A Comparative Investigation of the Antibacterial Proficiency Between Ayurvedic and Commercial Mouthwashes

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Herbal based products have gained an increasing amount of popularity over recent years due to its availability and natural origin. Ayurveda, an alternative medicine practice with roots from India, has been used for over thousands of years and is still widely used today in the Indian subcontinent As someone with an Indian background, I have developed an interest with the potential of this ancient medicinal system and whether it is comparable to western medicine in terms of antibacterial efficacy. Even though 25-50% of pharmaceuticals are derived from plants, very few are used as antimicrobials.

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This ought to be given special attention, as there may be an eco-friendly, side-effect-free alternative to our everyday practices. Moreover, there is an insufficiency of studies that support Ayurveda as being significantly stronger than the conventional European and American methods, which leaves room for further investigation in this area. This paper seeks to explore the research question: How do Ayurvedic mouthwashes (neem, clove, peelu) compare with commercial ones (Listerine, Paroex, Zendium) in terms of their antibacterial properties against Streptococcus mutans and Serratia marcescens, measured as the diameter of inhibition zones (mm) using the agar well diffusion assay?

With the continuous proliferation of antimicrobial resistance (AMR), scientists across the globe are on an ever more desperate search for new antibiotics. In many southern European places, it is possible to buy antibiotics prescription free without any regulations; approximately 40% of all pneumococcus are resistant in Spain. AMR threatens the effective treatment and prevention of bacterial infections which have saved millions of lives. The rapid occurrence of these resistant bacteria, or ‘superbugs’, are attributed to the increased misuse of medications in both the pharmaceutical and agricultural industries. 5 AMR spreads fast between bacterial populations through horizontal gene transfer called transformation – the uptake of resistance-gene-carrying plasmids by nearby bacteria. Since the 1980s, the development of new antibiotics has significantly decreased and the use of plant derivatives as antibiotics has been non-existent. It is therefore worth looking into what might be a more cost-efficient method to solve this crisis. I chose to narrow down my investigation by focusing on oral diseases. This is due to major health problems such as dental caries and periodontal diseases being among the most important global pathogenic diseases out there. These diseases are associated with the oral microflora which has been linked to other chronic conditions and systematic diseases in the body. As the gateway to our body, oral health influences our overall quality of life. An increased demand for sugar in today’s society is only worsening the situation. A study indicates that for 12-year-olds there is a significant positive correlation between the per capita availability of sugar and dental caries. In most industrialized countries, tooth decay affects 60-90% of schoolchildren as well as a substantial proportion of adults. 9Caries is the process of calcium and phosphate demineralization at the tooth enamel. The destruction may spread into to softer, more sensitive part of the tooth called the dentine. The weakening of the enamel happens as a result of the acids produced by the bacteria found in the oral cavity.

Oral microflora adhered to the biofilm of teeth (plaque) react with ingested cariogenic carbohydrates, mainly sucrose, which lead to the accumulation of these acids. When the saliva’s ‘healing’ properties are not enough to neutralize the excess acid and remineralize the enamel surface for a prolonged period of time, dental caries is likely to occur. 11 Periodontal disease (PD) is also related to the formation of plaque. PD affects the majority of Americans 30 years of age and older and is a common chronic condition. The swelling of gums is caused by the body’s response to toxins released by plaque-thriving bacteria that have accumulated on the biofilm of teeth. Although this inflammatory response is part of our defense system, it can promote serious problems. If not controlled, it can potentially lead to loss of teeth due to the destruction of periodontal ligamental and supporting bone. On top of this, a study suggests periodontal pathogens contribute to the pathogenesis of atherosclerosis. In fact, the evidence supports that PD is independently connected with arterial disease. Periodontitis-inducing-microbes may enter the general circulation which can result in conditions such as cardiovascular disease. Atherosclerosis, to this day, remains the largest cause of death. 12The treatment of oral infections is perhaps one of the most expensive diseases that the general public have to deal with. In the US the annual treatment of dental infections in 1977 was over a billion dollars and increase to around 24 billion dollars in 1984. Most of these expenditures being the restoration of tooth lost to dental decay. Therefore, the maintenance of good oral hygiene practices is extremely important. Mouthwashes have shown to effectively control plaque in combination with regular toothbrushing.

