Preliminary Study on the Antibacterial Activities and Antibacterial Guided Fractionation of Some Common Medicinal Plants Practices in Itum Bahal, Kathmandu Valley of Nepal

The aim of this study was to evaluate the antibacterial activities of selected medicinal plant practices by a traditional healer of the Newar community in Itum Bahal, Kathmandu, Nepal. The antibacterial activities of the methanolic extract (1 mg/disc) of fifteen medicinal plants were screened against two Gram-positive bacteria (Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633) and two Gram-negative bacteria (Escherichia coli ATCC 25922 and Salmonella typhi CCM 5445) using the disc diffusion method. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were calculated for the different fractions (hexane, chloroform, ethyl acetate, acetonitrile, and acetone) of the plants having a significant antimicrobial effect. Only Quercus infectoria G. Olivier (galls) and Mallotus repandus (Willd.) Müll.Arg. (seeds) exhibited prominent antibacterial effects. The acetone fraction from Q. infectoria had the strongest antibacterial effect, with a 41.00 mm inhibition zone against S. aureus. In contrast, the ethyl acetate fraction in M. repandus exhibited the highest efficacy, producing a 29.00 mm inhibition zone against S. typhi. In a similar manner, in the case of Q. infectoria, the acetoe fraction depicted the lowest MIC (0.19 mg/mL) and MBC (0.98 mg/mL) values against S. aureus, whereas the ethyl acetate fraction of M. repandus was most significant, showing the lowest MIC and MBC of 0.25 and 0.53 mg/mL, respectively, against S. typhi. This study suggested that the acetone extract of Q. infectoria galls can be used as a potential source against Gram-positive bacteria, whereas the ethyl acetate extract of M. repandus seeds could serve as a useful source to inhibit Gram-negative bacteria. Furthermore, extensive scientific investigation is mandatory to ensure the proper use of folk medicines.


Introduction
Conventional utilization of plants for remedial purposes anticipates a basis for the employment of particular plants for specifc medicinal conditions [1,2].Herbal medicine carries on furnishing health treatment for over 80% of the total population in the world, largely in developing countries [3][4][5][6].Terapeutic properties of plant materials have been documented and substantiated experimentally in diverse cultures since ancient times [7,8].Early civilizations such as Indian, Chinese, and the Middle East have recorded the medicinal values of thousands of plants.In recent times, numerous industries beyond the realm of food and medicine have shown increasing interest in exploring the potential of medicinal plants.Extensive research is being conducted to investigate the properties of these plants in various domains, focusing on their active ingredients for applications such as antioxidants, phenolic content, anticancer properties, DNA protection, antiproliferative efects, and antimicrobial activities.Tese studies aim at uncovering the valuable properties of medicinal plants that extend beyond their traditional therapeutic uses, opening up new possibilities and applications in diverse sectors [9][10][11][12].Nepal is a prosperous country in the biodiversity of medicinal plants.Approximately, 7000 species of medicinally signifcant plants have been discovered all around the world.In Nepal, 6,653 species of angiosperm plants have been documented.Among these, about 25%-50% are ethnomedicinally useful.Around 1,792-2,331 benefcial aromatic and medicinal plants are recorded in catalogs.Woefully, just a few of them are being practiced for their therapeutic value [13][14][15][16].Herbal medicine represents one of the most important felds of traditional medicine in Nepal especially in rural areas [17][18][19].A large number of traditional healers have been playing a pivotal role in narrowing the gap between rural and urban Nepal as they are more accessible than doctors having training in Western medicine [20,21].Te present paper deals with the investigation of the antibacterial potency of 15 diferent medicinal plants that have been used by the Newar community traditional healers of Itum Bahal, Kathmandu, Nepal, for the treatment of diferent kinds of diseases.
Even with substantial advancement in scientifc comprehension and medical technology, diseases caused by microbial infection persist as a prime cause of worldwide mortality and morbidity [22,23].Although deaths from bacterial infection have dropped in the developed countries, it is still a major cause of death in the developing world.According to WHO, 1.9 million children died worldwide due to respiratory infections with 70% of these deaths occurring in Africa and Asia in 2000 [24].In developed countries, food poisoning caused by the virulent strain of E. coli is still the major cause of death every year.Te exploration of antibiotics has suppressed the microbial infection that once devastated the humankind.Unfortunately, the irrational consumption of antimicrobial drugs for the treatment of infectious diseases has resulted in the development of multidrug-resistant microorganism strains [25][26][27][28].Unavailability of everlasting efective drugs and the exorbitant price of new generation antibiotics have resulted in an increased rate of mortality and morbidity accompanying the development of uncertainty regarding the prospects of antimicrobial drug use [29].Tis catastrophic circumstance has impelled us to investigate more successful antimicrobial agents utilizing plant material so that it will provide active therapeutic ingredients as well as lead molecules for the synthesis of perfect novel drugs [30].

