Antimicrobial Activity of Selected Medicinal Plants Against Oral Microflora

 

S. Nandakumar¹, M. G. Sanal Kumar², B. Bini³, Geethu G. Krishnan⁴

P.G. and Research Department of Zoology, N.S.S. College, Pandalam, Kerala, India -689501

 

ABSTRACT:

Antibiotic resistance and side effects of antibiotics are major problem in the medicinal field, in the present study four selected medicinal plants were used to find out the antibacterial activity against oral pathogen. Plant extracts of Azadirachta indica, Synzygium aromaticum, Piper nigrum and Mangifera indica were used for the study. By disc diffusion method these medicinal plant extracts produced clear and circular inhibition zones on the swab culture of oral microflora after 34 hrs of incubation. The inhibition zone for each medicinal plant varied in diameter. The inhibition zone indicates the destruction of microbial population in the culture by the antimicrobial activity of the medicinal plants. Three different treatments were conducted for each medicinal plant and each treatment shows a slight variation in diameter. Among four medicinal plants Azadirachta indica showed maximum zone of inhibition and Mangifera indica showed least inhibition zone diameter. One way ANOVA showed that there was no significant difference among the four medicinal plants in their antimicrobial effect on oral bacteria, there was a slight variation among them. As per the result from the present study, all of the selected medicinal plants showed their excellent efficiency against oral microflora. Since the tested extracts of selected plants were effective against pathogenic micro-organisms present in the oral cavity. The anti-microbial efficacy can be enhanced if the phyto constituents of these plant extracts are purified using different solvents like ethanol, methanol, acetone etc., antibacterial activity of these medicinal herbs, if translated into clinical practice would lead to the development of indigenous, chemical free, cost effective and holistic oral hygiene aids, which can be incorporated into various oral hygiene formulations like dentrifices, mouth rinses, gum paints etc.

 

 

INTRODUCTION:

Micro-organisms are found almost everywhere in our environment as well as on different part of eukaryotic body system. Symbiotic microbes associated with human body are of two types; resident floras are found inside the body, they are reestablished. Transient floras are found on skin and mucous membrane which are temporary.

 

Oral micro-biome is a complex ecological system where up to 700 species of microorganisms that have been identified. Oral microfloras are most commonly found in gingival crevices, tongue dorsum, buccal mucosa and saliva. Oral diseases continue to be a major health problem worldwide. Oral disease in an individual can be caused due to a combination of lack of oral hygiene and factors influencing the oral microbial community structure, such as diet. The most common oral bacteria include streptococci, lactobacilli, staphylococci, corynebacteria and various anaerobes. The most common type of pathological diseases in the mouth are periodontal diseases and dental caries (Marsh and Martin, 1992). These diseases are caused by various plaque forming bacteria such as Streptococcus mutans, Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus species and Fusobacterium sp., which reside in the oral cavity. Dental caries is an infectious microbial disease that results in localized dissolution and destruction of the calcified tissues of the teeth (Loesche, 1986). Streptococcus mutans is known as the causative bacteria in the formation of dental plaque and dental caries. If plaque is not removed thoroughly and routinely, tooth decay will not only begin but flourish (Hardie, 1992). Antibiotics such as penicillin and erythromycin have been reported to effectively prevent dental caries in animals and humans, but they are never used clinically because of many adverse (Kubo, et al., 1992). Many plants have been reported in the pharmacopeias which have efficacy to treat oral infections (Cowan, 1999; Hadissa et al.,  Kalemba et al., 2003; Wolinsky et al., 1996). Recent natural remedies with the use of medicinal plants, which are good reservoirs of chemo-therapeutants can be, contributed as an alternative for antibiotic effects such as hypersensitivity reaction, supra infections and teeth staining. Healthy teeth are fundamental for the proper functioning of the human body. Proper and regular hygiene is required to prevent dental problems.

 

MATERIALS AND METHODS:

The present study aimed to analyze the effect of various medicinal plants on oral bacteria. The selected medicinal plants are Azadirachta indica (Neem), Synzygium aromaticum (Clove), Piper nigrum (Black pepper) and Mangifera indica (Mango). The plants parts used for the extraction were, leaves from both neem and mango, flower bud from clove and seed from pepper.

