Chirality of the biomolecules enhanced its stereospecific action of dihydromyricetin enantiomers

Abstract The present study explores the effect of chirality of the biological macromolecules, its functional aspects, and its interaction with other food components. Dihydromyricetin (DHM) is a natural novel flavonol isolated from the vine tea (Ampelopsis grossedentata) leaves. However, limited progress in enantiopure separation methods of such compounds hinder in the development of enantiopure functional studies. This study is an attempt to develop a simple, accurate, and sensitive extraction method for the separation of the enantiopure DHM from vine tea leaves. In addition, the identification and purity of the extracted enantiopure (−)‐DHM were further determined by the proton nuclear magnetic resonance (1H‐NMR) and the carbon nuclear magnetic resonance (13C‐NMR). The study further evaluates the antimicrobial activity of isolated (‐)‐DHM in comparison with racemate (+)‐DHM, against selected foodborne pathogens, whereas the action mode of enantiopure (−)‐DHM to increase the integrity and permeability of the bacterial cell membrane was visualized by confocal laser scanning microscopy using green fluorescence nucleic acid dye (SYTO‐9) and propidium iodide (PI). Moreover, the morphological changes in the bacterial cell structure were observed through field emission scanning electron microscope. During analyzing the cell morphology of B. cereus (AS11846), it was confirmed that enantiopure (−)‐DHM could increase the cell permeability that leads to the released of internal cell constituents and, thus, causes cell death. Therefore, the present study provides an insight into the advancement of enantiopure isolation along with its antimicrobial effect which could be served as an effective approach of biosafety.


| INTRODUC TI ON
The antibiotic abuse causes multi-drug-resistant (MDR) bacteria, which were critical necessitate to put into practice effective strategy substitutes for antibiotics. Several series of bactericidal strategies (nanomaterial, cationic, and anionic coating) have been opting to overcome its effect (Zhu, Dong, Chang, Li, & Wang, 2019).
However, efficacy issues and irreversible biomolecules damage limit their application. Plants-derived bioactive compound flavonoids demonstrate a diversity of biological and functional perspectives. These chiral flavonoids are attractive applicants for the development of bactericidal effects and drug discovery (Zhu et al., 2018). However, only very few of these flavanols are asserted as a resolved enantiomer.
Dihydromyricetin (DHM), extracted from vine tea (Ampelopsis grossedentata), has been widely used for promoting health benefits. The presence of two asymmetry substituted carbon atoms in the DHM chemical structure leads to the formulation of four possible stereoisomers (Wang, Xiong, Perumala, Fang, & Sun, 2016).
The most common stereoisomer form of DHM is a racemate (±) DHM form rather than optically pure enantiomer, such as (+) DHM, and (−)-DHM. Because any change in the pH value during the extraction or crystallization process, or prolonged high-temperature exposure, or any direct in-contact with the metal surface, may result in the racemization process that ultimately affects the enantioselective functional property . So, it is imperative to develop a sensitive, reproducible, and semi-preparative extraction method for the extraction of the desired enantiomer with the required level of enantiomeric excess from the racemic mixture. Thereby the successful extraction of stereoisomer form of DHM is used to understand the enantioselective functional property that will significantly advance with other flavanols asserted as a resolved enantiomer. Chiral recognition and its interaction among other molecules is a significant step in natural biological processes. A recent development for enantiomers has highlighted the impact of specific stereochemistry of enantiomers and its configuration difference (due to repulsive forces between overlapping electron clouds) on the biological, metabolic, pharmacological, and toxicological activities in vitro and vivo (Lin et al., 2019;Zhou, Yu, & Zeng, 2014). It is also reported that enantiopure could play an essential role in drug discovery due to its effective biological compound coordination with receptor molecules, which can result in the desired effect with fewer side effects and more efficacious (Pinto et al., 2020). Another study has been reported that the selective adhesion and differentiation of the enantiomer of cells and proteins (Qiu et al., 2018) as chiral arrangement plays a crucial function in biological activity. Therefore, the enantioselective functional behavior of enantiopure in comparison with the racemate mixture is imperative. But, the selective adsorption of proteins could induce cell differentiation, and selective toxicity to bacteria of chiral molecules which has not reported yet.
So far, a minimum number of studies have performed to assess the biological and functional aspects of optically pure (+) DHM and (−) DHM in comparison with racemate mixture form (+)-DHM. In this, we have developed a simple, accurate, and sensitive semi-preparative HPLC extraction method for the isolation, separation and purification of enantiomer, such as (−)-DHM enantiomer and evaluated its antimicrobial capacity against selected foodborne pathogens. Further, the identity and the purity of the isolated enantiomer will be determined by proton nuclear magnetic resonance ( 1 H-NMR) and carbon nuclear magnetic resonance ( 13 C-NMR). Whereas, the stereospecific action of enantiomer compound against the integrity of the cell membrane was visualized by confocal laser scanning microscopy with green fluorescence nucleic acid dye (SYTO-9), and propidium iodide (PI). Moreover, bacterial cell morphology was observed through field emission scanning electron microscope. Lastly, the current study will also explore the effect of molecular chirality on the antioxidant capacity to further elucidate the enantioselective functional aspect of the enantiopure compound.

