The phytochemical composition of medicinal plants in Indonesia and their potential as antibacterial agents against Salmonella typhi ATCC 13311

Several Indonesian medicinal plants are commonly utilized as antibacterial agents, namely moringa (Moringa oleifera Lamk.), green tea (Camellia sinensis), binahong (Anredera cordifolia (Ten.) Steenis), and green meniran (Phyllanthus niruri L.). This investigation aimed to identify the chemical compounds found in plants and their antibacterial activity against Salmonella typhi. The simplicia was extracted by the maceration technique with a solvent of 70% ethanol. Chemical compounds are identified using various chemical reagents. The antibacterial activity test was conducted using the diffusion method. The data were analyzed using a one-way analysis of variance (ANOVA) test. The phytochemical analysis of plant samples revealed the presence of many bioactive compounds such as flavonoids, saponins, tannins, alkaloids, steroids, and phenolics. The antibacterial activity of plant samples against Salmonella typhi has been shown. The average values of the inhibition zones for moringa leaf extract, green tea, binahong, and green meniran were determined to be 20.33 mm, 18.33 mm, 22.67 mm, and 27.00 mm, respectively. The antibacterial activity of green meniran leaf extract is significantly more potent against Salmonella typhi than other plant.


Introduction
Pathogenic bacterial infections represent a significant health concern, frequently observed in Indonesia and globally [1].Typhoid fever, a rising infection, can infect individuals across all age groups.On a global scale, the incidence of typhoid fever amounts to approximately 13.5 million cases.In the Indonesian context, the incidence of typhoid fever was documented at approximately 148.7 cases per 100,000 individuals residing within the region [2].Typhoid fever is a pathological condition that can arise due to the presence and activity of the microorganism Salmonella typhi [3].The issue of bacterial infection is typically addressed through the administration of antibiotics to the affected individual [4].However, the elevated frequency of antibiotic usage is leading to a novel predicament, specifically bacterial resistance [5].The prevalence of bacterial resistance is becoming more extensive, encompassing not only the genera Pseudomonas, Acinetobacter, Staphylococcus but also the genus Salmonella [6].Antimicrobial resistance leads to a notable reduction in the efficacy of antibiotics, which 1312 (2024) 012035 IOP Publishing doi:10.1088/1755-1315/1312/1/012035 2 are currently employed as a therapeutic approach.Hence, it is imperative to explore alternative approaches to discover novel antibacterial agents, such as screening phytochemical compounds derived from indigenous plant species in Indonesia that exhibit promising medicinal properties [7].
As a tropical nation, Indonesia boasts a remarkable array of medicinal plants that exhibit rich diversity [8].These plants have been extensively utilized by diverse traditional healing communities within the region.Traditional medicine is pivotal in advancing contemporary medicine, as it harnesses the therapeutic potential of diverse botanical extracts [9].Moringa oleifera Lamk, a widely distributed botanical species, holds promise as a medicinal plant with global prevalence.Prior studies have demonstrated that the secondary metabolites found in the Moringa oleifera Lamk plant, specifically flavonoids, phenolics, terpenoids, tannins, and saponins [10], exhibit promising properties as agents with potential anticancer, antibacterial, antifungal, anti-inflammatory, antidiabetic, antioxidant, antihypertensive, and hepatoprotective effects [11].In addition to the moringa plant, the green tea plant (Camellia sinensis) exhibits promising characteristics as an antioxidant, antimutagenic, antitumor, antiviral, and anticoagulant, with potential benefits against heart diseases, blood pressure regulation, cancer prevention, and cholesterol reduction.The green components exhibit secondary metabolites such as xanthines, caffeine, tannins, theobromine, flavonoids, polyphenols, lipids, and vitamin C [12].
Another medicinal plant that warrants attention is binahong (Anredera cordifolia (Ten.)Steenis), which has garnered significant recognition in traditional medicine for its efficacy in addressing fever, gout, and digestive ailments.The binahong leaves possess a notable composition of phytochemicals, encompassing flavonoids, saponins, alkaloids, essential oils, and oleanolic acid.The correlation between the phytochemical content and its antibacterial properties is significant [13].Similarly Phyllanthus niruri L, commonly known as green meniran in Indonesia, is a botanical specimen with therapeutic properties that can thrive in both natural habitats and human-managed environments.The utilization of the green meniran plant has long been prevalent in managing conditions affecting the biliary system urinary tract, encompassing afflictions such as renal and cholecystic calculi [14].Based on prior research findings, it has been observed that the composition of phytochemicals present in green meniran encompasses alkaloids, flavonoids, lignin, terpenoids, lupeol, and tannin [15].Prior studies have demonstrated varying antibacterial efficacy of four medicinal plant extracts (Moringa oleifera, Camellia sinensis, Anredera cordifolia, Phyllanthus niruri) against Salmonella typhi.The petroleum ether extract of Moringa oleifera leaves, the n-hexane extract of Camellia sinensis, the essential oil extract of Anredera cordifolia, and the ethanolic extract of Phyllanthus niruri leaves have demonstrated inhibitory effects on the growth of Salmonella typhi.The inhibition zones observed for each extract are as follows: 11 mm, 15 mm, 6.25 mm, and 19.05 mm, respectively.The comparability of the study results is compromised due to variations in plant origins, as well as differences in eluents and extraction techniques employed.In order to ascertain the most effective plants in suppressing the growth of Salmonella thypi ATCC 13311 bacteria, it is imperative to conduct a comparative analysis of the phytochemical substances present in these four plants and evaluate their respective antibacterial activities.