 

Contents of Materials Used:

1.    Tryptone Soya Bean agar (TSBA)

 

2.    Muller Hinton Agar

Ingredients: Caesin acid hydrolysate -17.50gm/l, Beef heart infusion -2.00 gm/l, Starch (soluble) -1.50 gm/l, Agar -17.00 gm/l 

 

3. Medicinal Plants:

i)    Azadirachta indica (Neem):

Family: Maliaceae,

Scientific name : Azadirachta indica,

Part used : Leaves

Belongs to the family Maliaceae. It is a hardly quick growing ever green showy tree grows to a height of 12 m. Neem is used in traditional medicine as a source of many therapeutic agents in the Indian culture. Every part of neem tree may be used medicinally. Its twigs provide a chewing stick and are widely used in the Indian sub continents. Leaves are traditionally steeped for malaria.

 

ii)   Synzygium aromaticum (Clove)

Family: Myrtaceae,

Scientific name : Synzygium aromaticum,

Part used :flower bud

Cloves are the aromatic flower buds of a tree in the family Myrtaceae, Syzygium aromaticum. Cloves are one of the highly prized spices, widely recognized all over the world for their medicinal and culinary qualities. The active principles in the clove are known to have antioxidant, antiseptic, local anesthetic, anti inflammatory, rubefacient, carminative and anti-flatulent properties. The spice contains health benefiting essential oils such as eugenol. Eugenol has local anesthetic and antiseptic properties, hence; useful in dental care essentials as well as in treatment procedures. Clove is applied directly to the gums (used topically) for toothache, for pain control during dental work.

 

iii) Piper nigrum (Black pepper)

Family :Peperaceae,

Scientific name : Piper nigrum,

Part used : Seed

It is a perennial vine and climber that requires supporting tree to grow in height. Peppercorns contain an impressive list of plant derived chemical compounds that are known to have disease preventing and health promoting properties. Peppers have been in use since centuries for its anti-inflammatory, carmative, anti- flatulent properties. Peppers have been used therapeutically in dentistry as an antiseptic for tooth-decay and gum swellings.

 

iv)  Mangifera indica (Mango):

Family : Anacardiaceae,

Scientific name :Mangifera indica,

Part used : Leaves

The mango plant has several biological properties as like to treat mouth infections in children. Different parts of mango have been investigated for their antibacterial, antifungal and antioxidant activities. Leaves contain glucoside and mangiferin which is potent antimicrobial agent. Mangiferin has been demonstrated to possess antiviral activity against herpes simplex type 2 viruses, hypoglycemic, anti-hyper lipidemic and anti atherogenic activities (Zakaria et al., 2006).

 

Plant Collection and Extraction:

The plants were collected from different areas of Pathanamthitta district of Kerala and localities of Chunakkara area in Alappuzha district. These plant parts were selected on the basis of their use in medicine and on oral care. For the extraction, the plant parts were cleaned using tap water followed by sterile distilled water. The materials were air dried followed by grinding into a fine powder. For extraction with water, 25 g of powdered plant material was dissolved in enough sterilized distilled water.

 

Antibacterial Susceptibility Test Disc:

Test discs of 7mm diameter were made with Whatman No.1 filter paper and were sterilized in hot air oven at 160^ºC for one hour. For preparation of test discs with extract, the plant extract were taken in sterilized containers and the pre- sterilized test discs were soaked in the each plant extraction for 10-20 minutes and allowed to dry for preventing unwanted spreading of the content on bacterial lawn.

 

Pre Enrichment of Test Microbe:

Human oral micro flora were obtained through swab collection from the oral surfaces such as tongue surface, between the gum and the teeth etc and enriched in Tryptone Soya Bean agar with incubation at 37^o C for 24 hours.

 

Culture Media Preparation:

The culture media used was Muller Hinton agar. 3.8gm of agar was dissolved in 100ml distilled water, boiled on a hot plate ,to mix evenly, cotton plugged and sterilized in an autoclave at 121ºC and 15 lb/in² pressure for 15 minutes. Preserved at 4ºC for further use.

 

Antibacterial Susceptibility Test:

The antibacterial assay was carried out by using disc diffusion method (also known as Kirby- Bauer method) (Bauer et al., 1966) performed in laminar flow. Using a sterilized cotton swab, heterogeneous carpet culture of oral bacteria was made on the Muller Hinton agar plate with pre enriched inoculums. Antibacterial test discs were placed at sufficiently distances from each other in order to avoid the mixing of inhibition zone. Three replicas were also made with each test disc to minimize sampling error. Culture plates were then kept in incubator at 37^oC for 24 hours. After 24 hrs of incubation, the culture plates were observed to check antibacterial activity of selected medicinal plant extracts against the bacterial species. Inhibition zone diameters (IZDs) were measures using standard scale in nearest mm.