Materials and Methods
2.1.Plant Material.Dried raw material (not less than 500 g) of each selected plant variety (Table 1 and Figure 1) was collected from the Itum Bahal market of Nepal where a traditional healer practices all the collected plants for several cases of upper respiratory tract and lower urinary tract infection.Te scientifc name of all the materials was identifed from their local name with the help of a botanist (Homnath Pathak from Prithvi Narayan Campus, Nepal) and also compared with the literature.Te voucher specimens were carefully labeled and safely stored in the herbarium unit of Pokhara University, Kaski, Nepal.Te corresponding voucher numbers for all the specimens are given in Table 1.

Preparation of Extracts.
Cleaned shade-dried plant materials were comminuted into fne powders.Each dried sample (100 g) was extracted by double maceration using 500 mL methanol (MeOH) at room temperature for 48 hours.Te MeOH extracts were evaporated to dryness using a rotatory vacuum evaporator at 40 °C and stored in the refrigerator at a temperature of 4 °C until use.

2.3.
Antibacterial Bioassay-Guided Fractionation.Initially, methanolic extracts of all 15 samples were screened for the zone of inhibition shown by each extract.However, only Q. infectoria and M. repandus methanolic extract showed the measurable zone of inhibition.Hence, the methanolic extract of only these two plants were further partitioned with hexane, chloroform, dichloromethane, ethyl acetate (EtOAc), acetone, acetonitrile, and ethanol (EtOH) sequentially as shown in Figure 2.Each fraction thus obtained, including the fnal EtOH fraction, was evaporated to dryness and subjected to antibacterial activity.25% v/v of dimethyl sulfoxide (DMSO) was used as a solvent system.A stock solution of a concentration of 100 mg/mL was prepared for each extract.A sterile paper disc (6 mm diameter) was impregnated with 10 μL of stock and allowed to dry at 37 °C for 24 hours to give a fnal amount of 1 mg plant extract per disc.Amoxicillin 30 μg/disc and 2

Antimicrobial Screening by the Disc Difusion Method.
Preliminary antimicrobial screening for the methanolic extract of 15 diferent plants was carried out according to the disc difusion method described in a previous study.Te Mueller-Hinton agar (MHA) medium was prepared and sterilized at 121 °C for 20 minutes in the autoclave.About 25 mL sterilized media was poured into each 12 cm sterile Petri dishes under aseptic conditions and allowed to settle.Te bacterial suspension was lawn cultured over the agar medium using a sterile glass-L rod.Te extract disc was gently placed at equidistant on top of the agar layer.25% DMSO containing disc was used as a negative control.Te reference antibiotics disc of amoxicillin (30 μg/disc) and norfoxacin (10 μg) as positive controls were also placed at equidistant, on top of the agar layer.Plant extracts disc (1 mg/disc) and reference drugs were allowed to difuse for 1 hour into the plates and then incubated at 37 °C for 24 hours in an inverted position.Te results were obtained by measuring the zone of growth inhibition (mm) surrounding the discs.Each attempt was performed in triplicates [26,33].

Determination Minimum Inhibitory Concentration (MIC).
Seven vials were properly sterilized and labeled for the examination of individual extracts.Each vial was loaded with 750 µL of sterile Mueller-Hinton broth (MHB).In order to make the samples, a concentrated solution of 40 mg/ mL was prepared using DMSO.Te initial concentrated solution underwent a series of dilutions to produce seven distinct concentrations of the extract, spanning from 40 mg/ mL down to 0.625 mg/mL.Tis dilution process involved dissolving the concentrated solution in a 1 : 1 blend of   Te Scientifc World Journal DMSO and water.Following this, 250 µL of the resulting sample solution was meticulously transferred into the corresponding vial containing 750 µL of MHB.Consequently, the concentration of the sample solution in every vial varied from 10 mg/mL to 0.1562 mg/mL.Bacterial cultures with an initial concentration of around 1 × 10 5 CFU/mL were introduced into all vials, including solvent blanks, negative controls, and positive controls.Subsequently, the vials were tightly sealed and placed in an incubator set at 37 °C for a duration of 24 hours.After this incubation period, the MIC values were assessed.MIC denotes the lowest concentration of the plant sample capable of halting the observable growth of the bacterial culture.Te presence of viable bacterial cells was verifed through an assay utilizing 3(4,5 dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide (MTT).Tis process entailed subjecting the samples to further 2-hour incubation at 37 °C and visually examining the development of formazan, a byproduct that signifes the presence of viable cells [17,34,35].

