An In Vitro Evaluation of Antibacterial Efficacy of Black Pepper Against Periodontal Pathogens

Introduction

Periodontal disease is a chronic condition that starts with gingival inflammation and then progresses towards soft tissue and hard tissue destruction, ultimately resulting in tooth loss.¹ The primary cause of periodontal disease is the microbial invasion of the periodontal tissues, with diverse microorganisms linked to its development. Species such as Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Actinomyces viscosus, and Streptococcus oralis are particularly associated with periodontal diseases.² Periodontal disease has traditionally been treated with methods that disrupt these microbial flora, including mechanical debridement or the use of antimicrobial treatments.

Due to the rise in bacterial resistance to antibiotics, there is growing interest in finding new antimicrobials for infection treatment. Numerous plant extracts have been investigated for their potential as antibacterial agents.³ Black pepper extract is one such agent. Black pepper is a flowering vine of the Piperaceae family. It is native to India and has been prized as a spice since ancient times. Black pepper has many medicinal properties, like being used to treat vertigo, asthma, chronic indigestion, colon toxins, obesity, sinusitis, arthritic disorders, and cholera.⁴ However, there is no specific data on the effect of black pepper on periodontal pathogens. Hence, the current study was done to assess the inhibitory effect of black pepper on the periodontal pathogens, A. actinomycetemcomitans (Aa), P. gingivalis (Pg), A. viscosus, and S. oralis.

Materials and Methods

The research was approved by the Institutional Review Board. The test agent used in our study was a solvent (ethanol) extract of the commercially obtained dried, unripe berries of black pepper. Stock cultures of periodontal pathogens (A. viscosus, S. oralis, A. actinomycetemcomitans, and P. gingivalis) were obtained from a central research laboratory.

To determine the minimal inhibitory concentration of the ethanol extract of black pepper, the serial broth dilution method was used.

Materials

1. Himedia M210-500G

2. Brain heart infusion broth (BHI) 500 g. Ingredients: Calf brain infusion 200 g/litre, beef heart infusion

250 g/litre, proteose peptone 10 g/litre, dextrose 2 g/litre, sodium chloride 5 g/litre, disodium phosphate 2.5 g/litre, final pH (at 25ºC): 7.4+/- 0.2

Procedure

Minimum Inhibitory Concentration (MIC) Test (Aerobic)

1) Nine dilutions of each extract were done with BHI for MIC.

2) In the first tube, 20 microliters of extract was added to 380 microliters of BHI broth.

3) For dilutions, 200 microliters of BHI broth was added separately to each of the nine tubes.

4) Then, 200 microliters of BHI broth was transferred from the original tube to the first tube. This was regarded as a dilution of 10-1.

5) To create a 10-2 dilution, 200 microliters of the contents of 10-1 diluted tube was added to the second tube.

6) For each extract, the serial dilution process was repeated up to 10-9 dilution.

7) Five microliters of the aliquoted stock cultures of the required microorganisms were taken and added to two millilitres of BHI (brain heart infusion) broth.

8) 200 microliters of the aforementioned culture suspension was added to each of the serially diluted tubes.

9) After a 24-hour incubation period, the tubes were checked for turbidity.

MIC Test (Anaerobes)

1. For MIC, each extract had to be diluted nine times in Thioglycollate broth.

2. To the 380 microliters of Thioglycollate broth in the initial tube, 20 microliters of drug was added.

3. Each of the subsequent nine tubes received Thioglycollate broth (200 microliters) separately for dilutions.

4. Next, 200 microliters of the initial tube's contents were shifted to the first tube's Thioglycollate broth (200 microliters). This was regarded as dilution 10-1.

5. To create a 10-2 dilution, 200 microliters of solution from the tube with a 10-1 dilution was added to the second tube.

6. For each drug, the serial dilution was continued to achieve a 10–9 dilution.

7. Five microliters were taken from the required organism-maintained stock cultures and added to two millilitres of Thioglycollate broth.

8. 200 microliters of the above culture suspension was added to each of the serially diluted tubes.

9. The tubes were incubated for 48-72 hours in an anaerobic jar at 37°C and examined for turbidity.

To determine the MIC values, after incubation, a visual inspection was carried out. It was clear that the turbidity in the MIC tube suggested the growth of bacteria resistant to the ethanolic extract of black pepper. The MIC-sensitive tubes from the dilution tubes were plated, and after incubating for 24 hours, the colony count was recorded. MBC was used to determine whether the extract had a bactericidal or bacteriostatic impact on the chosen species. It was thought to have a bactericidal effect if there was no growth. If growth occurred, it was determined to be bacteriostatic.