 

Statistical Analysis:

Descriptive statistics: Mean (M), Standard deviation (SD) and Standard error (SE) for the values of IZDs produced for different treatments were calculated using MS excel software. One way ANOVA was conducted with IZD obtained through four treatments using MS excel software

 

RESULTS:

After 24 hours of incubation at 37⁰C, the colonies formed on nutrient agar medium. For analyzing the effect of four selected medicinal plants on oral microflora three different treatments were carried out for each medicinal plants  with an incubation period of 24 hours at 37⁰C. In the first treatment the cultures produced circular and clear zone of inhibition against four medicinal plants. These zones of inhibition were measured and noted, similarly two subsequent treatments were carried out and inhibition zone diameter in all cases (IZD) were measured and recorded. Zone of inhibition produced by Azadirachta indica in the three treatments are given in the table I.  Where   1^st and 2^nd treatment shows inhibition zone diameter of 8 mm, that is maximum zone of inhibition. In the 3^rd treatment, the inhibition zone is about 7 mm. The mean and standard deviation were calculated are 7.6 mm and 0.547723 respectively.

 

Synzygium aromaticum, the 2^nd treatment shows maximum zone of inhibition that is 8 mm and the 1^st and 3^rd treatment shows the minimum zone of inhibition, 7 mm. Mean and standard deviation were calculated and the values were 7.4 mm and 0.547723 respectively. Zone of inhibition produced by Piper nigrum on the microbe is shown in the table I & figure I. There was also a slight variation in each treatment. In 1^st treatments, it showed highest value that is 8 mm. the 2 ^nd and 3 ^rd treatment shows minimum zone of inhibition. The mean and standard deviation were calculated and the values were 7.4 mm and 0.547723 respectively. The measurements of inhibition zones produced by the leaf extraction of Mangifera Indica  at three different treatment is given in the table I, there is no variation in the diameter of inhibition zones in each treatment, the obtained value is 7mm. The mean and standard deviation calculated were 7 mm and 0.00 respectively. The IZD obtained for four selected medicinal plants through treatments summarized as column graph I. Two way ANOVA of IZD by four medicinal plants (Table 2) showed that the computed F value is lesser than the table value. Computed F value is 2.111111 which is lesser than the F value of 4.066181 for level of significance (P>0.05). Hence the result was not significant, ie, there was no significant difference between the effect of selected medicinal plants in their antimicrobial effect on oral microflora. Among the four medicinal plants analyzed,  Azadirachta indica (Neem) produce maximum zone of inhibition against oral pathogen. Two of them that is Piper nigrum and Synzygium aromaticum shows similar zone of inhibition and only one Mangifera indica shows least inhibition zone when compared to the other three.

 

Table 1: Inhibition zone produced by four medicinal plants against oral bacteria in five treatments

Name of plant used	Inhibition zone diameter (in mm)
	I	II	III	IV	V	Mean	SD
Azadirachta indica (Neem)	8 mm	8 mm	8 mm	7 mm	7 mm	7.6 mm	0.547723
Synzygium aromaticum (Clove)	7 mm	8 mm	8  mm	7 mm	7 mm	7.4 mm	0.547723
Piper nigrum (Pepper)	8 mm	7 mm	8  mm	7 mm	7 mm	7.4 mm	0.547723
Mangifera indica (Mango)	7 mm	7 mm	7 mm	7 mm	7 mm	7 mm	0

 

Table 2: Two way ANOVA showing the significance of variation of  IZD of different medicinal plants  in different concentrations

Source of Variation	SS	df	MS	F	P-value	F crit
Rows	0.95	3	0.316667	2.111111	0.152197	3.490295
Columns	1.8	4	0.45	3	0.0625	3.259167
Error	1.8	12	0.15
Total	4.55	19

 

Graph I: Inhibition zone diameters of four medicinal plants in five different treatments

 

DISCUSSION:

