Salivary ascorbic acid levels in betel quid chewers: A biochemical study.

BACKGROUND: Quid chewing practice has been a part of our tradition since centuries with little known evidence of oral cancer. However, recent trends show a rise in occurrence of oral cancer often associated with tobacco and arecanut usage. Ascorbic acid is an important salivary antioxidant. Betel leaf which is used in quid is known to contain ascorbic acid. AIM: The aim of our study was to assess the salivary levels of ascorbic acid in traditional quid chewers so as to determine whether the betel leaf has protective antioxidant action.
MATERIALS AND METHODS: Salivary ascorbic acid levels of 60 subjects were estimated using the Dinitrophenyl hydrazine method.
RESULTS: The results revealed that quid chewers who used betel leaf had higher salivary ascorbic acid content compared to nonbetel leaf quid chewers. This could possibly be due to the protective antioxidants in the betel leaf.

Introduction

Fresh leaves of piper betel (PB), popularly known as Paan in India, are consumed by about 15-20 million people in the country. It is cultivated following the traditional methods in India on about 55,000 hectares of land area with an annual production worth about Rs 9000 million.[1] The leaves of PB have long been in use in the Indian local system of medicine for their antioxidant and antimicrobial properties.[2] In a recent study saliva obtained after mastication of entire betel leaf reduced the microflora, approximately 56% as compared to control.[3] Betel leaves are good sources of vitamin B particularly nicotinic acid and antioxidants such as allylpyrocatechol, ascorbic acid and carotene.[4] Epidemiological studies carried out in the past have suggested that betel offers some protection against tobacco-induced carcinogenesis.[5]

Materials and Methods

Sixty subjects between the ages of 20 and 60 years attending the department of oral medicine and radiology were enrolled in the study. They were enrolled into three age and sex-matched study groups. The first group (BL) consisted of 20 subjects with chewing habit with betel leaf. The second group (NBL) consisted of 20 patients with chewing habit without betel leaf and the third group (CL) consisted 20 patients without chewing habits who formed the controls. Each of the study groups had five female subjects. Patients with systemic diseases, long-term drug intake and tobacco smoking habits were excluded from the study. Unstimulated saliva was collected from the study subjects between 9:00 AM to 12:00 PM to avoid diurnal variation. Subjects were requested not to eat, drink, perform oral hygiene activities or chew 60 minutes prior to the saliva collection procedure. Subjects were then seated in the dental chair and asked to spit in a graduated container every 1-min till 5 ml of saliva was obtained. During saliva collection subjects were instructed not to speak or swallow. The estimation of salivary ascorbic acid was done by dinitrophenyl hydrazine (DNPH) method. The procedure was as follows- 100 μL of sample saliva was taken in a clean test tube and 900 μL of 5% TCA was added to it and allowed to precipitate proteins for about ten minutes and centrifuged. One hundred microliters of the supernatant was separated and transferred into another test tube. To this, 200 μL of DTC reagent was added, the tube was plugged and the mixture was incubated at 60o C for 60 mins in a water bath. Simultaneously, a blank with 1 mL of TCA and 200 μL of DTC reagent was also incubated under similar conditions. Following 60 minutes of incubation, the reaction mixture was cooled in an ice bath and 1 mL of 4.5 M sulfuric acid was added to it and after it reached room temperature, the optical density was read at 540 nm against blank. The data obtained were subjected to statistical analysis using SPSS version 17 software. One-way ANOVA was used to compare the data among the groups and the differences were considered statistically significant if P values were 0.05 or less.

Results

The mean salivary ascorbic acid in Group BL was 0.22 ± 0.08 μg/mL. Whereas in group NBL group the mean salivary ascorbic acid was 0.13 ± 0.04 μg/mL. The mean salivary ascorbic acid level of the group CL was 0.24 ± 0.06 μg/mL. The mean salivary ascorbic acid of the male subjects in study groups BL, NBL and CL were 0.22 ± 0.11 μg/mL, 0.13 ± 0.08 μg/mL and 0.24 ± 0.09 μg/mL, respectively [Figure 1]. Whereas the mean salivary ascorbic acid of the female subjects in study groups BL, NBL and CL were 0.21 ± 0.07 μg/mL, 0.12 ± 0.09 μg/mL and 0.23 ± 0.09 μg/mL, respectively. When the mean salivary ascorbic acid of group BL was compared to group NBL a statistically significant difference (P = 0.001) was observed [Figure 2]. The mean salivary ascorbic acid level of group CL was higher than group BL but the difference was not statistically significant (P = 0.06). The mean salivary ascorbic acid levels of NBL group was significantly lower than the CL group (P = 0.001).

Figure 1

Salivary ascorbic acid levels in male and female subjects of the study groups

Figure 2

Intercomparison of mean salivary ascorbic acid levels of study groups

Discussion

The significance of PB in oral hygiene was indicated in several ancient literature and has also been shown in some recently conducted studies.[3] According to the universal classification of food, the areca nut and the betel leaf complement each other and are, therefore, in harmony. Since the areca nut is ‘hot’ and the betel leaf ‘cool’, they act together to keep the human body in balance. Some claim that the areca nut is an aphrodisiac, perhaps because of its classification as a ‘hot’ food. Conversely, the betel leaf, as a ‘cool’ food, is believed to relieve ‘hot’ illnesses such as headaches and fever.[6] Although studies have shown that quid chewing is carcinogenic, it is believed that unlike other constituents of the betel quid, the betel leaves devoid of carcinogenic effects and on the contrary possess cancer preventive effects including protective effects against the carcinogens present in tobacco.[7] In a prior study, cancer was induced in Swiss male mice with tobacco carcinogens. The study group which was treated with betel leaf extract showed resistance to cancer. This study proved that contents in the betel leaf extract had certain antioxidant products that neutralized the tobacco carcinogen-induced damage.[5] A recent study revealed that betel leaf extract contained important antioxidants.[8] Another recent study revealed unaltered levels of serum ascorbic and Vitamin E in experimental rats administered with betel leaf extract and challenged with hepatotoxic substance.[9] Similar methods were used in our study although no drug interventions were made to the study subjects. In our study subjects were divided into two groups based on the use of betel leaf in their quid chewing habit. The group using betel leaf in their quid showed near normal levels of salivary ascorbic acid. An experimental study conducted on rats revealed that levels of several serum antioxidants like ascorbic acid, vitamin E, superoxide dismutase, catalase and glutathione peroxidases were increased when they were administered betel leaf extract.[10] Studies have also revealed that betel leaf extract has radioprotective properties.[11]

In our study we evaluated the levels of salivary antioxidant ascorbic acid in individuals with quid chewing habits. We found that the ascorbic acid levels were near normal in individuals who use betel leaf in quid. This could probably be due to the protective effect of the betel leaf contents. This could also be the reason for decreased incidence of oral cancer in traditional quid chewers which is an age-old practice among our ancestors.