In vitro Cytotoxicity of Selected Medicinal Plant Extracts used for the Management of Gastroenteritis in Northern Namibia, and their Antibacterial Activity against Multidrug-resistant Pathogens - Journal of Pure and Applied Microbiology (2024)

Effect of extracting solvent on the percentage yields of crude extracts
Organic and aqueous plant extracts were prepared from selected four plants using methanol and distilled water, respectively. The percent extract yield of organic and aqueous plant extracts varies between 1.0 to 13.5% (Table 1). In most cases, the amount of residue extracted with methanol was higher than that of distilled water. However, the percentage yields for L. camara organic and aqueous seeds and twigs extracts and organic and aqueous twigs extracts were equal, respectively. Seed extracts yielded higher than twigs and root extracts (Table 1). Studies have linked the percentage yield of the crude extract to its efficacy and bioprospecting properties.²⁷

Table (1):
Percent (%) yield of various organic and aqueous plant extracts

Antibacterial activity of aqueous and organic extracts
The antibacterial activity of organic and aqueous extracts of G. tenax, A. anthelmintica, T. sericea, L. camara, and C. tridens were assessed against clinical and reference strains of E. coli and Salmonella. Organic extracts showed higher and broad-spectrum antibacterial activity in comparison to aqueous extracts (Table 2 and 3). Higher antibacterial activity was observed in organic seed extracts of L. camara against both clinical and reference strains of E. coli (20.67 ± 1.53 mm and 22.00 ± 1.00 mm) and Salmonella species (21.67 ± 0.58 mm and 20.33 ± 0.58 mm), respectively (Table 3). The L. camara (seeds) and C. tridens were the only aqueous extracts that showed antibacterial activity against clinical isolates of Salmonella spp. (11.33 ± 0.58 mm) and E. coli 157:H7 VTEC (11.67 ± 0.58 mm), respectively (Table 2). T. sericea twigs and seeds, L. camara seeds, and C. tridens also showed antibacterial activity against reference strains of E. coli, with higher activity depicted by L. camara seeds (12.33 ± 0.58 mm) and C. tridens whole plant (12.00 ± 1.00 mm). Among other, aqueous extracts, T. sericea twigs, and L. camara seeds, were the only aqueous extracts that showed antibacterial activity against the reference strain of S. Typhimurium (Table 2).

Table (2):
Antibacterial activity of aqueous extracts against clinical isolates and reference strains of Salmonella spp. and E. coli

Table (3):
Antibacterial activity of organic extracts against clinical isolates and reference strains of Salmonella spp. and E. coli

The results of this study help to validate the efficacy of 18 extracts prepared from medicinal plants used as alternatives to treat gastrointestinal conditions in Northern Namibian. In this study, L. camara organic extracts displayed higher antibacterial activity against Salmonella and E. coli strains. These findings are consistent with that of Sharma et al.,²⁸ who reported significantly higher activity in extracts of L. camara and C. tridens, respectively. Moreover, other studies have been conducted globally on the efficacy of T. sericea and G. tenax against diarrheal pathogens, but none of these studies tried to compare the efficacy in clinical and reference strains. This is important because, as observed in this study, there is a significant difference in the efficacy of extracts against clinical and reference strains; hence, the use of laboratory isolates alone will not fully reflect the efficacy of the extract in clinical isolates. Although reference strains are useful in susceptibility tests, their uses should not replace clinical isolates, and this is supported by the findings observed in this study. This is important, especially when analyzing the antibacterial effects of extracts that are being considered in clinical applications.

Isaiah and Arun et al.²⁷ reported that C. tridens had antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Klebsiella aerogenes, and Escherichia coli, the findings of this study also demonstrated broad-spectrum antibacterial activity against clinical and reference strains of Salmonella and E. coli were observed in its aqueous and organic extracts. Based on statistical analysis, there was a significant difference between the antibacterial activity of G. tenax roots, A. anthelmintica twigs, and T. sericea seeds (p < 0.001) to clinical and reference strains of Salmonella. Meanwhile, G. tenax roots, A. anthelmintic twigs, A. anthelmintic roots, T. sericea seeds, and T. sericea twigs showed a significant difference in their antibacterial activity against reference and clinical isolates of E. coli.

