Bactericidal activity of quercetin like-compounds isolated from Dendrophthoe pentandra leaves against Salmonella spp. and Escherichia coli: an in vitro experimental study

Bactericidal activity of quercetin like-compounds isolated from Dendrophthoe pentandra leaves against Salmonella spp. and Escherichia coli: an in vitro experimental study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bactericidal activity of quercetin like-compounds isolated from Dendrophthoe pentandra leaves against Salmonella spp. and Escherichia coli: an in vitro experimental study Lazuardi Mochamad, Aniek Setiya Budiatin, Wiwiek Tyas Ningsih, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8723177/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background Dendrophthoe pentandra L., Miq, a type of mistletoe that grows abundantly on Lansium domesticum , is estimated to possess antimicrobial activity against bacteria. This study focused on extracts of the plant, particularly quercetin-like compounds (QLC), with the aim of identifying potential analyte compounds as new antibiotics. The leaves of the mistletoe plant underwent methanol-ethyl acetate-n-hexane maceration, followed by QLC extraction using preparative high-performance liquid chromatography equipment. The extracts were subsequently analyzed using ultra-performance liquid chromatography-tandem mass spectrometry, Fourier transform infrared spectrophotometry, and proton analysis via nuclear magnetic resonance spectroscopy. The QLC extracts, ranging from 500 ppm to 10,000 ppm, were tested for antimicrobial activity against Salmonella species and Escherichia coli in vitro , based on colony growth inhibition screening. The results were further analyzed using probit analysis to determine the bactericidal values against these two enterobacteria. Results The findings indicated that the minimum inhibitory concentration required for 50% mortality of Salmonella species colonies was 359.283 µg/mL and that a 75% reduction occurred at an exposure of 1.6 mg/mL of QLC. Additionally, a 99% colony death rate was observed at a minimum bactericidal concentration of 61.9 mg/mL of QLC. In contrast, exposure to QLCs did not exhibit any colony-inhibiting or bactericidal effects on Escherichia coli . Conclusions The QLCs extracted from the leaves of Dendrophthoe pentandra growing on the host plant Lansium domesticum demonstrate significant potential for development as new antibiotics against Salmonella species. However, they are not recommended for use against Escherichia coli (P < 0.05). Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Drug discovery Biological sciences/Microbiology Biological sciences/Plant sciences Antibiotics Enterobacteriaceae Healthy and well-being Mistletoe Pullorum Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background It is well-established that secondary metabolite compounds (SMCs) derived from the leaves of mistletoe, specifically those growing on the Lansium domesticum host and known as Dendrophthoe pentandra L. Miq (BD), contain bioactive components. These components, purified from the flavonol fraction, are identified as quercetin-like compounds (QLCs) and have demonstrated antiviral properties in vitro , particularly against Newcastle disease. 1 This mistletoe, commonly referred to as "duku mistletoe" in Southeast Asia, especially among the Malay community, features leaves that are approximately 2–3 cm wide, with smooth edges and a rigid petiole. The species exhibits two subspecies: one with green leaves ( Dendrophthoe pentandra L. Miq) and another with reddish-green leaves ( Scurrula ferruginea ). The reddish-green subspecies is predominantly found in Indonesia, particularly in Bali and the regions of western and eastern Nusa Tenggara, as well as parts of East Timor, the Southern Philippines, Malaysia, and regions of Southern Africa, Northern Australia, and New Zealand. The duku mistletoe typically thrives in tropical regions characterized by relatively high rainfall. 2 Research has identified that these QLCs contain various elements, including 5,7,8,30,40-pentamethoxyflavonone, 7-hydroxy-1-methoxy-2-methoxyxanthone, and pelargonidin-3-glucoside. These three SMCs prominently dominate the QLC content and theoretically possess complex aromatic structures with additional electron-attracting groups such as = O=, -C-, and -N-, which may facilitate their penetration of lipid bilayers, particularly in eukaryotic cell membranes. Furthermore, these SMCs exhibit stability due to the resonance of their aromatic structures, rendering them less susceptible to reactions with other groups. This characteristic positions them as promising candidates for new "prodrugs" capable of interacting with the ribonucleic acid or deoxyribonucleic acid (DNA) of eukaryotic cells. In the context of bacterial cells, they hold potential as antibiotics by forming complexes with nucleic acid components that can bind to the complex aromatic structures of QLC. 2 , 3 The interactions among other QLC components, such as 5-hydroxy-6,7-dimethoxyflavone-4'-O-β D-glucoside, 7-hydroxy-1-methoxy-2-methoxy xanthone, ononin, morin, and quercetagine, may facilitate complexation with viral DNA components, acting as nucleic acid respiratory blockers. This mechanism can induce cell death while simultaneously functioning as an antibiotic through ionospheric action. 4 While the role of mistletoe plants as antibiotics remains relatively obscure, their potential benefits warrant exploration. These include (a) minimal disruption to ecosystems due to the selective parasitic nature of mistletoe on its primary host and (b) the SMCs' selective efficacy, which is highly dependent on the quality of the host. These factors may contribute to a more cost-effective and efficient extraction process for SMCs from mistletoe. Nonetheless, a significant challenge persists: the limited availability of these plants in the wild, despite efforts to cultivate them artificially. Figure 1 illustrates that following attempts to grow mistletoe in artificial media, seedlings typically develop within approximately 30 days, during which their root systems begin to establish. The artificial cultivation of mistletoe generally progresses through four phases: (A) the seedling phase, (B) the germination phase, (C) the maturation/preparation of plant stock, and (D) the emergence of small roots from the germinated stock. At this stage, the plants are ready to be transferred to soil, which must be kept consistently moist and well-aerated. However, phase (D) presents a challenge, as mistletoe plants often struggle to adapt when transitioned to non-artificial growing media, leading to mortality. To mitigate this issue, a longer development period is essential, allowing the emerging roots to strengthen and increasing their resilience during the transition to soil. Additionally, careful consideration of the soil media composition (50% original media and 50% new media) is crucial to ensure that the newly sprouted roots do not encounter an overly unfamiliar environment. 5 These findings create opportunities for the cultivation of mistletoe and facilitate the standardization of simplicial for the extraction of raw SMCs in diverse climatic conditions. The acquisition of SMCs from mistletoe aligns with the Sustainable Development Goals, particularly point 3, which focuses on health and well-being by minimizing the reliance on artificial chemical compounds in medicine and promoting the emergence of new therapeutic responses to non-natural drugs. Efforts to discover new antibiotics that circumvent antimicrobial resistance phenomena continue to be prioritized, drawing from both plant sources and semi-synthetic or synthetic origins. 6 The SMCs with antibiotic properties are predominantly located in the leaves of medicinal plants, with common components including plant alkaloids from various heterocyclic groups, such as pyrrolidine (hygroline), pyrrolizidine (senecionine), pyridine and piperidine (piperine, lobeline), tropane (cocaine), quinoline (quinine, quinidine), aporphine (boldine), quinolizidine (sparteine), indole or benzopyrrole (ergometrine), indolizidine (swainsonine), imidazole (pilocarpine), purine (caffeine), steroidal (solanidine), and terpenoid (aconitine). 7 Heterocyclic alkaloid groups rich in polyphenolic content, such as QLCs, contain numerous nitrogen ions bound to oxygen ions, potentially lowering the pH and enhancing lipid penetration, including through bacterial cell walls. Consequently, many SMCs in plants are not isolated specifically, as the pharmacological efficacy of an SMC can stem from a broad array of alkaloids. 8 , 9 Polyphenolic compounds in leaves theoretically possess bactericidal properties due to their heterocyclic structural elements, which can bind to the base pair elements of DNA, provided that the base pairs exhibit a strong affinity for the groups present in the SMC heterocycles. This binding occurs rapidly after macromolecules distribute SMCs from the blood of clinical subjects to areas of strong cellular affinity. This phenomenon may lead to the generation of novel compounds isolated from plants, which are often abundant and readily available in the wild. 10 Based on the aforementioned analysis, this research aims to investigate the potential benefits of QLCs as ionospheric antibiotic compounds with possible bactericidal action. This research is urgent to be conducted, considering the circulation of regulations prohibiting the use of antibiotics for animals consumed by humans in countries that support the provisions of the SDGs, particularly for bacteria of one of the Enterobacteriaceae, namely Salmonella, which causes pullorum disease in chickens. Methods Study design and mistletoe plant material This study was conducted using an exploratory model over a period spanning from late December 2022 to mid-2024, primarily at the Center for Testing and Application of Veterinary Pharmaceutical Sciences, Faculty of Veterinary Medicine, Universitas Airlangga. The mistletoe plant studied, identified as Dendrophthoe pentandra L. Miq, originates from Lansium Domesticum in the Muara Enim district, located at a latitude of -3.663223 and a longitude of 103.778161 in South Sumatra, Indonesia. The plant has received taxonomic confirmation from the National Research and Innovation Agency of the Republic of Indonesia via letter No. B-3286/II.6.2/IR.01.02/9/2024 . 11,12,13 The names of the officers who carried out the identification of plant materials come from the Directorate of Scientific Collection Management, National Research and Innovation Agency of the Republic of Indonesia was Mr. Wihermanto. The virtual visit to the mistletoe herbarium can be accessed at the link https://doi.org/10.6084/m9.figshare.31238056 . The mistletoe specimens that have been identified are stored at the National Research and Innovation Agency of the Republic of Indonesia at code BO 1738456 at September 11th, 2024. The collection of Mistletoe specimens was carried out after obtaining official permission from the Ministry of Research, Technology, and Higher Education of the Republic of Indonesia to conduct research under contract number 2344/B/UN3.LPPM/PT.01.03/2025 dated June 2, 2025.The mistletoe chosen for this research object is not a rare plant and still follows the rules of The International Union for Conservation of Nature. Flavonol extraction and isolation of QLC The technique employed for obtaining the analyte, QLC, was rapid, accurate, and precise, utilizing preparative High-Performance Liquid Chromatography (HPLC) as outlined in patent ID P000063522 [ https://patentscope.wipo.int/search/en/detail.jsf?docId=ID209010597] . 14,15 A total of 3 kg of BD leaf powder underwent mobile maceration, wherein 1 L of methanol (pro analysis, catalog number 106012, Merck Millipore) was added for every 500 g of BD powder. The mixture was agitated for 3 days using a rotary displacement method. The extract was subsequently filtered through a Buchner funnel, and the macerated juice was extracted using a flannel cloth. The resultant liquid was then dried using nitrogen vapor in a water bath maintained at 40°C. This process was repeated for the remaining 2,500 g of powder. Following this, the concentrated macerate was further treated with ethyl acetate (pro analysis, catalog number 109623, Merck Millipore) in a split tube, using a ratio of 100 g of macerate to 250 mL of ethyl acetate. The ethyl acetate solution was collected and dried under nitrogen vapor in a water bath at 40°C. The dried macerate was then subjected to additional maceration in a separate flask using n-hexane (catalog number 104374, Merck Millipore) and water (pro chromatograph, Merck Corp., catalog number 115333) in a 50:50 ratio. The final macerated product was dried using a rotary evaporator at 40°C, and the viscous compound was subsequently isolated as a QLC using a semi-preparative HPLC device (Agilent Infinity II, USA). The specifications for the device included a column with four units, a flow range of 1–50 mL/min, an injection volume range of 0.1–5000 µL, a maximum of 215 fractions, a binary pump system, and an operating pressure range of 20–420 bar, with a width of 941 mm. The preparative HPLC parameters were set at wavelengths of 250 nm, 254 nm, 261 nm, 370.4 nm, and 375.4 nm, utilizing an ODS C18 column (specification PN 440905-802, SN 71199, 250 x 10.0 mm, Agilent, USA) in isocratic mode with a mobile phase of water:methanol (30:70), adjusted to a flow rate of 0.5 mL/min for a 10-mL sample injection. Characterization of QLC using ultra-performance liquid chromatography mass spectrometry and Fourier transform infrared spectrophotometry The analysis of content QLC was conducted using Acquity ultra-performance liquid chromatography (UPLC) with a tandem quadrupole detector mass spectra/mass spectra (MS/MS) from Waters Corporation, USA. A C18 column with a particle size of 1.7 µm (serial number 04633408725164) was operated at an adjusted temperature of 25°C. The UPLC system was equipped with a library capable of identifying components and functional groups within one minute after the analyte detection. The gradient method employed utilized a mobile phase consisting of 0.1% (v/v) formic acid in H 2 O as Bottle A and Acetonitrile as Bottle B. The gradient conditions are detailed in Table 1 . The total run time was set to 16 minutes, with an injection volume of 1 µL and a sample column temperature maintained at 15°C. The MS/MS instrument configuration included an Lteff setting of 1800.0 and a Veff of 6336.21, with a resolution of 22,000 and a minimum peak point threshold of 2. The acquisition device (ADC) from Waters was set with a trigger threshold of -1.00 V and an input offset of -2.362 V. The average single ion intensity was measured at 25, while the ADC amplitude threshold was established at 10. The target enhancement delay coefficient was set to 2.1500, and the threshold for ion collision was 5.0. The MS/MS parameters were configured with