Materials
The leaves of moringa (Moringa oleifera Lamk.), green tea (Camellia sinensis), binahong (Anredera cordifolia (Ten.)Steenis), and green meniran (Phyllanthus niruri L.) plants were procured from the esteemed Research Center for Medicinal Plants and Traditional Medicine, under the auspices of the Ministry of Health Republic of Indonesia.The Salmonella typhi ATCC 13311 bacteria was acquired from a pure culture at the esteemed Microbiology Laboratory of Setia Budi University in Surakarta.The extraction materials and phytochemical identification reagents, including 70% ethanol, 2N HCl, FeCl3, Dragendroff's reagent, concentrated HCl, 96% ethanol, H2SO4, and 5% FeCl3, were procured from Sigma/Aldrich, Singapore.Various agar media are employed for bacterial cultivation and antibacterial assessments.These include Brain-heart infusion agar media, Muller-Hilton agar media, Bismuth Sulfite Agar media, Kligler's iron agar media, lysine iron agar media, sulfide indo motility (SIM) media, and Citrate media sourced from Merck, Darmstadt, Germany.The necessary materials for performing Gram staining, including Crystal violet, iodine, alcohol-acetone, and safranin, as well as immersion oil, distilled water, ethanol, and a negative control solution of DMSO 2%, were procured from Sigma Aldrich, Singapore.An antibacterial test was conducted, utilizing chloramphenicol at 5 µg/ml concentration as the positive control.

Moisture Content Evaluation of Moringa Leaf Powder, Green Tea, Binahong, and Green Meniran
The moisture content was measured with a Bidwel-Sterling instrument and xylene solvent.The powder was weighed at 20 g, and then 20 ml of xylene solvent was introduced to a flask with a rounded bottom.The condenser was mounted on a convex bottom flask containing the solution.This procedure was repeated thrice in circulation, with no water falling on the scale tube for 15 minutes [16].

Extraction of Moringa Leaf Powder, Green Tea, Binahong, and Green Meniran
The Moringa leaf powder, green tea leaf powder, green meniran powder, and binahong leaf powder weighed 300 grams.The maceration method was employed to extract each powder, utilizing a solvent of 70% ethanol.The ratio between the powder and solvent was maintained at 1:10.Three hundred grams of simplicia extract powder was carefully measured and subsequently transferred into a brown glass container.Following this, 3000 millilitres of 70% ethanol solution was introduced into the container.The container has been securely sealed and positioned in an area shielded from direct exposure to sunlight for approximately five days, with periodic homogenization.The maserate was subsequently subjected to filtration utilizing flannel cloth and filter paper, facilitating the separation of the ethanol liquid from the dregs.The resulting liquid extract was then concentrated employing a rotary evaporator operating at 60 revolutions per minute and a temperature of 50 0 C [17].

Phytochemical identification of Moringa leaf extract, green tea leaf, binahong, and green meniran.
The identification of phytochemicals in the test plant extracts was conducted by employing a range of chemical reagents as per established methodologies described in prior studies [16].The analysis identified phytochemical components, specifically flavonoids, saponins, tannins, steroids, phenolics, and alkaloids.The identification of flavonoid phytochemical components was conducted using a method that involved weighing 100 mg of extract and combining it with 2 ml of 95% ethanol.Subsequently, 0.05 grams of zinc powder and 2 ml of 2N HCl were added to the mixture.The solution was allowed to stand for 1 minute before adding 2 ml of concentrated HCl.Saponin was found using a process including the dissolution of 200 mg of extract in 10 mL of hot distilled water, followed by adding 2 mL of 2N HCl and subsequent shaking.Identifying tannins involved the dissolution of 200 mg of extract in 10 ml of hot distilled water, followed by the addition of 300 µl of a 1% FeCl3 solution.The identification of steroids involved the addition of 100 mg of extract to 2 ml of chloroform, followed by stirring and the subsequent addition of concentrated H2SO4.The phenolic detection procedure involved the dissolution of 200 mg of the extract in 10 ml of distilled water, followed by adding five drops of a 5% FeCl3 solution.The identification of alkaloids involved the preparation of a 200 mg extract, followed by adding 5 ml of hot hydrochloric acid (HCl) and 2 ml of Dragendroff's reagent [18].

