INTERNATIONAL JOURNAL OF BIOPHARMACEUTICAL SCIENCES (ISSN:2517-7338)

Introduction: Alveolitis is a circumscribed peripheral osteitis occurring 2-3 days after teeth extraction. Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum are among the incriminated bacteria. However, ill-adapted antibiotic therapy generates multi-resistance. Research is therefore focusing on the use of medicinal plants. The aim of the study was to evaluate the in vitro antibacterial activity of the essential oil of Azadirachta indica (Neem) on the said bacteria.

Methodology: This was an experimental study carried out at the FMBS in Yaoundé and the CSIR of Bruneton in Pretoria for 7 months in 2019. Chemical screening was done by CPG/SM. The antibacterial activity was studied according to the method of disc diffusion on this agar medium and the macro dilution technique. Statistical analysis was carried out using Graphpad Instat version 5.1 software.

Results: Chemical screening of Neem oil revealed the compounds responsible for its antibacterial activity, such as fatty acids including oleic acid (42.5%) and several volatile compounds including Lineoleoyl chloride (10.24%) and Methyl petroselinate (10.23%). Susceptible bacterial strains had diameters of 14.9 mm for A. actinomycetemcomitans, 11.3 mm P. gingivalis and 12.6 mm F. nucleatum. The MIC and MBC were 125 mg /ml and 500 mg /ml respectively; the MBC / MBC ratio was 4.

Conclusion: Neem oil has bacteriostatic activity on the germs Aggregatibacter actinomycetemcomitans, porphyromonas gingivalis and fusobacterium nucleatum; and would be an alternative for the management of alveolitis.

Neem essential oil; Azadirachta indica; Alveolite; Antibacterial activity

Osteitis is an inflammatory bone disease of bacterial infectious origin [1]. It can occur a few days after a tooth extraction, following a fracture with bone clearance or a severe periodontal infection leading to loss of attachment [2,3]. Alveolitis is a micro osteitis of the alveolar rim that can spread, characterised by severe, acute, radiating pain that is resistant to painkillers and requires appropriate and effective treatment [2]. Clinically, two forms of alveolitis are described: dry and suppurated [1,2]. The microorganisms incriminated are Gram+ (Streptococcus, staphylococcus, enterobacilli), Gram-pycyanic (Pseudomonas aeruginosa) and anaerobic (Fusobacterium nucleatum, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans) [2-4]. The diagnosis of alveolitis is clinical and the therapeutic means used are medico-surgical [2,3]. The most commonly used antibiotics are betalactam antibiotics, macrolides, cyclines and imidazoles [2,3]. However, the massive and repeated use of antibiotics generates an increase in bacterial resistance over time [5]. Today, this multi-microbial resistance poses major problems in a context such as ours where conventional antibiotic therapy used after exodontics may prove ineffective. In fact, very few antimicrobial agents remain effective against some multi-resistant microbes. Scientists are therefore looking for new antimicrobial products, including phytochemical compounds isolated from plants used in traditional medicine. Among them, Azadirachta Indica, commonly known as “Neem”, is known as a traditional medicine plant in many rural areas of Asia and Africa for its antiseptic, anti-inflammatory, hypoglycaemic, anti-ulcer, antibacterial, anti-malarial, and antiviral properties [8,9]. Used for aesthetic needs in the north of Cameroon, Neem oil can cause undesirable reactions [10]. Few studies have been conducted on the antibacterial activity of Neem in Cameroon. This work has made it possible to highlight its antibacterial effect in vitro on the periodontopathogenic bacteria of the essential oil of Azadirachta indica.

This was an experimental study conducted over a 7-month period from November 2018 to May 2019. The chemical screening of Neem oil from Faro in northern Cameroon (Figure 1) was carried out at the bio-analytical laboratory of the Scientific and Industrial Research Centre (CSIR) in Pretoria, South Africa. Patient samples were taken at the stomatology Department of the Centre Yaoundé University teaching hospital (CHUY). The evaluation of the antibacterial activity of Neem oil was carried out at the microbiology laboratory of the CHUY.

Included in the study were all consenting patients 18 years and above with aggressive periodontitis with periodontal pockets greater than 5 mm, located on a tooth with a degree 2 mobility. The patient had to have been free of periodontal treatment and topical or systemic antibiotics for at least six months.

