ORIGINAL RESEARCH |
https://doi.org/10.5005/jp-journals-10015-2360 |
Evaluation of Antimicrobial Activity of Nanoformulated Grape Seed Oil against Oral Microbes: An In Vitro Study
Department of Periodontics, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Chennai, Tamil Nadu, India
Corresponding Author: Jaiganesh Ramamurthy, Department of Periodontics, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences, Chennai, Tamil Nadu, India, e-mail: dr.r.jaiganesh@gmail.com
Received: 02 December 2023; Accepted: 04 January 2024 Published on: 20 February 2024
ABSTRACT
Aims and background: The aim of the current study is to assess the antimicrobial activity of the grape seed oil (GSO) gel infused with silver nanoparticle formulated under anaerobic condition against standard chlorhexidine gel.
Materials and methods: Subgingival plaque samples were obtained from individuals diagnosed with periodontitis. The plaque sample were transferred to the lab in an Eppendorf tube-containing thioglycollate broth. The samples were transferred to anaerobic chamber for incubation at room temperature for 24 hours. 20 µL of the cultured broth solution were further subcultured into the test group gel plate (GSO) and control group gel plate (chlorhexidine), the subcultured gels were of five different concentrations, namely 50, 100, 150, 200, and 250 µL. The subcultured plaque samples were transferred onto a Petri dish-containing brain heart transfusion agar. The Petri dishes were kept at 37° for 24 hours under anaerobic condition. Gas pack was placed in an anaerobic jar and evaluated after 24 hours for the number of colonies formed.
Results: From the obtained results, it was observed that the number of colonies formed at 50 µL concentration in the GSO group was 275 followed by 53 colonies at 100 µL concentration. The colony count reduced to 6, 4, and 7 at 150, 200, and 250 µL concentration, respectively. The colonies formed in the control group were 4, 2, 3, 16, and 2 at 50, 100, 150, 200, 250 µL, respectively. The control group with chlorhexidine showed less number of colonies at all five concentrations.
Conclusion: In conclusion, the findings highlight the noteworthy antimicrobial efficacy of GSO, particularly at higher concentrations, making it a promising alternative for managing periodontal disease. While chlorhexidine exhibited substantial microbial reduction even at lower concentrations, the natural extract of GSO offers the advantage of fewer side effects. This suggests a potential shift toward embracing GSO as a viable substitute for chlorhexidine gel in the pursuit of effective and minimally invasive periodontal care. Further research and clinical trials are warranted to solidify its role in enhancing dental health practices.
Clinical significance: Grape seed oil (GSO) is a natural polyphenolic compound and has less side effects and it could be a potential agent for plaque control and management of periodontal disease. Conventional antimicrobial agents like chlorhexidine have limitations like staining of teeth and taste alterations which leads to exploration of alternative biologics.
How to cite this article: Ramamurthy J. Evaluation of Antimicrobial Activity of Nanoformulated Grape Seed Oil against Oral Microbes: An In Vitro Study. World J Dent 2024;15(1):44-47.
Source of support: Nil
Conflict of interest: None
Keywords: Antimicrobial, Culture, Flavonoids, Grape seed oil, Herbal, Pathogens, Periodontal disease, Periodontitis.
INTRODUCTION
Periodontitis is a chronic inflammatory disease that alters the integrity of the tooth-supporting tissues, which include the gingiva, periodontal ligament, and alveolar bone.1 Periodontitis occurs as the inflammation reaches the periodontal ligament and alveolar bone which leads to tooth loss.2 The underlying cause of the disease is the presence of a polymicrobial biofilm that forms as plaque on the tooth surface. Periodontal pathogens generate deteriorating by-products and enzymes that dissolve the extracellular matrices as well as host cell membranes to generate nutrients for their growth and function.3
Antimicrobial drugs both systemic and in the form of local drugs have been administered and antimicrobial drugs have proved remarkably effective for the control of bacterial infections. However, some pathogens develop antibiotic resistance to many of the first-line effective drugs by protective biofilms, and horizontal gene transfer.4 Therefore, it is necessary to develop drugs that are effective against these resistant microorganisms.
