LC‐ESI‐QTOF/MS characterization of antimicrobial compounds with their action mode extracted from vine tea (Ampelopsis grossedentata) leaves

Abstract Vine tea (Ampelopsis grossedentata) is a tea plant cultivated south of the Chinese Yangtze River. It has anti‐inflammatory properties and is used to normalize blood circulation and detoxification. The leaves of vine tea are the most abundant source of flavonoids, such as dihydromyricetin and myricetin. However, as the main bioactive flavonoid in vine tea, dihydromyricetin was the main focus of previous research. This study aimed to explore the antibacterial activities of vine tea against selected foodborne pathogens. The antimicrobial activity of vine tea extract was evaluated by the agar well diffusion method. Cell membrane integrity and bactericidal kinetics, along with physical damage to the cell membrane, were also observed. The extract was analyzed using a high‐performance liquid chromatography‐diode array detector (HPLC‐DAD), and the results were confirmed using a modified version of a previously published method that combined liquid chromatography and electrospray‐ionized quadrupole time‐of‐flight mass spectrometry (LC‐ESI‐QTOF/MS). Cell membrane integrity and bactericidal kinetics were determined by releasing intracellular material in suspension and monitoring it at 260 nm using an ultraviolet (UV) spectrophotometer. A scanning electron microscope (SEM) was used to detect morphological alterations and physical damage to the cell membrane. Six compounds were isolated successfully: (1) myricetin (C15H10O8), (2) myricetin 3‐O‐rhamnoside (C21H20O12), (3) 5,7,8,3,4‐pentahydroxyisoflavone (C15H10O7), (4) dihydroquercetin (C15H12O7), (5) 6,8‐dihydroxykaempferol (C15H10O8), and (6) ellagic acid glucoside (C20H16O13). Among these bioactive compounds, C15H10O7 was found to have vigorous antimicrobial activity against Bacillus cereus (AS11846) and Staphylococcus aureus (CMCCB26003). A dose‐dependent bactericidal kinetics with a higher degree of absorbance at optical density 260 (OD260) was observed when the bacterial suspension was incubated with C15H10O7 for 8 h. Furthermore, a scanning electron microscope study revealed physical damage to the cell membrane. In addition, the action mode of C15H10O7 was on the cell wall of the target microorganism. Together, these results suggest that C15H10O7 has vigorous antimicrobial activity and can be used as a potent antimicrobial agent in the food processing industry.


| INTRODUC TI ON
Globally, six million people suffer from foodborne illness caused by pathogenic microorganisms (Basak et al., 2021). Food spoilage occurs due to the microbial growth and enzymes action from microbia (Delshadi et al., 2021). Food quality and safety are the chief concerns in the food industry (Cui, Li, et al., 2021). If these concerns are not addressed, food poisoning and other health issues arise (Yao et al., 2021).
Chemical preservatives have been used to prevent and control microbial growth in food (Hussain et al., 2021). However, there is an increasing consumer concern about the risks associated with the use of synthetic preservatives (El-Saber Batiha et al., 2021). Many studies have explored natural plant sources as antimicrobial agents (El-Saber Batiha et al., 2021;Molet-Rodríguez et al., 2021;Varghese et al., 2020). The World Health Organization's traditional medicine centers indicated that 80% of the total bioactive compounds used for ethno-medical purposes were derived from plant sources (Sivagurunathan Moni et al., 2021;Sopalun et al., 2021).
Recent works have focused on various functional aspects of dihydromyricetin, such as antimicrobial properties (Cui, Li, et al., 2021;Wu et al., 2017;Xiao et al., 2019), antioxidant capacity (Xie et al., 2019), flavor infusion (Carneiro et al., 2020), stability and stereo-specific action (Umair et al., 2020), pharmacological activities , the bioavailability of bioactive compounds (Sun et al., 2021), interaction with iron , and its optimized extraction  by using response surface methodology. The proposed antimicrobial mechanism of dihydromyricetin in previous reports (Farhadi et al., 2019;Liang et al., 2020;Shevelev et al., 2020;Zhang et al., 2018) is comprises on; the membrane permeability, functional changes in the cell membrane, cytoplasmic membrane damage, blockage in the energy metabolism, retardation in the nucleic acid synthesis process, porin inhibition on the cell membrane, and weakening of the pathogenicity (Farhadi et al., 2019;Xie et al., 2019;Xie et al., 2015).
Secondary metabolites are increasingly preferred as natural preservatives in food due to their antibacterial activity against a variety of microorganisms. Thus, it is critical to find safe and natural antibacterial chemicals that will inhibit foodborne microorganisms and improve food quality. Yet, so far, only a few studies have been done to explore the antimicrobial potential of vine tea extract, so this study aims to explore the antimicrobial activity of the methanolic extract prepared from vine tea leaves. To evaluate the isolate separation, a high-performance liquid chromatography-diode array detector was employed (HPLC-DAD). To validate the identity of these isolates, we used a well-established liquid chromatography method in conjunction with triple-quadrupole time-of-flight mass spectrometry with electrospray ionization in positive and negative modes. This study also focused on the antimicrobial activities of various isolates using the agar well diffusion method and its action mode against selected microorganisms.