The plant material was Azadirachta indica oil obtained by first cold-pressing seeds collected in the locality of Faro in northern Cameroon (Figure 2). The material for oil extraction was the oil press, model NF 500. One hundred mililitres (100 ml) of Neem oil was conditioned (dried on anhydrous sodium sulphate and stored at 4°C) and sent to the bioanalysis laboratory of CSIR for chemical characterization.

The microbiological material consisted of bacterial strains from the different cultures taken from the samples taken from patients meeting the inclusion criteria; and material for use in the microbiology laboratory. The culture media were chocolate + polyvitex agar, blood + vancomycin + Amicacycin agar, methylene blue eosin agar, chocolate + polyvitex + vancomycin + Amicacycin agar.

Procedure for the evaluation of antimicrobial activity

Samples were taken two hours before feeding and brushing the patient’s teeth. The choice of sampling site was made after evaluation of the clinical parameters of periodontal disease (bleeding on probing, gingival oedema, loss of attachments, pocket depth) [11]. Samples were taken quadrant by quadrant and pool by pool and placed in the same transport medium. The technique used was paper-tip sampling according to WOLF 2005 [11]. The supragingival plaque was first removed using a sterile curette, compress and sterile saline; then the specimen sites were isolated from the saliva with saliva cotton rolls and air-dried; afterwards, a sterile paper spike, grasped with a sterile tweezers, was inserted into the pouch until resistance was felt ; 10 to 20 seconds after the tip was removed, making sure that it was not in contact with the mucous membrane or saliva; finally, the tip was immediately inserted into a sterile bottle containing 1ml of heart-brain broth and transported directly to the bacteriology laboratory (Figure 3).

Bacteria culture

The samples were homogenised using a vortex at maximum power and then incubated for 15 minutes. The culture media were previously prepared according to the standards recommended by the manufacturers. After incubation, the bacterial inoculum was taken with a propette and spread using a sterile platinum handle according to the quadrant method and exhausted. The Petri dishes were placed in a transparent jar, a Genbox (anaerobiosis generators) bag was introduced into it to create anaerobic conditions at 37°c. Then, all this was incubated for 5 to 10 days.

Method for evaluating the antibacterial activity of Neem essential oil

Preparation of the bacterial inoculum: From a pure culture of the isolated and transplanted bacteria with a maximum of 24 hours, three to five colonies of the same morphological type and perfectly identical were scraped off using a platinum loop; the contents of this loop were then discharged into 5ml of sterile physiological saline and then homogenised in order to obtain a turbidity equivalent to that of the 0.5 standard of the Mc Farland range corresponding to 1 - 2 x 108 CFU/ml then diluted to 1/100th. The inoculum was sown in less than 15 min after its preparation. 

Sensitivity test: Agar disc diffusion method: To test the sensitivity of the bacteria, the principle used was that of the antibiogram [13]. A volume of 15 µL of Neem oil supplemented with 1% Tween 80 was introduced into a tube and homogenised by vortexing. Sterile discs of 6 mm diameter cut from Whatman No. 1 paper were then inserted. Once the discs were impregnated with Neem oil, they were removed and gently placed on the surface of a Mueller Hinton agar previously swabbed with the bacterial inoculum. At the same time, positive control antibiotic discs (Amoxicillin + Clavulanic Acid 30 μg, metronidazol 4 μg) were also deposited on the surface of the agar. The plates were then incubated for 24 hours under strict anaerobiosis. On leaving the incubator, the discs were surrounded by circular inhibition zones corresponding to an absence of culture, which allowed us to measure the inhibition diameters using a calliper. This was done for each bacterial strain three times during the three experiments.

The determination of the diameter of the inhibition zone allowed an estimation of a clinical categorisation (S, I, R) of the strain, based on the correlation between the results obtained by the diffusion method and the reference method [13]. Oil sensitivity is classified according to the diameter of the inhibition zones as follows: Non-sensitive (-) for diameter less than 8 mm; Sensitive (+) for diameter between 8-13.9 mm; Very sensitive (+) for diameter between 14-19 mm; Extremely sensitive (+++) for diameter greater than 19 mm. This sensitivity test provides qualitative results. The experiment was repeated three times.