Determination of Minimum Bactericidal Concentration (MBC).
To ascertain the MBC, refrigerated MHA plates were placed in an incubator at 37 °C for 45 minutes, after which they were moved to a sanitized laminar airfow (LAF) hood.Following this, aliquots from each diluted test tube, prepared using the same method as in the MIC assessment, were transferred onto MHA plates.Tese subcultured plates were subsequently placed in an incubator set at 37 °C for a period of 24 hours.Te MBC was characterized as the minimum concentration of the plant extract that entirely suppressed bacterial growth on the culture media's surface [16,33,34].

Statistical Analysis.
All the values and data were expressed as the mean ± SEM.All the experiments were carried out in triplicate (n � 3).

Results
A total of ffteen species of plants belonging to 15 diferent families (Table 1), every herb popular within traditional medicine of Nepal, were chosen for the antibacterial activity assay against four diferent microbial strains.Tis antibacterial potency of all samples was quantitatively confrmed by the absence or presence of an inhibition zone all over the disc loaded with extract.Te result confrmed that only M. repandus and Q. infectoria showed potent antibacterial efects in all tested organisms (Table 2).Te other thirteen  4 Te Scientifc World Journal plant extracts could not show a measurable zone of inhibition against all microorganisms.In this study, Q. infectoria was found to be more efective against Grampositive bacteria than Gram-negative bacteria, while M. repandus was more efective against Gram-negative bacteria than Gram-positive bacteria.Between the two Gram-positive bacteria, S. aureus was found to be more sensitive than B. subtilis to Q. infectoria extract, while B. subtilis was more susceptible than S. aureus to M. repandus extract.However, in the case of Gramnegative bacteria, both plant extracts exhibited more sensitivity towards S. typhi than E. coli.Both plant extracts performed strong growth inhibition of all strains with minimum inhibitory concentration (MIC) values ≤ 2.5 mg/mL and minimum bactericidal concentration (MBC) values ≤ 10 mg/mL, as given in Table 3.Furthermore, antibacterial assay-guided fractionation of Q. infectoria (Figure 3) and M. repandus (Figure 4) displayed a variable measure of inhibition zone among diferent fractions.Te acetone fraction of Q. infectoria showed the highest inhibition zone for S. aureus (ZOI-41 mm), whereas the maximum zone of inhibition against B. subtilis (ZOI-18.66mm), E. coli (ZOI-18.33 mm), and S. typhi (ZOI-23.66mm) was performed using its acetonitrile fraction.EtOH extract was reported to be least efective against all strains.As shown in Figure 4, M. repandus yielded six fractions, nonpolar fractions such as hexane and chloroform showed lesser activity towards all strains.Among the six fractions, the EtOAc fraction displayed the greatest inhibition zones of 20.33, 13.00, 22.66, and 29.00 mm against S. aureus, B. subtilis, E. coli, and S. typhi, respectively.Acetone fraction was also signifcant after EtOAc fraction.Te acetonitrile portion was more efective against Gramnegative bacteria than against Gram-positive bacteria.Oppositely, the EtOH fraction revealed greater activity against Gram-positive bacteria with mild activity against Gramnegative bacteria.
According to Table 3, MIC and MBC analyses of Q. infectoria revealed that the lowest values of MIC (0.19 mg/mL) and MBC (0.98 mg/mL) were shown by its acetone fraction against Gram-positive bacteria S. aureus, whereas, in the case of Gram-negative bacteria, its acetonitrile fraction was found to be more efective with MIC and MBC values of 0.61 mg/mL and 1.31 mg/mL, respectively.Similarly, in the case of M. repandus, the lowest value of MIC (0.25 mg/mL) and MBC (0.53 mg/mL) was exhibited by EtOAc fraction against Gram-negative bacteria S. typhi.Similarly, in the case of Gram-positive bacteria, EtOH fraction was found to be most prominent with MIC and MBC values of 0.59 mg/mL and 2.35 mg/mL, respectively, against S. aureus.