Data analysis

The gathered information was entered into an MS Office Excel worksheet and analysed with SPSS software version 17.0.

Results

The serial dilution of black pepper extract with the turbidity response of pathogens (Figure 1) is summarized in Table 1.

MIC results

The mean MIC value of black pepper’s ethanol extract showed sensitivity at 25 mg/mL for P. gingivalis, 6.25 mg/mL for A. actinomycetemcomitans, 12.5 mg/mL for A. viscosus, 6.25 mg/mL for S. oralis.

Minimum Bactericidal Concentration (MBC) results

A bacteriostatic effect indicates that the growth of microorganisms is inhibited, while a bactericidal effect means that microorganisms are killed, resulting in no growth (Table 2). In terms of MBC, the ethanolic extract of black pepper exhibited bactericidal activity against P. gingivalis at 25 mg/mL (Figure 2). The bacteriostatic activity (colonies were formed) was exhibited by the ethanolic extract of black pepper against A. actinomycetemcomitans, A. viscosus, and S. oralis at all concentrations (Figures 2).

Discussion

Despite the availability of a diverse range of antimicrobial agents for clinical use, the development of new ones remains crucial. Many studies are therefore focused on the discovery and development of novel antimicrobial agents.⁵ 

The combination of microorganisms and an inflammatory response is the cause of many diseases, including periodontitis, for which compounds having dual anti-inflammatory and antimicrobial activity may be desirable as therapeutic agents. Globally, plant extracts are employed for their antibacterial, antifungal, and antiviral activities.

The main component of black pepper is Piperine, which is an alkaloid responsible for antimicrobial activity. Black pepper causes alteration in membrane perme-ability, leading to leakage of intracellular materials, thus causing cell death.⁶

Aqueous decoctions of black pepper showed antibacterial activity against periodontal bacteria. The pro-inflammatory cytokines, GM-CSF, IL-6, TNF-, and IL1b were decreased by the administration of Piperine due to its anti-inflammatory action.⁷ Caryophyllene from black pepper exhibits anaesthetic activity. Piper nigrum has antipyretic activity. Black pepper also possesses anti-inflammatory activity.⁸ A previous study evaluated the effect of Piperine (Black pepper) for its antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Alternaria alternate, Aspergillus niger, Aspergillus flavus, and Fusarium oxysporum. Piperine showed antimicrobial activity against all tested bacteria, with a zone of inhibition ranging from 8 to 18 mm. The gram-positive bacteria S. aureus (18 mm) showed the maximum zone of inhibition and the minimum was against the gram- negative bacteria E. coli (8 mm). The results showed significant activity for Piperine, thus making it a natural antimicrobial agent.⁹

Black pepper, bay leaf, anise, and coriander were tested in another study for their antibacterial ability against 176 bacterial isolates from bacterial population (12 different genera). Overall, the black pepper (aqueous decoction) demonstrated 75% antibacterial activity, making it most hazardous to bacteria.¹⁰

The antibacterial efficacy of black pepper (Piper nigrum Linn.) and its method of action on several gram-positive and gram-negative bacteria were studied by Karsha and Lakshmi in 2010. The MIC in this investigation was determined to be 50-500 ppm. The outcomes showed excellent inhibition of gram-positive bacteria like Staphylococcus aureus, Bacillus cereus and Streptococcus faecalis. Pseudomonas aeruginosa, among the gram-negative bacteria, was more susceptible, followed by Salmonella typhi and Escherichia coli.¹¹

To the best of our knowledge, our study was the first in vitro study that investigated the antibacterial efficacy of black pepper against periodontal pathogens. We concentrated on determining the antibacterial activity of ethanolic extract of black pepper against P. gingivalis, A. actinomycetemcomitans, A. viscosus, and S. oralis (Two gram-positive and two gram-negative microorganisms).

Results of the present in vitro study showed the mean MIC value of black pepper to be sensitive at 25 mg/ mL for P. gingivalis, sensitive at 6.25 mg/mL for A. actinomycetemcomitans, sensitive at 12.5 mg/mL for A. viscosus, and sensitive at 6.25 mg/mL for S. oralis. Gram-positive organisms were more susceptible to black pepper at lower concentrations than gram-negative organisms.

