Medicinal plants have been used in traditional medicine to treat various infectious diseases since ancient times. In recent years, Sri Lankan traditional and Ayurvedic medicine has successfully excelled in treating patients with UTIs. The present study, investigated the antimicrobial properties of ten medicinal plants used in traditional and Ayurvedic medical practices for urinary tract infections (UTIs), focusing on their effectiveness against E. coli, S. aureus, and P. aeruginosa ATCC and pathogenic strains. Nine out of the ten selected plants exhibited antimicrobial activity against six organism strains, except C. melo. This highlights the potential of these medicinal plants in treating UTIs and demonstrates the importance of further exploring their therapeutic potential.

The present study used E. coli, S. aureus and P. aeruginosa strains that cause mainly complicated UTIs. The plant mixture was tested against these organisms’ ATCC and UTI-positive pathological strains. Antimicrobial effects of plants can be vary from country to country due to soil composition. The following plants Phyllanthus emblica, Ocimum tenuiflorum, Terminalia chebula, Zingiber officinale, Tribulus terrestris, Tragia involucrate, Aerva lanata. Boerhavia diffusa and Asparagus falcatus had an active profile [26].

According to the present study, B. diffusa methanolic roots extract is the best active extract identified for S. aureus ATCC25923 strain and P. aeruginosa ATCC27853 strains. T. chebula methanolic fruit extract is the best active extract identified for the S. aureus UTI-positive strain and P. aeruginosa UTI-positive strain. Additionally, P. emblica methanolic fruit extract is the best active extract identified for both E. coli ATCC25922 and E. coli UTI-positive strains. All plants except C. melo methanolic seed extract showed antimicrobial properties against six strains. They showed evident zones of inhibition in the agar well diffusion assay. The MIC values obtained from the present study indicated that the P. emblica methanolic fruits extract was more potent against E. coli ATCC25922; B. diffusa roots extract was more potent against S. aureus ATCC25923 and P. aeruginosa ATCC27853 strains. These results have some discrepancies in initial antimicrobial screening test results (agar well diffusion test). The differences in bacterial susceptibility between ATCC strains and pathogenic strains could be due to variations in the intrinsic tolerance of microorganisms. Bacteria can become resistant via genetic mutations or horizontal gene transfer. Mobile genetic elements such as plasmids and transposons are instrumental in transferring resistance genes horizontally [27]. Globally UTIs are treated with a variety of antibiotics, in this study Gentamycin was used as a reference antibiotic.

According to the previous literature, Silva et al. [28] showed the antimicrobial effects of the C. melo aqueous seed against these pathogens but in the present study, there were no antimicrobial properties observed. The present study’s results aligned with Narayanan et al. [6] findings for the methanolic extract of A. lanata against S. aureus, and P. aeruginosa. However, we observed a reduced antibacterial effect against E.coli. Narayanan et al. [6], reported the ethanolic extract showed poor or no antimicrobial effect against all organisms, while the ethyl acetate extract showed moderate activity than the methanolic extract against all the organisms. However bacterial strains were not specified [6]. We observed a higher antimicrobial effect for B. diffusa compared to the methanolic extract of Malhothra et al. [29]. The aqueous extract showed a lesser antimicrobial effect against the selected strains and no antimicrobial effect against P. aeruginosa [29]. P. emblica antimicrobial activity is similar to previous studies and not only methanolic extracts but also ethanolic extracts and essential oils showed good antimicrobial effects against P. emblica [30, 31]. Naik et al. [32] showed a significant effect against five organisms including E. coli and P. aeruginosa but their antibacterial effect was low compared to the present study. Rehman et al. [33], T. chebula did not show any antimicrobial effect against E.coli. However, the present study, T. chebula, showed antimicrobial effect for three selected organisms and showed the highest effect against S. aureus [33]. Sharma et al. 34, also supports the present study findings. The best antimicrobial effect was found in ethanol extract of Z. officinale and T. chebula against multi-drug resistance species, thus validating our finding [34]. Similar results were seen in Batoei et al. [35], Khalid et al. [36], and Ahamed et al. [37] where they have proven the antimicrobial effects against the T. terrestris plant. In this aspect our results were slightly different; it can be due to differences in selected parts and solvents. T. involucrata roots were used in the present study and it showed a significant effect against all selected organisms. Rajkumar et al. [38] used T. involucrata methanol, ethanol, aqueous, and chloroform extracts. All the extracts except aqueous showed a significant effect against P. aeruginosa. However, they used leaves and stems as the selected parts. Petroleum ether, acetone, chloroform, and aqueous extracts of T. involucrata roots did not show any antimicrobial effects against E. coli NCIM 2065 but leaves, stems and flowers showed the antimicrobial effects against T. involucrata [39]. Evidence was limited on the antimicrobial activity of A. falcatus roots, De Zoysa et al. [26] did not find any antimicrobial activity of A. falcatus, which we dispute as there was an antibacterial effect against three selected organisms and a higher antibacterial effect against both P. aeruginosa ATCC and pathogenic strains. Minor discrepancies could be attributed to different concentrations of solvents, and parts of the plants used.

