Evaluation of antimicrobial properties of acacia catechu (Khadir): an in-vitro studyEvaluation of antimicrobial properties of acacia catechu(Khadir): an in-vitro study

Introduction

Various plants and their products have been used in traditional medicine of India, Egypt and Greece to treat oral diseases such as dental caries and periodontal diseases.¹ Ayurvedic medications, have also been used for various ailments since time immemorial. They promote healing as they have naturally occurring active ingredients which offer benefits in least harmful way against the various ailments.²

Periodontal diseases are complex diseases of gingiva and tooth supporting structures, which are specified by microbe induced inflammatory destruction of them. If not treated, they can advance to considerable soft tissue harm, bone resorption, eventually leading to tooth loss.³ As our understanding of multi causal etiology and pathogenesis is increasing, the pattern of treatment concept is also changing. Antimicrobial therapy as an adjunct to mechanical therapy has grasped the attention of clinicians in present date.⁴

The herb, Khadir (Acacia catechu) is important historically because of its medicinal properties. It belongs to the family- Fabaceae and subfamilyMimosoideae and attains its genus name, ‘Acacia’, from the Greek word ‘akis’, meaning a point. The species name originates from the word 'cutch', which means a tanning extract which is separated from its heartwood.⁵ There are many synonyms in Ayurveda for this plant like balapatra (tiny leaved) vakrakanta (due to hooked spines), dantadhavana (useful for cleaning teeth), kanthi (helpful for throat), kusthaghna (anti dermatosis) etc. Meaning of Khadir in sanskrit means that which stabilizes the body and diseases. It is also known as Katha which is a common ingredient of pan (betel leaf mixture) chewed in India after meals.⁵

There are no reports of the antimicrobial action of Acacia catechu extract against periodontal pathogens according to the author’s knowledge. So this study was aimed to evaluate the antimicrobial property of Acacia catechu bark extract against Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans.

Materials and Methods

The herbal extract was prepared in Ayurved Mahavidyalaya. A certificate of genuineness was obtained from the concerned department for the authentication of extract preparation procedure which was done in accordance with the standard principles described under Kwatha kalpana by Sharangadharacharya in his classic text Sharangadhara Samhita. The bark of Acacia catechu was obtained from local market. It was ground into fine powder and one part of this powder (Dravya churna) was mixed with sixteen parts of water in an earthen pot and boiled over medium flame to reduce the mixture to one eighth part. The mixture was cooled to room temperature and filtered thoroughly, after which it was packed and stored.

This observational, prospective, cross sectional study was conducted in the Department of Periodontics and Oral implantology. Ethical clearance was obtained from the Institutional Ethical Committee and a signed written informed consent was obtained from all the participants before recruiting them in the study. Both male and female participants aged 18 to 60 years were included in the study who were diagnosed with Generalized periodontitis, Stage III, Grade B according to proceedings of 2017 World Workshop, and had 20 remaining natural teeth. Pregnant and lactating women, participants with systemic diseases that affect the periodontium, smokers and tobacco chewers and those who were not willing for participation in the study were excluded.

Pooled subgingival plaque samples from five patients were collected from three deepest pockets in four different quadrants using curettes after which it was immediately transferred into a sterile vial containing Phosphate Buffered Saline Solution (PBSS). The samples were then sent to laboratory and processed within next 24 hours.

'The samples were first vortexed and diluted in 1:10 ratio. They were inoculated on the culture medium selected on the basis of targeted organism.

For Porphyromonas gingivalis (Pg), blood agar was used as an enriched medium. After inoculation they were incubated at 370C for 3 to 4 days in anaerobic jar. For Aggregatibacter actinomycetemcomitans (Aa), dentaid agar was used as selective medium. After inoculation they were incubated at 370C in 5 to 10% carbon dioxide jar for 3 to 4 days.

After completion of incubation period the plates were removed and colony characters of the required organism were noted and colony count was done for quantification.

Microbial growth was noted in all the inoculated samples. For MIC, 9 dilutions of the herbal extract were prepared with thioglycollate broth. In the initial tube, 20μl of the extract was added to 380μl of broth. For dilutions 200μl of broth was added into the next 9 tubes separately after which from the initial tube 200μl was transferred to the first tube containing 200μl of broth. This was considered as 10-1 dilution. From 10-1 diluted tube, 200μl was transferred to second tube to make 10-2 dilution. Serial dilution was repeated up to 10-9 dilution. From the isolated cultures of Pg and Aa, 5μl was taken and added into 2ml of broth. In each serially diluted tube 200μl of above culture suspension was added. The tubes were then incubated for 48- 72 hours in anaerobic jar at 37°C and observed for absence of turbidity.

For time kill curve, equal quantity of the broth with organism and the herbal extract were mixed and incubated in carbon dioxide jar. It was immediately plated and the time was noted as 0 min. The broth was again kept in carbon dioxide jar till next time slot. At each pre determined time interval (5 mins, 10 mins, 30 mins and 120 mins) 10µl from the tube was cultured on culture plate according to the growth requirement. After 48-72 hrs of incubation the plates were removed and the colony count was performed.

