**7. Antioxidant activity of areca nut**

MMP9 [57].

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 EC50 value was acquired. The EC50 value is the extract concentration which was able to catch 50% free radical. The EC50 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 EC50 would be. The study showed that the EC50 value of areca nut extract was 15.95 μg/mL (**Figure 5**). The polyphenol could

#### **Figure 5.**

*Antioxidant activity estimated by DPPH method. The linear curve of areca nut extract has similar linearity with BHT control. The dosage used was 1–20 μg/mL.*

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

dispose of free radicals by becoming a hydrogen donor so the free radical chain reaction was broken. The EC50 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].