a minimum starting mass-to-charge ratio (m/z) of 50 and a maximum of 1200. The collision energy (CE) was set to a high value of 30 V and a low value of 10 V. Table 1 Gradient parameters for quercetin-like compounds as analytes Stage Time (min) Flow rate (mL/Min) Fraction A:B (%) Curve (unit) 1st Initial 0.300 95:5 Initial 2nd 10.00 0.300 10:90 6 3rd 13.00 0.300 10:90 6 4th 13.10 0.300 95:5 6 5th 16.00 0.300 95:5 6 The Fourier transform infrared spectrophotometer (FT-IR) analysis was performed using a SHIMADZU FT-IR Spectrometer Model IRTracer-100, manufactured by Shimadzu Corp., Japan. A sample weight of 0.5 mg of QLC was combined with 2 mg of FT-IR grade potassium bromide (KBr) and thoroughly homogenized. The resulting QLC-KBr mixture was placed onto the sample holder of the IR Tracer-100 accessory and scanned over a range of 4000 cm⁻¹ to 400 cm⁻¹, with results referenced against transmittance energy (% T). This process involved the interaction of the sample with infrared light, which was subsequently transformed from interferometric interference into a spectrum. The analysis focused on identifying functional groups within the molecular compounds of the QLC. Proton nuclear magnetic resonance analysis Proton nuclear magnetic resonance (¹H NMR) analysis was conducted using the JEOL Resonance Shimadzu ECS-400 instrument, with samples diluted in DMSO-d6. The parameters were set to a field strength of 9.389766 and a frequency of 400 MHz. Each analysis lasted for 1.6384 seconds. The proton examination procedure was conducted from point A to I as follows: Point A: The electronic console was connected to the computer by launching the Delta program. Point B: The NMR tube containing the sample solution was placed into the spinner, and the sample height was measured, ensuring it was ± 4 cm (between 3.8 and 4.2 cm). The tube was positioned using the gauge. Point C: The tube was inserted with the spinner into the superconducting magnet, the "Samples" tab was clicked, and a sample was added by clicking the "+" icon. Point D: The sample name was entered in the designated field, the type of solvent used was selected, and then "Verify" followed by "Share" were clicked. Point E: The sample was loaded by clicking on the notification labeled "Load," then "Interactive" was selected, and "Spinner" was clicked at approximately 15 Hz after the sample was inside the superconducting magnet. Point F: The autolock icon (located in the center) was clicked, followed by the automatic gradient shimming icon. Point G: Once the shimming process was complete, the file name was typed. "Create a Job" was clicked with this sample, and the type of analysis was selected as proton. Point H: The parameters for proton analysis were adjusted. The scan number was set to 8 and X_sweep to 20, then "Submit Job" was clicked. After a new window appears requesting tuning adjustments, the HF tune and match knobs were adjusted by turning the knobs located at the bottom of the superconducting magnet. Once the adjustments were complete, "Done" was clicked. After proceeding to point I, the scan process was waited for until finished. Once completed, the proton spectrum automatically appeared. The results of the proton NMR analysis of QLCs were compared to certified reference material, as referenced at https://go.drugbank.com/spectra/nmr_one_d/1582 . Preparation of analytes for injectable formulation The viscous compound withdrawn from QLC was prepared for injection by dissolving it in a solution of dimethyl sulfoxide (DMSO) obtained from Merck Millipore (catalog number 102952), with a solute-to-solvent ratio of 1:50. The analyte was sterilized to meet criteria for being free of pyrogens and isotonic, isoionic, and isohydric using Eagle media for CEF. Additionally, improvements were made by mixing with a homogenizer (NanoGenizer, US catalog number NG45K) and measuring viscosity (Rion VT06, Japan) using a viscometer. Acid-base balance was further improved using a 1% (w/v) NaOH (Kimia Farma, Indonesia) solution and 0.04 N HCl (Kimia Farma, Indonesia). 16 The pure QLC substance, serving as the test analyte, was subsequently diluted using DMSO in a sequential manner, starting from 150 ppm to 10,000 ppm. Isolation and identification of Salmonella spp. and Escherichia coli The bacteria were obtained from the Laboratory of Bacteriology at the Faculty of Veterinary Medicine, Airlangga University, and were suitable for use at the end of 2024. Samples were collected from cloacal swabs and inoculated onto tetrathionate broth from Merck Corp., USA, catalog number 1052850500, serving as enrichment media, and subsequently incubated at 37°C for 24 hours. Following this, the samples were cultured on selective Salmonella Shigella agar (SSA) from Agarindo Biological Laboratory, Indonesia (catalog number 999) using the streak method and incubated again at 37°C for 24 hours. The characteristics of Salmonella spp. colonies on SSA include being round, colorless with a black center, convex in shape, and possessing smooth edges. The suspected Salmonella spp. colonies were identified microscopically through Gram staining and biochemically. The biochemical tests employed the IMViC method, which included the Indole test using sulfide indole motility (SIM) media, the methyl red (MR) test, the Voges–Proskauer (VP) test, and the citrate test using Simmons citrate agar (SCA) from Merck Corp., USA, catalog number 103855. Following these tests, colony identification continued using triple sugar iron agar (TSIA) from Merck Corp., USA, catalog number 103915. 17 Samples for the isolation of Escherichia coli were collected from cloacal swabs and subsequently cultured on eosin methylene blue Agar (EMBA) media (from Merck Corp., USA, catalog number 103857) using the streak method and incubated at 37°C for 24 hours. The Escherichia coli colonies cultured on EMBA media exhibit distinctive characteristics, including round colony growth, metallic green coloration, convex surfaces, and smooth edges. Identification of Escherichia coli is performed through microscopic examination using Gram staining and IMViC biochemical tests. Suspected Escherichia coli colonies observed under Gram staining display a morphology of short rods and are Gram-negative. Further biochemical identification is conducted utilizing the IMViC test followed by the TSIA test. 18 , 19 Indole test using SIM medium The indole biochemical test is performed by adding 2–3 drops of Kovacs reagent (from Merck Corp., USA, catalog number 109293) to the media after incubation for 24 hours at 37°C. After conducting the indole biochemical test with Kovacs reagent, Salmonella species typically produce a colorless ring or a light pink hue. In contrast, Escherichia coli yields a positive result, indicated by the formation of a red to red-violet color in the reagent layer on top of the medium. MR-VP test The MR-VP test is conducted by inoculating colonies of Salmonella species and Escherichia coli into separate test tubes containing MR-VP media (Thermo Corp., US, catalog number CM0043B) in two separate test tubes and incubating them at 37°C for 24 to 48 hours. The MR test involves the addition of 2–5 drops of methyl red (Sigma Corp., US, catalog number: CAS 493-52-7) as an indicator. In contrast, the VP test is performed by adding 8–10 drops of alpha-naphthol solution (Merck Corp., US, catalog number CAS 90-15-3) along with 40% KOH (Merck Corp., US, catalog number CAS 1310-58-3). A positive result for Salmonella species and Escherichia coli in the MR test is indicated by a color change to red in the media, whereas the VP test yields a negative result, characterized by no change in color. Citrate utilization (SCA) and TSIA tests The citrate test assesses the ability of bacteria to utilize citrate as their sole source of carbon and energy. In this test, Salmonella species exhibits a positive result, while Escherichia coli shows a negative result, as indicated by no color change in the medium. The TSIA test evaluates carbohydrate fermentation (glucose, lactose, and sucrose) to determine acid or gas production. Salmonella species produces gas in the form of hydrogen sulfide (H 2 S), resulting in an alkaline medium. Conversely, Escherichia coli can ferment glucose, lactose, and sucrose, leading to a yellow color change in the TSIA medium. The colonies of Escherichia coli do not produce H 2 S in the TSIA test, indicating an acidic condition in the medium. Identification was conducted to differentiate between Salmonella species and Escherichia coli , using isolation and identification parameters as outlined in Table 2 . Table 2 Isolation and identification parameters related to specific tests for Salmonella spp. and Escherichia coli Parameter Test Type Bacteria Reference Salmonella spp. Escherichia coli Isolation Medium of Salmonella Shigella Agar Round, colorless colony with black center 19, 20, 21 Medium of Eosin Methylene Blue Agar Round colony, metallic green in color Identification Gram Staining Rod-shaped, Gram-negative Short rod, Gram-negative Indol Negative Positive Methyl Red Positive Positive Voges–Proskauer Negative Negative Citrate Positive Negative Triple Sugar Iron Agar Basic/Acid, with the presence of H 2 S Acid/Acid, H 2 S that is not attended Motilities Nonmotile Motile Antibacterial screening of QLC The bacterial tests identified the organisms as Salmonella typhimurium and Escherichia coli , each present in a suspension at a dilution of 1 × 10 5 . Screening tests were performed by inoculating 0.1 mL of the analytical serial QLC onto colony media for both bacteria at a concentration of 1 × 10 5 colony-forming units. 20 , 21 , 22 , 23 The minimum inhibitory concentration (MIC) test was conducted by incubating each bacterium with various concentrations of antibacterial substances in Mueller Hinton Broth (MilliporeSigma Corp., US, catalog number 70192) for 24 hours at 37°C. Observations were made of the last clear tube that showed no bacterial overgrowth. The growth of bacterial suspensions from the MIC test was then cultured on respective media ( Salmonella spp. on SSA medium and Escherichia coli on EMBA medium) to determine whether the antibacterial agent was bacteriostatic or bactericidal. After 24 hours of incubation at 37°C, bacterial growth was assessed. 24 The criteria for interpretation were as follows: if bacterial growth was observed from the clearest tube after inoculation in Mueller Hinton Broth, the analyte was classified as bacteriostatic; conversely, if no bacterial growth occurred, the analyte was deemed bactericidal. 25 , 26 , 27 If preliminary research indicated that QLC required a high concentration to eliminate bacterial colonies, the analyte was diluted to a concentration near the level required for colony elimination. For the positive control, kanamycin disulfate salt (Sigma-Aldrich Corp., CAS 64013-70-3) was utilized, along with clavamox, which contains amoxicillin trihydrate and potassium clavulanate, at a starting concentration ranging from 150 ppm to 10,000 ppm (Kalbemed Pharma Corp., Indonesia). The negative control employed sterile aqua pro injection solvent (Otsuka-Indonesia Corp.), characterized as sterile, pyrogen-free, isotonic, isoionic, and isohydric. 28 Statistical analysis Statistical analysis of QLC's effectiveness in eliminating colonies of Salmonella and Escherichia coli was conducted using a two-tailed Mann-Whitney nonparametric test at a significance level of 0.05. The independent variable in this analysis was the QLC treatment, while the dependent variable was the count of colonies on the microbial media for the two bacterial species. If the number of colonies could not be eliminated or counted, it was recorded as equal to or greater than 300 colonies on the growth media plate. This threshold was established to ensure that with a minimum of 300 colonies, it could be confirmed that all colonies growing on the media were resistant to QLC. The evaluation of the percentage of QLC analytical endpoints against the bacterial types from both Salmonella and Escherichia coli was performed using probit analysis with a significance level of 0.05%. The calculated minimum inhibitory concentration corresponds to a 50% inhibition of colony growth, while the subsequently defined MIC corresponds to a 99% reduction in colony viability. 29 , 30 Statistical analysis was conducted using IBM SPSS Statistics version 27.0 for Microsoft Windows, IBM Corp., Armonk, NY, USA. Results The chromatogram profile of QLC is presented in Fig. 2 , while the standard quercetin is depicted in Fig. 3 . The QLC sample exhibited several peaks at a wavelength of 250.4 nm, appearing at retention times (RTs) of 10.508, 10.868, 11.179, 11.452, and 13.56 minutes. At a wavelength of 255.4 nm, the appearance of additional peaks shifted slightly earlier by 0.06 to 0.12 minutes. At a wavelength of 370.4 nm, the number of peaks diminished, with RTs ranging from 10.534 to 11.624 minutes, 31 while a corresponding shift at 375.4 nm occurred approximately 0.42 minutes earlier. The quercetin standard at wavelengths of 250.4 nm, 255.4 nm, 370.4 nm, and 375.4 nm demonstrated minimal peak shift compared to the analyte, exhibiting nearly identical RTs. The primary HPLC chromatogram peak for QLC was observed at RTs of 14.60 to 14.61 minutes across wavelengths of 250.4 nm, 255.4 nm, 370.4 nm, and 375.4 nm (Fig. 2 ). When compared to the quercetin standard, which appears at RTs of 14.882 and 14.883 minutes, the QLC peak showed similar RTs. Thus, the secondary metabolite utilized as the analyte and isolated using the preparative HPLC system corresponds to the component with an RT of 14.61 minutes. Other chromatogram peaks at a wavelength of 250 nm occurred at RTs of 17.21, 19.41, 19.549, 21.082, 22.076, 22.33, 23.051, 23.391, 24.01, and 14.417 minutes, representing interfering compounds that were not collected. Interfering compounds also manifested at a wavelength of 255 nm, with RTs of 16.228, 16.668, 16.924, 18.736, 19.155, 20.406, 20.643, 21.382, 23.13, 23.332, 24.304, 24.584, 25.666, 25.838, 27.239, 28.157, 28.207, and 29.184 minutes. Observations at a wavelength of 370.4 nm reveal a decrease in interfering peaks, with RTs of 16.904, 19.154, 19.264, and 19.567 minutes. A similar decrease in interfering peaks was noted at a wavelength of 375.4 nm, with RTs of 16.664, 16.946, 19.155, and 19.587 minutes. The results indicate that the purification of QLC contains an aromatic complex, as illustrated in Fig. 4 . Out of 16 detected molecules (m/z), only seven SMCs exhibited percent intensity values exceeding 10%, as detailed in Table 3 . Table 3 Molecules of quercetin-like compounds at high percent intensity levels Formula Neutral mass (g/mol) Measured mass (m/z) Retention time (min) Monoterpenoid C 10 H 20 140.1565 140.150810918010 1.9 Methyl ecgonidine C 10 H 15 NO 2 181.1103 181.0921353141 2.00 3',6-Dimethylflavone C 17 H 14 O 2 250.09938 251.136081773 3.92 Quercetin C 15 H 10 O 7 302.236 303.0627359303 3.59 to 4.5 