| Sample preparation
Vine tea (Ampelopsis grossedentata) leaves were acquired from Moyeam Group Co., Ltd., in Changsha, Hunan, China (Grade 4 according to botanical classification). The voucher specimen of A. grossedentata was verified and deposited at Nanjing Agricultural University's College of Food Science and Engineering in Nanjing, Jiangsu, China. The material was air-dried in the dark at room temperature. Dried vine tea leaves were stored in a tightly sealed jar until further use.

| Isolation of compounds from vine tea extract
Dried ripe leaves of the vine tea plant were ground to fine powder. Fifty grams (50 g) of dried leaf powder was extracted with 500 ml of 75% aqueous methanol in 4 h at 25°C. The extract was then filtered through a filter paper to remove leaves and collect the extractant. The extractant was further concentrated under reduced pressure using a rotary evaporator (CCA-20 Low Temp Cooling Liquid Circulating Pump; RE-5299 Yarong) and stored for further phytochemical analysis. Sephadex LH-20 (Pharmacia biotech) resin was used for the preparative purification and removal of steroids, terpenoids, and lipids from methanol extract. The methanol extract was then chromatographed by eluting with the solvent mixture (methanol and chloroform) with increasing methanol up to 90%. All fractions were collected separately and used for further analysis.
Safety Control for their financial support for this study (No. 2015BAD16B04) cell wall of the target microorganism. Together, these results suggest that C 15 H 10 O 7 has vigorous antimicrobial activity and can be used as a potent antimicrobial agent in the food processing industry.
A TSQ-Quantum Access MAX LC/MS system (Thermo) was used for the LC-ESI-QTOF/MS analysis. Agilent 1200 HPLC and 6520 QTF-MS systems (Agilent Technologies) equipped with heated electrospray ionization (ESI) sources were used for the high-resolution mass spectrometry study. Positive and negative modes of identification were used, with mass spectra (m/z) ranging from 100 to 1200.
The mass spectrometry conditions were as follows: The column and conditions were identical to those described in HPLC-DAD with the exception of the injection sample volume (20 µl); the spray voltage source was set to 4.0 kV, the heated transfer capillary temperature was set to 350°C, and the argon vaporizer temperature was set to 200°C. With a collision pressure of 1.0, the sheath gas was set to 40 arbitrary units and the auxiliary gas to 5 arbitrary units.  The antimicrobial activity was determined by the agar well diffusion method (Valgas et al., 2007). Different concentrated solutions (256, 128, 64, 32, and 16 µg/ml) of each isolated compound (1-6) were tested. All bacterial strains were grown in LB (20% agar) medium for at least 24 h at 37°C, and molds were grown in potato dextrose agar (PDA) medium for 72 h at 28°C (200 g/L; agar 20 g/L; dextrose 10 g/L at 7.0 pH). Nisin (32 g/ml) and tetracycline (32 g/ ml) were used as positive controls, whereas methanol solution was used as a negative control. Triplicates of the assay were performed.

