Antioxidant Activity of Areca Nut to Human Health: Effect on Oral Cancer Cell Lines and Immunomodulatory Activity

Many herbs have been discovered to be potential sources of the antitumor and immunomodulatory drug. Areca nut (Areca catechu L.) has a high content of phenols and flavonoids and is highly related to antioxidant activity. Areca nut is a traditional herbal medicine that is popular around Indonesia, India, Thailand, and Taiwan. However, data on its effect on human health showed various results. This chapter’s aim to review the phytochemical and polyphenolic content, the molecular structure of bioactive compounds, the side effect of the crude extract, the role of catechin in cancer mechanism, the antioxidant activity, the cytotoxicity, and immunomodulatory activity of the areca nut. Areca nuts from Aceh province in Indonesia, contain flavonoids, phenolics, catechin, quercetin, and a small percentage of tannins which contribute to antioxidant activity. The areca nut has anticancer potential activity so it can be used in combination with chemotherapeutic agents to enhance the effect at lower doses and thus minimize chemotherapy-induced toxicity. Areca nuts also show immunomodulatory activity which can increase the body’s immune system.


Introduction
One of the plants with the potential to be developed as herbal medicine is the pinang plant (Areca catechu Linn; areca, Palmaceae). The term Areca catechu refers to the largest amount of the phytochemical compounds in areca nut that is arecoline and catechin. Areca nut is a traditional herbal medicine that is popular around Indonesia, India, Thailand, and Taiwan. This plant spreads widely in South East Asia, South Africa, and the Pacific Ocean Islands. It is usually used in betel chewing common among the Indians and Malays as a breath freshener, digestive aid, anthelmintic, aphrodisiac, and to maintain stamina [1]. Several kinds of research studied the functions of the areca plant showed that the stem can be used as antibacterial and antifungal [2]. Epidemiologic study and laboratory research found a large number of anticancer compounds obtained from the nuts. Some of them contained flavonoid, polyphenol, carotenoid, selenium, vitamin C, and E which showed chemotherapy and preventive activity. An antioxidant is one of the solutions to Characteristics of areca nut • Minerals: Calcium, phosphorus, iron, vitamin B6, and C.
• Apoptosis and cell cycle arrest in carcinoma cell lines [14].
• The arecolines can enhance Alzheimer's type of dementia.
Areca nut contains several alkaloids that belong to the pyridine group, especially arecoline. Arecoline affects the oculomotor nerve which can cause mydriasis, mild Areca plants [8].

Figure 2.
The ripe areca nuts. 3 Antioxidant Activity of Areca Nut to Human Health: Effect on Oral Cancer Cell Lines… DOI: http://dx.doi.org /10.5772/intechopen.96036 paralysis, and pupillary dilatation [15]. Several studies showed that arecoline can induce neurotoxicity through its action in oxidative stress and generating Reactive Oxygen Species (ROS), hepatotoxicity, testicular toxicity, oropharyngeal cancers, and oral submucous fibrosis [16,17]. Besides alkaloids, ent-catechin, and jacareubin are the major compound in areca nut [7]. Catechin and its analogs are antioxidant, anti-allergic, anticancer, anti mutation, and anti-inflammatory. It can improve liver function and scavenge free radicals [18]. The beneficial effect of catechins is reported in the treatment of cancer, cardiovascular diseases, diabetes, neurodegenerative diseases, and liver diseases [19]. Apparently, catechins have function not only as a powerful antioxidant [20], preventing oxidative damage in healthy cells [21], but also as an antiangiogenic, antitumor agent [22], and a modulator of tumor cell response to chemotherapy [23]. It can induce apoptosis by increasing caspases [24] and promotes cell growth arrest by altering the expression of cell cycle regulatory proteins [25]. Jacareubin is reported to have an anti-inflammatory property and increases the activity of H + ,K + -ATPase which is needed to gain the energy of the cells [26,27].
The majority of phytochemical ingredients in areca nut extract are phenolic compounds such as flavonoids, tannins, and alkaloids [28]. It was established that a great quantity of tannins has been found in dark dry fruits such as tea. The areca nut also has a reddish-brown color. The levels of tannin found in areca nut have different contents in different areas or regions [29]. It could be caused by climate conditions and environmental factors where it grows. Climatic factors such as temperature, weather, and rainfall. The environmental factors such as soil type and fertility, the height of growing, and plant maintenance.
The presence of catechin and quercetin mostly were identified through High-Performance Liquid Chromatography (HPLC) analysis. These two compounds are well-known antioxidants and could have contributed to the observed antioxidant activity [30]. Previous studies have identified several phenolic compounds in the areca nuts including trimer procyanidin, dimer procyanidin (B1 dan B2), catechin, and isorhamnetin 3-O-rutinoside [31]. Many of these compounds have antioxidants activities [20,32]. Catechin was proved to have strong antioxidant activity which could contribute towards the anti-cancer effects [33][34][35][36]. It has also been shown that polymerized catechin suppresses the activity of Staphylococcus aureus α-toxin and as an effective urease inhibitor in Staphylococcus saprophyticus strains. Although the level of quercetin is not much in areca nut extract, it also has been proved as an antioxidant. It possesses an anti-inflammatory potential that can be expressed in different cell types, both in animal and human models [37]. Quercetin is also able to inhibit the growth of cancer cells through induction of apoptosis and inhibitory proliferation in gastrointestinal, breast, esophagus, and ovary cancer [38].