4'-Acetamino-6-methylflavonone C 18 H 17 NO 3 294.30136 295.1636248876 None Flavone C 15 H 10 O 2 222.06808 223.1039497557 7.00 4'-Amino-5,7-dimethylflavanone C 17 H 17 NO 2 267.12593 267.1316147452 12.07 Table 3 reveals that not all compounds' RTs resulting from the adsorption-partition process using the UPLC-MS/MS column with gradient elution were detected and recorded in the equipment library. Of the seven detected compounds, one, 4'-acetamino-6-methylflavonone, could not be identified, resulting in a recorded RT of "none." However, this compound can be detected by the instrument in terms of mass (m/z), thus qualifying it as part of the QLC components. Mass spectrum analysis indicated nine unidentified masses with an intensity of less than 10% corresponding to (m/z) values of 237.1200, 209.0879, 169.0555, 141.154, 138.0986, 123.0484, 100.0817, 82.0705, and 71.9569. Monitoring the RTs of these nine unidentified SMCs on the integrator revealed unclear peaks and a broad area nearly equivalent to the impurities present in the QLC sample. The RT of quercetin in the QLC samples appeared to drift by 1–2 minutes, attributable to the gradient settings of the mobile phase and the percentage change over time, as outlined in Table 1 . The FT-IR spectrogram results comparing QLC and quercetin are depicted in Fig. 5 , focusing on the fingerprint region from 1400 cm⁻¹ to 400 cm⁻¹. In Fig. 5 a, the QLC sample displays water (H 2 O) elements in the wavenumber region of 3600 cm⁻¹ to 3200 cm⁻¹, which is comparatively higher than that of the quercetin standard shown in Fig. 5 b. Table 4 illustrates a relatively strong percent transmittance between wavenumbers (cm⁻¹) 400–1400, indicating the presence of SMC along with the functional groups attached to the molecular core (Fig. 6 a and Fig. 6 b). Table 4 Infrared finger print of quercetin-like compounds identified by high percent transmission compared to standard quercetin. Sample Quercetin-Like Compound Standard Quercetin Secondary metabolite structure Wavelength number (cm⁻¹) Transmission (%) Wavelength number (cm⁻¹) Transmission (%) 401.1937 83.7422 401.1937 62.9112 Cl–C = O in-plane deformation 403.1225 85.1477 403.1225 62.6879 601.7905 82.1267 601.7905 54.5052 In-plane ring deformation, naphthalene structure 603.7194 82.1778 603.7194 55.4690 619.1499 82.9565 619.1499 66.3706 806.2450 85.8510 835.1773 72.1019 1,2,4-trisubstituted benzenes, triazines 898.8282 91.1739 900.7570 85.9532 R–NH 2 primary amines 921.9740 89.1173 904.6147 86.2411 CH=CH 2 in vinyl compounds, CH = CH in trans disubstituted alkenes, single aromatic C 6 H 5 977.9097 87.9884 974.0520 84.7275 1001.0555 84.9080 1001.0555 60.2916 1028.0589 83.8100 1028.0589 81.5640 Carbon ring in cyclic compounds, in cyclic alcohols, C = S in thiocarbonyl compounds, C–O–C in aliphatic ethers, C–OH in secondary or tertiary alcohols 1029.9877 83.3049 1029.9877 81.3669 1228.6557 84.2667 1228.6557 48.2723 1390.6763 81.5295 1390.6763 51.4070 COO⁻ group in carboxylic acid salts, SO 2 in sulfonyl chlorides 1400.3204 82.019843 1400.3204 54.9228 Results from administering positive controls on Salmonella spp. and Escherichia coli are summarized in Additional file 1, while the MIC test of the QLC analytes against Salmonella spp. and Escherichia coli is presented in Additional file 2. In Additional file 1, the positive control ranged from a concentration of 156.25 µg/mL to 10,000 µg/mL, informed by preliminary studies indicating the elimination potency of kanamycin and clavamox beginning at a level of 150 ppm. Consequently, the positive control concentration initiates slightly above the 150 ppm threshold. In Additional file 2, the QLC analyte begins at a concentration of 625 µg/mL, based on preliminary research findings showing the analyte's elimination capacity commencing at 600 ppm. Thus, the series of test concentrations extends from 625 µg/mL to 10,000 µg/mL. Furthermore, the results of the QLC analysis regarding the elimination capacity of the two types of colonies, assessed using the Mann-Whitney test, are presented in Additional file 2 at p < 0.05. Probit analysis at a 95% confidence limit indicated that the antibacterial effect of the analyte against Salmonella species colonies was bactericidal, with 50% of the colonies expected to perish following exposure to 359.283 µg/mL QLC. Colony death of 75% occurred at an exposure of 1.6 mg/mL QLC, while 99% colony death occurred at an exposure of 61.9 mg/mL QLC as a bactericide. Conversely, exposure to QLC did not yield any inhibitory effect on Escherichia coli bacteria. The inhibitory effect of QLC on Salmonella colonies is further illustrated in Fig. 7 . Discussion The results of the isolation and identification of QLC using preparative HPLC, UPLC-MS/MS, and FT-IR indicate the presence of components within the analyte that include identifiable and non-identifiable SMC. The QLC components exhibiting antibacterial potential consist of a group of SMCs characterized by either a single aromatic or a complex aromatic core ring that is bonded to a strong electronegative group (-O-, -N-, -S-). These SMCs have demonstrated effectiveness in eliminating colonies of Salmonella spp.; however, they are not effective against Escherichia coli colonies, as presented in Tables 3 and 4 and Additional file 2. 32,33 The SMC components that exhibit antibacterial activity against Salmonella spp. include complex aromatic structures containing sulfur and carboxyl ions. As illustrated in Fig. 2 , SMC appears at an RT (min) of 14.61 to 14.808, closely resembling the quercetin standard peak (Fig. 3 ) at 14.882 to 14.883. Furthermore, the UPLC-MS/MS analysis identified SMC components such as monoterpenoid, methyl ecgonidine, 3',6-dimethylflavone, quercetin, 4'-acetamino-6-methylflavonone, flavone, and 4'-amino-5,7-dimethylflavanone (Table 3 , Fig. 4 ). These components are characterized by complex aromatic rings linked to strongly electronegative groups. 34 , 35 Among the seven SMCs, monoterpenoids exhibited a high percentage of transmittance, and these elements theoretically possess a Log P value of less than 3, rendering them hydrophilic and highly soluble in water. Such characteristics facilitate infiltration into the hydrophilic surfaces of bacterial cells and promote interactions with the bacteria's internal structures. 36 Ultimately, this leads to the inability of the bacteria to regulate their biochemical systems, resulting in gradual cell death. The other six SMCs demonstrated similar average transmittance values, generally exhibiting aromatic molecular structures characterized by high stability due to the resonance of ion charges in each atom, and are attached to strong electron-withdrawing groups. 37 Some were also identified as being ionically bound to central molecules such as sulfonyl chlorides, carboxylic acid salts, thiocarbonyl compounds, benzene, and naphthalene (Table 4 ). Figure 6 demonstrates that QLCs, when compared to the standard quercetin at fingerprint wavenumbers, produce similar molecular ions. Wavenumbers (cm − 1 ) 401–403 indicate the presence of C and O bonds that bind Cl. 38 This SMC has the potential to form bonds with the positively charged inductive groups present in bacterial cytoplasm. Similarly, the wavenumbers 601–619 indicate that the lipophilic naphthalene structure has the capacity to penetrate the lipid bilayer of the bacterial membrane, thereby forming bonds with bacterial DNA components. The resultant effect of these bonds inhibits the bacterial cell's respiratory system, ultimately slowing down and eventually halting bacterial growth and colony formation. Other components, such as 1,2,4-trisubstituted benzenes, triazines R–NH 2 , and CH=CH 2 , CH = CH– bound to alkenes or single aromatics (wavenumbers 806–1001 cm − 1 , Fig. 5 ), are lipophilic and thus possess the potential to penetrate bacterial membranes. 39 Likewise, SMCs containing carbon rings in cyclic compounds, cyclic alcohols, C = S in thiocarbonyl compounds, C–O–C in aliphatic ethers, C–OH in secondary or tertiary alcohols, as well as the COO⁻ group in carboxylic acid salts and SO 2 in sulfonyl chlorides (detected at wavenumbers 1200–1400 cm − 1 ), exhibited lipophilic properties that enable complexation with the core acidic elements of bacteria, either through electron-withdrawing or inductive groups. This phenomenon accelerated bacterial death, impacting bacterial colonies significantly. In Fig. 6 a, the percentage transmittance of SMC QLC at wavenumbers (cm − 1 ) 401–619 has an average intensity 1.5 times stronger compared to the quercetin standard shown in Fig. 6 b, indicating that the SMC QLC component at these wavenumbers likely possesses strong antibacterial activity. However, the percentage transmittance at wavenumbers (cm − 1 ) 806–1001 for MSC QLC was slightly higher compared to the same element found in standard quercetin. Under these conditions, the penetration capability of this element yielded antibacterial effects, albeit not very potent. Nevertheless, the antibacterial efficacy was enhanced in elements characterized by high percentage transmittance. Conversely, the SMC QLC at the wavenumbers (cm − 1 ) 1028–1029 exhibited percentage transmittance values similar to standard quercetin, although still higher for QLC. Thus, the antibacterial potency of elements at these wavenumbers (cm − 1 ) was relatively weak. This contrasts with elements appearing at wavenumbers (cm − 1 ) 1228–1400, where the percentage transmittance value was twice as high in QLC compared to standard quercetin. 40 At this wavenumber, SMC QLC elements that exhibit significant antibacterial activity were produced. The results of the NMR analysis of the analyte revealed (Fig. 6 a) that at chemical shifts (ppm) between 7.9059 and 7.8875, the H7 of protonated QLC was present, along with H6 (7.6349) and H4 (7.1089). The chemical shifts (Fig. 6 b) for the standard H7 (7.7344) and H6 (7.6202), as well as H4 (7.6149), with a standard deviation of 0.217, indicated a minor difference in the protonation of the C15H10O7 content of QLC. Such differences are expected when natural materials are dissolved in a solvent. Meanwhile, the protonation of the analyte solvent (Fig. 6 a) was detected to match the chemical shift of the standard (Fig. 6 b). However, the protonation of C15H10O7 at the H atoms in positions 1, 2, 3, 5, 8, 9, and 10 (Fig. 6 a) could not be detected by the NMR detector, likely due to the QLC isolate being too small for monitoring. If the NMR device operates at an energy level above 400 MHz, it may be possible to detect all protons. The analytical chemical shifts (Fig. 6 a) at (ppm) 7.1089 and 7.0872, including 1.2888, likely correspond to protons from components such as monoterpenoid, methyl ecgonidine, 3',6-dimethylflavone, 4'-acetamino-6-methylflavonone, flavone, and 4'-amino-5,7-dimethylflavanone. A significant difference is evident when compared to Fig. 6 b, which represents a single compound standard, whereas the QLC analyte is a complex compound, indicating that the QLC compound contains multiple constituent elements. In summary, the results indicate that the QLC content contains the element C15H10O7 and has the potential to exhibit biological activity against bacterial colonies. The analysis of colony death demonstrated that 50% of Salmonella spp. bacteria died following exposure to QLC at a concentration of 359.283 µg/mL, while 75% death occurred at 1.6 mg/mL and 99% at 61.9 mg/mL, suggesting that SMC QLC possesses antibacterial activity against Salmonella spp. (Fig. 7 ). However, exposure of SMC QLC to Escherichia coli colonies did not yield antibacterial activity (p < 0.05). Additional files 1–3 indicate that QLC is ineffective in eliminating bacterial colonies, as the number of colonies remained at or above 300. The reduction in Salmonella bacterial colonies due to QLC exposure ranged from 2.93% to 99.6%. Nevertheless, Additional file 1, as the positive control, illustrates that both bacterial strains are highly sensitive to kanamycin and clavamox. The results from the negative control analysis reveal that the colonies of both bacteria continue to grow, averaging 300 colonies or more. The antibacterial activity of kanamycin against Salmonella spp. was observed at 5 mg/mL, while against Escherichia coli , it was noted at a concentration of 10 mg/mL. The antibacterial efficacy of clavamox against Salmonella spp. was identified at a concentration of 1.2 mg/mL, whereas its activity against Escherichia coli was found at 2.5 mg/mL (Additional file 1). This table indicates that the Salmonella strain used exhibits greater resistance to antibiotics compared to Escherichia coli . Consequently, the infection potential of the Salmonella spp. is considered more significant than that of Escherichia coli . 41 The antibacterial activity of QLC against Salmonella spp. was 12.5 times lower than that of kanamycin and 6.2 times lower than that of clavamox. These results suggest that SMC QLC, at an exposure concentration of 61.9 mg/mL or 62 mg/mL against Salmonella spp., has not yet reached toxic levels in plasma concentrations within the body in vivo. Therefore, its potential as an anti- Salmonella agent is highly promising. The ineffectiveness of QLC against Escherichia coli colonies may be attributed to the components in QLC being unable to form complexes with the cytoplasmic elements of bacterial cells and/or unable to interact with the nucleic acids of these cells. Theoretically, effective antibacterial activity against Escherichia coli would necessitate more lipophilic chemical compounds containing elements capable of damaging the bacterial cell wall. Such efforts to disrupt the bacterial cell wall could lead to cytoplasmic damage and potentially harm the bacterial nucleic acid chains. The differences in cell wall biosynthesis between Salmonella species and Escherichia coli suggest that the biosynthesis control system regulating the metabolism of saccharide lipoproteins essential for the cell wall is more complete in Salmonella . Consequently, SMC that can complex with the complete elements of Salmonella spp. can easily form complexes. 