**8. The cytotoxicity and immunomodulatory activity of areca nut to the human health**

#### **8.1 Cytotoxicity activity of areca nut**

The majority of the community prefer to choose herbal medicine because the natural ingredient is considered to be safer and cheaper than chemical drugs. The precipitating factors of the increasing use of herbal medicine in developed countries are the side effects of chemical drugs, the high cost of modern medicine, and the increasing life expectancy when the prevalence of chronic disease had increased, so herbal medicine becomes an alternative treatment that is believed to cover all classes of the community especially in Indonesia [65]. In Taiwan and South-Eastern Asia, areca nut chewing has been associated with the development of oral squamous cell carcinoma (OSCC) through epidemiological studies and has been classified as a human carcinogen by the IARC (2004) [6].

MTS assay was done to observe the cytotoxicity effect of areca nut extract in HSC-2, HSC-3, and HaCat cells. Areca nut has a stimulation effect on mitochondria's activity, so it could be more efficient in creating energy and preventing free radicals. It can increase the cell viability graph over 100% in HaCat cell line by MTS assay. The areca nut extract in a certain dose could increase respiration and metabolism in the HaCat cell line [28]. Cytotoxicity of the extract was displayed with the viability percentage. The result showed that IC50 of areca nut extract in HSC-2 cells was 629.50 μg/mL and the IC50 value in HSC-3 occurred in lower concentration which was 164.06 μg/mL. The cytotoxicity effect started from the smallest concentration which was between 160–640 μg/mL, but at a concentration higher than 1280 μg/mL, the extract started to show proliferative activity. Areca nut extract provided a stronger cytotoxicity effect in HSC-3 cells than in HSC-2 cells. The cytotoxicity graph of the three cell lines is shown in **Figure 6.**

MTS assay showed increased cellular viability in doses higher than IC50, which was 16.2% followed by 4.5% in HSC-3 cells and an increase of 13.8% and 4% in HSC-2 cells. This was caused by strong antioxidant activity in the extract. Polyphenol had a direct stimulation effect on mitochondria's activity, so it was more efficient in producing energy and free radical scavenging. This was probably caused by the high extracellular formazan intensity from tetrazolium reduction by dehydrogenase succinate enzyme in cells performing respiration and metabolism. The more energy and cellular respiration product, the more formazan would be formed. Areca nut extract did not cause cytotoxicity in HaCat cell lines. This study showed that areca nut could induce formazan in large numbers so it showed a large increase in cell number. This was not caused by the high HaCat cells viability, but because of the high absorbance value by following the dense formazan color which was influenced by cellular respiration and metabolism. This absorbance value was read

**Figure 6.**

*The result of MTS assay in HSC-2, HSC-3, and HaCat cell lines. Areca nut extract has a stronger cytotoxicity effect in HSC-3 cells (b) than in HSC-2 cells (a). The extract does not show a cytotoxicity effect in HaCat cells (c).*

based on 490 nm wavelength. The denser the color of formazan's product, the higher the absorbance value would be. Although this study did not perform the test for Nicotinamide Adenosine Dinucleotide Hydrogen (NADH) content in the extract, it is possible that areca nut extract might contain NADH which could provide additional energy for the cells. NADH is an active coenzyme form of vitamin B3 (Niacin) which has an important role in the nervous system. This

#### *Antioxidant Activity of Areca Nut to Human Health: Effect on Oral Cancer Cell Lines… DOI: http://dx.doi.org/10.5772/intechopen.96036*

vitamin is found in all living cells of plants, animals, and humans. Tharakan et al. showed that *Tricopus zeylanicus* containing NADH had antioxidant activity by inducing lipid peroxidase and inhibiting lipoxygenase activity [66]. Further studies are needed to detect NADH content in areca nut.

