Anti-Microbial Efficacy Of Ficus Benghalensis And Azadirachta Indica Formulation - An In Vitro Study

Keywords

Neem; Banyan; Antimicrobial Solution.

Introduction

The most common oral health issues are dental caries and periodontal disease; however, other conditions such as oral cancer and oral mucosal lesions are also causes for concern.[1]. Dental caries is a widespread oral disease caused by Gram-positive bacteria such as Streptococcus mutans, Streptococcus sobrinus, Lactobacillus spp., and some non-mutans streptococci forming plaque biofilms on tooth surfaces.[2, 3] Root caries and periodontal infections are caused by various bacterial species such as Actinomyces spp. and Enterococcus faecalis [4]. The rise in disease incidence (particularly in developing countries), increased resistance by pathogenic bacteria to currently used antibiotics and chemotherapeutics, opportunistic infections in immunocompromised individuals, and financial considerations all contribute to the global need for alternative prevention and treatment options and products for oral diseases that are safe, effective, and cost-effective.

Ficusbenghalensis, is commonly known as the banyan and Indian banyan tree. Ficusbenghalensis is the national tree of India. Ficusbengalensis methanol and chloroform extracts have antibacterial activity against Streptococcus mutans and Actinomycesviscosus bacteria. The antibacterial activity on the extract is due to the presence of different phytochemicals. Sterols and flavonoids abound in Ficusbenghalensis Linn. These phytochemicals are thought to be responsible for the plant's antibacterial properties.[5]

Azadirachtaindica, is commonly known as neem, nim tree or Indian lilac. It is typically grown in tropicaland subtropical regions. As opposed to other dental caries-causing species including S. salivarius, S. mitis, and S. sanguinis, dried chewing sticks of Neem displayed the most antibacterial activity against S. mutans [6]. Muco adhesive dental gel containing Azadirachtaindica has been found to be more effective than chlorhexidinegluconate mouthwash in reducing plaque index and salivary bacterial count [7].

In this study, the most common oral microbial species were considered: staphylococcus aureus is a gram-positive, round-shaped bacteria, streptococcus mutans is a facultatively anaerobic, grampositive bacteria that occurs in the oral cavity, candida albicans is a yeast-like fungi that is common in human gut flora, and enterococcus faecalis is a gram-positive bacterium that can cause a variety of oral diseases [8]. Enterococcus faecalIs is gram-positive bacterium that can cause a variety of nosocomial infection of urinary tract [10, 11]

The aim of the study was to investigate the antimicrobial efficacy of ficusbenghalensis and azadirachtaindica against four oral pathogens. Previously our team has a rich experience in working on various research projects across multiple disciplines [12-26] Now the growing trend in this area motivated us to pursue this project.

Materials And Method

Preparation of herbal solution

Azadirachta indica and Ficus benghalensis leaves were collected from a university campus in Chennai, Tamil Nadu, in December. To remove dirt and dust from the surface of the leaves, they were thoroughly washed in running water.. They were dried for 15 days and kept in the hot air oven at 600°C for 24-48 hours. These leaves were then ground to a fine powder. 1g Neem and banyan leaf extract powder were mixed with 100 mL distilled water and kept in an orbital shaker for 1 day. The solution was boiled for 10 minsat 70 to 80c. The solution was heated and reduced upto 10 ML. It was filtered using whatman no 1 filter paper.

Cytotoxic effect of newly introduced herbal solution

Cytotoxicity effect determines whether the bioactive compound is toxic to cells. Assay for the lethality of brine shrimp was assessed. The crustacean salina is a dependable and convenient method for assessing the cytotoxic effect of bioactive chemicals. Aquatic Remedies in Chennai supplied the brine shrimp eggs. In a hatching chamber, artificial sea water was created by combining 36 g of iodine-free salt with 1000 ml of water that had been distilled. The hatching chamber was divided into a dark area where shrimp eggs were added, as well as lighting the area with the lamp above. The formalised paraphrase Brine shrimp hatch in two days and mature in two weeks. The hatched nauplii were used to evaluate the cytotoxic effect of herbal solution. In a 6 well ELISA (Enzyme Linked Immunosorbent Assay) plate, 10-12 mL of saline was added. E.faecalis, S. aureus, S. mutans were incubated at 37degree celsius for 24 hours.C. albicans, a yeast like fungi, was incubated at 37 degree celsius for 48 hours. Fours groups of microbial culture were included with the measurement of 25µL, 50 µL, 100 µL and standard (amoxyrite). 10 nauplii were added at each well and the number of live nauplii observed after 24h incubation.