41 , 42 In contrast, the specific biosynthesis system required for Escherichia coli makes it difficult for SMCs to bind to its interior, rendering the bacteria resistant to QLC exposure. Conclusion The findings of this study indicate that QLC is capable of exhibiting bactericidal effects against Salmonella spp. colonies at a concentration of 62 mg/mL. However, QLC is ineffective as a bactericide against Escherichia coli colonies. QLCs extracted from the leaves of Lansium domesticum demonstrate significant potential for utilization as new antibiotics targeting Salmonella spp. It is recommended that for antibiotic application, QLC be administered in liquid form as a solution. List of abbreviations ADC acquisition device BD Dendrophthoe pentandra L. Miq CE collision energy DMSO dimethyl sulfoxide EMBA eosin methylene blue agar HPLC high-performance liquid chromatography MIC minimum inhibitory concentration MR methyl red NMR nuclear magnetic resonance QLC quercetin-like compounds RT retention times SCA Simmons citrate agar SIM sulfide indole motility SMC secondary metabolite compounds SSA Salmonella Shigella agar TSIA triple sugar iron agar UPLC ultra-performance liquid chromatography VP Voges–Proskauer Declarations Ethics approval and consent to participate: Not applicable Consent for publication: Not applicable Availability of data and materials: https://doi.org/10.6084/m9.figshare.31112728.v2 https://doi.org/10.6084/m9.figshare.31238056 Competing interests: The authors declare that they have no competing interests. Funding: This work was supported by the Directorate General of Research and Development, Ministry of Higher Education, Science, and Technology for the 2025 fiscal year [DIPA SP Number 139.04.1.693320/2025, revision 04, dated April 30, 2025]. Authors' contributions: Acknowledgements: The authors express their gratitude for the assistance received from the following individuals and groups: (1) the Research Group in Veterinary Pharmacy Science, Faculty of Veterinary Medicine, Universitas Airlangga, (2) the staff of the Laboratory of Veterinary Pharmacy Science, Faculty of Veterinary Medicine, Universitas Airlangga, and (3) Student faculty of veterinary medicine Universitas Airlangga especially Rafif Praptama Iskandar, Zahra Alyssa Ekaputri, Annida Annafiri Rakhman, Nazwa Afifah Pujihati Muluk, Baiq Gina Rahayu Putri, Christine Jayanti . 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Supplementary Files AdditionalFile1.docx Additional file 1.docx (table): Antibacterial activity of kanamycin and clavamox against Salmonella spp. and Escherichia coli AdditionalFile2.docx Additional file 2.docx (table): Concentration of quercetin-like compounds and the corresponding inhibitionof Salmonella spp. and Escherichia coli colonies AdditionalFile3.docx Additional file 3.docx (table): Bactericidal activity of quercetin-like compounds versus kanamycin and clavamox against Salmonella spp. and Escherichia coli DataMistletoeLIPI2024.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 30 Mar, 2026 Reviewers agreed at journal 30 Mar, 2026 Reviewers agreed at journal 26 Feb, 2026 Reviewers invited by journal 09 Feb, 2026 Editor assigned by journal 05 Feb, 2026 Editor invited by journal 05 Feb, 2026 Submission checks completed at journal 03 Feb, 2026 First submitted to journal 03 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8723177","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":589350015,"identity":"823dc800-73b8-4d63-bc2a-f49cd0624018","order_by":0,"name":"Lazuardi Mochamad","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIie2RsWrDMBCGJQSXRVjrGaf0FVQMpsWEvIpCwH6D0qlkui5+h7xIBoNA2Tq3UEiyeMqQMaUdKjvN0EGh3TroG04a9HH67xiLRP4hyekQUgkHw/VUGfJFQIGzklL1R4Vp962cCSujdYfvq3KcO6i2nN6uk6zlhyO7vQoq0lRp09WycLDWnLobSoxIG4Z5+GPGoWytLDZPhJwsJ8lY5rPMgoraUfrplZxGgzL1ivi4qOAcsr6LBnC9MvMKXO6CnSjHbS3Rx0fzbOckOd01OpxFqXr3um/LqSIo8HBvJ8tG2Jfjw2NwYj8xQ+03on/1PhKJRCIBvgBoD0jnG3JAlwAAAABJRU5ErkJggg==","orcid":"","institution":"Airlangga University","correspondingAuthor":true,"prefix":"","firstName":"Lazuardi","middleName":"","lastName":"Mochamad","suffix":""},{"id":589350016,"identity":"81c881fa-8ad4-4030-81ca-0161796b11a0","order_by":1,"name":"Aniek Setiya Budiatin","email":"","orcid":"","institution":"Airlangga University","correspondingAuthor":false,"prefix":"","firstName":"Aniek","middleName":"Setiya","lastName":"Budiatin","suffix":""},{"id":589350017,"identity":"b2ac6fa9-83a1-4376-8407-23506b075138","order_by":2,"name":"Wiwiek Tyas Ningsih","email":"","orcid":"","institution":"Universitas Airlangg","correspondingAuthor":false,"prefix":"","firstName":"Wiwiek","middleName":"Tyas","lastName":"Ningsih","suffix":""},{"id":589350019,"identity":"1bbda6a0-451d-4aeb-8761-3c2f5e6700d5","order_by":3,"name":"Gandul Atik Yuliani","email":"","orcid":"","institution":"Airlangga University","correspondingAuthor":false,"prefix":"","firstName":"Gandul","middleName":"Atik","lastName":"Yuliani","suffix":""}],"badges":[],"createdAt":"2026-01-28 16:08:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8723177/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8723177/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102616214,"identity":"ab984413-e0aa-4811-a6c2-06b8fe835590","added_by":"auto","created_at":"2026-02-13 15:40:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":25004785,"visible":true,"origin":"","legend":"\u003cp\u003eDevelopment of \u003cem\u003ein vitro\u003c/em\u003emistletoe from \u003cem\u003eLansium domesticum\u003c/em\u003e over a 30-day period.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8723177/v1/82135156a3cdfcf87c4b73ca.jpg"},{"id":102616193,"identity":"3fae8328-b953-49d5-a2ce-38f274459571","added_by":"auto","created_at":"2026-02-13 15:40:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18621428,"visible":true,"origin":"","legend":"\u003cp\u003eChromatogram of quercetin-like compounds. Retention times were 14.61 minutes at a wavelength of 255.4 nm, 14.508 minutes at a wavelength of 370.4 nm, and 14.808 minutes at a wavelength of 375.4 nm.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8723177/v1/2870e79479cf4711652902c9.jpg"},{"id":102616124,"identity":"ca0a14a3-a112-408a-9cdf-6d2754836a30","added_by":"auto","created_at":"2026-02-13 15:40:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":13652997,"visible":true,"origin":"","legend":"\u003cp\u003eChromatogram of the standard quercetin. Retention times were approximately 14.883 minutes at a wavelength of 255.4 nm, 14.882 minutes at a wavelength of 370.4 nm, and 14.882 minutes at a wavelength of 375.4 nm.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8723177/v1/ddf1136fd5050269749dff62.jpg"},{"id":102616219,"identity":"ff721a7b-cf25-4ecd-bbb8-fbc295a1e555","added_by":"auto","created_at":"2026-02-13 15:40:51","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":14070656,"visible":true,"origin":"","legend":"\u003cp\u003eProfiling of mass spectra for quercetin-like compounds (m/z) with intensity values (%). Quercetin-like compounds were dissolved in methanol at a concentration of 50 μg/mL using Acquity ultra-performance liquid chromatography-tandem quadrupole detector mass spectrometry with a Xevo Generation 3 detector.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8723177/v1/10d88950e42d8f2533d0e901.jpg"},{"id":102616162,"identity":"92fd35bb-a050-40ce-94ba-1a98ca1f4709","added_by":"auto","created_at":"2026-02-13 15:40:41","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":16792075,"visible":true,"origin":"","legend":"\u003cp\u003eProfiling via Fourier transform infrared spectrophotometry. (a) Quercetin-like compounds and (b) standard quercetin at a concentration of 0.5 mg of analytes and 2 mg of potassium bromide pellets were mapped using the Shimadzu Model IRTracer-100 across a wavelength range of 400 cm\u003csup\u003e-1\u003c/sup\u003e to 4000 cm\u003csup\u003e-1\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8723177/v1/c72810bcc104a19b49ade767.jpg"},{"id":102616194,"identity":"e72f5100-1f18-4c4d-a5ff-1aa88533a423","added_by":"auto","created_at":"2026-02-13 15:40:47","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6868291,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eH nuclear magnetic resonance spectrophotometry using a JEOL Shimadzu ECS-400. (a) Chemical shifts of quercetin-like compounds and (b) standard quercetin at a concentration of 0.09 μg/mL, dissolved in dimethyl sulfoxide-d6. Parameters were processed at a transmittance strength of 9.389766, with a frequency of 400 MHz , and each analysis lasted 1.6384 seconds.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8723177/v1/981ceea46c82d87a297546b6.jpg"},{"id":102748169,"identity":"fcf2f1a6-e3af-4274-8d24-52c0f312d6bb","added_by":"auto","created_at":"2026-02-16 09:06:11","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":7856915,"visible":true,"origin":"","legend":"\u003cp\u003eConcentrations of quercetin-like compounds demonstrating antimicrobial activity against \u003cem\u003eSalmonella\u003c/em\u003e spp., with a 50% effective concentration of 359.283 μg/mL and a 90% effective concentration of 61.9 mg/mL.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8723177/v1/8eda3350904ae95d98dcb9eb.jpg"},{"id":102616158,"identity":"27be1a5e-1544-488a-99dc-f1df8ed8109f","added_by":"auto","created_at":"2026-02-13 15:40:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":35320,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 1.docx (table): Antibacterial activity of kanamycin and clavamox against \u003cem\u003eSalmonella\u003c/em\u003e spp. and \u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e","description":"","filename":"AdditionalFile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8723177/v1/0d3e7090650accc64852807e.docx"},{"id":102616189,"identity":"107673dc-f728-4ba5-8262-099e6fdffc3a","added_by":"auto","created_at":"2026-02-13 15:40:45","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":28659,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 2.docx (table): Concentration of quercetin-like compounds and the corresponding inhibitionof \u003cem\u003eSalmonella\u003c/em\u003e spp. and \u003cem\u003eEscherichia coli\u003c/em\u003e colonies\u003c/p\u003e","description":"","filename":"AdditionalFile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8723177/v1/d92224eabdf57265436bbee7.docx"},{"id":102616188,"identity":"672eaee7-f3b3-4f01-b2e5-34e0e9881ed8","added_by":"auto","created_at":"2026-02-13 15:40:45","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":25481,"visible":true,"origin":"","legend":"\u003cp\u003eAdditional file 3.docx (table): Bactericidal activity of quercetin-like compounds versus kanamycin and clavamox against \u003cem\u003eSalmonella\u003c/em\u003e spp. and \u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e","description":"","filename":"AdditionalFile3.docx","url":"https://assets-eu.researchsquare.com/files/rs-8723177/v1/0f9a770ec9d82cb608d7a014.docx"},{"id":102616218,"identity":"1bfab210-6328-4e21-9197-3b5d4e3c17ea","added_by":"auto","created_at":"2026-02-13 15:40:51","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":200335,"visible":true,"origin":"","legend":"","description":"","filename":"DataMistletoeLIPI2024.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8723177/v1/b998348d53e0895981f9620f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bactericidal activity of quercetin like-compounds isolated from Dendrophthoe pentandra leaves against Salmonella spp. and Escherichia coli: an in vitro experimental study","fulltext":[{"header":"Background","content":"\u003cp\u003eIt is well-established that secondary metabolite compounds (SMCs) derived from the leaves of mistletoe, specifically those growing on the \u003cem\u003eLansium domesticum\u003c/em\u003e host and known as \u003cem\u003eDendrophthoe pentandra\u003c/em\u003e L. Miq (BD), contain bioactive components. These components, purified from the flavonol fraction, are identified as quercetin-like compounds (QLCs) and have demonstrated antiviral properties \u003cem\u003ein vitro\u003c/em\u003e, particularly against Newcastle disease.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e This mistletoe, commonly referred to as \"duku mistletoe\" in Southeast Asia, especially among the Malay community, features leaves that are approximately 2\u0026ndash;3 cm wide, with smooth edges and a rigid petiole. The species exhibits two subspecies: one with green leaves (\u003cem\u003eDendrophthoe pentandra\u003c/em\u003e L. Miq) and another with reddish-green leaves (\u003cem\u003eScurrula ferruginea\u003c/em\u003e). The reddish-green subspecies is predominantly found in Indonesia, particularly in Bali and the regions of western and eastern Nusa Tenggara, as well as parts of East Timor, the Southern Philippines, Malaysia, and regions of Southern Africa, Northern Australia, and New Zealand. The duku mistletoe typically thrives in tropical regions characterized by relatively high rainfall.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eResearch has identified that these QLCs contain various elements, including 5,7,8,30,40-pentamethoxyflavonone, 7-hydroxy-1-methoxy-2-methoxyxanthone, and pelargonidin-3-glucoside. These three SMCs prominently dominate the QLC content and theoretically possess complex aromatic structures with additional electron-attracting groups such as =\u0026thinsp;O=, -C-, and -N-, which may facilitate their penetration of lipid bilayers, particularly in eukaryotic cell membranes. Furthermore, these SMCs exhibit stability due to the resonance of their aromatic structures, rendering them less susceptible to reactions with other groups. This characteristic positions them as promising candidates for new \"prodrugs\" capable of interacting with the ribonucleic acid or deoxyribonucleic acid (DNA) of eukaryotic cells. In the context of bacterial cells, they hold potential as antibiotics by forming complexes with nucleic acid components that can bind to the complex aromatic structures of QLC.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e The interactions among other QLC components, such as 5-hydroxy-6,7-dimethoxyflavone-4'-O-β D-glucoside, 7-hydroxy-1-methoxy-2-methoxy xanthone, ononin, morin, and quercetagine, may facilitate complexation with viral DNA components, acting as nucleic acid respiratory blockers. This mechanism can induce cell death while simultaneously functioning as an antibiotic through ionospheric action.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e While the role of mistletoe plants as antibiotics remains relatively obscure, their potential benefits warrant exploration. These include (a) minimal disruption to ecosystems due to the selective parasitic nature of mistletoe on its primary host and (b) the SMCs' selective efficacy, which is highly dependent on the quality of the host. These factors may contribute to a more cost-effective and efficient extraction process for SMCs from mistletoe. Nonetheless, a significant challenge persists: the limited availability of these plants in the wild, despite efforts to cultivate them artificially. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrates that following attempts to grow mistletoe in artificial media, seedlings typically develop within approximately 30 days, during which their root systems begin to establish.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe artificial cultivation of mistletoe generally progresses through four phases: (A) the seedling phase, (B) the germination phase, (C) the maturation/preparation of plant stock, and (D) the emergence of small roots from the germinated stock. At this stage, the plants are ready to be transferred to soil, which must be kept consistently moist and well-aerated. However, phase (D) presents a challenge, as mistletoe plants often struggle to adapt when transitioned to non-artificial growing media, leading to mortality. To mitigate this issue, a longer development period is essential, allowing the emerging roots to strengthen and increasing their resilience during the transition to soil. Additionally, careful consideration of the soil media composition (50% original media and 50% new media) is crucial to ensure that the newly sprouted roots do not encounter an overly unfamiliar environment.