Antibacterial Effect of Berberis Aquifolium Homeopathic Mother Tincture against Biofilm Formers from Dental Plaque

Contents

List of Figures

List of Tables

Abbreviations

Abstract

1. Introduction
1.1. Objective of the Study

2. Review of Literature
2.1 Teeth and its structure
2.2 Pathogenicity of dental infection
2.3 Dental caries
2.4 Microbial etiology of caries
2.5 Treatment of caries

3. Materials and Methods
3.1 Chemicals
3.2 Preparation of powdered form of Homeopathic mother tinctures (HMT)
3.3 Collection of dental caries samples
3.4 Isolation and screening of bacteria
3.5 Morphological and biochemical identification of isolates
3.6 Identification of Streptococcal Isolates
3.7 Physical characterization
3.8 Biochemical characterization
3.9 Anti-microbial screening of extracts
3.10 MIC determination

4. Results
4.1. Isolation of bacteria from plaque samples based on their colony morphology
4.2. Biochemical identification of Colony 2
4.3. Biochemical identification of Colony 8
4.4. Biochemical identification of Colony 9
4.5. Identification of Colony 1 (budding Yeast cells) by Germ tube assay
4.6. Screening of DMSO (Negative control) and standard drugs (Positive control)
4.7. Inhibitory effects of B. aquifolium against dental biofilm producers and normal dental
4.8. Minimum inhibitory concentration (MIC) of B. aquifolium against dental pathogens

5. Discussion

6. Summary and Conclusion

References

ANTIBACTERIAL EFFECT OF BERBERIS AQUIFOLIUM HOMEOPATHIC MOTHER TINCTURE AGAINST BIOFILM FORMERS FROM DENTAL PLAQUE

Nirmal Ghosh OS1 , Ratheesh Mohanan2*, Sulumol Thomas2, Sheethal Sreevallabhan2, Svenia P. Jose2, Asha Sukumarapillai2, Parvathy UJ3, Sandya Sukumaran4, Sony Rajan1

1- Advanced Research Laboratory (ARL), Government Homeopathy College, Thiruvananthapuram, Kerala, India
2- Department of Biochemistry, St. Thomas College, Palai, Kottayam, Kerala, India
3- Department of Microbiology, PGM College Kangazha, Kottayam, Kerala, India.
4- Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore, Karnataka, India.

List of Figures

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List of Tables

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Abbreviations

MIC Minimum Inhibitory Concentration

CFU Colony Forming Unit

DMSO Dimethyl sulphoxide

Flc Fluconazole

mg Milligram

R Resistance

S Sensitive

SDA Sabouraud Dextrose Agar

MHA Muller Hinton Agar

MS Mitis Salivary

Ak Amikacin

I Imipenam

P Penicillin

HMT Homeopathic mother tincture

HOMCO The Kerala State Homoeopathic Co-operative Pharmacy

NCCLS National Committee for Clinical Laboratory Standards

BIA Blood Infusion Agar

MR Methyl Red

VP Voges Proskauer

TSI Triple Sugar Iron

mm Millimeter

μg Microgram

Abstract

Dental infection is being considered as one of the six most widespread non-communicable diseases throughout the world. Berberis aquifolium have shown positive antimicrobial, antifungal and anti-inflammatory actions in several microbial studies. So, this study was conducted to determine the antimicrobial efficacy of B. aquifolium against dental biofilm forming Streptococcus mutans and common oral pathogens.

The dental caries samples were collected from 5 patients of age group 7-35yrs from Kottayam Dist., Kerala, under aseptic condition. All the collected plate samples were cultured on nutrient, blood and Mitis Salivary Agar plates and colonies were counted. Microbial species were identified on the basis of morphological and biochemical studies. Microbiological assay (agar well diffusion method and minimal inhibitory concentration (MIC) to determine inhibition zone against oral pathogens were performed.

Thus this study concluded that the ethanolic extracts of B. aquifolium showed significant MIC against S. mutans, Candida albicans and Pseudomonas aeruginosa compared to that of standard drug.

1. Introduction

The world health sectors may face remarkable challenges due to the development of non- communicable diseases. This includes chronic oral diseases, becoming the fastest cause of disability and mortality in developing countries. Mouth and teeth function as vital part of the body and it helps in the basic human functions like chewing, supporting, and assists in smiling, socializing, speaking, eating etc (Glick et al, 2016; Petersen, 2003). In the developing countries about 60-90% of adults and children are affected by oral teeth decay or dental diseases (Petersen & Scheie, 2000).