| Sample preparation
Plant material (ripe leaves of vine tea, 4-Grade based upon the part of the plant classification) Ampelopsis grossedentata was collected from Moyeam Group Co., Ltd, from the region of Changsha, Hunan, China. The voucher specimen of A. grossedentata was confirmed and deposited at the College of Food Science and Engineering, Nanjing Agricultural University, Nanjing, Jiangsu, 210095, China. The collected material was air-dried in darkness at room temperature and was stored in a air tight-seal jar until needed.
Dried ripe leaves of vine tea (2017 production) plants (as mentioned above) were ground to make a fine powder. Fifty gram (50 g) dried, fresh, and sound leaves powder of vine tea with 8.53% moisture content on a dry matter basis (DB) was further used in the experiment. Racemate and enantiopure DHM standards were bought from Aladdin (Shanghai, China). HPLC grade ethanol and methanol were of HPLC-grade used whereas, all other chemicals were of analytical grade. All the chemicals were correctly stored during handling, as mentioned for safety and storage instruction in the product manufacturer's instruction manual.

| Preparation of DHM by solvent extraction
First of all, fifty grams (50 ± 0.8 g dry matter based) vine tea powder was extracted with 1.5 L of metal-free ethanol. After this, ultrasound mixing was done at 60°C for 25 min, followed by the filtration process. Then discard the sediments in collecting filtrates. The filtrates were further concentrated using a rotavap and then freeze-dried using lyophilizer to get 7.441 g of compound A.

| Preparation of DHM by the crystallization process
Fifty grams (50 ± 0.8 g dry matter based) of dried vine tea leaf powder were extracted with 2.5 L distilled water for 8 hr and then filter to collect the filtrate. The filtrate was further settled down for two more hours and repeated the filtration process. This final filtrate was further precipitated by hot deionized water until the recrystallization process forms white crystals (repeat the recrystallization process five times) and then collected the crystals and weighted them before drying (33.24 ± 0.12 g). After drying, about 2.66 g of dry matter of compound B was obtained. The schematic diagram for the extraction of (−)-DHM enantiopure, and the racemate (±) DHM by solvent extraction and recrystallization process, respectively, as depicted in Figure 1. After this, the identification of these two isolated compounds A and B from vine tea extract was carried out.

| NMR analysis
Nuclear magnetic resonance (NMR) (Bruker, Fallanden, Switzerland) 1 H and 13 C NMR spectral data of DHM in CH 3 DO, detected at 500.130 MHz and 125.758 MHz, by gradient equipped inverse 5 mm triple probe with Pi/2 pulses of 6.3 and 14.2 microseconds, respectively.

| Quantitative analysis
For the calibration curve, commercially available DHM (as mentioned in the sample preparation section) was used to prepare a stock solution of various concentrations (0.0012-1.002 mg/ml).

| DPPH radical-scavenging ability
The DPPH radical-scavenging ability of racemate DHM was measured in comparison with enantiopure DHM, such as (+) DHM and (−) DHM (Li, Ji, et al., 2014) with slight modification. The determination of the antioxidant ability of the compound depends on the ability of a compound for donating a hydrogen atom to eliminate DPPH radical, as reported (Suttirak & Manurakchinakorn, 2014). The stock solution of ascorbic acid with various concentrations was prepared after dissolving in deionized water and used as a reference sample.
Of 0.5 ml DPPH solution (0.4 mM, ethanol), 1.0 ml sample (DHM) was added in the glass test tube and added 2.0 ml water. After mixing the test tube vigorously, incubated it for 30 min and measured the absorbance at 517 nm using a UV-spectrophotometer. Then, the scavenging activity % was calculated by following Equation 1.