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThese findings create opportunities for the cultivation of mistletoe and facilitate the standardization of simplicial for the extraction of raw SMCs in diverse climatic conditions. The acquisition of SMCs from mistletoe aligns with the Sustainable Development Goals, particularly point 3, which focuses on health and well-being by minimizing the reliance on artificial chemical compounds in medicine and promoting the emergence of new therapeutic responses to non-natural drugs.\u003c/p\u003e \u003cp\u003eEfforts to discover new antibiotics that circumvent antimicrobial resistance phenomena continue to be prioritized, drawing from both plant sources and semi-synthetic or synthetic origins.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e The SMCs with antibiotic properties are predominantly located in the leaves of medicinal plants, with common components including plant alkaloids from various heterocyclic groups, such as pyrrolidine (hygroline), pyrrolizidine (senecionine), pyridine and piperidine (piperine, lobeline), tropane (cocaine), quinoline (quinine, quinidine), aporphine (boldine), quinolizidine (sparteine), indole or benzopyrrole (ergometrine), indolizidine (swainsonine), imidazole (pilocarpine), purine (caffeine), steroidal (solanidine), and terpenoid (aconitine).\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Heterocyclic alkaloid groups rich in polyphenolic content, such as QLCs, contain numerous nitrogen ions bound to oxygen ions, potentially lowering the pH and enhancing lipid penetration, including through bacterial cell walls. Consequently, many SMCs in plants are not isolated specifically, as the pharmacological efficacy of an SMC can stem from a broad array of alkaloids.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Polyphenolic compounds in leaves theoretically possess bactericidal properties due to their heterocyclic structural elements, which can bind to the base pair elements of DNA, provided that the base pairs exhibit a strong affinity for the groups present in the SMC heterocycles. This binding occurs rapidly after macromolecules distribute SMCs from the blood of clinical subjects to areas of strong cellular affinity. This phenomenon may lead to the generation of novel compounds isolated from plants, which are often abundant and readily available in the wild.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Based on the aforementioned analysis, this research aims to investigate the potential benefits of QLCs as ionospheric antibiotic compounds with possible bactericidal action. This research is urgent to be conducted, considering the circulation of regulations prohibiting the use of antibiotics for animals consumed by humans in countries that support the provisions of the SDGs, particularly for bacteria of one of the Enterobacteriaceae, namely Salmonella, which causes pullorum disease in chickens.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and mistletoe plant material\u003c/h2\u003e \u003cp\u003eThis study was conducted using an exploratory model over a period spanning from late December 2022 to mid-2024, primarily at the Center for Testing and Application of Veterinary Pharmaceutical Sciences, Faculty of Veterinary Medicine, Universitas Airlangga. The mistletoe plant studied, identified as \u003cem\u003eDendrophthoe pentandra\u003c/em\u003e L. Miq, originates from \u003cem\u003eLansium Domesticum\u003c/em\u003e in the Muara Enim district, located at a latitude of -3.663223 and a longitude of 103.778161 in South Sumatra, Indonesia. The plant has received taxonomic confirmation from the National Research and Innovation Agency of the Republic of Indonesia via letter No. B-3286/II.6.2/IR.01.02/9/2024 .\u003csup\u003e11,12,13\u003c/sup\u003e The names of the officers who carried out the identification of plant materials come from the Directorate of Scientific Collection Management, National Research and Innovation Agency of the Republic of Indonesia was Mr. Wihermanto. The virtual visit to the mistletoe herbarium can be accessed at the link \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.6084/m9.figshare.31238056\u003c/span\u003e\u003cspan address=\"10.6084/m9.figshare.31238056\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. The mistletoe specimens that have been identified are stored at the National Research and Innovation Agency of the Republic of Indonesia at code BO 1738456 at September 11th, 2024. The collection of Mistletoe specimens was carried out after obtaining official permission from the Ministry of Research, Technology, and Higher Education of the Republic of Indonesia to conduct research under contract number 2344/B/UN3.LPPM/PT.01.03/2025 dated June 2, 2025.The mistletoe chosen for this research object is not a rare plant and still follows the rules of The International Union for Conservation of Nature.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFlavonol extraction and isolation of QLC\u003c/h3\u003e\n\u003cp\u003eThe technique employed for obtaining the analyte, QLC, was rapid, accurate, and precise, utilizing preparative High-Performance Liquid Chromatography (HPLC) as outlined in patent ID P000063522 [\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://patentscope.wipo.int/search/en/detail.jsf?docId=ID209010597]\u003c/span\u003e\u003cspan address=\"https://patentscope.wipo.int/search/en/detail.jsf?docId=ID209010597]\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003csup\u003e14,15\u003c/sup\u003e A total of 3 kg of BD leaf powder underwent mobile maceration, wherein 1 L of methanol (pro analysis, catalog number 106012, Merck Millipore) was added for every 500 g of BD powder. The mixture was agitated for 3 days using a rotary displacement method. The extract was subsequently filtered through a Buchner funnel, and the macerated juice was extracted using a flannel cloth. The resultant liquid was then dried using nitrogen vapor in a water bath maintained at 40\u0026deg;C. This process was repeated for the remaining 2,500 g of powder. Following this, the concentrated macerate was further treated with ethyl acetate (pro analysis, catalog number 109623, Merck Millipore) in a split tube, using a ratio of 100 g of macerate to 250 mL of ethyl acetate. The ethyl acetate solution was collected and dried under nitrogen vapor in a water bath at 40\u0026deg;C. The dried macerate was then subjected to additional maceration in a separate flask using n-hexane (catalog number 104374, Merck Millipore) and water (pro chromatograph, Merck Corp., catalog number 115333) in a 50:50 ratio. The final macerated product was dried using a rotary evaporator at 40\u0026deg;C, and the viscous compound was subsequently isolated as a QLC using a semi-preparative HPLC device (Agilent Infinity II, USA). The specifications for the device included a column with four units, a flow range of 1\u0026ndash;50 mL/min, an injection volume range of 0.1\u0026ndash;5000 \u0026micro;L, a maximum of 215 fractions, a binary pump system, and an operating pressure range of 20\u0026ndash;420 bar, with a width of 941 mm. The preparative HPLC parameters were set at wavelengths of 250 nm, 254 nm, 261 nm, 370.4 nm, and 375.4 nm, utilizing an ODS C18 column (specification PN 440905-802, SN 71199, 250 x 10.0 mm, Agilent, USA) in isocratic mode with a mobile phase of water:methanol (30:70), adjusted to a flow rate of 0.5 mL/min for a 10-mL sample injection.\u003c/p\u003e\n\u003ch3\u003eCharacterization of QLC using ultra-performance liquid chromatography mass spectrometry and Fourier transform infrared spectrophotometry\u003c/h3\u003e\n\u003cp\u003eThe analysis of content QLC was conducted using Acquity ultra-performance liquid chromatography (UPLC) with a tandem quadrupole detector mass spectra/mass spectra (MS/MS) from Waters Corporation, USA. A C18 column with a particle size of 1.7 \u0026micro;m (serial number 04633408725164) was operated at an adjusted temperature of 25\u0026deg;C. The UPLC system was equipped with a library capable of identifying components and functional groups within one minute after the analyte detection. The gradient method employed utilized a mobile phase consisting of 0.1% (v/v) formic acid in H\u003csub\u003e2\u003c/sub\u003eO as Bottle A and Acetonitrile as Bottle B. The gradient conditions are detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The total run time was set to 16 minutes, with an injection volume of 1 \u0026micro;L and a sample column temperature maintained at 15\u0026deg;C. The MS/MS instrument configuration included an Lteff setting of 1800.0 and a Veff of 6336.21, with a resolution of 22,000 and a minimum peak point threshold of 2. The acquisition device (ADC) from Waters was set with a trigger threshold of -1.00 V and an input offset of -2.362 V. The average single ion intensity was measured at 25, while the ADC amplitude threshold was established at 10. The target enhancement delay coefficient was set to 2.1500, and the threshold for ion collision was 5.0. The MS/MS parameters were configured with a minimum starting mass-to-charge ratio (m/z) of 50 and a maximum of 1200. The collision energy (CE) was set to a high value of 30 V and a low value of 10 V.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGradient parameters for quercetin-like compounds as analytes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTime (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFlow rate (mL/Min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFraction A:B (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCurve\u003c/p\u003e \u003cp\u003e(unit)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1st\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInitial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95:5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInitial\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2nd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10:90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3rd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10:90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4th\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95:5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5th\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95:5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe Fourier transform infrared spectrophotometer (FT-IR) analysis was performed using a SHIMADZU FT-IR Spectrometer Model IRTracer-100, manufactured by Shimadzu Corp., Japan. A sample weight of 0.5 mg of QLC was combined with 2 mg of FT-IR grade potassium bromide (KBr) and thoroughly homogenized. The resulting QLC-KBr mixture was placed onto the sample holder of the IR Tracer-100 accessory and scanned over a range of 4000 cm⁻\u0026sup1; to 400 cm⁻\u0026sup1;, with results referenced against transmittance energy (% T). This process involved the interaction of the sample with infrared light, which was subsequently transformed from interferometric interference into a spectrum. The analysis focused on identifying functional groups within the molecular compounds of the QLC.\u003c/p\u003e\n\u003ch3\u003eProton nuclear magnetic resonance analysis\u003c/h3\u003e\n\u003cp\u003eProton nuclear magnetic resonance (\u0026sup1;H NMR) analysis was conducted using the JEOL Resonance Shimadzu ECS-400 instrument, with samples diluted in DMSO-d6. The parameters were set to a field strength of 9.389766 and a frequency of 400 MHz. Each analysis lasted for 1.6384 seconds.\u003c/p\u003e \u003cp\u003eThe proton examination procedure was conducted from point A to I as follows:\u003c/p\u003e \u003cp\u003ePoint A: The electronic console was connected to the computer by launching the Delta program.\u003c/p\u003e \u003cp\u003ePoint B: The NMR tube containing the sample solution was placed into the spinner, and the sample height was measured, ensuring it was \u0026plusmn;\u0026thinsp;4 cm (between 3.8 and 4.2 cm). The tube was positioned using the gauge.\u003c/p\u003e \u003cp\u003ePoint C: The tube was inserted with the spinner into the superconducting magnet, the \"Samples\" tab was clicked, and a sample was added by clicking the \"+\" icon.\u003c/p\u003e \u003cp\u003ePoint D: The sample name was entered in the designated field, the type of solvent used was selected, and then \"Verify\" followed by \"Share\" were clicked.\u003c/p\u003e \u003cp\u003ePoint E: The sample was loaded by clicking on the notification labeled \"Load,\" then \"Interactive\" was selected, and \"Spinner\" was clicked at approximately 15 Hz after the sample was inside the superconducting magnet.\u003c/p\u003e \u003cp\u003ePoint F: The autolock icon (located in the center) was clicked, followed by the automatic gradient shimming icon.\u003c/p\u003e \u003cp\u003ePoint G: Once the shimming process was complete, the file name was typed. \"Create a Job\" was clicked with this sample, and the type of analysis was selected as proton.\u003c/p\u003e \u003cp\u003ePoint H: The parameters for proton analysis were adjusted. The scan number was set to 8 and X_sweep to 20, then \"Submit Job\" was clicked. After a new window appears requesting tuning adjustments, the HF tune and match knobs were adjusted by turning the knobs located at the bottom of the superconducting magnet. Once the adjustments were complete, \"Done\" was clicked. After proceeding to point I, the scan process was waited for until finished. Once completed, the proton spectrum automatically appeared.\u003c/p\u003e \u003cp\u003eThe results of the proton NMR analysis of QLCs were compared to certified reference material, as referenced at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://go.drugbank.com/spectra/nmr_one_d/1582\u003c/span\u003e\u003cspan address=\"https://go.drugbank.com/spectra/nmr_one_d/1582\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003ePreparation of analytes for injectable formulation\u003c/h3\u003e\n\u003cp\u003eThe viscous compound withdrawn from QLC was prepared for injection by dissolving it in a solution of dimethyl sulfoxide (DMSO) obtained from Merck Millipore (catalog number 102952), with a solute-to-solvent ratio of 1:50. The analyte was sterilized to meet criteria for being free of pyrogens and isotonic, isoionic, and isohydric using Eagle media for CEF. Additionally, improvements were made by mixing with a homogenizer (NanoGenizer, US catalog number NG45K) and measuring viscosity (Rion VT06, Japan) using a viscometer. Acid-base balance was further improved using a 1% (w/v) NaOH (Kimia Farma, Indonesia) solution and 0.04 N HCl (Kimia Farma, Indonesia).