Determination of minimum bactericidal concentrations (MBC): The Minimum Bactericidal Concentration (MBC) is the lowest concentration of substance capable of killing more than 99.9% of initial bacterial inoculum (i.e. less than 0.11% survivors) after 24 hours incubation at 37°C [14]. Thus, their determination is based on subculture from the MIC on an agar medium.

For each of the tubes devoid of bacterial pellets (visible growth not observed) and positive control of the concentration range achieved for the MIC determination, we performed streak plating on Mueller Hinton agar in Petri dishes. The Petri dishes were incubated for 24 hours at 37°C. The BMC of Neem oil is inferred from the lowest concentration at which no culture is observed on Mueller Hinton agar [13]. This procedure was repeated three times for each bacterium.

CMB/CMI ratio: The CMB/CMI ratio made it possible to define the bacteriostatic or bactericidal character of our Neem oil. An essential oil is said to be bacteriostatic when this ratio is greater than or equal to 4 and bactericidal when this ratio is less than 4 [13].

The mechanical pressure of 1000g of Azadirachta indica seeds allowed us to obtain 186g of oil, a yield of 18.6%.

Macroscopic evaluation of Neem oil showed the brown colour, characteristic pungent garlic-repellent odour, viscous consistency, with a density of 37.30-0.85 at 20°C and a refractive index of 30.6621-1.6331 at 40°C. Its iodine value was 447.0 -81.0, its Azadiratchine content by HPLC (A+B) was 110-247 PPM and its sap content 168-221 (Figure 4).

Chemical characteristics of the essential oil of Azadirachta indica

The centesimal fatty acid composition of Azadirachta indica oil has highlighted its richness in acids, of which oleic acid is the predominant one with an average content of 42.5%, followed respectively by palmitic acid with a value of 22-36%, stearic acid of 13-22%, linoleic acid of 18% and palmitoleic acid of 13%.

The volatile compound composition of Azadirachta indica oil by Gas Chromatography coupled with Mass Spectrometry revealed the presence of 17 compounds. The results show that the most important compounds are respectively Lineoleoyl chloride 10.24%, Methyl petroselinate 10.23%, Phytol 9.6%, Hentriacontane 8.8%, 7-hexyl Eicosane 8.1% (Table 1)

Bacterial strain identifications [12]

After 7 days, a morphological identification oriented by the appearance of the colonies was made as well as the staining of control Gram, then a selective transplanting with the isolated bacteria to obtain only pure cultures. The Gram test allowed us to differentiate Gram positive from Gram negative. The transplanted bacteria were used for biochemical and enzymatic identification tests.

•  Actinobacillus actinomycetemcomitans

It is a Gram-negative, capnophilic, non-mobile coccobacillus that adheres strongly to the agar. Biochemical tests: positive catalysis, negative oxidase, negative indole. 

• Porphyromonas gingivalis

Small size bacilli, gram-negative, strict anaerobic non-sporing and immobile colony of 1 to 3mm, round, creamy, irregular edges, smooth, hydrophobic, haemolytic and brown; catalase-negative and oxidase-negative.

• Fusobacterium nucleatum

Long bacillus, Gram negative, strict anaerobic. Catalase negative, oxidase negative, indole positive.

Biochemical tests were done, strain 1 had the enzyme catalase positive, there was no colour shift to purple, so all isolates are oxidase-free. Indole production has been demonstrated for some bacteria, notably strains 2 and 3. Strain 2 was sensitive to vancomycin unlike strain 3. All these characteristics enabled us to identify three bacterial strains, namely Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum (Table 2).

In vitro evaluation of the antibacterial activity of Azadirachta indica oil

Azadirachta indica oil inhibition zone diameters: The three identified bacterial strains were tested for sensitivity to control antibiotics and Neem oil. All bacterial strains tested were found to be sensitive to our Neem oil. We obtained diameters of 14.9 ± 0.26mm Actinobacillus actinomycetemcomitans, Fusobactrium nucleatum 12.3±0.81mm and Porphyromonas gingivalis 11.3 ± 0.51mm. 

All these bacterial strains were also sensitive to amoxicillin clavulanic acid. However, apart from Fusobactrium nucleatum which was sensitive to metronidazole with a diameter of 26.7mm the other bacterial strains showed resistance to metronidazole (Table 3).