Dental infection or dental caries can be defined as the destruction of homeostatic condition in the localized region of mouth assisted by the formation of dental plaque or dental biofilm. Biofilm producer Streptococcus mutans are the main causative agent of dental caries. Also, there are other microbes which can cause dental infections such as Lactobacillus acidophilus, other Streptococci species and Candida albicans. Dentine is the hardest part found in the tooth structure. Generally, the food waste particle found in teeth, are the richest source of sugars like monosaccharide, disaccharides etc. The fermentation of these sugars leads to the formation of acid by-product onto to the surface of the tooth. Accumulation of these acid end products cause dentine destruction and thereby increase tooth decay. Development of dental plaque is dynamic processes accompanied with fluctuations in pH, gradually caused de-mineralization and re-mineralization of the tooth structure (Fejerskov et al, 2003; Selwitz et al, 2007).

The medical sectors try to overcome oral diseases by the administration of antibiotics like penicillin and vancomycin. The fluorides in various preparations like tooth paste, Chlorhexidine and triclosan in mouthwashes and gels involved in the management of tooth decay. Unfortunately, the high fluoride content in these preparations may cause serious side effects. Fluoride and chlorhexidine are very dangerous to children below 6 years and also fluorine is found to be toxic to humans (Harris & Garcia-Godoy, 2009; Fatemeh et al, 2015). Increased use of antibiotics gradually facilitates the development of multi drug resistant bacteria, and thereby decreases the normal flora in the oral region.

Homeopathic treatment strategies are very effective in the treatment of dental caries. Because it has lesser side effects and possesses potentially active components. Berberis aquifolium is an herbal species which are spread all over the world and is broadly used as tinctures, root bark, fruits as food, extracts, and stem bark ointments for a huge variety of active substances. The active phytochemical found in B. aquifolium is Berberine. Its biosynthesis is enhanced by berberine bridge enzyme. Berberine is a pharmaceutically important isochinoleinic alkaloid helps Berberis from pathogen attack (Cornelia et al, 2020). The current study explores the potential antimicrobial effects of B. aquifolium against oral pathogens, since this plant extract are widely used in homeopathic therapies.

1.1. Objective of the Study

Dental infection becomes the most common infectious disease throughout the world day by day. Dental decay is a complex infectious disease associated with interactions of cariogenic bacteria with the tooth surface. Fermentable carbohydrate food material accumulates on the tooth surface gradually facilitate the growth of acidogenic bacteria. Tooth infections directly correlated with the health and hygiene of peoples. Lack of knowledge towards dental hygiene practices is one of the major factors of dental infection. Therefore, increasing knowledge towards better dental practices can prevent dental infections.

Some of the commercially used drugs can unbalance oral flora and have adverse side-effects such as vomiting, diarrhea and tooth staining. Hence, there is a need for investigating homeopathic plants used as alternative medicines in the treatment of dental infection. Therefore, this study entails a divergent approach to provide a scientific validation of homeopathic mother tincture of B. aquifolium against dental pathogens.

The main objective of the study: -

Antibacterial effect of B. aquifolium homeopathic mother tincture against biofilm formers from dental plaque

The specific objectives are;

- Isolation and characterization of dental biofilm producer S. mutans and common dental pathogen from 5 volunteers of age group between 7- 35.
- Morphological and Biochemical characterization of isolated pathogens up to species level.
- Evaluation of agar well diffusion method for assessing inhibitory effects of B. aquifolium and Standard drugs.
- Determining the MIC of B. aquifolium against dental biofilm producer S. mutans and common dental pathogen .

2. Review of Literature

2.1 Teeth and its structure

Odontogenesis or tooth development is a complex process usually begins with thickening of the oral epithelium. The tooth not only functions in chewing but also helps in speaking. Teeth are covered by calcium rich outer layer called enamel. Enamel is the most mineralized tissue in the human body primarily consists of hydroxyapatite. The hardest part of the human body is considered as teeth enamel. During tooth development the cells of the body produces a mineral called hydroxyapatite- consists of crystalline calcium phosphate.

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Figure 1. Teeth anatomy

The structure of human tooth includes enamel, dentine, gums, cement and pulp (Figure 1). Enamel is the calcified outer part of the tooth mainly functions as breakdown of food particles, and also forms protective layer around tooth crowns. The largest mineralized tissue found in mammals is dental enamel. The enamel is originated from epithelial layers of tissue whereas dentin and cementum were originated from mesenchymal layers. Dentine is tough and elastic, also most vulnerable part of the teeth. Cementum is an unnerved nonvascular mineralized tissue found between dentine and periodontal ligament, which attach the teeth to the jaw sockets (Sasaki and Garant, 1996; Mitsiadis & Papagerakis, 2013).

2.2 Pathogenicity of dental infection

Oral cavity harbors saliva and numerous microbiota, which plays an important role in the development of dental biofilm or plaque. Vast majority of dental infections are arisen due to poor dental hygiene practices. Fluorine, zinc, lead and iron deficiencies in enamel of teeth also increase the risk of dental infection.