The minimum inhibitory concentration of different extracts with antibacterial activity
Results obtained from the validation and quality control assay for MIC by resazurin were within the range of recommended MIC values of the Clinical and Laboratory Standards Institute¹⁵ as shown in Table 4. Clinical diarrheal pathogens showed higher sensitivity to organic extracts in comparison to reference strains. Among the aqueous extracts, L. camara showed the lowest MIC value of 125 µg/mL against the clinical isolate of Salmonella (Table 5). Moreover, L. camara organic seed extract showed the lowest MIC and minimum bactericidal concentration (MBC) of 31.25 µg/mL against clinical E. coli 157:H7 VTEC and reference strain of E. coli (Table 5). A. anthelmintica organic and aqueous extracts showed less sensitivity to clinical and reference strains in this study. Distilled water and 99% methanol that were used as negative controls did not show any inhibitory properties against the test organisms.

Table (4):
Determination of the MIC for ATCC Gram-negative strains for quality control purposes

While aqueous extracts are normally used in the ethnomedicinal setting to prepare decoctions for treating gastrointestinal, the findings of this study showed minimal efficacy in aqueous extracts in comparison to organic extracts. This could be because bioactive compounds extracted using water require further metabolism to perform bacterial inhibitory properties.^28,29 Moreover, while roots are reported to have potent antibacterial activity in other studies, in this study, seeds of the studied plants have shown potent antibacterial activity with significantly lower MIC values. This is important especially since according to the global conservative estimate, the current loss of plant species is between 100 and 1000 times higher than the expected natural extinction rate, and the Earth is losing at least one potential major drug every 2 years²⁹; hence, using plant parts that prevent uprooting and killing plants while still offering maximum therapeutic benefits is very important.

Sharma et al.²⁸ reported the MIC of G. tenax root extracts to be 62.5 and 125 µg/ml for aqueous and organic extracts, respectively. However, in this study, only the organic twig extract of G. tenax showed antibacterial activity with the lowest MIC of 62.5 against clinical Salmonella isolate (Table 5). Moreover, other studies have reported MIC values of L. camara than the MIC of 31.5 µg/mL that are reported in this study. Opportunistic infections such as gastroenteritis are quite inevitable among children under the age of 5 and immunocompromised patients; hence, finding alternative medicine to eliminate them is crucial and one way to mitigate the challenge of microbial resistance.^30,31 The findings of this study are the first to use a resazurin assay to evaluate MIC values of methanol and aqueous plant extracts. These findings have shown the easy reproducibility and accuracy of MIC evaluation by resazurin and agree with the findings of Elshikh et al.³¹ These findings also show the potential of the resazurin-based 96-well plate microdilution method as an interpretational tool for testing the MIC of natural products.

Table (5):
MIC of organic and aqueous plant extracts against clinical and laboratory isolates of Salmonella and E. coli by Resazurin microtiter dilution assay

C.E= Clinical E. coli; R.E= Reference E. coli; C.S= Clinical Salmonella; R.S= Reference Salmonella; MIC values are expressed in µg/mL; SEM: standard error of the mean; n = 3; (-): mean MIC above 500 µg/mL, *p < 0.05 significant between the difference in MICs different parts of the same plant

A comparison of the antibiogram activity of selected antibiotics against clinical and reference bacteria causing gastroenteritis
A higher antibiotic multidrug resistance pattern was observed among all 4 pathogens tested in this study. The reference strain of E. coli showed resistance to antibiotics such as amoxicillin 10 µg, ciprofloxacin 10 µg, tetracycline 30 µg/mL, erythromycin, penicillin, vancomycin, and ampicillin (Table 6). Among the 10 antibiotics used in the antigram assay, clinical E. coli 157:H7 VTEC has shown resistance to 70% with sensitivity only observed in tetracycline (20.33 ± 0.58 mm), gentamycin (10.00 ± 0.00 mm), and ciprofloxacin (15 ± 0.00 mm) (Table 6). Meanwhile, the S. Typhimurium reference strain showed resistance to 50% of the standard antibiotics. Furthermore, a 70% resistance was also observed in clinical isolates of Salmonella with sensitivity only observed in ciprofloxacin (10 ± 0.00 mm), gentamicin (9 ± 0.00 mm), and tetracycline (18 ± 0.00 mm) (Table 6). Overall, the clinical isolates of Salmonella and E. coli 157:H7 VTEC showed the highest resistance pattern with activity only observed in 3/10 antibiotics tested in comparison to the reference strains. This agrees with the findings of the lowest MIC value for tetracycline that was used as a positive control in the MIC test was 2.0 ± 0.00 µg/mL (Table 2).