Two main constituents of therapeutic mouth rinses are chlorohexidine and fluoride. 16 Fluoride has shown to prevent caries by increasing the rate of remineralization by saliva as well as interfering with the glycolysis of bacteria. Glycolysis is the metabolizing of sugars by cariogenic bacteria which produces acid as a by-product and is proportional to bacterial growth. However, there are some unwanted side-effects of excessive fluoride exposure. Long-term ingestion of fluoride can lead to enamel fluorosis during tooth development in children. This results in white stains and pits on the enamel. Ingested fluoride can also react with gastric acid producing hydrofluoric acid in the stomach which results in immediate effects such as abdominal pain, excessive saliva, nausea, and vomiting.

Chlorohexidine (CHX) is considered the ‘gold standard’ of antiplaque agents. It works as a bacteriostatic or bactericidal by progressively damaging the membrane. The positively charged CHX is attracted to the negatively charged bacterial cell wall. By changing the integrity of the cell membrane, CHX binds to the phospholipids in the inner membrane, disrupting it and causing the cytoplasm to leak. The coagulation and precipitation of the cytoplasm cause the cell to die. CHX has shown antibiotic efficacy for both gram-negative and gram-positive bacteria. Similarly to fluoride, CHX also possesses undesirable side-effects. This includes the brown discoloration of teeth and an altered taste perception; the positively charged molecules may adhere to the negatively charged glycoproteic enamel causing disruption. CHX is not well absorbed if swallowed and may cause stomach irritation or nausea. In some cases, its symptoms can also include difficulty in breathing or severe rash due to allergy. Consequently, non-allopathic therapeutics that may contain side-effect-free phytochemicals, should be explored. The quintessential components of Ayurveda include Azadirachta indica (neem), Syzygium aromaticum (clove) and Salvadora persica (peelu)1; all of which have shown a somewhat inhibitory effect towards bacteria. Clove has been found to contain eugenol: a phenolic compound considered to be bacteriostatic. Numerous studies suggest antibacterial proficiency in Eugenol. Through histological procedures, it was observed that eugenol denatures cytoplasmic protein and forms nonspecific pores on the plasma membrane. This lead to the swelling and eventual cell necrosis of the bacteria. Peelu, also referred to as the toothbrush tree, is widely used for oral health, not only in India. A study done on a Sudanese population suggested that the periodontal status for people chewing peelu sticks were better than those of toothbrush users. Lastly, neem has often been compared to CHX. An experiment using the agar well diffusion assay indicated that at certain concentrations neem had a higher inhibitory effect against Streptococcus mutans than chlorhexidine did.

A pilot study was done preceding the main experiment in order to evaluate potential bacteria and mouthwashes. It was also performed to familiarize with the protocol. The results indicated that mouthwashes did possess some sort of antibacterial proficiency against most bacteria tested. The oral microflora contains around 500 bacterial species, most of which are anaerobic and thrive in the gingival and periodontal pockets of the mouth. Streptococcus mutans (S. mutans) is a gram-positive, anaerobic bacterium that is relatively small and circular in shape. It is found mostly in the oral cavity and is considered the main contributor to dental caries. Due to its presence in the oral microbiome and availability in the lab, S. mutans was utilized as a model bacterium for this investigation. It was used to represent the gram-positive bacteria that are said to induce tooth decay. Periodontal diseases, on the other hand, has been linked to gram-negative bacteria. Serratia marcescens (S. marcescens) is an example of this, of which students have access to in the lab. This rod-like-shaped bacterium is widely found in the environment, in places such as soil and water. It has said to be found on the biofilm of teeth and is connected to PD. S. marcescens produces an orange-reddish pigment called prodigiosin, which may cause staining to the teeth. Due to its relevancy and availability, this bacterium was chosen as a model for this experiment to represent gram-negative bacteria that contribute to PD.

The aim of this investigation was to compare the antibacterial efficacy between western and Ayurvedic mouthwashes with the agar well diffusion method. The mouth rinses will be tested upon a gram-positive and a gram-negative bacterium which possess structural differences and are supposed indicators of oral health quality. This study is important due to the inevitable AMR crisis, the importance of oral health, the costliness of treatment and the ramifications of commercial mouthwashes. Now with a plentitude of herbal mouthwashes available, it is the need of the hour to validate their recommendation and potential use.

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