The flavonoid in areca nut extract has antioxidant and also pro-oxidant activity. These two activities were also possessed by other herbal plants, *curcumin* [59, 60]. Lower doses of *curcumin* (12.5 μM) has the properties of reactive oxygen species scavenging, anti-inflammatory, apoptosis induction, and proliferation inhibition in myeloid leukemic cells, but in a higher dose and long term, the metabolite contents of *curcumin,* which is lipophilic or water-insoluble, could increase the level of cellular reactive oxygen species that causes carcinogenic potential through oxidative DNA damage or metal-mediated DNA damage at P450 cytochrome [61, 62]. This damage occurred because of the presence of Cu(II)-CYP2D6 which caused the damage of the DNA, especially 5-TG-3, 5-GC-3, and GG sequences [61]. *Curcumin* could induce lung cancer by increasing reactive oxygen species resulting in disarray between mitogen-activated protein kinase, NF-jb, and p53, causing genetic mutation and oxidative stress [63]. This finding concluded that the antioxidant effect which was started by an oxidative stimulus, depending on time, dose, and certain cancer type, could also cause unwanted side effects [63].

The principle of the MTS assay method was determined by a reduction–oxidation reaction occurring in cells. MTT/MTS reagent was reduced into formazan salts by succinate dehydrogenase enzyme found in living mitochondria cells. This reaction was allowed to take place for 4 hours then stopper reagent was added in MTT assay. The stopper reagent will lysate the cell's membrane so that formazan salts could get outside of the cell, and it could be dissolved. The MTS assay does not need a stopper reagent because formazan could dissolve in the culture medium with the addition of PMS. The formazan salt, that was in extracellular, was quantified with a spectrophotometer and measured in form of absorbance (**Figure 7**).

The higher the absorbance, the higher cell viability would be. The IC50 value between HSC-2 and HSC-3 cells had quite a large difference range. This difference showed the selective toxicity difference and type of cell death in some OSCC cell lines during exposure by natural anticancer or synthetic agent. The flavonoid compound showed weak cytotoxicity activity against HSC-2 cells so the IC50 value was higher [67, 68]. The factors of the substance in plants that influenced the cytotoxicity capability were the presence or absence of hydrophilic and hydrophobic groups in one same molecule, the presence or absence of isoprenyl groups, the presence or absence of polycyclic and/or halogen structure, the most condensed structure (low molecular weight is more cytotoxic) and lipophilicity. The factors from inside the cells which could probably cause this cytotoxicity was the difference

**Figure 7.** *The cells preparation in MTS assay.*

of protein expression which was resistant against some anticancer agent and expression of the drug's metabolism enzyme or natural substances [68]. Environmental factors were serum type, the presence of metal ion, oxygen concentration, and external pressure [68]. The success of the MTS assay depended greatly on a cell's metabolism and respiration capability. This study also showed the morphology of HSC-2 cells which was different from HSC-3 cells. The HSC-2 cells had bigger, wider cytoplasm and tighter cell junction than HSC-3 cells that probably could cause weaker extract's cytotoxicity activity than in HSC-2 cells so the IC50 value of HSC-2 was higher than HSC-3 cells.

Analysis of apoptotic cells using flow cytometry demonstrated that areca nut can induce apoptosis in oral cancer cell lines, HSC-2 and HSC-3 cell lines [14]. The caspase-3 activity as an effector caspase is shown to be related to late apoptosis activity because of the increase of caspase-3 with increasing late apoptotic cells percentage in both cells after areca nut treatment. Analysis of caspase activity confirmed that apoptosis might be the major mechanism of cell death induced by areca nut. As far as we know, there is no similar report regarding the caspase-3 activity induced by areca nut extract, but this result is similar to several previous studies that used plants containing flavonoids to increase caspase activity in cancer cells [41–43]. This finding may have biological implications in cancer treatment. The caspases inside cells are in an inactive form (procaspase), but activation induces the production of other caspases leading to cell death through proteolytic activity [44, 45]. Caspase-3 activation is a crucial component in the apoptotic signaling cascade. The apoptosis pathway involved in areca nut-induced cell death in both cancer cell lines may be through the extrinsic and intrinsic pathways. The areca nut also caused the cell-cycle arrest in HSC-3 and HSC-2 cell lines. The areca nut inhibited cell proliferation by the enhancement of Ki-67 after 24 hours of extract treatment in both cells [69].