Quantitative data of polyphenolic content in areca nut
Various studies have been conducted to calculate the content of polyphenolic areca nut from various regions around the world. The results also showed various results. This difference shows that the variation in polyphenolic content depends on the geographical conditions in which the plant grows so that it affects the quality and quantity of the phytochemical composition in areca nut. This variation is also influenced by the species and type of the areca nuts used such as freshness, maturity, and form of the nut drying process. One of the polyphenolic content derived from areca nut on the island of Sumatra in Indonesia is shown in Table 1. The total phenolic and flavonoid contents of areca nut are expressed as Tannin Acid Equivalents (TAE) and Catechin Equivalents (CE).

Phenolic compounds
Phenolic compounds are the largest part of the phytochemicals in plants, especially fruit, seeds, and leaves. This collection of compounds provides many health benefits because they contain many antioxidants that can scavenge oxidative stress due to reactive oxygen species. Intake of fruit, vegetables, and whole grains that are rich in phenolics can lower the risk of cardiovascular disease, chronic inflammation, cancer, and neurodegenerative diseases. Polyphenolics contain an aromatic hydroxyl ring derived from L-phenylalanine. Phenolic compounds comprise one (phenolic acids) or more (polyphenols) aromatic rings with attached hydroxyl groups in their structures. Several types of polyphenols that are classified as phenolic acids are hydrolyzed tannins, lignans, stilbene, and flavonoids. The intact polyphenolic form is usually absorbed directly from the digestive tract.
Microorganisms can cause the degradation of polyphenolics into aglycones and aromatic acids. Polyphenolics are detected in all tissues, especially the digestive tract and oral mucosa. All types of polyphenolics are excreted in the urine and bile ducts. The bioavailability of polyphenolics is low, and the remaining at urinary excretion is 0.3% for anthocyanins and 43% for isoflavones. The absorption of phenolic compounds in food is determined by the chemical structure which influences the degree of glycosylation and acylation, basic structure, conjugation with other phenolic compounds, molecular size, polymerization, and solubility. The maximum concentration in plasma rarely exceeds 1 mM after ingestion of 10-100 mg of a single phenolic compound. Polyphenolics that are mostly absorbed by the body are isoflavones, gallic acid, flavanones, catechins, and quercetin glycosides, while proanthocyanidins, anthocyanins, and gallolylated catechins are less widely absorbed.

Flavonoid
The total flavonoids in ripe areca nuts were found to be the highest in levels. Flavonoids belong to a group of natural substances with variable phenolic structures  and are found in vegetables, tea, flowers, fruit, grains, vegetables, grains, and wine. This natural ingredient is known to have many health benefits. Flavonoids are a group of more than 4000 polyphenolic compounds that occur naturally in foods of plant origin. These compounds possess a common phenylbenzopyrone structure (C6-C3-C6), and they are categorized according to the saturation level and opening of the central pyran ring, mainly into flavones, flavanols, isoflavones, flavonols, flavanones, and flavanonols. Flavonoids are polyphenolic compounds that consist of several types based on their structure, namely flavonols (quercetin and kaempferol), flavones (luteolin and wogonin), flavanols (catechin, gallocatechin), isoflavones (genistein), flavanones, and flavanonols. Flavonoids have an antibacterial, anti-inflammatory, antioxidant, allergy, antimutagenic, antiviral, antineoplastic, anti-thrombotic, vasodilatory, and antihepatotoxic activities. Flavonoids can cause cell cycle arrest, thereby inhibiting the activity of Cyclin-Dependent Kinases (Cdks), Phosphorylation Kinases (PKs), and signal transduction of cell proliferation. Flavonoids are also able to modulate Mitogen-Activated PKs (MAPKs). The Cdk enzyme is an enzyme that functions to control the activity of the phosphorylase enzyme which regulates every phase of the cell cycle, especially during DNA replication and chromosome formation. The anti-tumor properties of flavonoids can inhibit the release of prooxidant enzymes, modulate carcinogen metabolism, suppress protein tyrosine kinase activity, antiproliferation, anti-metastasis, inhibit some drug resistance, are antioxidant and anti-angiogenesis, induce apoptosis and cell cycle retention. The chemical structures of flavonoids in areca nut can be seen in Figure 3.