Recently, in the field of drug development, the interest for herbal medicine and herbal formulations using various plant fractions and extracts has increased consistently. The reasons behind the development of herbal products were the reduction in antimicrobial drug resistance of some microbes to existing antimicrobial agents, and avoidance of unwanted side effects, and high cost of the treatments.5
Numerous techniques can be used to increase the quantities of bioactive metabolites in plants and produce chemically standardized extracts; plants are the source of hundreds of phytochemicals.6,7 Grapes are rich in nutrients and bioactive compounds and its seeds and leaves are considered to have medicinal value. The phytochemical Acylated procyanidin obtained from grape seeds is bioactive in nature.
Polyphenols are significant secondary metabolites produced by grapes and have significant antioxidant potential. They counteract the free radicals produced by the body during inflammation and stress. The health benefits of grape polyphenols include their antibacterial, anticancer, anti-inflammatory, anti-allergic, and antioxidant capabilities.8,10Vitis vinifera or grape seeds contain lipids, proteins, carbohydrates, and 5–8% polyphenols.11 Between 74 and 78% of oligomeric proanthocyanidins and about 6% of flavanol monomers are present in grape seed extracts (GSE).12,13 Anastasiadi et al.14 suggested that the antibacterial activity was attributed to significant amounts of phenolic acids and flavonoids in grape stems, and flavonoids and their derivatives in grape seeds. Ferrazzano et al.15 reported that high polyphenol content in grape seeds had anticariogenic effects against Streptococcus mutans.
Oxidative stress is the disturbance in the prooxidant and antioxidant balance, resulting in potential tissue damage. Plaque buildup allows the growth of anaerobic bacteria, which eventually leads to the recruitment and activation of neutrophils. This further results in the upregulation of pro-inflammatory cytokines and also leads to the release of neutrophilic enzymes and reactive oxygen species. Prolonged exposure of the connective tissue to these insults results in the degradation and subsequent loss of ligamentous support and alveolar bone, eventually leading to tooth loss. To combat the oxidative stress, all the cells in the body are equipped with an intrinsic store of molecules known as “antioxidants.” Antioxidants may be regarded as “those substances which when present at low concentrations, compared to those of an oxidizable substrate, will significantly delay, or inhibit oxidation of that substrate.” They function by scavenging free radicals as and when they form, thereby preventing oxidative stress.10 Since grapes have antioxidant potential, they can be used effectively in the treatment of periodontal disease. Furthermore, it has been proposed that GSE inhibits the growth of anaerobic bacteria linked to periodontal disorders, such as Porphyromonas gingivalis and Fusobacterium nucleatum. There is a growing interest to use natural extracts instead of conventional antimicrobials as they have added health benefits. Hence, the aim of the current study was to assess the antimicrobial activity of the grape seed oil (GSO) gel infused with silver nanoparticle formulated under anaerobic condition against standard chlorhexidine gel.
MATERIALS AND METHODS
The in vitro study was conducted in the Department of Nano Pharmacy Microbiology and the Department of Periodontics, Saveetha Dental College and Hospitals, Chennai, India from February to April 2023 and approved by the scientific review board and ethical committee of the Saveetha Dental College and Hospitals.
Formulation of Grape Seed Oil Gel Infused with Silver Nanoparticle
In a flask, 9 mL of distilled water were added to 1 mL of GSO which was commercially obtained from Green Leaf Organics as medical grade and these components were heated at 90°C until mild color change was noted. To 90 mL of distilled water, silver nitrate was added (1 mmol) to this mixture the grape seed solution was added and the conical flask was placed over a magnetic stirrer. The color change in the solution was noted and the centrifugation process was done for 48 hours and the solution was subjected to ultraviolet (UV) spectrometric analysis. The amount of absorbance was analyzed, once the optimum value of 650 nm was achieved, the solution was centrifuged at 8000 rpm. Since the nanoparticle base was oil-based the supernatant solution was air dried for 48 hours in a hot air oven post 48 hours the dried concentrate was subjected to transmission electron microscopy (TEM) scanning. The physicochemical properties of nanoparticles were important for their behavior, biodistribution, safety, and efficacy. Therefore, characterization of silver nanoparticles was important in order to evaluate the functional aspects of the synthesized particles. Further, the concentrate was added to Carbopol. Once the Carbopol was thoroughly dissolved in 50 mL of distilled water in a beaker, the concentrated GSO extract was added and continuously stirred until the gel consistency was reached. The prepared nanoformulated GSO gel was preserved in sterile environment.