Table (6):
Antigram activity of standard antibiotics against clinical and reference strains of Salmonella spp. and E. coli spp.

In Namibia, morbidity, and mortality due to drug-resistant Gram-negative gastrointestinal pathogens are of great concern due to the lack of surveillance data for antibiotics. Moreover, antimicrobials prescription without following the Namibian treatment guidelines for local and regional antimicrobial sensitivity data is also reported in Namibia.³² Although antibiotics such as amoxicillin, sulphathiazole, ciprofloxacin, and ampicillin are used in Namibia as first-line drugs to treat E. coli 157:H7 VTEC and Salmonella infection, their uses may not always be effective due to the higher resistance pattern observed in this study. Comparative studies looking at the effectiveness of medicinal plants and antibiotics against both clinical isolates and reference strains are therefore important as they help to eliminate the limitation possessed using only reference strains in drug development. Antibiotic resistance develops as an evolutionary response; hence, it must be visualized as an evolving phenomenon that demands constant surveillance and continuous efforts to identify the degree of resistance to available antibiotics while creating strategies to combat this problem.^33,34 The higher antibiotic resistance pattern against 10 antidiarrheal antibiotics, especially against clinical isolate reported in this study, is alarming and this makes the plant extracts especially organic extracts of L. camara, T. sericea, G. tenax, and C. tridens with broad-spectrum and potent inhibitory activity against MDR gastrointestinal pathogens worth further analysis in formulating alternative plant-based medicine for gastroenteritis however the toxicity profile of these extracts have to be determined.

Cytotoxicity effect against NIH/3T3 cell line
Making assumptions that plant-based extracts are safe due to their long history of use could be misleading and dangerous. Hence, cell-based assays are often used for screening novel formulations to determine, if the test molecules are having direct cytotoxic effects. MTT assay was used in the present study. With this assay, viable cells with active metabolism can convert MTT into a purple-colored formazan with a maximum absorbance of 590 nm. When cells die, they lose the ability to convert MTT into formazan; thus, color formation is the marker of only viable cells.^35,36

The cytotoxic activities of the methanol extracts of L. camara, C. tridens, G. tenax, and T. sericea were determined at various concentrations on 3T3 cells. Using the absorbance from the microplate reader values of percentage cell viability of the 3T3 cells at different concentrations of the extracts after 48 hours’ incubation are shown in Figure 1. The values for cell viability with methanol extracts at the highest concentration of 100 µg/mL (Figure 1) were 38.78% for G. tenax, 63.08% for T. sericea, and 50.52% for C. tridens, and 51.68% for L. camara. These values were higher than the recorded 29.81% cell viability of the negative control (10% triton-x + media) which is known to kill cells by lysing the cell membrane and solubilizing mitochondrial membranes releasing mitochondrial matrix proteins. At the lowest concentration of 1.56 µg/mL for all extracts, the percentage viability was between 80.05 ± 0.06% and 97.63 ± 0.14%.

This study is the first to demonstrate the effect of L. camara, C. tridens, G. tenax, and T. sericea methanol extracts on 3T3 cell lines. The results showed that all L. camara, C. tridens, G. tenax, and T. sericea extracts showed a decrease in cell viability of the 3T3 cell line as the concentration increased. In this study, a threshold of the cytotoxic concentration (IC₅₀) below

20 µg/mL was considered to be toxic, and above 20 µg/mL to be non-toxic.^37-39 A study by Koley and Bard,⁴⁰ on the effect of Triton-x on HeLa, has also demonstrated toxicity due to changes in cell morphology, membrane permeability, and viability caused by the presence of Triton-x. Among the four studied extracts, G. tenax showed the highest average half-maximal inhibitory concentration (IC₅₀) values of 91.50 ± 0.14 µg/mL after 48 hours. Meanwhile, the T. sericea extract showed a lower IC₅₀ of 65.85 ± 0.07 µg/mL as depicted in Figure 2. It is important to note that while L. camara is used in Namibia in the traditional setting to treat different diseases, other studies such as Pour et al.²⁵ have reported on the toxicity of L. camara in livestock, because it contains secondary metabolites such as naphthoquinones, irdoid, glycosides, and the most poisonous lantadenes. Lantadenes are pentacyclic triterpenes that are also reported to cause hepatotoxicity and jaundice, as well as photosensitization. However, since the IC₅₀ of all the extracts is above 20 µg/mL which was set as the threshold for cytotoxicity, the extracts are considered not toxic.