Catechin
Catechins are flavonoids (flavanols) that are included in the polyphenol group, which have high concentrations in vegetables, fruits, and beverages. Catechins contain two aromatic rings and several hydroxyl groups. Catechins are divided into two groups, namely free catechins and esterified catechins. Catechin, gallocatechin, epicatechin, epigallocatechin are non-esterified catechins, whereas epigallocatechin gallate, epicatechin gallate, gallocatechin gallate are classified as esterified. The chemical structure of catechins can be seen in Figure 4.
Several in vitro studies have proven the role of catechins as protection for several diseases such as degenerative, heart disease, and bacterial infection. The catechins in green tea are able to inhibit carcinogenesis of the skin, lungs, esophagus, stomach, liver, small intestine, colon, and mammary glands in animal experiments.

The side effect of crude extract of areca nut
Among the benefits of consuming areca nut, other studies have shown that these nuts are carcinogens so that they can cause oral malignancy lesions. Arecoline is the most common alkaloid found in areca nut which is known to cause cytotoxicity in mammalian cells in vivo and cause carcinogenicity. Some of the oral lesions that can be caused by crude areca nuts include:

Oral submucous fibrosis
Oral submucous fibrosis (OSF) is a chronic disease that produces hyperkeratosis, epithelial atrophy, tissue fibrosis, and precancerous lesions. Pathological characteristics include chronic inflammation, excessive collagen deposition in the connective tissues below the oral mucosal epithelium, local inflammation in the lamina propria or deep connective tissues, and degenerative changes in the muscles. OSF patients experience a severe burning sensation in the mouth after ingesting spicy foods. Other symptoms of OSF include dry mouth, pain, taste disorders, restricted tongue mobility, trismus, dysphagia, and altered tone. This disease contributes significantly to mortality because of its high malignant transformation rate (1.5-15%). Previous research has shown that the arecoline in areca nuts can induce COX-2 expression in humans. It also suggests contributing to pathogenesis of OSF in betel chewers. Consistently, the elevated expression level of COX-2 has also observed in arecolinetreated HGF-1 cells and primarily cultured HGF cells in the study, suggesting these cells of different origins derived from oral cavity might have similar inflammatory responses upon exposure to arecoline, which in turn promote oral lesions and tumorigenesis.