Anaerobic Microculture
The study group was divided into test and control groups. GSO gel was considered the test group and chlorhexidine gel was considered the control group. After getting informed consent, samples of subgingival plaque were taken from the deepest pocket of five patients diagnosed with chronic periodontitis. Using Gracey curettes, the plaque was removed, and the sample was quickly placed into an Eppendorf tube filled with thioglycollate broth. The growth of the anaerobic microcolonies was encouraged by the use of anaerobic gas chambers containing anaerobic gas packs. The samples were incubated for 24 hours at 37° in an anaerobic environment. One milliliter of distilled water was used to dilute both test and control gels, into five distinct concentrations (50, 100, 150, 200, and 250 µL), as indicated. A total of 20 µL of the cultured broth was subcultured into the control group chlorhexidine gel (Hexigel) and test group gel (GSO gel) after a 24-hour period. Using an L-rod, a 20 µL individual sample was distributed into the solidified brain heart infusion agar medium on a Petri dish. The Petri dishes were incubated in an anaerobic environment for 24 hours at 37° and colony count were measured by manual counting method. The number of colonies in test and control groups was tabulated and compared in graphical data.
RESULTS
In the current study, it was observed that the number of colonies formed at 50 µL concentration in the GSO group was 275 followed by 53 colonies at 100 µL concentration. The colony count reduced to 6, 4, and 7 at 150, 200, and 250 µL concentration, respectively. The colonies formed in the control group were 4, 2, 3, 16, and 2 at 50, 100, 150, 200, and 250 µL, respectively. The control group with chlorhexidine showed less number of colonies at all five concentrations with a slight increase of 16 in 200 µL concentration which could be due to the type of microbes formed. At 150 µL, a significant reduction in microbial load was detected in the GSO group, indicating that the optimum concentration required to kill the microbes might be higher than 150 µL. Chlorhexidine showed a reduction in microbial load from the 50 µL concentration itself indicating the potent nature of the antimicrobial. The results are tabulated in Table 1 and graphically represented in Figure 1.
| Concentration of the gel | 50 µL | 100 µL | 150 µL | 200 µL | 250 µL |
|---|---|---|---|---|---|
| GSO gel | 275 | 53 | 6 | 4 | 7 |
| Chlorhexidine gel | 4 | 2 | 3 | 16 | 2 |
µL, microliters; GSO, grape seed oil
Fig. 1: The figure represents the number of colonies formed at five different concentrations between GSO group and chlorhexidine gel group
DISCUSSION
Periodontitis is a multifactorial disease in which microorganisms play an important role in initiating and progressing the disease. Numerous antimicrobials have been tested to evaluate the effect on microbes but they are not free of side effects and microbial resistance. Natural extracts having innate potential to eradicate microbes with less impact on resistance have been the subject of interest. GSO showed antimicrobial, and antioxidant effects in existing literature.16 The impact of GSO gel infused with silver nanoparticles at varying concentrations against anaerobic microbes has been assessed in this study and to check their antibacterial effect, chlorhexidine gel was taken as control. Grape seeds are considered rich source of polyphenolic compounds,17 grape seed proanthocyanidins extract, which is a rich source of several bioflavonoids, is extracted from grape seeds.18 Polyphenols are proven to have bactericidal activities against numerous pathogenic bacteria like Streptococcus mutans and Aggregatibacter and are considered the vital ingredient of GSO.19,20