Characterization of Salmonella thypi ATCC 13311 bacterial isolate
Salmonella typhi ATCC 13311 bacteria were characterized using microscopic observations, colony formation, and biochemical properties.Gram staining was employed for the microscopic characterization of microorganisms.Growing bacteria on bismuth sulphate agar media allowed for characterizing bacteria colonies.Bacteria were cultured on Kligler iron agar (KIA), Sulfide Indole Motility (SIM), Lysine Iron Agar (LIA), and Simmons Citrate Medium for biochemical identification [19].

The Antibacterial Activity Test of Moringa Leaf Extracts, green tea leaves, binahong, and green meniran shall be conducted using the Disc Diffusion Method.
The antibacterial activity test was conducted on moringa leaf extract, green tea leaf, binahong, and green meniran, all at a concentration of 60%.The antibacterial testing for each plant extract was conducted in three repetitions, with a positive control utilizing chloramphenicol and a negative control using DMSO 3%.The S. typhi bacterial isolate was cultured in a 5 ml volume of Brain Heart Infusion (BHI) media and subjected to incubation for 24 hours at a temperature of 37°C within an incubator.The bacterial culture on BHI media has been standardized using the 0.5 McFarland method [20].The paper disks have been meticulously prepared to assess the antibacterial properties of moringa leaf extract, green tea leaf, binahong, and green meniran.The paper disks were duly immersed in each test extract, with a concentration of 60%, and subsequently subjected to an incubation period of 24 hours at ambient temperature.The bacterial colonies observed to proliferate on BHI media are meticulously streaked uniformly onto Muller-Hilton Agar (MHA) media within a petri dish.The MHA Petri dish has been divided into six sections.Four sections have been allocated for paper disk samples soaked in the test extract.One section is reserved for the positive control (chloramphenicol), and another is designated as the negative control (DMSO 3%).The petri dishes were placed in an incubator at 37°C and incubated for 24 hours.The activity test results were observed by measuring the inhibition zone that formed around the disc paper sample [21] .

Analyses of data
Statistical tests were used to analyze the data obtained from investigating the antibacterial activity of ethanol extracts of Moringa leaves, green tea leaves, meniran leaves, and binahong leaves against Salmonella typhi ATCC 13311 by well diffusion method.The data obtained from measuring the area of the clear zone formed around the well opening were then examined for normality and homogeneity.SPSS 17 software is then used to conduct parametric tests on normally distributed data utilizing the One-Way ANOVA test.

