the Methodology section. PrestoBlue has been developed for detecting cell-mediated cytotoxicity in vitro. It is a resazurin-based compound converted to the reduced form by mitochondrial enzymes of viable cells in the tested systems. Bonder etal. [ 25 ] stated that PrestoBlue assay offers a new alternative to MTT for analyzing cell viability. The assay was fast, simple, and allows continuous monitoring of cultures. Thus, we decided to use the Presto Blue assay method for cytotoxic activity assessment.  (Fig. 3 A) gave a 97 -09 Pg/mL value. Treatment of EPMC on B16 cells showed cell growth inhibition in a concentrationdependent manner. Interestingly, we did not observe the decrease of A 549 cell growth treated with EPMC with an IC 50 value of 1407.75 pg / mL. This IC 50 value can be obtained even though the concentration tested was 1000 pg / mL due to poorly linear regression of EPMC concentration toward corrected absorbance of A 549 cells (Fig. 4 A). Meanwhile, we evaluated cisplatin's cytotoxic activity against A 549 cells and B16 cells, given an IC 50 value of 18 pg / mL 53 pg / mL, respectively. Prayong et al. [26] stated that cytotoxic activity was categorized into three depending on the IC 50 value, which are potential cytotoxic (IC 50 < 100 pg / mL), moderate cytotoxic (100 pg / mL < IC 50 < 1000 pg / mL), and weak cytotoxic activity (IC 50 > 1000 pg / mL). According to that statement, we suggest that EPMC had no cytotoxic activity on A 549 cells. Simultaneously, it was cytotoxic on B16 cells, even though the IC 50 value of EPMC against B16 cells was still higher than cisplatin. However, EPMC has the potential to be developed as an anticancer agent on B16 cells.  .IS ' ' ?!

. Conclusion
microscope morphology on B16 cells. An increase in EPMC concentration caused more cells to undergo morphological alteration. Viable cells exhibited epithelial shape, but after being treated with a specific concentration of samples, the cells exhibited cell shrinkage and rounding (Figure 3 B). Supports its noncytotoxic activity, treated cells of A 549 did not show any changes in cell morphology (Figure 4 B). A 549 cells retain their original morphology that showed a long fusiform shape, small size, clear cell boundaries, welladherent pebble-like growth, placental cytoplasm, and less cytoplasmic granules.
we have isolated ethyl-pmethoxycinnamate from K. galanga, with the percentage of yield was 49 -99 %. XH-NMR and 13 C-NMR analysis confirmed the structure of EPMC, and the spectrum was similar to the reference. The cytotoxic activity of EPMC was evaluated against two cancer cells, B16 cells, and A 549 cells. EPMC showed a cytotoxic effect on B16 cells with an IC 50 value of 97 -09 Pg/mL. However, EPMC showed no significant cytotoxic activity of A 549 lung cancer cells with an IC 50 value of 1407.75 pg / mL.
Considering that EPMC can be an anticancer agent against B16 cells, further research on the cytotoxic mechanism of EPMC against B16 cells and its selectivity on normal cells needs to be investigated.

In this research
Several studies of EPMC cytotoxic activities on different cancer cells have been reported. Amuamuta et al. [18] reported the IC 50 value of EPMC against CL-6 and OUMS cancer cells were 49.16 pg / mL and 103.18 pg / mL. Anticancer activity of EPMC against the HepG2 cell line given IC 50 value of 27.1 pg / mL exhibits antiproliferative properties, inducing the significant increase of subGo