(%)=1-Asample-A0Ablank100
A 0 can be determined by following the above procedure, but the only change was deionized water instead of the DPPH solution. The blank reading was noted as A blank , measured by observing the absorbance values at 517 nm (with the addition of the sample).

| Reducing power ability
The ability of compound for reducing power was determined by the method (Wang et al., 2014) with slight modification, and ascorbic acid was used as a positive control. Simply, 2.5 ml sample was mixed with 2.5 ml of 0.1% phosphate ferricyanide (w/v) and 2.5 ml of 0.2 mM phosphate buffer (pH = 6.62) in the glass tube and heated the glass tube mixture at 50°C for next 20 min. Then, 2.5 ml of 10 % of trichloroacetic acid (w/v) was added. Then, the above-prepared mixture was centrifuged for 10 min at 6,000 × g. After this centrifugation process, supernatants were collected and discarded the sediments. Then 5 ml of supernatant was added into a separate tube and mixed with 0.5 ml of 0.1% ferric chloride (w/v). Absorbance was determined at 700 nm using a UV-spectrophotometer (Shimadzu Co., Kyoto, Japan).

| Antimicrobial activity
Agar well diffusion method was used with slight modification for antimicrobial activity of (+) DHM, (−)-DHM, and (±) DHM (Valgas, Simone, Elza, & Smania, 2007). The assay was carried out in triplicates, and then, measurements of inhibition zone (ZOI) were taken from the top of the well to the clear zone in millimeters (mm).

| Determination of MIC and MBC
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by using two serial dilution methods (Zhao, Hong, Dong, Meng, & Mu, 2013)

| Determination of Cell membrane integrity of B. cereus (AS11846)
The integrity of the bacterial cells was observed by determining the leakages of the constituents in the supernatants, as described by (Cui, Zhang, Zhou, Zhao, & Lin, 2015). First of all, the supernatant was collected for each sample, and then, the absorption was noticed at 260 nm (OD 260 ) at a serial of time intervals. The exponential phase (1 × 10 7 log CFU/ml) bacterial cells of B. cereus (AS11846) harvested and centrifuge at 5,000 × g for 5 min at 4°C and put in phosphate buffer (PBS 0.1 M, pH 7.0). Consequently, the microbial cells were treated with (32, 64 μg/ml) for 1, 2, 4, and 8 hr, whereas the bacterial suspension in the absence of DHM was treated as a control.

| Determination of physical damage on the cell membrane
Scanning electron microscopy analysis (SEM) was performed according to the method of (Li, Xing, et al., 2014).

| Statistical analysis
The data were statistically analyzed as a mean of standard deviation (±SD). One-way analysis of variance (ANOVA) and the Student t test was used to analyze the data at p < .05 significance level. All the data were analyzed in triplicate by Statistical Packages for Social Sciences (SPSS) 17.0.5 version.

| Dry matter contents
Five grams of the vine tea powder was weighed and hot air-dried at 103 ± 2°C for at least 16 hr in an oven to get constant weight and repeated the experiment three times.

| Semi-preparative separation of DHM enantiomer
The development of a high-efficient extraction method should consider both the quality and quantity of isolates. For the further identification of the physical properties of the compound solution stability test analysis was carried out. Results indicated that (−)-DHM was found to be more soluble in ethanol than water, and it reaches 165-170 mg/ml at room temperature. Moreover, in the case of water as a solvent to dissolve (−)-DHM, the finding showed that the sample (−)-DHM was more soluble in hot water (15-18 mg/ml at 75-80°C) than cold water (2-3 mg/ml at 25°C). The Better absorption of (−)-DHM was showed in ethanol than water because the ethanol-dihydromyricetin adduct possesses higher acidity than water-dihydromyricetin, as reported earlier (Kalíková, Martínková, Schmid, & Tesařová, 2018;Lesellier & West, 2015). So results proved that (−)-DHM has better absorption in the organic solvent than water. It is noteworthy to mention that (−)-DHM can be easily oxidized by high-temperature exposure or/and direct in-contact with the metal surface. As a result, the racemization process may increase the quantity of the racemate mixture (±) DHM and affect the quality of desire enantiopure (−)-

DHM. A similar finding has been reported by another researcher,
where the author has found the acceleration of the racemization process at a higher temperature and metal concentration (Qingquan, Yuan, & Zeng, 2014;.

| HPLC-ESI/MS Analysis for isolates
The isolation of compounds A and B was achieved successfully at In comparison to our study, Du and Gao also have reported the (−)-DHM extraction method at a high purity of over 99% with a preparatory of triple-column counter-current chromatography.
However, this method is challenging to put in industrial use (Du, Cai, Xia, & Ito, 2002;Zhu et al., 2019). However, the extraction method optimized by HPLC analysis of (−)-DHM has 95% purity in this case, and it could be obtained by the re-crystallizing process of vine tea leaves for five times (as depicted in the method section Figure 1), which was a useful technology for industrialization. The extraction process adopts in the current study is much more proficient and controllable than that of the prevailing process. Therefore, the finding of the current study can be used as a base for analytical scale to semi-preparative scale study, which further can be explored for the industrial scale. Du and Gao have reported a similar finding when the chiral separation of DHM has been done by using supercritical fluid chromatography and keeping the view of the influence of different chiral separating solvent, co-solvents, and the flow rate (Speybrouck & Lipka, 2016;. Moreover, the presence of two chiral-centers (C-2 and C-3) in the molecular structure of dihydromyricetin (DHM) and its possible two enantiopure compounds has been explained in Figure 3.