Results and Discussion
Characterization and standardization of medicinal plants such as moringa leaves, green tea leaves, binahong leaves, and green meniran leaves were accomplished by measuring the flour's water content the extract's yield and identifying phytochemical compounds.Table 1 displays the water content measurement results for powder.The water content of medicinal plant powders demonstrates that all plants satisfy the Indonesian herbal pharmacopoeia standards, as indicated by water content values below 10%.The yield value of the extracts from moringa leaves, green tea, binahong, and green meniran was used to characterize the extracts.The yield of the extract is a percentage of the extraction results.The higher the extract yield percentage, the more optimal extraction is occurring in plants.Table 2 displays the yield results for moringa leaf extract, green tea, binahong, and green meniran.Compared to other plants, green meniran has the maximum extract yield (23%), according to the findings of this study.The large extract yield indicates that green meniran extract is more active than extracts from other plants [20].The phytochemical compounds flavonoids, saponins, tannins, steroids, phenolics, and alkaloids have been identified in moringa leaf, green tea, binahong, and green meniran extracts.These phytochemical compounds are known to have biological activities, including antibacterial, antifungal [22], antidiabetic, anti-inflammatory [23], and anticancer properties [24].Table 3 displays the results of identifying phytochemical compounds in plants.The Salmonella typhi ATCC 13311 bacterial strain, obtained from the microbiology laboratory collection at Setia Budi University, was subjected to characterization and identification procedures.The process of characterizing and identifying bacteria involves the utilization of microscopic techniques, colony observation, and biochemical analysis.Characterizing bacterial colonies cultivated on bismuth sulfite media reveals that these colonies exhibit a circular shape, tiny size, brown coloration with a central black dot, and a silver-white periphery around the colonies.The gram staining results indicate that the bacteria possess the following attributes: they exhibit gram-negative features, display a red coloration, possess a rod-shaped morphology, and exhibit a dispersed arrangement.The colony characterization and gram staining outcomes are depicted in Figure 1.  4 presents the biochemical characteristics of the Salmonella typhi ATCC 13311.The findings from the biochemical characterization indicate that the identification of Salmonella typhi aligns with the reference standards.The study's findings indicated that extracts derived from moringa, green tea, binahong, and green meniran leaves exhibited inhibitory effects on Salmonella typhi ATCC 13311 bacteria growth.The inhibitory effects of these extracts on bacteria exhibited considerable variability.Table 5 presents the antibacterial efficacy of moringa leaf extract, green tea, binahong, and green meniran against Salmonella thypi ATCC 13311.The study findings revealed that the antibacterial activity of green meniran leaf extract was the most potent, as evidenced by the average resistance value reaching 27 mm.The extract of green meniran is composed of many phytochemical components, including flavonoids, tannins, saponins, steroids, phenolics, and alkaloids.In addition, the research findings indicated that the highest yield in this study was obtained from the extract of green meniran leaves.According to prior research findings, it has been established that flavonoid compounds represent the predominant category of phenolic compounds, exhibiting inhibitory effects on the proliferation of fungi, bacteria, and viruses [25].Flavonoids have antibacterial properties through three primary mechanisms: interference with bacterial energy metabolism, nucleic acid synthesis disruption, and cell wall development inhibition.These actions result in the leakage of plasma and subsequent bacterial lysis.Alkaloids are a class of chemicals characterized by a basic nitrogen-containing group, which undergoes reactions with the amino acids constituting cell walls and bacterial DNA.This chemical reaction induces alterations in the configuration of amino acids, thereby perturbing the genetic equilibrium inside the DNA strand.Consequently, this disruption promotes cellular harm and lysis, ultimately resulting in the demise of the bacterial cell [26].Tannin is a polyphenolic molecule that exhibits antibacterial properties through its ability to disrupt enzymatic activity, protein transport, and cell wall polypeptides.This disruption leads to the formation of an incomplete cell wall, rendering the cell susceptible to lysis.Saponins consist of lipophilic molecules capable of dissolving fat and hydrophilic molecules that can attract water.This dual property of saponins reduces cell surface tension, ultimately resulting in the destruction of bacterial cells.Phenolics exhibit antibacterial properties by deactivating cellular enzymes through penetration into cells and disruption of cell membrane permeability.The antibacterial compounds react with cell membranes, damaging the integrity of the cytoplasmic membrane.This damage causes the release of ions and macromolecules from the cell, resulting in further impairment [27].Additionally, phenolics disrupt the function of lipoprotein components in the membrane, compromising the cell's ability to protect itself against osmotic pressure.Steroids and triterpenoids have antibacterial properties by establishing robust polymer bonds and inducing detrimental effects on porins, which are trans-membrane proteins located on the outer membrane of bacterial cell walls [28].The impairment of porins will decrease the permeability of bacterial cell walls, hence impeding the transportation of essential nutrients and other substances necessary for bacterial growth.Consequently, the compromised bacterial cells will ultimately undergo cell death [29].

Conclusion
Moringa, green tea, binahong, and green meniran extracts contain the following phytochemical compounds: flavonoids, saponins, tannins, steroids, phenolics, and alkaloids.The extract with the maximum yield was an ethanolic extract of green meniran.Each of the test extracts inhibited the growth of Salmonella typhi ATCC 13311.Green meniran ethanolic extract inhibited the proliferation of Salmonella typhi ATCC 13311 more effectively than other plants.

Figure 1 .
Figure 1.Characterization of Salmonella typhi ATCC 13311.a: Characteristics of the S. thypi ATCC 13311 bacterial colony on bismuth sulfite agar.b: Results of microscopic identification using gram staining.

Table 1 .
Results of measuring the quantity of water granules in medicinal plants

Table 2 .
Calculated yield values for plant extracts

Table 3 .
Results of plant phytochemical compound identification

Table 5 .
Antibacterial test results of medicinal plant extracts against S. thypi ATCC 13311 bacteria