Zone of inhibition

The antibacterial activity was carried out by disc diffusion method. Nutrient agar medium plates were prepared, sterilized and solidified. After solidification bacterial cultures were swabbed on these plates. The sterile discs were dipped in the solution and placed in the nutrient agar plate in (25 µL, 50 µL, 100 µL and standard (AMOXYRITE)) and kept for incubation at 37° for 24 hours and then zones of inhibition were measured.

Result And Discussion

Based on the cytotoxic results, it has shown that 25µL herbal solution had 10 shrimpsnauplii still alive. The 50µL herbal solution showed 9 nauplii still viable and the 100µL had 7 nauplii viable. As shown in table 1, a minimum of 10mm zone of inhibition was observed for three bacterial species E.faecalis, C.albicans and S.aureus, whereas S.mutans showed a minimum of 15mm in diameter.

This results shows zone of inhibition is higher in s.mutans. These preliminary data indicated that neem and banyan extract have antibacterial activity.

Herbal medicines, according to WHO, serve the health needs of approximately 80% of the world's population, particularly millions of people in developing countries' rural areas.The beneficial medicinal effects of plant materials, including antibacterial activity, are typically attributed to secondary products present in the plant, rather than a single compound or a combination of metabolites [10, 27, 9].

Though the precise mechanism by which the active components of plant materials contribute to antibacterial activity is unknown, the antimicrobial effect could be mediated by one of several mechanisms, including inhibition of cell wall synthesis, cell membrane damage, inhibition of nucleic acid synthesis, inhibition of protein synthesis, and so on [28]. Antimicrobial phytochemicals are classified into several groups, including phenolics, polyphenols, flavones, flavonoids, flavonols, quinones, tannins, coumarins, terpenoids, essential oils, alkaloids, lectins, and polypeptides. Ficusbenghalensis Linn. is rich in sterols and flavanols. These phytochemicals are attributed to the plant’s antibacterial activity. Flavones, flavonoids, and flavonols complex with bacterial proteins and cell walls and exhibit antimicrobial activity [28, 29]. Ethanolic leaf extract of Azadirachta indica shows significant antibacterial activity against selected acidogenic oral bacteria. Presence of gallotannins during the early stages of plaque formation could effectively reduce number of bacteria [11, 27, 30].

Zone inhibition of neem and banyan leaf extract is demonstrated against bacterial cultures. The antibacterial activity was done against the pathogenic bacteria such as streptococcus aureus, staphylococcus aureus, candida albicans and enterococcus faecalis [30]. There were three different concentrations (25 µL, 50 µL, 100 µL and standard (AMOXYRITE)) which were taken to kill the pathogenic bacteria. Overall, this study reported that herbal formulation mediated by azadirachta indica and ficus benghalensis plant extract demonstrated good antibacterial activity.

Zone of inhibition (ZOI) of neem and banyan extract demonstrated against microbial cultures. The zone of inhibition increased with concentration of herbal formulation,therefore,for streptococcus mutans 15MM shows that highest in 100 µL/ml, while the lowest ZOI for streptococcus aureus was 9MM shows that lowest in 100 µL/ml. Both herbal extraction was against all the major pathogens that cause dental caries [27, 30]. Our institution is passionate about high quality evidence based research and has excelled in various fields [16, 31-40].

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Figure 1. Preparation of plant extracts.(a) 1gm of neem and banyan extract were mixed with 100 ml distilled water (B) boiled for 10 mins using mandel until the solution is reduced to 10ml

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Figure 2. Cytotoxic effect observation.ELISA plate wells with different concentrations of neem and banyan extract observed for presence or absence of live nauplii after 24 hour incubation effect.

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Figure 3. Antibacterial activity of Herbal formulations against pathogens by agar well diffusion method. (E.Faecalis,S. mutans).

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Figure 4. Antibacterial activity of Herbal formulations against pathogens by agar well diffusion method. (S.aureus,C.albicans).

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Figure 5. Anticariogenic activity of banyan and neem leaf extract.

Conclusion

Our studies confirmed the antimicrobial effect of these natural products of Ficus benghalensis and Azadirachta indica extract. Antimicrobial activity against all the four oral microbial Pathogens with the highest effect at 100 µL of the extract. However, in the case of this plant, future research should focus on an alternative mechanism of synergistic effects of various natural substances when used in combination with other antibiotics.

Acknowledgement And Declarations

The authors would like to acknowledge the institution and all the staff members of the Department of Conservative Dentistry and Endodontics for their support towards completion of this research. The authors deny any conflicts of interest associated with this paper.