ABSTRACT
Introduction:
Tridax procumbens has long been used as an insect deterrent, anticoagulant, and for the healing of wounds. In some areas of India, traditional healers also employ T. procumbens to treat injuries, blisters, and boils. A widespread rod-shaped, gram-negative, strictly aerobic bacteria called Pseudomonas aeruginosa can infect both plants and animals, including humans. It can be found in a variety of infections, particularly those that have a tendency to become chronic such as gingivitis and periodontitis. This study aims to assess the minimum inhibitory concentration (MIC) assay of T. procumbens stem extract-based chitosan gel against P. aeruginosa.
Materials and Methods:
The extract was produced from powdered T. procumbens stem part. To create the wound-healing gel, medium molecular weight chitosan was added to the T. procumbens stem extract to form a homogenous gel. Time-kill assay was conducted to determine the time-kill kinetics of the T. procumbens stem extract-based chitosan gel against P. aeruginosa.
Results:
The absorbance values decreased progressively over time at all concentrations (25 μL, 50 μL, and 100 μL) showing its inhibitory effect. With the increase in concentration, the inhibitory effect of T. procumbens stem extract-based chitosan gel against P. aeruginosa also increased.
Conclusion:
T. procumbens stem extract-based chitosan gel has revealed potent antimicrobial activity against P. aeruginosa and therefore the gel can be applied for efficient wound healing.
KEYWORDS: Antimicrobial activity, chitosan gel, disease, innovation, minimal inhibitory concentration, T. procumbens
INTRODUCTION
Various rural and tribal tribes in many tropical areas have long used a few native plants as remedies to alleviate illnesses or injury.[1,2,3] Tridax procumbens is a well-known traditional medicinal herb that is frequently used to treat a wide range of illnesses.[4] Studies have demonstrated the dose-dependent healing potential of T. procumbens leaf juice, with high doses inducing an inflammatory response.[1]
The plant’s pharmacological properties have been documented, and Indian traditional medicine uses it extensively to treat bronchial catarrh, diarrhea, and dysentery as well as an anticoagulant, insect repellant, and antifungal. Moreover, it stimulates hair growth and has wound-healing properties.[5,6] The plant is used for hair restoration, diarrhea, dysentery, and bronchial catarrh. The leaf extract has parasiticidal, insecticidal, and antiseptic qualities. It is employed to arrest bleeding from wounds, bruises, and cuts. The plant’s aqueous extract causes reflex tachycardia and briefly lowers blood pressure relative to normal. It is used as a herbal remedy for several conditions, including jaundice. The herb is utilized in Ayurveda and possesses hepatoprotective properties as well.[7,8]
Gram-negative, aerobic rod bacteria belonging to the Pseudomonadaceae family (a subfamily of Gammaproteobacteria) is the bacteria named Pseudomonas aeruginosa. It is frequently found in soil and water, as well as in plants and humans, much like other members of the genus. Crucially, P. aeruginosa is now recognized in clinical settings as an emerging opportunistic pathogen.[9] Its nosocomial pathogen status has been demonstrated by recent epidemiological research, especially for the strains that have developed greater resistance to antibiotics.[10] The oral cavity could be a key source of respiratory infections serving as a reservoir of P. aeruginosa, especially in the presence of poor oral hygiene and periodontal diseases.[11]
The minimum inhibitory concentration (MIC) is the lowest possible concentration of an antimicrobial substance that prevents the observable proliferation of a micro-organism following overnight incubation.[12] One of the most crucial aspects of treating chronic periodontitis is eliminating the periodontal pockets, and gram-negative, anaerobic bacteria such as P. aeruginosa are often the initial cause. These issues can therefore be avoided by using a local drug delivery system that enters the inflammation site directly, such as chitosan gel, a hydrophilic polymer derived from the alkaline deacetylation of chitin.[4]
Because of its healing qualities, T. procumbens extract has long been used to treat cutaneous wounds and injuries. To provide an efficient antibacterial agent for oral infections such as periodontitis, a new chitosan gel infused with T. procumbens stem extract was created in the current study. The present study aims to evaluate the minimal inhibitory concentration assay of T. procumbens stem extract-based chitosan gel against P. aeruginosa.
MATERIALS AND METHODS
Extract preparation
Dried T. procumbens stem parts were crushed well and ground to powder. One gram of powder was heated for 15 min in 100 mL of distilled water. After passing the extract through a 4.75 μm diameter sieve, the filtrate was boiled to concentrate it to 5 mL. Then, 1 mL of T. procumbens stem extract and 9 mL of medium molecular weight chitosan solution were combined. To eventually achieve homogeneous T. procumbens stem extract-based chitosan gel, it was then placed in a magnetic stirrer for 24 h.
Minimum inhibitory concentration
Muller–Hinton broth was prepared and 6 mL of the broth was added to each of the five test tubes. Overnight P. aeruginosa bacterial suspension was added to all five tubes at a range of 5 × 105 CFU/mL. Different concentrations of T. procumbens stem extract-based chitosan gel such as 25 μL, 50 μL, and 100 μL to three tubes. The fourth tube contains standard sodium fluoride and the fifth tube contains an aerobic suspension to serve as a positive control. Incubation is performed under aerobic conditions at 37°C for various time intervals such as 1 h, 2 h, 3 h, 4 h, and 5 h. The percentage of dead cells was then calculated at periodic intervals at 600 nm wavelength.