Oral squamous cell carcinoma (OSCC)
Betel (areca nut) quid chewing is a widely prevalent habit correlated with a high incidence of oral cancer. A Previous study showed that the carcinogenic mechanism of action of betel quid chewing is caused by DNA damage induced by arecaidine and Cu (II). The Cu (II) is an important structural metal ion in chromatin; however, it is reported to be an important factor in DNA damage induced by many organic compounds. It was found that arecaidine alone had no significant effect on inducing DNA damage, but it caused significant DNA double strand breaks in the presence of Cu (II) ions under alkaline conditions. Further studies showed that reactive oxygen species were generated and Cu (II) was formed in the reaction. Areca nuts contain arecoline and arecaidine, both of which can cause mutation. When chewing areca nut, arecoline dissolves in saliva and its concentration can reach 140 g/mL. arecoline can cause cytotoxicity and genotoxicity for multicultured human cell lines and inhibits oral mucosal and gingival fibroblast which can cause epithelial damage and delayed wound healing.
The areca nut does not contain carcinogenic substances, but this carcinogenic effect is caused by nitrosamines product in the long term and uncontrollable. Nitrosamines are produced by nitrosation of the alkaloids in dried stored nuts, when in the mouth, and especially in the acid conditions of the stomach, in the presence of nitric oxide generated by bacteria [39]. The combination with nitric oxide, produced by bacteria, causes the production of methylnitrosaminoproprionitrile which is proved to cause carcinogenesis in animal studies [39]. This endogen nitrosation is higher significantly in a patient with bad oral hygiene [40]. If areca nut is combined with tobacco, then chewed by people with bad oral hygiene, there will be a very high accumulation of nitrosamines product [41]. This process usually occurs continuously for the long term because the seeds have addictive properties. Some studies also reported increased reactive oxygen species such as hydroxyl oxide in the oral cavity caused by a combination of polyphenol auto-oxidation from areca nut with high alkaline pH of slaked lime (Calcium hydroxide paste) [42]. If the areca nut is chewed with Piper betel and slaked lime, these two materials will cause mucous membrane erosion, so that carcinogenic substance could easily penetrate cells through the mucous membrane [43]. Some part of the community in India and Pakistan use industrial packaging areca nut called gutka and pan masala. Gutka contains areca nut, Piper betel, tobacco, and slaked lime, while pan masala was prepared without tobacco. Approximately 40% of gutka and pan masala packaging are contaminated with aflatoxin which has carcinogenic properties from Aspergillus flavus, Aspergillus niger, and Rhizopus spp. fungi [44]. The occurrence of the OSCC's risk depends greatly on the composition of the compound which determines the quality of the seeds, the method of using seeds which are associated with oral hygiene, duration of use, the presence or absence of toxin caused by fungi contamination in the seeds, and the presence or absence of other carcinogenic substances such as tobacco and slaked lime. The occurrence of OSCC could also be influenced by several factors such as intrinsic factors (tumor suppressor gene abnormality or mutation and oncogene) and extrinsic factors (tobacco smoking, vitamin A and iron deficiency, candidiasis, viral infection, and immunosuppression).
The dried areca nut, gambir, and calcium hydroxide wrapped in betel leaf are usually used as symbol of respect for guests in traditional ceremonies in Aceh, Indonesia.

Role of catechin in cancer mechanisms
The term catechins are commonly used to refer to the family of flavonoids and the subgroup flavan-3-ols or simply, flavanol. Catechins are differentiated from the ketone-containing flavonoids such as quercetin and rutin, which are called flavonols. High concentrations of catechin can be found in fresh tea leaves, red wine, broad beans, black grapes, apricots, and strawberries [45].

Initiation Stage
• Catechins neutralize the procarcinogens by inhibiting the activity of cytochrome P450 enzyme and modulating free radicals [46]. • Catechins inhibit the activity of Nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome c reductase. • Epigallocatechin gallate (EGCG) could interact with hepatic cytochrome P450 and inhibit the P450-dependent mixed-function oxidase enzymes in the skin and liver [47]. • The epicatechin derivative structure is capable of inhibiting microsomal enzyme system derived from the catechin hydroxyl group. The pyrogallol structure causes catechin molecules to have a strong metal-chelating ability that can bind to metal transition ions and act as preventive antioxidants [48]. • The EGCG reduces cell proliferation and induces apoptosis in low-dose H 2 O 2 (10 M)-treated colon carcinoma cells and downregulates 12-Otetradecanoylphorbol-13-acetate-mediated oxidative stress in cervical carcinoma cells [49]. • The EGCG can suppress the growth of HepG2 human HCC cells. The oral administration of green tea for 1 year can inhibit the progression of high-grade prostate intraepithelial neoplasia to prostate cancer [50].

Promotion Stage
• The inhibitory mechanism of the promotion stage is divided into three processes, namely the intervention of intracellular signaling pathways, increases the caspase activity, and cell cycle modulation. • Catechins inhibit phosphorylation of extracellular signal-regulated protein kinases (ERK)-1 and 2 and suppress the activity of p38 MAPKs in human fibrosarcoma cells [51]. The ERK enzymes are important transducers of proliferation signals. • Catechin hydrate exhibits anticancer effects by blocking the proliferation of MCF7 cells and inducing apoptosis in part by modulating expression levels of caspase-3, À8, and À 9 and p53 [52]. • (À)-Epigallocatechin-3-gallate has potential as a novel therapeutic agent for patients with B-cell malignancies including multiple myeloma via induction of apoptosis mediated by modification of the redox system. • Catechins can also inhibit the cell cycle. The cell cycle is controlled by numerous mechanisms ensuring correct cell division [53]. The mechanisms are regulation of cyclin-dependent kinases (CDK) by cyclins, CDK inhibitors, and phosphorylating events [53]. • Cell-cycle dysregulation is a hallmark of tumor cells. The ability of normal cells to undergo cell-cycle arrest after damage to DNA is crucial for the maintenance of genomic integrity [54]. • The biochemical pathways that stop the cell cycle in response to cellular stressors are called checkpoints. • Defective checkpoint function results in genetic modifications that contribute to carcinogenesis. The regulation of checkpoint signaling also has important clinical implications because the abrogation of checkpoint function can alter the sensitivity of tumor cells to chemotherapeutics.