Discussion
Te study of ethnobotanical information about the plants that were used in this study revealed that all the plants are being used for the treatment of the same class of pathogens in diferent areas of Nepal by diferent tribes.For example, the juice from the root of M. repandus is used to treat bacterial pathogen-associated problems such as stomach complaints such as indigestion, external gouty rheumatism, diarrhea, and dysentery [15].
Te preliminary fnding obtained from the investigation of the crude methanolic extracts recommended that all plants having ethnomedicinal use may not ensure its therapeutic efcacy.Tus, further investigation is necessary to minimize the irrational use of the medicinal plants.In this study, only Q. infectoria and M. repandus showed a worthwhile antibacterial activity, wherein the antibacterial activity of Q. infectoria MeOH extract showed a greater inhibition zone in Gram-positive bacteria than Gram-negative bacteria.Generally, plant extracts are more active against Grampositive bacteria than Gram-negative bacteria due to the presence of lipopolysaccharides in the multilayered cell wall Te Scientifc World Journal  6 Te Scientifc World Journal composition of Gram-negative strains [31,35].It is interesting to note that M. repandus MeOH extract showed a greater inhibition zone in Gram-negative bacteria than the Gram-positive bacteria.
Te MIC values shown by the MeOH extracts of the Q. infectoria galls against S. aureus, B. subtilis, S. typhi, and E. coli were 0.31 mg/mL, 0.62 mg/mL, 1.25 mg/mL, and 2.5 mg/mL, respectively.Te lower MIC values of the extract against Gram-positive bacteria, in comparison to Gramnegative bacteria, suggest that the extracts of the Q. infectoria galls can suppress Gram-positive bacteria more efciently [27,36].Furthermore, bioassay-guided fractionation of Q. infectoria yielded only four fractions, namely, acetone, ethyl acetate, acetonitrile, and ethanol.Among them, the acetone fraction showed the greatest inhibition zone against S. aureus, whereas the EtOH fraction had the least inhibition zone against all strains.Acetone and EtOAc fractions had a similar inhibition zone to B. subtilis, while the ethyl acetate fraction showed a lesser efect on S. aureus than the acetone fraction but greater activity than the other remaining fractions.Te EtOAc fraction had a better inhibition zone in Gram-negative bacteria than the acetone fraction.Acetonitrile fraction showed a smaller inhibition zone than acetone and EtOAc fraction to S. aureus but formed a higher inhibition zone to B. subtilis, E. coli, and S. typhi.Te MIC values of EtOH and MeOH extract in the study were found to be lower than results of the previous study, against S. aureus and E. coli [37].
Te MBC values for all the fractions were greater than the MIC values against all the bacteria, which advises that the bioactive chemicals in the extract have higher extent of bacteriostatic efect as compared to bactericidal, against all bacteria strains.In contrast, MIC and MBC for acetone extract of Q. infectoria galls were reported to be the same in a previous study [27].By observing the data of zone of inhibition (Table 2 and Figure 3) and MIC and MBC (Table 3), we can conclude that the acetone extract of Q. infectoria (gall parts) is most suitable to inhibit the S. aureus bacteria, whereas the acetonitrile extract can be the best option to achieve a broad-spectrum activity.Many scientifc investigations have asserted that the presence of abundant hydrolysable and condensed tannins in this plant might be responsible for broad-spectrum antibacterial activity as they can stop protein synthesis required for bacterial wall synthesis through the formation of an irreversible complex with proline-rich protein [27,38,39].
Bioassay-guided fractionation of M. repandus yielded six fractions: hexane, chloroform, acetone, EtOAc, acetonitrile, and EtOH.As shown in Figure 4, the EtOAc fraction exhibited the highest inhibitory efect against both Gramnegative bacterial strains.Similarly, the inhibitory efect of acetone and acetonitrile against Gram-negative S. typhi was almost similar to that of the EtOAc fraction.Oppositely, the EtOH fraction was found to be most efective against Grampositive strains.Furthermore, the inhibitory efect of the EtOAc fraction against S. aureus was almost comparable with the ethanol fraction.Nonpolar fractions of hexane and chloroform were reported to be least efective against all the bacterial strains.Tis may implicit that the pharmacologically active antimicrobial molecules might be hydrophilic in  Te Scientifc World Journal nature.MIC and MBC of all the fractions (Table 3) showed a proportional correlation with the data obtained from the disc difusion method.According to a study conducted in Bangladesh, seeds of M. repandus have a moderate inhibition efect against S. typhi, B. subtilis, and E. coli [39].However, there is no study on the antibacterial activity of diferent fractions and their MIC and MBC.Furthermore, this study has explored the potent inhibitory efect of different fractions against Gram-negative bacteria S. typhi for the frst time.
Te seeds of M. repandus are a rich source of antifungal and antitumor maytansinoids such as trewiasine, dehydrotrewiasine, treforine, trenudine, and N-methyl trewiasine [40,41].Besides these, seeds also contain potent antimicrobial compounds such as alkaloids, ricinidine and neolignans, glyceride oil, and pyridinone alkaloids [42].In a previous study, fve neolignans were isolated from the seed endothelium of M. repandus, among which 9′-butyl americanol A and americanin showed the antibacterial activity against Gram-positive bacterium S. aureus and Gramnegative bacterium Mycobacterium tuberculosis [43].A previous study has reported that neolignans present in Piper regnellii have a potent antibacterial activity against S. aureus and B. subtilis [44].
In a previous study on P. guajava, it is revealed that its methanolic extracts showed little antibacterial efect against two Gram-negative bacteria (E. coli and S. enteritidis) and two Gram-positive bacteria (S. aureus and B. cereus) [45], but in our study, there was no zone of inhibition.Essential oils obtained from the hydrodistillation of V. negundo fresh leaves showed promising antibacterial efects against B. subtilis and E. coli [46].According to a previous study on C. repens, S. aureus was more susceptible to the EtOH extracts of C. repens [47].MeOH extracts of C. repens showed a signifcant antibacterial activity against S. aureus, B. subtilis, E. coli, and P. aeruginosa [48].However, in this study, the methanolic extract of C. repens was used, and hence, there was no zone of inhibition observed.Te leaves [49] and bark [50] of A. catechu showed antibacterial activity against Streptococcus mitis, Streptococcus sanguis, and Lactobacillus acidophilus, but in this study, we used resin of respective plants, and no zone of inhibition was observed.Similarly, the chloroform extract of F. religiosa fruit was found to be inefective against the bacterial strains used in this study, although it has a moderate efect against other strains such as S. faecalis, B. megaterium, and Azotobacter chrooeoccum [51].It has been reported that the chloroform extract of T. sinensis fower has an inhibitory efect against Pseudomonas aeruginosa [52].Furthermore, the acetone extract of E. serratus fruit was found to be slightly efective against S. aureus bacteria [53].Although the antimicrobial efcacy of N. nucifera fower extract has been verifed against both Gram-positive and Gram-negative bacteria, the efect of seeds has not yet been justifed [54].Seed oil of C. sativa has revealed a measurable zone of inhibition against both Gram-positive and Gram-negative bacteria, but the MeOH seed extract could not exhibit a measurable bacterial inhibitory efect in this study [55].Moreover, the MeOH extract of B. utilis bark has shown a moderate bacterial inhibitory efect in a previous study [56].
Discussing the study's limitations, it is important to note that the potent fractions, specifcally the acetone fraction of Q. infectoria and the EtOAc fraction of M. repandus, could not undergo chromatographic separation to isolate the distinct active compounds.To enhance the standardization of plant extracts and fractions, employing HPLC analysis would be advisable.