The fact is that not all the plaque is harmful to tooth; some commensal plaque plays an important role in preventing tooth decay by reducing acid attack and control mineralization. The bacteria along with the salivary molecules absorbed on to the tooth surface and began to multiply themselves. The teeth polymer matrix serves as the skeleton for the formation of natural plaque. The homeostasis condition within the oral region is always associated with commensal microflora including Streptococcal species. This commensal microflora prevents the colonization of pathogenic species. On the other hand, this homeostasis condition can be leads to the destruction of oral ecosystem. Ultimately this may result in tooth mineralization by the over dominance of Gram positive carbohydrate- fermenting bacteria (Scannapieco, 2013; Kaidonis &Townsend, 2016).

The process of dental plaque biofilm development can be divided into three categories (figure 2):

1. Attachment of oral pathogens to a solid surface called dental plaque
2. Formation of bacterial colonies on enamel of teeth
3. Formation of plaque biofilms on outer layer of teeth

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Figure 2. Overview of caries pathogenesis

Pathogenesis of oral microbiota begins with the formation of pellicle. Pellicle is a thin proteinaceous covering found on the outer layer of teeth. It will act like double sided tape, in which one side is attached to the teeth surface whereas other side providing a sticky surface to the bacterial attachment. Fimbriae are a special structure found in bacteria, which helps the adherence of bacteria to pellicle. After 2 days of bacterial colonization, with no more proper washing occurs, the tooth surface is primarily colonized by Streptococcus sanguis, Streptococcus mutans and Actinomyces viscosus. The attachment of fimbrial protein to pellicle protein is a type of “lock and key” mechanism.

The accumulated food particles are the richest source of sugars favor the growth of dental pathogens. Dental plaque can be defined as a thin, tenacious biofilm found on the outer region of tooth. The biofilm producing bacteria and other dental pathogens ferment the accumulated food materials. Thus, forming acid end-products, cause demineralization of inorganic substances. The change in pH due to the fermentation process enhances demineralization and remineralisation of tooth structure (Naseem, 2018; Keyes, 1960).

2.3 Dental caries

Dental caries might be considered to be the most common oral disease worldwide (Selwitz et al, 2007; Costa et al, 2012). They are microscopic deposits found primarily on the surface of the teeth, finally invade the inner region of teeth, this includes dentin and innermost pulp.

Biofilm producing cariogenic bacteria ferment dietary carbohydrates results in the production of lactic acid leads to dental enamel breakdown, called enamel caries. Progressive accumulation of cariogenic bacteria on enamel of teeth gradually leads to dentin caries. This may cause infection in pulp cavity, associated with teeth ache, swelling, difficulty in chewing and speaking. Finally, this infection leads to chronic inflammation of gingiva and associated tissue usually characterized by swelling, bleeding and erythematous. Periodontitis is chronic inflammation caused by the persistence of bacteria on biofilm (figure 3) (Jin et al, 2016; Petersen & Ogawa, 2005; Scannapieco, 2013).

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Figure 3. Dental caries in various stages

2.4 Microbial etiology of caries

The etiology of dental decay disease is multifactorial. Dental caries will not occur if the oral cavity is free of pathogenic biofilm producing bacteria. The surface of teeth is covered by yellowish coloured material called dental plaque which harbors biofilm producing bacteria. According to acidogenic theory (Miller's chemo-parasitic theory), “Dental decay or dental infection is a chemico-parasitic process. Decalcification of enamel and decalcification of dentin are the two stages of tooth decay, followed by dissolution of softened residue. Primary decalcification is emerged by the action of acids produced from the fermentation of starch and sugars” (Malvin, 2002).

The commonly found dental pathogens are S. mutans, Veillonella spp., Actinomyces spp and Lactobacillus spp. These bacteria can be transferred from one generation to other by means of mother to child. These bacteria over dominate normal oral flora and thereby producing tooth decay. Dental pathogens utilize the waste material produced by the metabolic reactions in oral regions and produce acids which leads to pH alteration in oral cavity; this will trigger mineralization of tooth. Primary dental decay is always characterized by severe dental pain, swelling and finally to headache (Khushbu & Satyam, 2016).

2.5 Treatment of caries

At present the current treatment strategies used across the world for dental decay is the administration of antibiotics. Antibiotics are considered as the keystone for all infectious disease. Most of the dentists use metronidazole, penicillin, and antimicrobials like fluorides and chlorhexidine in the treatment of dental decay. Depending on the dental decay there are also different treatment methods are available- Fillings, Root canal, Crown and Extraction. Unfortunately, all these treatment strategies produce adverse side effects to human health. Therefore, researches are focusing on natural based medicine for the treatment of dental plaque and associated health problems.