\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e The pure QLC substance, serving as the test analyte, was subsequently diluted using DMSO in a sequential manner, starting from 150 ppm to 10,000 ppm.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and identification of Salmonella spp. and Escherichia coli\u003c/h2\u003e \u003cp\u003eThe bacteria were obtained from the Laboratory of Bacteriology at the Faculty of Veterinary Medicine, Airlangga University, and were suitable for use at the end of 2024. Samples were collected from cloacal swabs and inoculated onto tetrathionate broth from Merck Corp., USA, catalog number 1052850500, serving as enrichment media, and subsequently incubated at 37\u0026deg;C for 24 hours. Following this, the samples were cultured on selective \u003cem\u003eSalmonella Shigella\u003c/em\u003e agar (SSA) from Agarindo Biological Laboratory, Indonesia (catalog number 999) using the streak method and incubated again at 37\u0026deg;C for 24 hours. The characteristics of \u003cem\u003eSalmonella\u003c/em\u003e spp. colonies on SSA include being round, colorless with a black center, convex in shape, and possessing smooth edges. The suspected \u003cem\u003eSalmonella\u003c/em\u003e spp. colonies were identified microscopically through Gram staining and biochemically. The biochemical tests employed the IMViC method, which included the Indole test using sulfide indole motility (SIM) media, the methyl red (MR) test, the Voges\u0026ndash;Proskauer (VP) test, and the citrate test using Simmons citrate agar (SCA) from Merck Corp., USA, catalog number 103855. Following these tests, colony identification continued using triple sugar iron agar (TSIA) from Merck Corp., USA, catalog number 103915.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSamples for the isolation of \u003cem\u003eEscherichia coli\u003c/em\u003e were collected from cloacal swabs and subsequently cultured on eosin methylene blue Agar (EMBA) media (from Merck Corp., USA, catalog number 103857) using the streak method and incubated at 37\u0026deg;C for 24 hours. The \u003cem\u003eEscherichia coli\u003c/em\u003e colonies cultured on EMBA media exhibit distinctive characteristics, including round colony growth, metallic green coloration, convex surfaces, and smooth edges. Identification of Escherichia coli is performed through microscopic examination using Gram staining and IMViC biochemical tests. Suspected Escherichia coli colonies observed under Gram staining display a morphology of short rods and are Gram-negative. Further biochemical identification is conducted utilizing the IMViC test followed by the TSIA test.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIndole test using SIM medium\u003c/h3\u003e\n\u003cp\u003eThe indole biochemical test is performed by adding 2\u0026ndash;3 drops of Kovacs reagent (from Merck Corp., USA, catalog number 109293) to the media after incubation for 24 hours at 37\u0026deg;C. After conducting the indole biochemical test with Kovacs reagent, \u003cem\u003eSalmonella\u003c/em\u003e species typically produce a colorless ring or a light pink hue. In contrast, \u003cem\u003eEscherichia coli\u003c/em\u003e yields a positive result, indicated by the formation of a red to red-violet color in the reagent layer on top of the medium.\u003c/p\u003e\n\u003ch3\u003eMR-VP test\u003c/h3\u003e\n\u003cp\u003eThe MR-VP test is conducted by inoculating colonies of \u003cem\u003eSalmonella\u003c/em\u003e species and \u003cem\u003eEscherichia coli\u003c/em\u003e into separate test tubes containing MR-VP media (Thermo Corp., US, catalog number CM0043B) in two separate test tubes and incubating them at 37\u0026deg;C for 24 to 48 hours. The MR test involves the addition of 2\u0026ndash;5 drops of methyl red (Sigma Corp., US, catalog number: CAS 493-52-7) as an indicator. In contrast, the VP test is performed by adding 8\u0026ndash;10 drops of alpha-naphthol solution (Merck Corp., US, catalog number CAS 90-15-3) along with 40% KOH (Merck Corp., US, catalog number CAS 1310-58-3). A positive result for \u003cem\u003eSalmonella\u003c/em\u003e species and \u003cem\u003eEscherichia coli\u003c/em\u003e in the MR test is indicated by a color change to red in the media, whereas the VP test yields a negative result, characterized by no change in color.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCitrate utilization (SCA) and TSIA tests\u003c/h2\u003e \u003cp\u003eThe citrate test assesses the ability of bacteria to utilize citrate as their sole source of carbon and energy. In this test, \u003cem\u003eSalmonella\u003c/em\u003e species exhibits a positive result, while \u003cem\u003eEscherichia coli\u003c/em\u003e shows a negative result, as indicated by no color change in the medium. The TSIA test evaluates carbohydrate fermentation (glucose, lactose, and sucrose) to determine acid or gas production. Salmonella species produces gas in the form of hydrogen sulfide (H\u003csub\u003e2\u003c/sub\u003eS), resulting in an alkaline medium. Conversely, \u003cem\u003eEscherichia coli\u003c/em\u003e can ferment glucose, lactose, and sucrose, leading to a yellow color change in the TSIA medium. The colonies of \u003cem\u003eEscherichia coli\u003c/em\u003e do not produce H\u003csub\u003e2\u003c/sub\u003eS in the TSIA test, indicating an acidic condition in the medium. Identification was conducted to differentiate between \u003cem\u003eSalmonella\u003c/em\u003e species and \u003cem\u003eEscherichia coli\u003c/em\u003e, using isolation and identification parameters as outlined in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIsolation and identification parameters related to specific tests for \u003cem\u003eSalmonella\u003c/em\u003e spp. and \u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameter Test\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eType\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eBacteria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e spp.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIsolation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedium of \u003cem\u003eSalmonella Shigella\u003c/em\u003e Agar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRound, colorless colony with black center\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003e19, 20, 21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedium of Eosin Methylene Blue Agar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRound colony, metallic green in color\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"6\" rowspan=\"7\"\u003e \u003cp\u003eIdentification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGram Staining\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRod-shaped, Gram-negative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eShort rod, Gram-negative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIndol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethyl Red\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVoges\u0026ndash;Proskauer\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCitrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTriple Sugar Iron Agar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBasic/Acid, with the presence of H\u003csub\u003e2\u003c/sub\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAcid/Acid, H\u003csub\u003e2\u003c/sub\u003eS that is not attended\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMotilities\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNonmotile\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMotile\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAntibacterial screening of QLC\u003c/h2\u003e \u003cp\u003eThe bacterial tests identified the organisms as \u003cem\u003eSalmonella typhimurium\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e, each present in a suspension at a dilution of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e. Screening tests were performed by inoculating 0.1 mL of the analytical serial QLC onto colony media for both bacteria at a concentration of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e colony-forming units.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e The minimum inhibitory concentration (MIC) test was conducted by incubating each bacterium with various concentrations of antibacterial substances in Mueller Hinton Broth (MilliporeSigma Corp., US, catalog number 70192) for 24 hours at 37\u0026deg;C. Observations were made of the last clear tube that showed no bacterial overgrowth. The growth of bacterial suspensions from the MIC test was then cultured on respective media (\u003cem\u003eSalmonella\u003c/em\u003e spp. on SSA medium and \u003cem\u003eEscherichia coli\u003c/em\u003e on EMBA medium) to determine whether the antibacterial agent was bacteriostatic or bactericidal. After 24 hours of incubation at 37\u0026deg;C, bacterial growth was assessed.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e The criteria for interpretation were as follows: if bacterial growth was observed from the clearest tube after inoculation in Mueller Hinton Broth, the analyte was classified as bacteriostatic; conversely, if no bacterial growth occurred, the analyte was deemed bactericidal.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e If preliminary research indicated that QLC required a high concentration to eliminate bacterial colonies, the analyte was diluted to a concentration near the level required for colony elimination.\u003c/p\u003e \u003cp\u003eFor the positive control, kanamycin disulfate salt (Sigma-Aldrich Corp., CAS 64013-70-3) was utilized, along with clavamox, which contains amoxicillin trihydrate and potassium clavulanate, at a starting concentration ranging from 150 ppm to 10,000 ppm (Kalbemed Pharma Corp., Indonesia). The negative control employed sterile aqua pro injection solvent (Otsuka-Indonesia Corp.), characterized as sterile, pyrogen-free, isotonic, isoionic, and isohydric.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis of QLC's effectiveness in eliminating colonies of \u003cem\u003eSalmonella\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e was conducted using a two-tailed Mann-Whitney nonparametric test at a significance level of 0.05. The independent variable in this analysis was the QLC treatment, while the dependent variable was the count of colonies on the microbial media for the two bacterial species.\u003c/p\u003e \u003cp\u003eIf the number of colonies could not be eliminated or counted, it was recorded as equal to or greater than 300 colonies on the growth media plate. This threshold was established to ensure that with a minimum of 300 colonies, it could be confirmed that all colonies growing on the media were resistant to QLC.\u003c/p\u003e \u003cp\u003eThe evaluation of the percentage of QLC analytical endpoints against the bacterial types from both \u003cem\u003eSalmonella\u003c/em\u003e and \u003cem\u003eEscherichia coli\u003c/em\u003e was performed using probit analysis with a significance level of 0.05%. The calculated minimum inhibitory concentration corresponds to a 50% inhibition of colony growth, while the subsequently defined MIC corresponds to a 99% reduction in colony viability.\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Statistical analysis was conducted using IBM SPSS Statistics version 27.0 for Microsoft Windows, IBM Corp., Armonk, NY, USA.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eThe chromatogram profile of QLC is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, while the standard quercetin is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The QLC sample exhibited several peaks at a wavelength of 250.4 nm, appearing at retention times (RTs) of 10.508, 10.868, 11.179, 11.452, and 13.56 minutes. At a wavelength of 255.4 nm, the appearance of additional peaks shifted slightly earlier by 0.06 to 0.12 minutes. At a wavelength of 370.4 nm, the number of peaks diminished, with RTs ranging from 10.534 to 11.624 minutes,\u003csup\u003e31\u003c/sup\u003e while a corresponding shift at 375.4 nm occurred approximately 0.42 minutes earlier. The quercetin standard at wavelengths of 250.4 nm, 255.4 nm, 370.4 nm, and 375.4 nm demonstrated minimal peak shift compared to the analyte, exhibiting nearly identical RTs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe primary HPLC chromatogram peak for QLC was observed at RTs of 14.60 to 14.61 minutes across wavelengths of 250.4 nm, 255.4 nm, 370.4 nm, and 375.4 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). When compared to the quercetin standard, which appears at RTs of 14.882 and 14.883 minutes, the QLC peak showed similar RTs. Thus, the secondary metabolite utilized as the analyte and isolated using the preparative HPLC system corresponds to the component with an RT of 14.61 minutes. Other chromatogram peaks at a wavelength of 250 nm occurred at RTs of 17.21, 19.41, 19.549, 21.082, 22.076, 22.33, 23.051, 23.391, 24.01, and 14.417 minutes, representing interfering compounds that were not collected. Interfering compounds also manifested at a wavelength of 255 nm, with RTs of 16.228, 16.668, 16.924, 18.736, 19.155, 20.406, 20.643, 21.382, 23.13, 23.332, 24.304, 24.584, 25.666, 25.838, 27.239, 28.157, 28.207, and 29.184 minutes. Observations at a wavelength of 370.4 nm reveal a decrease in interfering peaks, with RTs of 16.904, 19.154, 19.264, and 19.567 minutes. A similar decrease in interfering peaks was noted at a wavelength of 375.4 nm, with RTs of 16.664, 16.946, 19.155, and 19.587 minutes.\u003c/p\u003e \u003cp\u003eThe results indicate that the purification of QLC contains an aromatic complex, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Out of 16 detected molecules (m/z), only seven SMCs exhibited percent intensity values exceeding 10%, as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMolecules of quercetin-like compounds at high percent intensity levels\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFormula\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNeutral mass (g/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMeasured mass (m/z)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRetention time (min)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonoterpenoid\u003c/p\u003e \u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e140.1565\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e140.150810918010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethyl ecgonidine\u003c/p\u003e \u003cp\u003eC\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eNO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e181.1103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e181.0921353141\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3',6-Dimethylflavone\u003c/p\u003e \u003cp\u003eC\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e250.09938\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e251.136081773\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuercetin\u003c/p\u003e \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e302.236\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e303.0627359303\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.59 to 4.