Results

Aa and Pg were detected in all the subgingival plaque samples collected and the water extract of Acacia catechu showed antimicrobial activity against both tested microbes Pg & Aa. Four samples were sensitive for undiluted Acacia extract and two samples were sensitive for dilutions upto 0.4μg/ ml when tested for Pg. All samples were sensitive for Acacia catechu extract dilutions upto 0.4 μg/ ml. One sample was sensitive for extract dilutions upto 0.2μg/ml (Table I).

Within the time intervals tested, Acacia catechu bark extract had shown bacteriostatic effect against the tested organisms as there was no 3 log 10 fold decrease in the bacterial count (Graph I and II).

Discussion:

Although herbal medicines have been in use since numerous centuries, it is in recent evidence- based era that structured and meticulous approaches to study their properties and clinical applications have been reinstalled. This has set off extensive application of these herbal extracts in almost all healthcare specialities including periodontics. These products present as an alluring alternative to presently available adjuncts to non surgical therapy. The dawn of research in this field has set in.⁴

Acacia catechu extract is having many beneficial properties such as anti-bacterial, anticancer, anti-diarrhoeal, anti-inflammatory, antimicrobial, antioxidant, antipyretic, anti-ulcer, antisecretory, hepatoprotective, hypoglycaemic etc. The chemical fractions of catechu mainly are flavanoids (catechin, epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, rocatechin, phloroglucinol, procatechuic acid, catecutannic acid, quercetin, quercitrin), alkaloids (kaempferol, dihydrokaempferol, taxifolin, afzelchin gum), glycosides (poriferasterol, poriferasterol acylglucosides), tannins (gallic acid, phlobatannins), and sugars (d-galactose, d-rhamnose and larabinose).⁵

Periodontitis is omnipresent and is an infectious disease of microbial origin. The oral microbial alcove includes more than 700 different species, with approximately 400 types found in the subgingival plaque.⁶ Aa, Tannerella forsythia and Pg have been identified as distinct periodontal pathogens in the causation of periodontal disease.³ In the present study, the antimicrobial activity of Acacia catechu extract was tested against Pg and Aa from subgingival plaque samples obtained by curette sampling which is a reliable and reproducible method.⁷ Pooled subgingival plaque samples were used in the present study as it increases the likeliness of detecting an organism.⁸

Clinical microbiology laboratories play a crucial role in the performance of antimicrobial susceptibility testing of important microbes that are associated with the disease. The aim of this testing is to assure susceptibility to drugs of choice and also the detection of drug resistance.⁹ The laboratories choose from among various manual or instrumentbased methods available for performing these susceptibility tests. These include the disk diffusion test, antibiotic gradient methods, agar dilution method, broth microdilution method and short incubation automated instrument methods.

The broth microdilution susceptibility test with commercially prepared antibiotic panels is popular test among the currently available methods.¹⁰ In the present study broth microdilution test was performed as it offers many advantages over other tests, like convenience of performing the test and identification of organism in the same tray, the generation of a quantitative result.¹⁰

In our investigation, Acacia catechu bark extract exhibited antimicrobial property at various concentrations against Pg and Aa. Lakshmi T et al had found the antimicrobial activity of Acacia catechu against Streptococcus mitis, Streptococcus sanguis and Lactobacillus acidophilus.¹¹ Similar finding was reported for grampositive organisms such as Staphylococcus aureus, Bacillus subtilis and gram-negative organisms like Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa and Candida albicans.¹² Lakshmi T et al had found the antibacterial activity of ethanolic bark extract of Acacia catechu willd at different concentrations by disc diffusion technique against gram negative bacilli, Shigella dysentriae, Escherichia coli, Klebsiella pneumonia, Vibrio cholerae, Pseudomonas aeruginosa and Staphylococcus aureus (gram positive cocci).¹³ This action might be because of the alkaloid taxifolin present in heartwood extract of Acacia catechu which is the active ingredient for its antibacterial action. It is also proclaimed to possess broad spectrum antimicrobial and antifungal activity.¹⁴

Time kill test was used in this investigation to determine the bactericidal or bacteriostatic effect of Acacia catechu bark extract. It is an important test to attain data regarding interaction between the antimicrobial agent and the microbe. It also reveals time dependent or a concentration dependent antimicrobial effect. The bactericidal activity is determined by the percentage of dead cells relative to the growth control by determination of number of living cells of each tube by agar plate counting method.¹⁵ The bactericidal activity was defined as being equal to 3log10 CFU/ml or greater reduction in the viable colony count relative to the baseline counts.¹⁵ In the present study the herbal extract exhibited bacteriostatic effect as there was no 3log 10 CFU/ml reduction in the bacterial counts within the time intervals measured. No reports have been observed till date according to authors to test the bactericidal or bacteriostatic effect of Acacia catechu extract against periodontopathogens by time kill curve. Bactericidal action of Acacia catechu can be investigated at higher concentrations of this extract. Future studies using Acacia catechu can be done invivo to check wound healing, immunomodulatory, anti inflammatory and antioxidant properties.

Conclusion

Acacia catechu bark had bacteriostatic effect and exhibited antibacterial activity against the bacterial strain tested i.e. Pg and Aa at different concentrations.