Antioxidant and cytoprotective activities of Piper betle, Areca catechu, Uncaria gambir and betel quid with and without calcium hydroxide

Nordin Nur Sazwi, Thurairajah Nalina, Zubaidah Haji Abdul Rahim, Nordin Nur Sazwi, Thurairajah Nalina, Zubaidah Haji Abdul Rahim

Abstract

Background: Betel quid chewing is a popular habit in Southeast Asia. It is believed that chewing betel quid could reduce stress, strengthen teeth and maintain oral hygiene. The aim of this study was to investigate the antioxidant and cytoprotective activities of each of the ingredients of betel quid and compared with betel quid itself (with and without calcium hydroxide). The correlation of their cytoprotective and antioxidant activities with phenolic content was also determined.

Methods: Five samples (betel leaf, areca nut, gambir, betel quid and betel quid containing calcium hydroxide) were extracted in deionized distilled water for 12 hours at 37°C. Antioxidant activities were evaluated for radical scavenging activity using DPPH assay, ferric reducing activity using FRAP assay and lipid peroxidation inhibition activity using FTC assay. Total phenolic content (TPC) was determined using Folin-Ciocalteu procedure. Phenolic composition was analyzed using LC-MS/MS. Cytoprotective activity towards human gingival fibroblast cells was examined using MTT assay.

Results: Among the ingredients of betel quid, gambir demonstrated the highest antioxidant (DPPH - IC50 = 6.4 ± 0.8 μg/mL, FRAP - 5717.8 ± 537.6 μmol Fe(II)/mg), total phenolic content (TPC - 1142.5 ± 106.8 μg TAE/mg) and cytoprotective (100.1 ± 4.6%) activities. Betel quid when compared with betel quid containing calcium hydroxide has higher antioxidant (DPPH - IC50 =59.4 ± 4.4 μg/mL, FRAP - 1022.2 ± 235.7 μmol Fe(II)/mg), total phenolic content (TPC - 140.0 ± 22.3 μg TAE/mg), and cytoprotective (113.5 ± 15.9%) activities. However, all of the five samples showed good lipid peroxidation inhibition compared to vitamin E. LC-MS/MS analysis revealed the presence of quinic acid as the major compound of gambir and betel quid. A positive correlation was observed between TPC and radical scavenging (r = 0.972), reducing power (r = 0.981) and cytoprotective activity (r = 0.682).

Conclusions: The betel quid has higher TPC, and antioxidant and cytoprotective activities than betel quid with calcium hydroxide. The quinic acid in betel quid may play an important role in the oral health protection.

Figures

Figure 1
Figure 1
Layout of the 96-well plate for cytoprotective analysis. Different colours indicate different extract tested. Ascorbic acid was used as positive control.
Figure 2
Figure 2
Effects of extracts on human gingival fibroblast cells. Cell viability was determined by MTT assay. Values are expressed as means ± SD (n = 3). *Significant difference at p < 0.05 (t-test).
Figure 3
Figure 3
Effects of hydrogen peroxide on human gingival fibroblast cells. Cell viability was determined by MTT assay. Values are expressed as means ± SD (n = 9).
Figure 4
Figure 4
Cytoprotective effects of the extracts against hydrogen peroxide-induced oxidative stress cells. Values are expressed as means ± SD (n = 3). Ascorbic acid was used as positive control. *Significant difference within the same samples at p < 0.05 (t-test). **Significant difference when compared to H202 at p < 0.05 (t-test).

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