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4'-Acetamino-6-methylflavonone\u003c/p\u003e \u003cp\u003eC\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e294.30136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e295.1636248876\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFlavone\u003c/p\u003e \u003cp\u003eC\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e222.06808\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e223.1039497557\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4'-Amino-5,7-dimethylflavanone\u003c/p\u003e \u003cp\u003eC\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eNO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e267.12593\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e267.1316147452\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e reveals that not all compounds' RTs resulting from the adsorption-partition process using the UPLC-MS/MS column with gradient elution were detected and recorded in the equipment library. Of the seven detected compounds, one, 4'-acetamino-6-methylflavonone, could not be identified, resulting in a recorded RT of \"none.\" However, this compound can be detected by the instrument in terms of mass (m/z), thus qualifying it as part of the QLC components.\u003c/p\u003e \u003cp\u003eMass spectrum analysis indicated nine unidentified masses with an intensity of less than 10% corresponding to (m/z) values of 237.1200, 209.0879, 169.0555, 141.154, 138.0986, 123.0484, 100.0817, 82.0705, and 71.9569. Monitoring the RTs of these nine unidentified SMCs on the integrator revealed unclear peaks and a broad area nearly equivalent to the impurities present in the QLC sample. The RT of quercetin in the QLC samples appeared to drift by 1\u0026ndash;2 minutes, attributable to the gradient settings of the mobile phase and the percentage change over time, as outlined in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe FT-IR spectrogram results comparing QLC and quercetin are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, focusing on the fingerprint region from 1400 cm⁻\u0026sup1; to 400 cm⁻\u0026sup1;. In Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, the QLC sample displays water (H\u003csub\u003e2\u003c/sub\u003eO) elements in the wavenumber region of 3600 cm⁻\u0026sup1; to 3200 cm⁻\u0026sup1;, which is comparatively higher than that of the quercetin standard shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb. Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates a relatively strong percent transmittance between wavenumbers (cm⁻\u0026sup1;) 400\u0026ndash;1400, indicating the presence of SMC along with the functional groups attached to the molecular core (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea and Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInfrared finger print of quercetin-like compounds identified by high percent transmission compared to standard quercetin.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eSample Quercetin-Like Compound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eStandard Quercetin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSecondary metabolite\u003c/p\u003e \u003cp\u003estructure\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWavelength number (cm⁻\u0026sup1;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTransmission (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWavelength number (cm⁻\u0026sup1;)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTransmission (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e401.1937\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e83.7422\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e401.1937\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62.9112\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCl\u0026ndash;C\u0026thinsp;=\u0026thinsp;O in-plane deformation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e403.1225\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e85.1477\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e403.1225\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62.6879\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e601.7905\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e82.1267\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e601.7905\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e54.5052\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIn-plane ring deformation, naphthalene structure\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e603.7194\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e82.1778\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e603.7194\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e55.4690\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e619.1499\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e82.9565\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e619.1499\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.3706\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e806.2450\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e85.8510\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e835.1773\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e72.1019\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1,2,4-trisubstituted benzenes, triazines\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e898.8282\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e91.1739\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e900.7570\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e85.9532\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eR\u0026ndash;NH\u003csub\u003e2\u003c/sub\u003e primary amines\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e921.9740\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e89.1173\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e904.6147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e86.2411\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCH=CH\u003csub\u003e2\u003c/sub\u003e in vinyl compounds,\u003c/p\u003e \u003cp\u003eCH\u0026thinsp;=\u0026thinsp;CH in trans disubstituted alkenes, single aromatic C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e977.9097\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e87.9884\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e974.0520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e84.7275\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1001.0555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e84.9080\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1001.0555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e60.2916\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1028.0589\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e83.8100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1028.0589\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e81.5640\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCarbon ring in cyclic compounds, in cyclic alcohols, C\u0026thinsp;=\u0026thinsp;S in thiocarbonyl compounds, C\u0026ndash;O\u0026ndash;C in aliphatic ethers, C\u0026ndash;OH in secondary or tertiary alcohols\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1029.9877\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e83.3049\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1029.9877\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e81.3669\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1228.6557\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e84.2667\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1228.6557\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e48.2723\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1390.6763\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e81.5295\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1390.6763\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e51.4070\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCOO⁻ group in carboxylic acid salts, SO\u003csub\u003e2\u003c/sub\u003e in sulfonyl chlorides\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1400.3204\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e82.019843\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1400.3204\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e54.9228\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eResults from administering positive controls on \u003cem\u003eSalmonella\u003c/em\u003e spp. and \u003cem\u003eEscherichia coli\u003c/em\u003e are summarized in Additional file 1, while the MIC test of the QLC analytes against \u003cem\u003eSalmonella\u003c/em\u003e spp. and \u003cem\u003eEscherichia coli\u003c/em\u003e is presented in Additional file 2. In Additional file 1, the positive control ranged from a concentration of 156.25 \u0026micro;g/mL to 10,000 \u0026micro;g/mL, informed by preliminary studies indicating the elimination potency of kanamycin and clavamox beginning at a level of 150 ppm. Consequently, the positive control concentration initiates slightly above the 150 ppm threshold.\u003c/p\u003e \u003cp\u003eIn Additional file 2, the QLC analyte begins at a concentration of 625 \u0026micro;g/mL, based on preliminary research findings showing the analyte's elimination capacity commencing at 600 ppm. Thus, the series of test concentrations extends from 625 \u0026micro;g/mL to 10,000 \u0026micro;g/mL. Furthermore, the results of the QLC analysis regarding the elimination capacity of the two types of colonies, assessed using the Mann-Whitney test, are presented in Additional file 2 at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eProbit analysis at a 95% confidence limit indicated that the antibacterial effect of the analyte against \u003cem\u003eSalmonella\u003c/em\u003e species colonies was bactericidal, with 50% of the colonies expected to perish following exposure to 359.283 \u0026micro;g/mL QLC. Colony death of 75% occurred at an exposure of 1.6 mg/mL QLC, while 99% colony death occurred at an exposure of 61.9 mg/mL QLC as a bactericide. Conversely, exposure to QLC did not yield any inhibitory effect on \u003cem\u003eEscherichia coli\u003c/em\u003e bacteria. The inhibitory effect of QLC on \u003cem\u003eSalmonella\u003c/em\u003e colonies is further illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of the isolation and identification of QLC using preparative HPLC, UPLC-MS/MS, and FT-IR indicate the presence of components within the analyte that include identifiable and non-identifiable SMC. The QLC components exhibiting antibacterial potential consist of a group of SMCs characterized by either a single aromatic or a complex aromatic core ring that is bonded to a strong electronegative group (-O-, -N-, -S-). These SMCs have demonstrated effectiveness in eliminating colonies of \u003cem\u003eSalmonella\u003c/em\u003e spp.; however, they are not effective against \u003cem\u003eEscherichia coli\u003c/em\u003e colonies, as presented in Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Additional file 2.\u003csup\u003e32,33\u003c/sup\u003e The SMC components that exhibit antibacterial activity against \u003cem\u003eSalmonella\u003c/em\u003e spp. include complex aromatic structures containing sulfur and carboxyl ions. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, SMC appears at an RT (min) of 14.61 to 14.808, closely resembling the quercetin standard peak (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) at 14.882 to 14.883. Furthermore, the UPLC-MS/MS analysis identified SMC components such as monoterpenoid, methyl ecgonidine, 3',6-dimethylflavone, quercetin, 4'-acetamino-6-methylflavonone, flavone, and 4'-amino-5,7-dimethylflavanone (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These components are characterized by complex aromatic rings linked to strongly electronegative groups.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e Among the seven SMCs, monoterpenoids exhibited a high percentage of transmittance, and these elements theoretically possess a Log P value of less than 3, rendering them hydrophilic and highly soluble in water. Such characteristics facilitate infiltration into the hydrophilic surfaces of bacterial cells and promote interactions with the bacteria's internal structures.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Ultimately, this leads to the inability of the bacteria to regulate their biochemical systems, resulting in gradual cell death. The other six SMCs demonstrated similar average transmittance values, generally exhibiting aromatic molecular structures characterized by high stability due to the resonance of ion charges in each atom, and are attached to strong electron-withdrawing groups.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e Some were also identified as being ionically bound to central molecules such as sulfonyl chlorides, carboxylic acid salts, thiocarbonyl compounds, benzene, and naphthalene (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e demonstrates that QLCs, when compared to the standard quercetin at fingerprint wavenumbers, produce similar molecular ions. Wavenumbers (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) 401\u0026ndash;403 indicate the presence of C and O bonds that bind Cl.\u003csup\u003e38\u003c/sup\u003e This SMC has the potential to form bonds with the positively charged inductive groups present in bacterial cytoplasm. Similarly, the wavenumbers 601\u0026ndash;619 indicate that the lipophilic naphthalene structure has the capacity to penetrate the lipid bilayer of the bacterial membrane, thereby forming bonds with bacterial DNA components. The resultant effect of these bonds inhibits the bacterial cell's respiratory system, ultimately slowing down and eventually halting bacterial growth and colony formation. Other components, such as 1,2,4-trisubstituted benzenes, triazines R\u0026ndash;NH\u003csub\u003e2\u003c/sub\u003e, and CH=CH\u003csub\u003e2\u003c/sub\u003e, CH\u0026thinsp;=\u0026thinsp;CH\u0026ndash; bound to alkenes or single aromatics (wavenumbers 806\u0026ndash;1001 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), are lipophilic and thus possess the potential to penetrate bacterial membranes.