Progression Stage
• Metastasis requires down-regulation of cell adhesion receptors necessary for tissuespecific, cell-cell attachment, as well as up-regulation of receptors that enhance cell motility. • Inhibition of migration and invasion of tumor cells could be a target of anticancer therapy. • Catechins can inhibit MMP-2 and MMP-9 in endothelial cells [55]. MMP-2 and MMP-9 secretions are elevated in several types of human cancers and their elevated expression has been associated with poor prognosis. • Angiogenesis, the development of new capillaries from preexisting blood vessels, is required in physiological processes such as wound healing and pathological conditions including tumor growth and metastases.

Role of catechin in cancer mechanisms
• Tumor angiogenesis is a complex process that consists of several steps including the secretion of angiogenic factors by tumor and host cells, activation of proteolytic enzymes, endothelial cell migration, invasion, endothelial cell proliferation, and capillary formation. • Vascular endothelial growth factor (VEGF) and its receptors have been known as important angiogenic factors and are commonly overexpressed in several types of human cancers. • Catechins especially EGCG is proved to inhibit tumor growth and angiogenesis by the down-regulation of VEGF expression in serum-deprived HT29 human colon cancer cells [56]. • Catechin in green tea extract inhibits angiogenesis partly through the disruption of STAT3-mediated transcription of genes, including VEGF [57].

Antioxidant activity of areca nut
Antioxidants protect cells from deleterious effects of oxidation and are also employed as dietary supplements to neutralize the adverse effects of oxidative stress. Many of the natural antioxidants of interest are of plant origin and belong to bioactive compounds in the phenolic and polyphenolic class. The phytochemical contents of areca nut mostly come from phenolic and flavonoid content which produce antioxidant activity. Total phenolic content test using the Folin-Ciocalteu method was performed based on oxidation-reduction mechanism. Methanol is the best solvent for areca nut compared to petroleum ether, ethyl acetate, and water, and was used in this study [58]. The value of total phenolic concentration in 1 gram extract is 80.3 mg TAE/gr extract. The highest phenolic concentration was usually found in methanol, acetonide, and water solvent. This concentration is depended on how big the solvent's polarity is used at extraction. The high phenolic solubility in the polar solvent showed a high concentration of phenolic content in the extract. When compared with literature using the same method, the phenolic content of areca nut in the study is lower than that of a study conducted in Assam, India (146.7 mgTAE/g extracts) and Hainan province, Taiwan (167.70 mgTAE/g extracts) [5,58]. Total flavonoid test using Dowd modification method also revealed a higher content of flavonoid (238.5 mg CEmg/gr extract). This number is higher than that reported by Zhang et al. and Wang et al., which was 77.36 and 10.45-142.65 mg CE mg/gr extract, respectively. This indicates that the variation of polyphenol content depends on the geographical locations where the plant grows. This variation is also affected by species and the characteristics of the nut used in the study, including freshness, maturity, and methods of drying.
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].
Catechins are phytochemical compounds found in high concentrations in a variety of plant-based foods and beverages. Catechins are classified as flavanols. Catechin is the highest phenolic compound in the areca nut extract (2.79 mg/g). In comparison with catechin, quercetin is found in much smaller amounts in the areca nut extract (0.14 mg/g). The areca nut extract from Aceh, Indonesia, has a low level of tannin (0.007%) through the titrimetric analysis. This finding showed different results with less amount than areca nut from various districts in Karnataka, India, showed 1.13%-3.39% tannin in areca nut extract with the same technique [29]. Tannin is a polyphenol compound of plant origin, bitter in taste, which reacts with and coagulates protein, or various other compounds including amino acids and alkaloids.
The main characteristic of an antioxidant is its ability to trap free radicals. The antioxidant activity of an extract can be measured by 2,2-diphenyl-1-picrylhydrazyl-hydrate (DPPH) free radical method. DPPH is a stable nitrogen-centered free radical, the color of which changes from violet to yellow upon reduction by either the process of hydrogen-or electron-donation. The substance which is performs this reaction can be considered as antioxidants and therefore radical scavengers [64]. The DPPH test is a direct and reliable method for determining radical scavenging action. The reducing properties are generally associated with the presence of reductones, which have been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom. The result of the antioxidant activity curve showed linear line formula so the EC 50 value was acquired. The EC 50 value is the extract concentration which was able to catch 50% free radical. The EC 50 value was measured from the association curve between the percentages of radical catcher DPPH against the concentration of the treatment's solution. This value is inversely proportional to antioxidant extract capability. The higher the antioxidant activity, the lower EC 50 would be. The study showed that the EC 50 value of areca nut extract was 15.95 μg/mL (Figure 5). The polyphenol could  of free radicals by becoming a hydrogen donor so the free radical chain reaction was broken. The EC 50 value of the extract was smaller than Butylated Hydroxytoluene (BHT); synthetic antioxidant posing as control. The result of the study showed stronger antioxidant activity of areca nut compared to BHT control, so it could be concluded that the activity potency of the combination of several phenolic compounds in the extract could work synergic and resulted in more potent antioxidant than the activity of one isolate in the extract [38]. Polyphenolics had 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 human keratinocyte (HaCat) cell line [28].