Conclusion
Our study concluded that all the plants used in folk medicine may not have medicinal value; thus, the use of evidencebased traditional medicine is necessary for the rational and efective use of ethnomedicinal plants.Among 15 diferent plants that were used by traditional healers of Nepal, only the galls of Q. infectoria and seeds of M. repandus showed signifcant antibacterial properties against both Grampositive and Gram-negative bacteria.Q. infectoria was reported to be more active against Gram-positive bacteria, whereas M. repandus showed more efectiveness against Gram-negative bacteria.Te acetone fraction of Q. infectoria was more active against Gram-positive bacteria, whereas the ethyl acetate fraction of M. repandus was the most active against Gram-negative bacteria.
Based on our research fndings, we recommend the utilization of extracts derived from Q. infectoria galls and M. repandus seeds as potential inhibitors of both Grampositive and Gram-negative bacteria.In the future, it is essential to conduct bioassay-guided analysis of these plants in order to identify the specifc bioactive compounds responsible for their antibacterial activity.In addition, we propose conducting an investigation into the antibacterial properties of all the plants indigenous to Nepal that have been traditionally used for treating various infection-related diseases.Tis comprehensive study is necessary as not all plants may possess genuine antibacterial efects, and it will help identify and validate the plants with signifcant antibacterial potential.

Table 1 :
List of selected medicinal plants with their medicinal parts.

Table 2 :
Measurement of the bacterial zone of inhibition shown by ffteen investigated plant samples.
All the values represented mean ± SEM (n � 3).ND indicates ZOI are not detected at the examined concentration.