| Identification and purity analysis of enantiomer of DHM by NMR spectroscopy
The identity and purity of homemade and commercially available enantiopure DHM were further tested through UV, ESI-MS, and

| DPPH radical-scavenging ability
The DPPH radical-scavenging ability was measured at 517 nm, as mentioned in the method sections. A higher DPPH radical-scavenging activity was observed for both of (−)-DHM and (±) DHM samples in a concentration manner. The finding of the present study depicted that (−)-DHM could reduce oxidative damage. A similar observation of dihydromyricetin for preventative oxidative damage has been reported in previous studies, where the author has proposed that DHM could follow one of the following mechanism to reduce oxidative damage mechanism such as by direct radical-scavenging mode, acting as Fe 2 -chelation and increasing the superoxide dismutase (SOD) enzyme activity which mainly catalyzes the dismutation of superoxide anion O 2 (−) to molecular oxygen (Song et al., 2017), and/ or by activating phosphatidylinositol 3-kinase (PI3k/Akt) and modulating the nuclear transcriptional factor-erythroid 2-related factor 2 (Nrf2), that take part in antioxidant activity and gene encoding detoxification (Hu, Zhang, Yi, Zhou, & Mi, 2018;Luo et al., 2017). More interestingly, the finding of our study also suggested that enantio-

| Reducing power capacity of DHM enantiomer
The reducing power can serve as a significant parameter to reflect the antioxidant activity of a compound. The reducing power was determined at 700 nm by the method (Zou, Lu, & Wei, 2004). All samples showed different degrees of reducing power. Noticeably, the reducing power of (+)-DHM and (−)-DHM was comparable to the commercially available synthetic antioxidant TBHQ used as the food industry ( Figure 6). The findings of the current study are similar to the conclusion of the previously reported studies (Gao et al., 2009).

| Antimicrobial activity of enantiopure DHM
The different concentration of enantiopure (−)-DHM has demon- positions with their antimicrobial properties (Liu, Du, Zeng, Chen, & Niu, 2009). This is further supported by another study, where the finding of the study revealed that the steric-effect of a compound could enable it for improved antimicrobial activity (Hongxia, Jing, Jiayi, Wenjing, & Jianhao, 2019). That is why the present study has proposed the unique antimicrobial mechanism of (−)-DHM enantiopure as compared to racemate (+)-DHM involved against foodborne pathogens.

| Integrity of the cell membrane
The  and structure of the cell membrane. However, it might be possible that there is some difference in cell membrane permeability.
As it is reported, the permeability of the bacterial cell membrane might have increased as time passes (Cui et al., 2015).

| Determination of morphological changes in B. cereus (AS11846) through CLSM
Confocallaser scanning microscopy (CLSM) was further used to explore the action mode of enantiopure (−)-DHM involved in antimicrobial activity. The finding of the current study suggested that the (−)-DHM can increase cell membrane permeability and cause injury of the microbial cell surface, which releases essential internal constituents and thus causes cell death, as can be seen in Figure 8.
Also, better penetration capacity of PI with more prolonged exposure of time (8 hr) produced red color after PI could enter into double-stranded DNA through the damaged cell membranes. This red color indicated the rigorous injury by the action of (−)-DHM on the cell membrane, while SYTO-9 could enter through the cell membrane to bind the DNA of live cells and produce green fluorescence (Sun et al., 2015). It is noted that the SYTO-9 stains will cover the intact cell membrane bacteria and produce green fluorescence, whereas; PI will penetrate through the decade cell membrane of dead bacteria and produce red fluorescence. Therefore, we suggest that this work has the potential for the development of chiral bacteriostatic material because stereo-configuration will provide insight into the development of novel antimicrobial compounds with better biosafety.

| CON CLUS ION
The findings of the current study suggested that vine tea leaves are rich in dihydromyricetin. The compound (−)-DHM, was found to be more potent antimicrobial and antioxidant as compared to synthetic antimicrobial (nisin) and antioxidant agents (TBHQ).

| E THI C AL RE VIE W
This study does not involve any human or animal testing.

| INFORMED CON S ENT
Written informed consent was obtained from all study participants.

ACK N OWLED G EM ENT
The author would like to say thanks to the National Research 2015BAD16B04.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.