Minimum inhibitory and bactericidal concentration of Neem oil: The minimum inhibitory and minimum bactericidal concentrations and their MBC/MIC ratio of Neem oil are shown in Table 3. The MIC for all three strains was 125mg/ml and the MBC 500mg/ml. The MIC/MBC ratio gave a value of 4.

The present work has made it possible to evaluate the antibacterial activity of Azadirachta indica oil on bacteria responsible for periodontal pathologies. The aim of this study was to investigate whether Neem essential oil could be an alternative for the effective treatment of alveolitis. Volatile compounds such as Lineoleoyl chloride and many others, saturated and unsaturated fatty acids were identified by the GPC of the oil. Three bacteria were identified, namely Aggregatibacter actinomycetemcomitans, porphyromonas gingivalis and fusobacterium nucleatum with diameters of 14.9 mm; 12.3 mm and 11.3 mm sensitivity respectively. The ratio CMB to CMI was 4, which showed that Neem oil had antibacterial bacteriostatic properties.

An extraction yield of 18.6% of Azadirachta indica oil was obtained by cold mechanical pressing without using chemicals that could alter the oil. Extraction by cold mechanical pressure avoids any alteration of the oil. This result is similar to that obtained by Sagoua et al. in 2009 [15] in Senegal, which had a yield of 18.3%. Chemical screening of Azadirachta indica oil revealed a light brown colour, a pungent garlic smell, the presence of fatty acids with a level of 42.5% oleic acid, 13% for palmitoleic acid. This result differs from that obtained in Senegal in 2009 by Sagoua et al. [15] which had a light green oil. The GPC also showed many volatile compounds with high content of Lineoleoyl chloride 10.24%, Methyl petroselinate 10.23%, Phytol 9.6%, Hentriacontane 8.8%, 7-hexyl Eicosane 8.1% and many other compounds that were poorly represented with percentages below 3% . The presence of volatile compounds would be at the origin of the antibacterial activity of this Neem oil. However, the volatile compounds in our oil are different from those found by Fokunang Charles et al, in 2019 in Cameroon who found a predominance of methyl14methylpentadecanoate (38.12%) followed by m-toluylaldehyde (22.76%), and hentriacontane (13.98%) [16]. This could be explained by the difference in the location where the Neem seeds are harvested. The Neem oil used in this study was harvested in the locality of Faro in the northern region of Cameroon while the Neem oil used by Fokunang et al. [16] in 2019 came from the commercial centre of Maroua [16].

The search for bacteria after successive cultures and transplanting has made it possible to identify three bacterial strains. This result is different from that found by Lakhssassi et al. [17] in 2005 in Toulouse who found more than ten bacterial strains. This difference could be justified by the use of culture media specific to certain bacteria. The bacteria identified on the basis of cultural and biochemical characteristics corresponded to Actinobacillus actinomycetemcomitans, Fusobactérium nucleatum and porphyromonas gingivalis. This result is in agreement with the one found by Criton in 2007 at the Nancy academy [12].

All these isolated bacterial strains were susceptible to Neem oil to varying degrees. Thus, the determination of the inhibition diameter gave us a result of 12.3 mm for Fusobacterium nucleatum. This diameter is smaller than that found by Packia Lekshmi et al. [18] in 2012 in India who had obtained a diameter of 16mm. This difference could be explained by the fact that they had used the Soxhlet extraction method using chloroform and ethanol as solvent. However, the resulting diameter shows that Fusobacterium nucleatum is sensitive to Neem oil. Actinobacillus actinomycetemcomitans showed an inhibition diameter of 14.9mm and Porphyromonas gingivalis showed 11.3mm. According to Moreira’s classification [19] both strains are susceptible to Faro Neem oil.

In a nutshell, neem oil contains volatile compounds such as Lineoleoyl chloride, Methyl petroselinate, Phytol, Hentriacontane,7-hexyl Eicosane and fatty acids which are believed to be responsible for its bacterial growth inhibiting properties of the strains Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum tested. Neem du Faro oil showed bacteriostatic antibacterial activity on periodontal bacteria. It could therefore be a good alternative in the treatment of post-extraction alveolitis in order to reduce bacterial resistance due to antibiotics. However, it seems important to evaluate the acute and sub-acute toxicities of  Neem essential oil in order to secure its use and to formulate an Improved Traditional Medicine.

This research has not received any grant from any international or public organisation.

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