The manual MIC method may produce false positive results due to difficulty in determining the MIC value using the naked eye. Therefore, incorporating quality control measures and adopting more reliable methods for MIC assay such as ELISA can minimize errors and improve the accuracy of the results. MBC results of the present study have discrepancies with MIC values. MIC values are in lower concentrations but most of the MBC values are closer to the stock solution concentration. Proper sterilization methods such as microfilters, freeze-drying can minimize variability in results. Following a systematic approach can ensure consistency, reproducibility, and desired outcomes.

When selecting plant material, it should be appropriate for the intended purpose and have desired properties. Considering factors such as the plant’s therapeutic potential, safety, availability, and compatibility with other ingredients, all the plants must be authenticated. Correct identification ensures the use of the intended plant and prevents potential misinterpretations or adverse effects. The plant preparation should be devoid of dirt, debris, or impurities.

The plant mixture was prepared according to the antimicrobial properties of each plant material. In this process, quality control measures were used to ensure the consistency and quality of the plant mixture. Quality control tests such as chromatography or fingerprinting techniques can be employed to assess the presence of specific compounds or marker substances. Storage and preservative methods should also be considered in further studies. In the present mixture, P. emblica, T. chebula, T. terrestris, B. diffusa, T. involucrata, A. lanata, O. tenuiflorum, Z. officinale, A. falcatus were added in the weight ratio of 1:1:4:1:4:4:2:2:3. Constituents of the combined extract were also tested individually for above six bacterial strains. P. aeruginosa ATCC and P. aeruginosa UTI-positive strains showed the highest antimicrobial effect with the plant mixture. However, the herbal formula showed satisfactory inhibition zones in agar well diffusion assay with 50 mg/mL stock solution of nine herbal plants except C. melo for all strains mentioned above.

Plant toxicity studies play a crucial role in ensuring the safety of herbal medicines, evaluating potential risks associated with plant consumption, and identifying plant toxins. In toxicity assessments, traditional methods such as animal studies are commonly used. The brine shrimp lethality assay (BSLA) is a widely used and cost-effective screening tool for assessing the toxicity of various substances, including plant extracts and compounds. It is a comparatively easy and cheap method, which provides the basic information useful to extend further cytotoxicity tests. This assay utilizes the sensitivity of brine shrimp (Artemia salina) nauplii to evaluate the lethality and potential cytotoxic effects of test samples [22]. Toxicity index values (LC50) of extracts were used to find out whether the extracts were toxic or non-toxic. If the LC50 value of an extract is greater than 1000 µg/ml, that extract is considered as a non-toxic extract [21, 24]. LC50 values of all samples were lower than 1000 µg/ml and are considered toxic according to the BSLA. If LC50 of a plant is between 0 and 100 µg/ml, it is considered as a highly toxic species. T. involucrate showed the lowest LD50 value (4.967 µg/ml) and plant mixture was the second most toxic substance (8.69 µg/ml). While the BSLA offers a rapid and cost-effective screening tool, it is important to consider certain limitations. The assay’s reliance on a single species (brine shrimp) as a toxicity indicator may not fully represent the complexity of mammalian systems. Additionally, factors such as variations in the nauplii quality, temperature, and salinity can influence the assay results. Hence, the BSLA should be complemented with additional toxicity studies and mechanistic investigations to obtain a comprehensive understanding of plant toxicity.