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 IC 50 of areca nut extract in HSC-2 cells was 629.50 μg/mL and the IC 50 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 IC 50 , 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 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

cells (b) than in HSC-2 cells (a). The extract does not show a cytotoxicity effect in HaCat cells (c).
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 IC 50 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 IC 50 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 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 IC 50 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][42][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].

Immunomodulatory activity
The areca nut extract was found to increase the white blood cell count postchallenge with Staphylococcus aureus induction significantly indicating that the extract could stimulate the hemopoietic system. Another study found that areca nut extract can induce calcium signals in at least three immune cell lines and human primary immune cells (PBMCs), inducing the production of pro-inflammatory cytokines. Further separation of the PBMCs into T cells, B cells, and monocytes would potentially be of interest in elucidating specific responses to each cell subtype [70]. However, areca nut extract can also induce antigen-specific immune responses and promote inflammatory reactions in vivo, which may contribute to immune deregulation associated with areca-related diseases [71]. In fact, the study of areca nut as an immunomodulatory drug still causes various results. It depends on the content of polyphenolic compound in the areca nut extract which greatly affects the efficacy of the nut.
The areca nut can also increase the activity and capacity of macrophages. The process by which a cell ingests and disposes of foreign material, including microorganisms is called phagocytosis. In normal conditions, most phagocytes are circulating in the blood and when there is an inflammation, the phagocytes will leave the bloodstream and migrate to the site of inflammation. The circulation in the capillaries and venules is rapidly moving with the red blood cells in the mainstream and neutrophils and other leukocytes tending to flow more slowly along the vessel's periphery. Monocytes and macrophages have the same functions as neutrophils but for a longer time and in a later stage of the inflammatory response. Monocytes are produced in the bone marrow, enter the circulation, and migrate to the inflammatory site, where they develop into macrophages. Macrophages are more active as phagocytes than their monocytic precursors. Macrophages, particularly those residing in the tissue, are often important cellular initiators of the inflammatory response.
Several bacteria are resistant to killing by granulocytes and can even survive inside macrophages. Microorganisms such as Mycobacterium tuberculosis, Salmonella typhi, and Mycobacterium leprae can remain dormant or even multiply inside the phagolysosomes of macrophages. However, the bactericidal activity of macrophages can be markedly increased with the help of inflammatory cytokines produced by cells of the acquired immune system (a subset of T lymphocyte) or cells activated through Toll-like receptors. Macrophage activation results in increased phagocytic activity, the size of itself, plasma membrane area, glucose metabolism, and a number of lysosomes. The activation of leukocytes, monocytes, and macrophages which is induced by S. aureus and treated with areca nut is shown in Figure 8.
The areca nut extract probably stimulates the proliferation of macrophages, which in turn leads to the activation of macrophage activity. Further study is needed to find out how extract can increase macrophage activity and capacity.