Grape seed oil (GSO) gel infused with silver nanoparticle was used at various concentration against putative periodontal pathogens present in dental plaque. In the present study, plaque sample from the deepest pockets were used to assess the number of colony-forming units. It was observed that the number of colonies formed decreased gradually as the concentration of the GSO gel increased. The results are in accordance with a study conducted by Furiga et al. who reported the antibiofilm activities of extracts obtained from Vitis vinifera or grape against oral biofilms mainly composed of Streptococcus mutans, Porphyromonas gingivalis, and Fusobacterium nucleatum. Red wine extract (1.6 gm/L) enriched either with GSE (2.5 gm/100 mL) or inactive dry yeast (IDY, 0.4 gm/L) were screened against biofilms. The biofilms were treated with the extract for 2 minutes three times a day over a period of 1 week. As a result, the total bacterial count of Fusobacterium nucleatum was decreased by treatment in both groups. The same group further conducted a few other studies that used GSE as a chief constituent and assessed its antimicrobial activity. GSE (2000 μg/mL), either alone or enriched with fluoride, was incubated with biofilms for 1 minute, at 4-hour intervals. The combination of GSE and fluoride exhibited good antibiofilm properties and biofilms were inhibited by 97·4% of the insoluble glucan synthesis by glucosyltransferases.21
The concentration-dependent variation in antimicrobial effect was reported in other studies also. In a study done by Baydar et al., it was seen that the antibacterial effectiveness of the extract concentrations followed the sequence: 10% > 5% > 2.5% > 1%. Methanol was used as a control in this study and it had no inhibitory effects against the bacteria tested in their study. The GSEs at 10% concentration exhibited the most antibacterial effect against the bacteria.22 Similarly, the results of the current study showed that there was increased antibacterial activity at 100 µL concentration of the GSO gel, and the antibacterial activity increases as the concentration of the GSO increases.23 In the present study, it was seen that the colonies were higher at lower concentration of GSO gel 275 colonies at 50 µL and it reduced to 7 colonies at 250 µL. Similar results have been obtained by Swadas et al. who mentioned that when compared to the control, GSE at a lower concentration (125 µg/mL) did not significantly inhibit the growth of bacteria.24 Similarly a study by Mirkarimi et al. mentioned that GSE with lower concentration has not shown any bactericidal or bacteriostatic effects against gram-positive organism, Streptococcus mutans.25 On the other hand, Thimothe et al. proved that the phenolic chemicals in grapes suppress the biological activity of Streptococcus mutans, supporting the antibacterial efficacy of GSE at 500 and 250 µg/mL concentrations.26
Results of the prior studies show significant antibacterial activity, but periodontitis is a disease that is worsened majorly by gram-negative anaerobic bacteria, prior studies have focused on the antibacterial activity against gram-positive aerobic bacteria.27 In this study, the organisms were cultured under anaerobic condition. It was seen that GSO gel was effective against anaerobic microorganisms at higher concentrations of the gel. Chlorhexidine which is a standard has shown significant antibacterial activity at all concentration. Chlorhexidine, even though effective, has its own side effects like stain formation on the teeth and altered taste sensation. Natural extracts like GSEs with their potent photochemical compounds can be a sustainable source and alternative for the treatment of periodontal diseases. The limitations of the present study were that it did not evaluate the specific serotypes of microorganisms and that could have added more information about the antimicrobial effect of GSO. GSO gel can be used as local drug delivery for the management of periodontitis. However, animal studies and long-term clinical studies are required to validate the in vitro results.
CONCLUSION
In conclusion, the findings highlight the noteworthy antimicrobial efficacy of GSO, particularly at higher concentrations, making it a promising alternative for managing periodontal disease. While chlorhexidine exhibited substantial microbial reduction even at lower concentrations, the natural extract of GSO offers the advantage of fewer side effects. This suggests a potential shift toward embracing GSO as a viable substitute for chlorhexidine gel in the pursuit of effective and minimally invasive periodontal care. Further research and clinical trials are warranted to solidify its role in enhancing dental health practices.
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