RESULTS
The present study evaluated the minimum inhibitory concentration (MIC) of T. procumbens stem extract-based chitosan gel against P. aeruginosa, a pathogenic bacterium. The MIC assay was conducted, and the absorbance readings were measured at 1 h, 2 h, 3 h, and 4 h to determine the inhibitory effect of the gel at different concentrations [Table 1]. At a concentration of 25 μL, the absorbance readings decreased progressively over time, indicating the potential antimicrobial activity of the gel. The absorbance values were 0.574 at 1 h, 0.376 at 2 h, 0.347 at 3 h, and 0.310 at 4 h. Similarly, at a concentration of 50 μL, the gel displayed inhibitory effects on P. aeruginosa growth. The absorbance values decreased over time and were recorded as 0.422 at 1 h, 0.376 at 2 h, 0.321 at 3 h, and 0.298 at 4 h. Furthermore, at a concentration of 100 μL, the gel exhibited significant inhibitory properties against P. aeruginosa. The absorbance values continued to decrease, indicating a higher inhibition rate. The readings were 0.356 at 1 h, 0.328 at 2 h, 0.315 at 3 h and 0.265 at 4 h. In comparison, the standard, used as a reference, displayed relatively lower inhibitory effects. The absorbance values for the standard were measured as 0.310 at 1 h, 0.201 at 2 h, 0.212 at 3 h, and 0.196 at 4 h. To validate the growth control, a positive control was included, which showed increasing absorbance values over time. The absorbance readings for the positive control were 0.583 at 1 h, 0.624 at 2 h, 0.669 at 3 h, and 0.702 at 4 h. These results indicate that the T. procumbens stem extract-based chitosan gel possesses potential inhibitory activity against P. aeruginosa at all tested concentrations (25 μL, 50 μL, and 100 μL) [Figure 1]. The decreasing absorbance values suggest the ability of the T. procumbens stem extract-based chitosan gel to inhibit bacterial growth
Table 1.
Time kill kinetic assay from 1 to 4 h of T. procumbens stem extract-based chitosan gel against P. aeruginosa
| Concentration | 1 h (absorbance) | 2 h (absorbance) | 3 h (absorbance) | 4 h (absorbance) |
|---|---|---|---|---|
| 25 μL | 0.574 | 0.376 | 0.347 | 0.310 |
| 50 μL | 0.422 | 0.376 | 0.321 | 0.298 |
| 100 μL | 0.356 | 0.328 | 0.315 | 0.265 |
| Standard | 0.310 | 0.201 | 0.212 | 0.196 |
| Positive control | 0.583 | 0.624 | 0.669 | 0.702 |
Figure 1.
MIC assay of T. procumbens stem extract-based chitosan gel against P. aeruginosa
DISCUSSION
T. procumbens is a highly valuable medicinal plant that is utilized in a variety of ayurvedic therapeutic agents. Previous literature evaluated the flower extract of methanolic extract of T. procumbens for its antibacterial activity against clinical bacterial isolates, specifically Escherichia coli and Staphylococcus aureus. The inhibitory zones identified in the disc-diffusion assay revealed that the flower extract had good antibacterial action against these bacteria. The experiment mediums were nutrient agar and Mueller–Hinton agar, with methanol employed for extraction and the extract had a minimum inhibitory concentration (MIC) of 67.3 mg/mL against E. coli and 48.2 mg/mL against S. aureus.[13] In another study, T. procumbens was shown to be effective against gram-negative water-borne bacteria. The inhibitory effect of T. procumbens can be seen due to the presence of compounds such as alkaloids, tannins, flavonoids, and saponins.[14]
The phytochemicals, antioxidants, cytotoxic, and antibacterial properties of T. procumbens leaves were examined by Syed et al.[15] Polyphenols, carbohydrates, alkaloids, and tannins were found in the leaves. Methanol, ethanol, and ethyl acetate extracts of the leaves demonstrated good antibacterial action. The present study had a few limitations such as the identification of phytocompounds in the stem part responsible for the biological activity and the mechanism by which the T. procumbens stem extract inhibits bacterial growth were not analyzed. Only the antibacterial efficacy of the test gel against particular bacteria (P. aeruginosa) was assessed in this investigation. Future studies may explore the antimicrobial efficacy against a wide variety of pathogens and other biological properties.
CONCLUSION
T. procumbens stem extract-based chitosan gel showed potent antimicrobial activity against P. aeruginosa. This shows that the gel could be a good candidate for creating new antibacterial therapies, notably against P. aeruginosa infections. More clinical studies are needed to properly understand its therapeutic potential and safety profile.
Ethical approval
Approval No. SRB/SDC/FACULTY/24/PERIO/242.
Conflicts of interest
There are no conflicts of interest.
Acknowledgment
The authors would like to thank Saveetha Dental College, Chennai, India for providing the platform and support system to carry out this work.
Funding Statement
Nil.
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