Revealing anti-cariogenic efficacy of smokeless tobacco: A pilot study.

BACKGROUND: The tobacco plant, Nicotiana tabacum, has been responsible for more deaths than any other herb. However, the literature has also been endowed with its use as "holy herb" since the pre-Columbian era. Used for treating pain, poisonous bites, ulcers, nasal polyps, and basal cell carcinoma; it also acts as an important ingredient of commercially available toothpastes; and even used as tobacco vaccines against Streptococcus species as highlighted in the literature. AIMS AND
OBJECTIVES: (1) To elicit the anti-microbial property of tobacco against Streptococcus mutans, if any, in raw smokeless tobacco. (2) To study the relationship of duration and growth inhibition efficacy of smokeless tobacco.
MATERIALS AND METHODS: Extracts were prepared by centrifugation of mixed raw smokeless tobacco with Ringer's lactate solution and with saliva. The extracts were placed in wells prepared on Mitis salivarius culture plate and incubated at 37°C for 24 h after 0 h, 1 h, and 2 h of extract preparation. The inhibition zones were measured on the underside of plate using the vernier calipers.
RESULTS: Smokeless tobacco has a statistically significant zone of inhibition, which proves its anti-microbial activity against S. mutans. However, the mean zones of inhibition were greater for Ringer's lactate and tobacco group as compared to test samples (saliva and tobacco) with subsequent reduction of inhibition zones with an increase in duration.
CONCLUSION: The anti-microbial property of extensive tobacco resources can be utilized from their extracts in order to balance the deterioration it had caused to mankind.

INTRODUCTION

Oral diseases are major health problems with dental caries and periodontal diseases among the most important preventable global infectious diseases. Oral health influences the general quality of life, and poor oral health is linked to chronic conditions and systemic diseases. The association between oral diseases and the oral microbiota is also well established. The development of dental caries involves acidogenic and aciduric gram-positive bacteria (mutans streptococci, Lactobacilli, and Actinomycetes), which metabolize sucrose to organic acids (mainly lactic acid) that dissolve the calcium phosphate in teeth, causing decalcification and eventual dental decay.[1] Streptococci of the mutans group are closely associated with dental caries, mainly those involving smooth surfaces. Production of acid and extracellular polysaccharides due to hydrolysis of sucrose facilitates their adhesion to tooth surfaces.[2]

Tobacco, however, is a very important economic crop.[3] When the use of Nicotiana by the indigenous populations in the new world was first observed by Columbus and the plant was brought to Europe, all herbs were considered to have potential therapeutic properties. Indeed, Nicotiana acquired a reputation as a panacea, to the extent of being called the “holy herb” and “God's remedy.”[4] Medicinal plants including tobacco have been used as traditional treatments for numerous human diseases for thousands of years and in many parts of the world.[1] Tobacco variously called petum, betum, cogioba, cohobba, quauhyetl, picietl, or yietl, appeared later in herbals or pharmacopoeias.[4]

The initial interaction of nicotine with the human body occurs most often in the oral cavity, where it is expected to be most active and its exposure to be most intense. Its possible effect on immunity (especially near mucosal surfaces) has been described, while the effects of its interaction with the normal, indigenous microflora (especially in the oropharynx) is unknown; yet relatively high salivary concentrations (70-1,560 μg/ml) of nicotine are achievable for those using tobacco-based products. Along these lines, little has been reported on the ability of nicotine to support or suppress the growth of micro-organisms, and such studies have provided only inconsistent results[5] with numerous conflicting reports on the effect of local smokeless tobacco products on bacterial growth. This preliminary study was therefore, carried out to elicit the anti-microbial property of smokeless tobacco against Streptococcus mutans, if any, and to study the relationship of duration and growth inhibition efficacy of smokeless tobacco.

MATERIALS AND METHODS

An extract of smokeless tobacco was prepared by placing 7.5 mg of raw smokeless tobacco [Figure 1] in 7.5 ml Ringer lactate solution, and 7.5 ml of saliva from subjects with no tobacco habits and zero DMFT at 37°C for 2 h [Figure 2]. The mixture was stirred intermittently. After 2 h, the mixture was centrifuged beyond 2,500 rpm for 5 min and the supernatants obtained were used as extracts [Figure 3]. Mitis salivarius culture plates were prepared, and wells were made. The extracts were placed in the wells at 0 h, 1 h, and 2 h of extract preparation, respectively. The zones of inhibition were measured using vernier calipers on Mitis salivarius culture plate after incubation at 37°C for 24 h [Figure 4]. These inhibition zones were later compared among the groups and with control (anti-microbial disc). The growth on the culture plate was confirmed to be of S. mutans using Bio ID Strep kit (HiMedia) for sorbitol, mannitol, and catalase test, respectively.