Oral hygiene is an integral part of health of a person and when neglected it results in various oral disorders. Due to high emergence of oral infections, the fast developing resistance by bacteria to antibiotics, the adverse effects of the chemical based remedies currently used in dentistry and financial considerations there is a need for alternative prevention and treatment options that are safe, economical and effective. Oral infections have been linked to bacterial species especially by mutants group. Unhealthy diet pattern and lack of oral hygiene promote the growth of these bacteria. A number of methods were used to treat oral infections but it is important to look for the role that plants play in maintenance of oral hygiene since many of them have medicinal properties. There is a long history of the use of plants to improve dental health and promote oral hygiene. When compared to chemical based remedies plants always keep an upper hand. In many Indian and African homes even today teeth are cleaned using stems and barks of certain plants. Natural tooth brush sticks could be used by majority of people who cannot afford the western toothbrush and toothpaste. Therefore due to adverse effect of chemical based remedies and high cost western toothpaste the search for the alternative products still continues and the phyto-chemicals isolated from the plants proves to be a good alternatives. The present review was an attempt to create an interest among people regarding the potential of natural plants in treating the infections and disorders of oral activity. The anti-bacterial activity of medicinal plants are due to the presence of potential bioactive compounds, which help to reduce bacterial load in the oral cavity and thus prevent the formation of plaque, dental caries and ulcers. Use of indigenous plants in oral health and hygiene has a long history in different parts of the world. Therefore, this knowledge is likely to vanish soon as many of these ethno-phototherapeutic remedies are followed only by a few in rural areas. New generation is ignorant of this traditional knowledge. Because of younger generations lack of knowledge on the identification, collection, preservation and processing of the plant species for medicinal use it is therefore very crucial to conserve these ethno-cultural practices before they are lost definitively. The selected four medicinal plants which treated against the oral bacteria culture produce clear and discrete zone of inhibition. The zone of inhibition produced by the selected plants slightly varied in diameter among each other. The bacterial growth around the diffusion disc of medicinal plants disappears and thus the inhibition zones were developed. Hence it was found that each medicinal plant had varied their antimicrobial effect on the oral bacteria. During the treatment, Azadirachta indica was found to be having maximum effect ie., 7.66 mm average inhibition zone diameter. It is one of the oldest medicinal plants in the world and has been used in indigenous medicinal system for thousands of years. (Biswas et al., 2000) have recently reviewed the biological activities of some of the neem compounds, pharmacological action of neem extract, clinical study and medical applications of neem. Oils from neem have shown a wide spectrum of antibacterial activity against gram negative bacteria. Every part of neem tree may be used medicinally. It has antibacterial and also anti fungal activity. It has proved its antidiabetic activity too (Khan and Khanum, 2005) Neem has been proved to be effective against Enterococus faecalis and Candida albicans. Its antioxidant and antimicrobial properties makes it a potential agent for root canal irrigation as an alternative to sodium hypochlorite (Vanka et al., 2001). Synzygium aromaticum and Piper nigrum produced average inhibition zone diameters of 7.4 mm. They showed almost similar antimicrobial activity against the oral bacteria. These two spices are widely used for their antibacterial effect against various bacteria.  The phenolic present in the species play an important role in anti bacterial activity. Use of spices reduces or inhibits the growth of micro organisms before they produce any toxins. In the traditional Indian system of medicine Ayurvedha and Siddha various spices and herbs are described to possess medicinal properties, such as being anti oxidant, anti bacterial, anti thrombotic, anti atherosclerotic, hypolidermic, hypoglycemic, anti inflammatory, anti arthritic etc. It was found to be helpful in reducing pain, chills, flu, colds, fever and muscular aches. Piperine is the major chemical constituent is responsible for the antimicrobial activity. Clove is proved to have antiseptic, anti bacterial, anti fungal. antiviral, spasmolytic, local anesthetic, anti stress, antipyretic, anti platelet, anti-inflammatory activities. The phenolic compounds present in clove can react with cell membrane phospholipids and inhibits a great number of gram negative and gram positive bacteria. The extract from the leaves of Mangifera indica produce minimum inhibition zones of 7 mm in all treatments. This was the least inhibition zone diameter produced among the four selected medicinal plants. Thus it is clear that these extracts are not highly effective against the oral bacteria. The IZD produced by each medicinal plants show only a small variation and two way ANOVA conducted reveal that there was no significant variation among the medicinal plants (Table 2). Among the four medicinal plants selected for the study almost all of them has effect on the microbe. Hence they can be used for the treatment of infections by the test microbe at their in habitual areas. Continuous use of the single medicine among them is not advisable but alternative use recommended so as minimizing the chance of developing resistant varieties and also for the better restriction of the growth of the microbe in patient.

 

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Accepted on 06.11.2016        © RJPT All right reserved

Research J. Pharm. and Tech 2016; 9(12):2271-2275