In terms of MBC, black pepper's ethanolic extract showed bactericidal efficacy against P. gingivalis at a concentration of 25 mg/mL. Since colonies developed, the ethanolic extract of black pepper showed bacteriostatic action against A. actinomycetemco-mitans, A. viscosus, and S. oralis at all concentrations. Considering MBC results, except for P. gingivalis, the ethanolic extract of black pepper exhibited bacteriostatic activity.

Black pepper extracts (aqueous and ethanol) displayed antibacterial activity against S. aureus strain which was penicillin-G-resistant (Perez and Anesini, 1994).¹²

Piperine enhances the bioavailability of various structurally and therapeutically diverse drugs. Martins et al. (2015), in an in vitro study, showed that curcumin combined with piperine suppresses osteoclastogenesis.¹³ Also, black pepper has been tested in various clinical studies when combined with other agents.

Jayashankar et al. (2011) showed that brushing with an herbal toothpaste containing Piper nigrum, Syzyium aromaticum, and Zinziber officinale as essential elements for a period of 12 weeks showed a significant improvement in gingival bleeding, oral hygiene, salivary anaerobic bacteria count, demonstrating an overall improvement in oral hygiene.¹⁴

The subgingival irrigation with herbal extracts consisting of Punica granatum Linn. (pomegranate), Piper nigrum Linn. (Black pepper), and detoxified copper sulphate (test group) was done in a study conducted by Abullais et al. (2015). It was found to be a simple, safe, and non-invasive technique reporting no serious adverse effects. It also reduced the percentage of microorganisms in the periodontal pockets.¹⁵

Due to its multiple therapeutic properties, black pepper can be considered a safe, nontoxic, and effective alternative therapy in inflammatory conditions like periodontitis. Nonetheless, more clinical testing is required.

Conclusion

The minimum bactericidal concentration (MBC) test is used as a supplement to the minimum inhibitory concentration (MIC) test in antibacterial testing. The MBC indicates the lowest concentration of antimicrobial agent required to kill microorganisms, while the MIC test shows the lowest concentration needed to inhibit their growth. Black pepper ethanol extract demonstrated inhibitory effect against the chosen bacteria, but exhibited bactericidal activity only against P. gingivalis. This study provides significant insights into the therapeutic effectiveness of black pepper against periodontal pathogens, suggesting its potential as an alternative therapy. To corroborate this finding, nevertheless, additional clinical trials must be done. This study marks a new step in exploring black pepper as a potential antimicrobial treatment for periodontitis patients.

Ethical approval

This research was approved by the Institutional Review Board of Coorg Institute of Dental Sciences.

Source of Support

Nil

Conflicts of interest

None

Acknowledgement

The authors express heartfelt gratitude to Dr. Kishore G. Bhat (Director), Central Research Lab, Maratha Mandals Nathajirao G Halgekar Institute of Dental Sciences and Research Centre, Belagavi, for the support in conducting microbiological assays. The test agent used in this study was a solvent (Ethanol) extract of the dried unripe berries of black pepper, obtained commercially from Synthite Valley, Kerala.

a simple, safe, and non-invasive technique reporting no serious adverse effects. It also reduced the percentage of microorganisms in the periodontal pockets.¹⁵

Due to its multiple therapeutic properties, black pepper can be considered a safe, nontoxic, and effective alternative therapy in inflammatory conditions like periodontitis. Nonetheless, more clinical testing is required.

Conclusion

The minimum bactericidal concentration (MBC) test is used as a supplement to the minimum inhibitory concentration (MIC) test in antibacterial testing. The MBC indicates the lowest concentration of antimicrobial agent required to kill microorganisms, while the MIC test shows the lowest concentration needed to inhibit their growth. Black pepper ethanol extract demonstrated inhibitory effect against the chosen bacteria, but exhibited bactericidal activity only against P. gingivalis. This study provides significant insights into the therapeutic effectiveness of black pepper against periodontal pathogens, suggesting its potential as an alternative therapy. To corroborate this finding, nevertheless, additional clinical trials must be done. This study marks a new step in exploring black pepper as a potential antimicrobial treatment for periodontitis patients.

Ethical approval

This research was approved by the Institutional Review Board of Coorg Institute of Dental Sciences.

Source of Support

Nil

Conflicts of interest

None

Acknowledgement

The authors express heartfelt gratitude to Dr. Kishore G. Bhat (Director), Central Research Lab, Maratha Mandals Nathajirao G Halgekar Institute of Dental Sciences and Research Centre, Belagavi, for the support in conducting microbiological assays. The test agent used in this study was a solvent (Ethanol) extract of the dried unripe berries of black pepper, obtained commercially from Synthite Valley, Kerala.