Evaluation Of Synergistic Anti-Bacterial Effect of Azadirachta Indica and Solanum Trilobatumthrough In-Vitro Studies

Pharmacognosy plays a significant role in the modern era of drug discovery, especially in the search for new antibacterial agents. The importance of pharmacognosy in the development of antibacterial compounds cannot be overstated, especially considering the global issue of antibiotic resistance. Antibiotic resistance has become one of the most pressing challenges in medicine today. With the continuous emergence of multidrug-resistant (MDR) bacteria, there is an urgent need to explore alternative treatments. This has led researchers to focus on natural compounds that have demonstrated antibacterial properties as potential candidates for therapeutic development. The role of synergy in antibacterial therapy is especially significant in addressing bacterial resistance. Synergistic antibacterial activity refers to the combined effect of two or more substances, where their interaction results in an enhanced antibacterial effect compared to when used separately. This strategy is particularly useful in combating bacteria that have developed resistance to conventional antibiotics. By combining different compounds that target multiple

bacterial pathways, synergistic therapy can enhance efficacy, reduce toxicity, and delay the development of resistance.

Mechanisms of Synergistic Antibacterial Activity

Synergistic antibacterial combinations work through various mechanisms. Some of the primary mechanisms include:

• Disruption of cell wall synthesis: Several bioactive compounds from natural sources can weaken the bacterial cell wall, which is essential for maintaining the integrity and survival of the bacteria.

• Inhibition of protein synthesis: Certain compounds target bacterial ribosomes, halting protein production, which is crucial for bacterial growth and survival.

• Disruption of nucleic acid synthesis: Other compounds interfere with bacterial DNA replication and transcription processes, preventing bacterial reproduction.

• Prevention of biofilm formation: Many bacteria form biofilms, protective layers that shield them from the effects of antibiotics. Synergistic agents can help prevent or disrupt biofilm formation, making bacteria more vulnerable to treatment. These mechanisms of action can be harnessed effectively in synergistic combinations, enhancing the overall antibacterial activity. The combination of multiple agents that target different bacterial functions increases the likelihood of a successful therapeutic outcome.

Importance of Synergistic Combinations in Antimicrobial Therapy The combination of multiple antibacterial agents holds great potential in combating bacterial infections. By using synergistic formulations, it is possible to reduce the overall dosage of each individual agent while maintaining or even increasing the therapeutic effect. This not only helps in minimizing potential side effects but also makes it less likely for bacteria to develop resistance against the combined agents. As bacterial resistance to conventional antibiotics continues to rise, synergistic antibacterial combinations represent a promising approach to overcoming this challenge. Research into the synergistic effects of different bioactive compounds, especially from natural sources, has shown that such combinations can be more effective than single-agenttreatments. These combinations may offer broader spectrum activity, targeting a range of bacterial species, including those that are resistant to multiple drugs.

Aim: To evaluate the synergistic antibacterial effect of Azadirachtaindica and Solanumtrilobatum through in-vitro studies against selected bacterial strains.

Objectives: To assess the individual and synergistic antibacterial effect of the combined extracts against bacterial strains using disc diffusion method.

MATERIALS AND METHODS:

1. Collection of plant material

 Procurement of fresh Solanum trilobatum leaves and Azadirachta indica leaves from a reliable source.

2. Identification and authentication of plant material

The collected specimens were botanically identified and authenticated by Dr.N.K. Sunil Kumar, Research officer, Department of Pharmacognosy, Siddha Central Research institute, Chennai - 600106. The samples were identified as Azadirachtin indica belongs to the family Meliaceae and Solanum trilobatum belongs to the family Solanaceae.

3. Plant profile

Azadirachta indica

Vernacular Names

Table: 1 Indian Names

Table :2 International Names

Taxonomical Classification

Table 3: Taxonomical Classification

Figure 1: Leaves of Azadirachta indica

Solanum Trilobatum

Vernacular Names

Table:4 Indian Names

Table:5 International names

Taxonomical Classification

Table 6: Taxonomical Classification

Figure 2: Leaves of Solanum trilobatum

4. Extraction of plant material

The leaves of plant were collected from a healthy tree, ensuring that only mature, disease-free leaves are selected. The leaves are washed and dried under shade to prevent photochemical degradation. The dried leaves are ground into a fine powder using a mechanical grinder. Weigh 50gm of coarsely powdered air-dried Azadirachita indica and Solanum trilobatum leaves and transferred into the beaker. It was treated with (50%) ethanoli.e 1:1 ethanol and distilled water and macerate for 48-72 hours, with occasional stirring. The extract was filtered using Whatman filter paper no.1, and concentrated under water bath at 40-45°C.

5. Phytochemical Screening

Preliminary qualitative analysis of bioactive compounds such as alkaloids, flavonoids, tannins, saponins, and phenols.

6. Antibacterial Testing

Selection of Bacterial Strains: Gram-positive and Gram-negative bacteria will be selected for testing.

Disc Diffusion Method: Individual and combined extracts will be tested for antibacterial activity.

Synergistic Effect Analysis: Combination studies of Solanum trilobatum and Azadirachta indica extracts to evaluate enhanced antibacterial activity. That can be compared with streptomycin.

Antibacterial Assay-Disc Diffusion method

Antibacterial screening

Medium: Mueller Hinton agar.

Composition of Mueller Hinton agar

1. Beef infusion – 30g/l
2. Casein and hydrolysate - 17.5g/l
3. Starch - 1.5g/l
4. Agar - 17g/l

Preparation of agar medium

3.8g of Mueller Hinton agar was weighed and dissolved in 100ml of distilled water and adjust the pH 7.3±0.2 sterilized by autoclave at 121°C for 15mins and used for the sensitivity test.

Preparation of sterile swabs

Cotton wool swab on wooden applicator or plastics were prepared and sterilized by autoclaving or dry heat (only for wooden swabs) by packing the swabs in culture tubes, papers or tins etc.,

Sterilization of forceps

Sterilize forceps by dipping in alcohol and burning off the alcohol.

Procedure

After preparing and solidifying the Mueller-Hinton Agar (MHA) plates, the bacterial culture is evenly spread across the agar surface using a sterile cotton swab. The swabbing is done in three different directions to ensure uniform distribution of the bacterial suspension. The plates are then left undisturbed for 5 minutes to allow proper absorption of the inoculum into the agar. Next, sterile filter paper discs (6 mm diameter) are impregnated with the extracts at the desired concentrations and carefully placed on the agar surface using sterile forceps. The plates are then incubated at 37°C for 18–24 hours, allowing the test compounds to diffuse into the agar and exert their antibacterial effects. After incubation, the zones of inhibition (clear areas around the discs indicating bacterial growth inhibition) are measured using a millimeter ruler or caliper. The diameter of these inhibition zones is recorded, and the antibacterial activity of the extracts is evaluated by comparing the results with the standard antibiotic.

Figure 3: Zone Of Inhibition

Invitro Antimicrobial Studies

Antibacterial Screening by Disc Diffusion Method

The ethanolic extract of azadirachita indica and solanum trilobatumwas screened against various gram-positive organisms like Staphylococcus aureus and pseudomonusauroginosa gram-negative organisms Escherichia coli. The spectrum activity was compared with streptomycin as standard.