| Antimicrobial activity assay
Additionally, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using the technique described in Zhao et al. (2013). A solution of Tween-80 (5% w/v) was added to the antibacterial agent. Tween-80 was generated in this experiment at concentrations ranging from 16 to 256 mg/ml using a twofold serial dilution of sterile nutritional broth medium. Six compounds were administered to identical bacterial strains in 96-well microplates at the same dose and volume. In addition, the plates were incubated at 37°C for 24 h, during which time visible turbidity was observed inside the plates. MBC was measured by subculturing 10 ml of the MIC solution on PDA for 24 h at 37°C.

| Cell membrane integrity
The integrity of B. cereus (AS11846) bacterial cells was evaluated by determining component leakages in supernatants using optical density at 260 nm (OD 260 ), as described by Cui et al. (2015). The supernatant from each sample was collected and tested for absorbance at OD 260 at different time points. Thus, exponentially growing bacteria were collected and centrifuged at 5000 g for 5 min at 4°C before being added to phosphate buffer (PBS 0.1 M, pH 7.0). Finally, the microbial cells were treated for 1, 2, 3, and 4 h with 5,7,8,3,4-pen tahydroxyisoflavone (C 15 H 10 O 7 ; 32 g/ml), whereas the bacterial suspension without the extracted chemical (compound 3 in this case) served as a control. E. coli (ATCC25922) cell suspensions at a concentration of 32 g/ml (1MIC) for 1, 2, 4, and 8 h, followed by centrifugation at 5000 g for 5 min at 4°C (Eppendorf centrifuge 5804R AG). These were then suspended in phosphate-buffered saline (PBS; pH 7.0), the pallets were washed three times with buffer, and the separated compounds were reacted to a final concentration of 32 g/ml prior to being transferred to the cover slip. The control sample did not include the C 15 H 10 O 7 molecule. Cells mounted on slides were fixed with a glutaraldehyde solution (2.5% w/v). The SEM (SU-8010, Hitachi) showed physical damage to the cell membrane at 30 kV of the electric beam.

| Statistical analysis
All analyses were performed in triplicate. The data were presented as mean ± standard deviation (SD). Microsoft Excel software (Microsoft, USA) was used to generate graphics. One-way analysis of variance and t-tests were used at the level of significance of p < .05. The Statistical Package for the Social Sciences (SPSS) 17.0 was used to examine all of the data.

| HPLC and LC-ESI-QTOF spectroscopic mass analysis
The results of the HPLC-DAD analysis were used for the analytical characterization of the methanolic vine tea extract phytoconstituents. Separation was based on various parameters like retention time, physical properties, molecular weight, and molecular formula.
Next, a MIC was established for six isolated products against all tested microorganisms as presented in Table 2 spectrum at 220 nm ( Figure 4). This finding is similar to that cited in the tea polyphenol database (TMBD; Xiang et al., 2017;Yue et al., 2014). Therefore, in this study, we only focus on C 15 H 10 O 7 by testing it in an in vitro assay.