The current study focused the inhibitory effect of B. aquifolium against dental biofilm forming bacteria. Homeopathic medicine has great source of natural antimicrobial compound against dental diseases, which can be used to treat dental biofilms. Homeopathic medicinal plant and its product were used as adjuvant in the treatment of oral disease because they reduce the adverse effects of antibiotics.

B. aquifolium is a homeopathic medicinal herb species distributed throughout the world and is widely used as medicine and its fruits are used as food. The active component present in B. aquifolium is berberine; it is a type of isochinoleinic alkaloid located in the rhizome, stem bark and root bark, which helps against a pathogen attack. It has anti-diabetic, antidiarrheal, antimicrobial, immune-stimulating hypotensive and anti-inflammatory properties (Fazel et al, 2010; Andres & Chantalat, 2014).

Abbildung in dieser Leseprobe nicht enthalten ⁴ Figure 4. Berberis aquifolium

3. Materials and Methods

3.1 Chemicals

All the analytical grade chemicals were purchased from Merck India Pvt. Ltd. Microbiological media, including Sabouraud Dextrose Agar (SDA) , Muller Hinton Agar (MHA), MS Agar (Mitis Salivary) and Standard drugs such as Amikacin (AK), Penicillin (P), Imipenam (I), Fluconazole (FLC) and were purchased from Hi-Media Laboratories, Pvt. Ltd., India.

3.2 Preparation of powdered form of Homeopathic mother tinctures (HMT)

Mother tinctures of B. aquifolium was collected from HOMCO Ltd. Kerala. 100 mL of four different HMT’s was taken in a conical flask. The solvent from the HMT’s was removed by using rotary vacuum evaporator with the water bath temperature of 60°C. Finally, the residues were collected and refrigerated at 4 ºC until further analysis.

3.3 Collection of dental caries samples

The dental caries samples were collected from 5 patients of age group 7-35years from Kottayam District, Kerala, under aseptic condition. Before to the collection, the buccal cavity of each patients was rinsed with sterile water, followed by 3% hydrogen peroxide and then cleaned with decontaminating solution (2.5% sodium hypochlorite) (Jebashree et al, 2011). Using a sterile swab, the caries samples were collected from the patients and were introduced into 2ml of normal saline in sterile screw cap tubes. The samples were transported to the laboratory for further study and stored at 4ºC.

3.4 Isolation and screening of bacteria

The samples in each tube was inoculated separately into 25ml of nutrient broth and incubated at 35°C ± 2°C for 48 hours. After incubation grown culture was serially diluted up to 10-8 with sterile distilled water. 0.1ml of serially diluted samples was spread plated over NA, BIA and Mitis Salivary Agar plates. The plates were incubated both aerobically and anaerobically at 370C. Candle Jar method was used for the anaerobic culturing of isolates. Count of more than 250 colonies (104cells/ml) was considered as positive samples (Friedrich, 1981; Chandrabhan et al, 2012). The predominant and morphologically different colonies were streaked on nutrient agar slant for pure culture preservation.

3.5 Morphological and biochemical identification of isolates

The bacteria were gram stained and observed under light microscope (100X). Motility was checked using hanging drop method (Aneja, 1996). Biochemical properties of the isolates were tested according to Bergey’s Manual of Systematic Bacteriology (Holt et al, 2000).

3.6 Identification of Streptococcal Isolates

After estimation of positive samples on MS-agar medium, small colonies were sub-cultured on BIA plates for further purification and incubated aerobically and anaerobically for two days at 37°C. Modified MS Agar culturing was used for the easy way of identification of Streptococcus species. (Gold et al, 1973; Carlsson, 1968). The following methods were used for initial characterization of the isolates:

a. Morphological appearance on MS-agar and blood agar.
b. Microscopic examination.
c. Catalase test.

3.7 Physical characterization

i) Gram staining: Gram staining of the isolates was performed as described in Textbook of Medical Laboratory Technology (Godkar and Godkar, 2003) and the Gram natures of the isolates were recorded.
ii) Germ tube identification.
iii) Colony morphology: Colony characteristics of overnight culture of the isolate on BHI agar media was observed and recorded.
iv) Cell morphology: The Gram stained smears of the isolates were viewed microscopically under a magnification of 1000 X using the oil immersion lens to determine the morphological characteristics of the cells.

3.8 Biochemical characterization

All the bacteria were widely differing in both biochemical aspects and morphological aspects. These differences in various reactions can be identified by different tests which are listed below (Collee et al, 1996).

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¹ Author’s own creation

² Author’s own creation

³ Author’s own creation

⁴ Author’s own creation