Figure 1

Raw smokeless tobacco

Figure 2

Mixtures of tobacco with Ringer's lactate solution and saliva

Figure 3

Extracts after centrifugation

Figure 4

Streptococcus mutans colonies over Mitis salivarius culture plate growing beyond the zones of inhibition

RESULTS

There was a statistically significant difference among the groups. The mean zones of inhibition were of maximum size for anti-microbial disc and of minimum size for saliva tobacco at 2 h of extract preparation [Table 1]. The mean difference as regards to the zones of inhibition between and within the groups gave statistically significant results at 0.05 level using the ANOVA test [Table 2]. Multiple comparisons also revealed a statistically significant difference among groups except for saliva with tobacco at 0 h versus saliva with tobacco at 1 h [Table 3].

Table 1

Comparison of mean zones of inhibition under different treatment conditions

Table 2

ANOVA test results

Table 3

Multiple comparisons

DISCUSSION

The tobacco plant, Nicotiana has probably been responsible for more deaths than any other herb. Undoubtedly, tobacco is the most important avoidable cause of premature death and disease in the world. Tobacco leaves and the smoke generated when they are burned contain over 4,000 chemicals, the best known of which is nicotine, first isolated from tobacco leaves in 1828 by Posselt and Reimann.[4]

Over 20% of tobacco resources are discarded as processing waste, which pollute the environment and cause a big waste. In fact, the discarded tobacco leaves are economically valuable because of abundant bioactive compounds in them. Therefore, it is important to investigate and utilize the resource of tobacco leaf.[3]

Interference with S. mutans ability to colonize teeth surfaces is an important strategy of dental caries prevention. In addition to being a vaccination target, inhibition of Glucosyl transferases GTFs and sucrose-dependent S. mutans colonization has been a subject of many in vitro studies in which different agents, including monoclonal and polyclonal antibodies, plant extracts, natural substances, andchemical reagents, were shown to possess such inhibitory properties. The active componentspresent in plant extracts are tannins and other polyphenols.[6] However, the major classes of compounds identified in tobacco include aliphatic and aromatic hydrocarbons, aldehydes, ketones, alcohols, phenols, amines, amides, alkaloids, metals, and radioelements. Tobacco-specific nitrosamines are formed from alkaloids during the processing of tobacco leaves.[7]

Smokeless tobacco in our study had statistically significant zones of inhibition which proves their anti-microbial activity against S. mutans. However, the mean zones of inhibition were greater for Ringer's lactate and tobacco as compared to test samples (saliva and tobacco). There was a subsequent reduction of inhibition zones with an increase in duration suggesting the rapid breakdown of the anti-microbial substances like polyphenols (chlorogenic acid and rutin) in tobacco[3] probably due to the activity of lytic enzymes. Although tannins, polyphenols, oils, and others have been identified as the effective component for the bacterial damaging or killing action in the in vitro system, many of these compounds failed in human clinical trials to determine their therapeutic effectiveness.[8] Therefore, in vitro observations from this study may not necessarily be obtained in vivo as such effect can be counteracted by the natural immune defense system.[7]

Contrary to this, Keen and Johnson reported a biphasic dose-dependent effect of nicotine on the growth of cariogenic S. mutans and suggested that the concentration of nicotine (10−3 M) reported within the saliva of smokeless tobacco users could actually stimulate growth of S. mutans and possibly place the user at risk for the dental caries. It is commonly known that loose-leaf tobacco contain sweeteners such as molasses or sugar, whereas, moist snuff contains few sweeteners. The sugar contents of tobacco products have been shown to vary from one form-to- another form, brand-to-brand, and region-to-region.[9] Studies published have suggested that chewing tobacco products contain very high levels of fermentable sugars (30-40% by weight). The sugars in these products are believed to contain glucose, fructose, sucrose, maltose, and isomaltose, which can increase the in vitro growth of the cariogenic bacteria, S. mutans.[10] Hence, raw smokeless tobacco was employed in the study in contrast to the smokeless tobacco products available in the market in order to avoid any bias due to the reported evidence of added sugar contents.

CONCLUSION

Tobacco has a strong anti-cariogenic effect. Despite of this fact, the use of smokeless tobacco product is not advised as an anti-caries measure in any raw form due to the known carcinogenic potential of tobacco. The findings of the aforementioned study are preliminary and hence, more samples are required to validate the results. Furthermore, to use smokeless tobacco as an anti-caries measure, an attempt should be made to examine the tobacco leaves systematically for substances of high therapeutic value. Fractional distillation for specific anti-cariogenic substances in the tobacco leaves would therefore, help to analyze commercial viability of smokeless tobacco product as an anti-cariogenic measure.