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e Likewise, SMCs containing carbon rings in cyclic compounds, cyclic alcohols, C\u0026thinsp;=\u0026thinsp;S in thiocarbonyl compounds, C\u0026ndash;O\u0026ndash;C in aliphatic ethers, C\u0026ndash;OH in secondary or tertiary alcohols, as well as the COO⁻ group in carboxylic acid salts and SO\u003csub\u003e2\u003c/sub\u003e in sulfonyl chlorides (detected at wavenumbers 1200\u0026ndash;1400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), exhibited lipophilic properties that enable complexation with the core acidic elements of bacteria, either through electron-withdrawing or inductive groups. This phenomenon accelerated bacterial death, impacting bacterial colonies significantly. In Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, the percentage transmittance of SMC QLC at wavenumbers (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) 401\u0026ndash;619 has an average intensity 1.5 times stronger compared to the quercetin standard shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, indicating that the SMC QLC component at these wavenumbers likely possesses strong antibacterial activity. However, the percentage transmittance at wavenumbers (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) 806\u0026ndash;1001 for MSC QLC was slightly higher compared to the same element found in standard quercetin. Under these conditions, the penetration capability of this element yielded antibacterial effects, albeit not very potent. Nevertheless, the antibacterial efficacy was enhanced in elements characterized by high percentage transmittance. Conversely, the SMC QLC at the wavenumbers (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) 1028\u0026ndash;1029 exhibited percentage transmittance values similar to standard quercetin, although still higher for QLC. Thus, the antibacterial potency of elements at these wavenumbers (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was relatively weak. This contrasts with elements appearing at wavenumbers (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) 1228\u0026ndash;1400, where the percentage transmittance value was twice as high in QLC compared to standard quercetin.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e At this wavenumber, SMC QLC elements that exhibit significant antibacterial activity were produced.\u003c/p\u003e \u003cp\u003eThe results of the NMR analysis of the analyte revealed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) that at chemical shifts (ppm) between 7.9059 and 7.8875, the H7 of protonated QLC was present, along with H6 (7.6349) and H4 (7.1089). The chemical shifts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb) for the standard H7 (7.7344) and H6 (7.6202), as well as H4 (7.6149), with a standard deviation of 0.217, indicated a minor difference in the protonation of the C15H10O7 content of QLC. Such differences are expected when natural materials are dissolved in a solvent. Meanwhile, the protonation of the analyte solvent (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) was detected to match the chemical shift of the standard (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). However, the protonation of C15H10O7 at the H atoms in positions 1, 2, 3, 5, 8, 9, and 10 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) could not be detected by the NMR detector, likely due to the QLC isolate being too small for monitoring. If the NMR device operates at an energy level above 400 MHz, it may be possible to detect all protons. The analytical chemical shifts (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) at (ppm) 7.1089 and 7.0872, including 1.2888, likely correspond to protons from components such as monoterpenoid, methyl ecgonidine, 3',6-dimethylflavone, 4'-acetamino-6-methylflavonone, flavone, and 4'-amino-5,7-dimethylflavanone. A significant difference is evident when compared to Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb, which represents a single compound standard, whereas the QLC analyte is a complex compound, indicating that the QLC compound contains multiple constituent elements.\u003c/p\u003e \u003cp\u003eIn summary, the results indicate that the QLC content contains the element C15H10O7 and has the potential to exhibit biological activity against bacterial colonies. The analysis of colony death demonstrated that 50% of \u003cem\u003eSalmonella\u003c/em\u003e spp. bacteria died following exposure to QLC at a concentration of 359.283 \u0026micro;g/mL, while 75% death occurred at 1.6 mg/mL and 99% at 61.9 mg/mL, suggesting that SMC QLC possesses antibacterial activity against \u003cem\u003eSalmonella\u003c/em\u003e spp. (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). However, exposure of SMC QLC to \u003cem\u003eEscherichia coli\u003c/em\u003e colonies did not yield antibacterial activity (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additional files 1\u0026ndash;3 indicate that QLC is ineffective in eliminating bacterial colonies, as the number of colonies remained at or above 300. The reduction in \u003cem\u003eSalmonella\u003c/em\u003e bacterial colonies due to QLC exposure ranged from 2.93% to 99.6%. Nevertheless, Additional file 1, as the positive control, illustrates that both bacterial strains are highly sensitive to kanamycin and clavamox. The results from the negative control analysis reveal that the colonies of both bacteria continue to grow, averaging 300 colonies or more. The antibacterial activity of kanamycin against \u003cem\u003eSalmonella\u003c/em\u003e spp. was observed at 5 mg/mL, while against \u003cem\u003eEscherichia coli\u003c/em\u003e, it was noted at a concentration of 10 mg/mL. The antibacterial efficacy of clavamox against \u003cem\u003eSalmonella\u003c/em\u003e spp. was identified at a concentration of 1.2 mg/mL, whereas its activity against \u003cem\u003eEscherichia coli\u003c/em\u003e was found at 2.5 mg/mL (Additional file 1). This table indicates that the \u003cem\u003eSalmonella\u003c/em\u003e strain used exhibits greater resistance to antibiotics compared to \u003cem\u003eEscherichia coli\u003c/em\u003e. Consequently, the infection potential of the \u003cem\u003eSalmonella\u003c/em\u003e spp. is considered more significant than that of \u003cem\u003eEscherichia coli\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e The antibacterial activity of QLC against \u003cem\u003eSalmonella\u003c/em\u003e spp. was 12.5 times lower than that of kanamycin and 6.2 times lower than that of clavamox. These results suggest that SMC QLC, at an exposure concentration of 61.9 mg/mL or 62 mg/mL against \u003cem\u003eSalmonella\u003c/em\u003e spp., has not yet reached toxic levels in plasma concentrations within the body in vivo. Therefore, its potential as an anti-\u003cem\u003eSalmonella\u003c/em\u003e agent is highly promising. The ineffectiveness of QLC against \u003cem\u003eEscherichia coli\u003c/em\u003e colonies may be attributed to the components in QLC being unable to form complexes with the cytoplasmic elements of bacterial cells and/or unable to interact with the nucleic acids of these cells. Theoretically, effective antibacterial activity against \u003cem\u003eEscherichia coli\u003c/em\u003e would necessitate more lipophilic chemical compounds containing elements capable of damaging the bacterial cell wall. Such efforts to disrupt the bacterial cell wall could lead to cytoplasmic damage and potentially harm the bacterial nucleic acid chains. The differences in cell wall biosynthesis between \u003cem\u003eSalmonella\u003c/em\u003e species and \u003cem\u003eEscherichia coli\u003c/em\u003e suggest that the biosynthesis control system regulating the metabolism of saccharide lipoproteins essential for the cell wall is more complete in \u003cem\u003eSalmonella\u003c/em\u003e. Consequently, SMC that can complex with the complete elements of \u003cem\u003eSalmonella\u003c/em\u003e spp. can easily form complexes.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e In contrast, the specific biosynthesis system required for \u003cem\u003eEscherichia coli\u003c/em\u003e makes it difficult for SMCs to bind to its interior, rendering the bacteria resistant to QLC exposure.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe findings of this study indicate that QLC is capable of exhibiting bactericidal effects against \u003cem\u003eSalmonella\u003c/em\u003e spp. colonies at a concentration of 62 mg/mL. However, QLC is ineffective as a bactericide against \u003cem\u003eEscherichia coli\u003c/em\u003e colonies. QLCs extracted from the leaves of \u003cem\u003eLansium domesticum\u003c/em\u003e demonstrate significant potential for utilization as new antibiotics targeting \u003cem\u003eSalmonella\u003c/em\u003e spp. It is recommended that for antibiotic application, QLC be administered in liquid form as a solution.\u003c/p\u003e"},{"header":"List of abbreviations","content":"\u003cp\u003eADC acquisition device\u003c/p\u003e\u003cp\u003eBD \u003cem\u003eDendrophthoe pentandra\u003c/em\u003e L. Miq\u003c/p\u003e\u003cp\u003eCE collision energy\u003c/p\u003e\u003cp\u003eDMSO dimethyl sulfoxide\u003c/p\u003e\u003cp\u003eEMBA eosin methylene blue agar\u003c/p\u003e\u003cp\u003eHPLC high-performance liquid chromatography\u003c/p\u003e\u003cp\u003eMIC minimum inhibitory concentration\u003c/p\u003e\u003cp\u003eMR methyl red\u003c/p\u003e\u003cp\u003eNMR nuclear magnetic resonance\u003c/p\u003e\u003cp\u003eQLC quercetin-like compounds\u003c/p\u003e\u003cp\u003eRT retention times\u003c/p\u003e\u003cp\u003eSCA Simmons citrate agar\u003c/p\u003e\u003cp\u003eSIM sulfide indole motility\u003c/p\u003e\u003cp\u003eSMC secondary metabolite compounds\u003c/p\u003e\u003cp\u003eSSA \u003cem\u003eSalmonella Shigella\u003c/em\u003e agar\u003c/p\u003e\u003cp\u003eTSIA triple sugar iron agar\u003c/p\u003e\u003cp\u003eUPLC ultra-performance liquid chromatography\u003c/p\u003e\u003cp\u003eVP Voges–Proskauer\u003c/p\u003e"},{"header":"Declarations","content":"\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003ehttps://doi.org/10.6084/m9.figshare.31112728.v2 https://doi.org/10.6084/m9.figshare.31238056\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing\u0026nbsp;interests.\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was supported by\u0026nbsp;the Directorate General of Research and Development, Ministry of Higher Education, Science, and Technology for the 2025 fiscal year [DIPA SP Number 139.04.1.693320/2025,\u0026nbsp;revision 04,\u0026nbsp;dated April 30, 2025].\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eThe authors express their gratitude for the assistance received from the following individuals and groups: (1) the Research Group in Veterinary Pharmacy Science, Faculty of Veterinary Medicine, Universitas Airlangga, \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;(2) the staff of the Laboratory of Veterinary Pharmacy Science, Faculty of Veterinary Medicine, Universitas Airlangga, and (3) Student faculty of veterinary medicine Universitas Airlangga especially Rafif Praptama Iskandar, Zahra Alyssa Ekaputri, Annida Annafiri Rakhman, Nazwa Afifah Pujihati Muluk, Baiq Gina Rahayu Putri, Christine Jayanti . 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Antibacterial activity of kanamycin and clavamox against \u003cem\u003eSalmonella\u003c/em\u003e spp. and \u003cem\u003eEscherichia coli\u003c/em\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Antibiotics, Enterobacteriaceae, Healthy and well-being, Mistletoe, Pullorum","lastPublishedDoi":"10.21203/rs.3.rs-8723177/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8723177/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003e \u003cem\u003eDendrophthoe pentandra\u003c/em\u003e L., Miq, a type of mistletoe that grows abundantly on \u003cem\u003eLansium domesticum\u003c/em\u003e, is estimated to possess antimicrobial activity against bacteria. This study focused on extracts of the plant, particularly quercetin-like compounds (QLC), with the aim of identifying potential analyte compounds as new antibiotics. The leaves of the mistletoe plant underwent methanol-ethyl acetate-n-hexane maceration, followed by QLC extraction using preparative high-performance liquid chromatography equipment. The extracts were subsequently analyzed using ultra-performance liquid chromatography-tandem mass spectrometry, Fourier transform infrared spectrophotometry, and proton analysis via nuclear magnetic resonance spectroscopy. The QLC extracts, ranging from 500 ppm to 10,000 ppm, were tested for antimicrobial activity against \u003cem\u003eSalmonella\u003c/em\u003e species and \u003cem\u003eEscherichia coli in vitro\u003c/em\u003e, based on colony growth inhibition screening. The results were further analyzed using probit analysis to determine the bactericidal values against these two enterobacteria.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe findings indicated that the minimum inhibitory concentration required for 50% mortality of \u003cem\u003eSalmonella\u003c/em\u003e species colonies was 359.283 \u0026micro;g/mL and that a 75% reduction occurred at an exposure of 1.6 mg/mL of QLC. Additionally, a 99% colony death rate was observed at a minimum bactericidal concentration of 61.9 mg/mL of QLC. In contrast, exposure to QLCs did not exhibit any colony-inhibiting or bactericidal effects on \u003cem\u003eEscherichia coli\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe QLCs extracted from the leaves of \u003cem\u003eDendrophthoe pentandra\u003c/em\u003e growing on the host plant \u003cem\u003eLansium domesticum\u003c/em\u003e demonstrate significant potential for development as new antibiotics against \u003cem\u003eSalmonella\u003c/em\u003e species. However, they are not recommended for use against \u003cem\u003eEscherichia coli\u003c/em\u003e (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e","manuscriptTitle":"Bactericidal activity of quercetin like-compounds isolated from Dendrophthoe pentandra leaves against Salmonella spp. and Escherichia coli: an in vitro experimental study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-13 15:39:39","doi":"10.21203/rs.3.rs-8723177/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-03-30T15:42:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"44180414901288924034499337980783170364","date":"2026-03-30T14:17:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"232506409803488211747623919159125842497","date":"2026-02-26T06:33:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-10T04:58:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-06T04:20:06+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-05T11:33:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-04T00:48:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-02-04T00:40:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"390cec9c-4641-40a5-9278-caec7dc6fb68","owner":[],"postedDate":"February 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":62705668,"name":"Biological sciences/Biological techniques"},{"id":62705669,"name":"Biological sciences/Biotechnology"},{"id":62705670,"name":"Biological sciences/Drug discovery"},{"id":62705671,"name":"Biological sciences/Microbiology"},{"id":62705672,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-02-13T15:39:40+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-13 15:39:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8723177","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8723177","identity":"rs-8723177","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}