| Zone of inhibition
The antimicrobial activity of the six isolated compounds tested against different selected foodborne pathogens is described in Table 2. Different concentrations of six isolated compounds were tested against selected microorganisms and it was found that and B. cereus (AS11846) were 32 μg/ml (Table 3). Plant-derived flavonoids are increasingly being studied for their antibacterial properties (Cui, Zeng, et al., 2021). Previously, other flavonoids (dihydromyricetin) in vine tea were isolated and reported for their antimicrobial activity . It is also reported that presence of a hydroxyl group in an aromatic ring of flavonoids' structure can boost the antimicrobial activity. Whereas, the methylation of active hydroxyl groups may result in the reduction of antimicrobial activity. Furthermore, the lipophilicity of ring A is critical for chalcone action (Senan et al., 2021). Prenyl groups, alkylamino chains, alkyl chains, and nitrogen or oxygen containing heterocyclic moieties are all hydrophobic substituents that increase the activity of flavonoids. Flavonoids are thought to work against bacteria by inhibiting nucleic acid production, cytoplasmic TA B L E 1 Compounds 1-6 isolated and purified by using LC-ESI-QTOF/MS in membrane permeability, and reduction in pathogenicity (Xie et al., 2015).
Previous research has shown that hydroxylation at the fifth and seventh sites is important for flavonol antibacterial activity against bacteria (Echeverría et al., 2017;Umair et al., 2020;Zapadka et al., 2019). Furthermore, another study examined the link between flavonoid structure and antibacterial properties, finding a similar hydroxylation site to be associated with antimicrobial activity (Farhadi et al., 2019) and antibiotic resistance (Abdelgader et al., 2018). During 12 h of exposure to bacterial cells, tetracycline (128 g/ml) can reduce the number of bacterial cells (by 1 log CFU/ ml). Simultaneously, the same efficiency may be obtained using C 15 H 10 O 7 (64 g/ml for 8 h). The viable count was decreased to 1 log CFU/ml, which is half of the tetracycline concentration ( Figure 5).
It has been shown that 2R, 3R-dihydromyricetin (DHM), a flavonoid isolated from vine tea, had good inhibitory action against S. aureus (Umair et al., 2020). In terms of the structure-activity

| The integrity of the cell membrane
Results indicated that the isolated compound C 15 H 10 O 7 has the potential to inhibit the bacterial cell. The effect of the isolated compound on the integrity of the cell membrane was noticed when the bacterial suspension was incubated with C 15 H 10 O 7 at 32 μg/ ml for 2 h. A significant increase in absorbance value (OD 260 ) was observed in supernatants. This might be due to the leakage of essential cell components which ultimately increases the OD 260 value (Mohamed et al., 2018). It is widely established that increases in absorbance at OD 260 reflect nucleic acid and protein leaks from cells and a change in membrane permeability . Other investigations showed that a chemical with a high affinity for the cell surface and the capacity to change the lipid bilayer was capable of altering the permeability and integrity of the cell membranes and was eventually detrimental to cell membranes (Peng et al., 2020).
Taken together, these findings suggest that the isolated chemical examined has the ability to impact cell membranes and limit cell development. A similar dosage and time-dependent antibacterial action of flavonoids was previously described, with the authors emphasizing the rise in OD 260 and implying that it was caused by cell membrane leakage (Xie et al., 2015). However, when the supernatant was treated for 4 h, the OD 260 readings indicated a rapid reaction and a significant increase. Additionally, incubation for 8 h had a significant impact on cell membrane breakdown, as shown by a significant increase in the OD 260 value (Table 4)  are comparable to those seen in prior research (Wu et al., 2017).
In addition, smaller charged particles were leaked out before from the cell than the higher charged sized particles such as proteins and nucleic acid (Liao et al., 2014;Zhu et al., 2019). Therefore, it can be inferred that the hydroxylation of C 15 H 10 O 7 at 5 th and 7 th positions plays an essential role in antimicrobial activity against bacteria, making it different from the other isolated compounds and giving a solid reason for the current study.

| Observing morphological changes through SEM
The in form may be related to a differential in the cell's permeability.
According to the study, the permeability of the bacterial cell membrane may have grown over time (Li et al., 2014). When B. cereus (AS11846) and E. coli (ATCC25922) bacterial cells were incubated with C 15 H 10 O 7 at 32 g/ml (1MIC) for 2 h, more irregularities, bleb projections, and concave formation with faster shrinkages were observed on the cell membrane (Figures 6b and 7b).
This may be due to the interaction between ionic proteins and lipids. Although phospholipids are the main constituents of the cell membrane, they perform other functions in addition to creating lipid bilayers (Chi et al., 2020). Acidic phospholipids, for example, create microdomains in the plasma membrane and may interact ionically with proteins through polybasic sequences, potentially affecting the protein's function (Ashraf & Gerke, 2021). Under certain circumstances, these acidic phospholipids may diffuse heterogeneously and form micro-or nanodomains .
Many proteins have lipid-binding domains capable of interacting with acidic phospholipids . Additionally, these domains often have well-defined globular shapes and include positively charged pockets or surfaces that interact with the acidic phospholipids' negatively charged head groups (Kulma & Anderluh, 2021). Positive charges may be provided by basic residues within the domain or by Ca 2+ ions attached to the domain (Li et al., 2014). to diffuse (Mironov et al., 2020). Therefore, any changes in the protein transmembrane domains also affect the transmembrane asymmetry and eventually cause cell surface deformation (Ahmed et al., 2021). The cell structure was completely flat with no bleb and completely lost its formal shape when B. cereus (AS11846) and E. coli (ATCC25922) cells were incubated with C 15 H 10 O 7 at 32 μg/ ml (1 × MIC) for 8 h (Figures 6d and 7d).
The present research hypothesized that the bactericidal impact of C 15 H 10 O 7 on gram-positive and gram-negative bacteria was due to changes in cell permeability, which may alter the interaction of membrane proteins and lipids, disrupting the ionic protein-lipid link (Li et al., 2014). Thus, it has an effect on, and disrupts the structure and function of, proteins (Ashraf & Gerke, 2021 (Chi et al., 2020). They provide a functional purpose for certain acidic phospholipids by forming a microdomain in the plasma membrane and interacting with protein ionically through polybasic sequences as reported by Sarmento, Hof, et al. (2020) and Sarmento, Ricardo, et al. (2020). Additionally, the existence of transmembrane holes indicated the formation of ion channels, which resulted in cell membrane rupture, as shown in Nisin targets the membrane-bound cell wall, interacts with the precursor of lipid II, and retards the outer cell membrane (peptidoglycan) (Panina et al., 2020). This results in the formation of pores on the cell wall, which eventually cause cell lysis (Gharsallaoui et al., 2016). in a previous study, where the antibacterial activity and mechanism of monolauroyl-galactosylglycerol against B. cereus was explored (Diao et al., 2018).
Based on the above findings, we hypothesize that C 15 H 10 O 7 alters the permeability of gram-positive and gram-negative bacteria by affecting membrane protein and lipid interactions, thereby disrupting the ionic protein-lipid bond and ultimately disrupting protein structure and function. However, the presence of a higher hydrophobic membrane core in gram-positive bacteria also showed an altered pathway for cell inhibition by the binding of lipid II. Thus, the current study has depicted that C 15 H 10 O 7 has solid antibacterial activity as observed in vitro. Further research is needed to develop natural antibacterial drugs as a better alternative to antibiotics against gram-positive and gram-negative bacteria.

| CON CLUS ION
This study explored the antimicrobial properties of vine tea (A. grossedentata) against disease-causing foodborne pathogens. Successful RP-HPLC isolation of six bioactive molecules was carried out, which was further confirmed through LC-MS/MS. Among these bioactive compounds, C 15 H 10 O 7 was found to have intense antimicrobial activity against B. cereus and S. aureus. A dose-dependent effect on the bactericidal kinetics with a higher degree of OD 260 absorbance altered the membrane permeability. The alteration in membrane permeability could facilitate the transport of cell material. SEM analysis further confirmed the formation of ion channels and transmembrane pores that result in increased membrane permeability and cytoplasm leakage. However, the presence of a higher hydrophobic membrane core in gram-positive bacteria also suggested that another action mode of C 15 H 10 O 7 is involved in the bio-static pathway and affect the ionic protein-lipid bond, which ultimately disturbs the protein structure and its normal functioning. The potent antimicrobial properties of vine tea (C 15 H 10 O 7 ) need to be explored further for the development of effective drugs to counter bacterial pathogens.

Dr Muhammad Umair would like to thank the Chinese Government
Scholarship Council (CSC) for a doctorate degree scholarship. Dr Muhammad Umair would also like to thank the respected editor, reviewers, and the Journal Central Accounts Team for their valuable comments, effort, and support for their valuable contributions.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

E TH I C S A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
Not applicable.

CO N S E NT FO R PU B LI C ATI O N
Not applicable.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed during this study are available from the corresponding author upon reasonable request. However, all the data explained here are enough to support the results and findings of the manuscript.