Relationship between antimicrobial activity and amphipathic structure of ginsenosides

Industrial Crops & Products 143 (2020) 111929

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Relationship between antimicrobial activity and amphipathic structure of ginsenosides

T

Peng Xuea,*, Xiushi Yangb, Lei Zhaoa, Zhaohua Houc, Ruoyu Zhanga, Fengxiang Zhanga,*, Guixing Renb,** a

School of Public Health and Management, Weifang Medical University, Weifang, 261053, People’s Republic of China Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, People’s Republic of China c College of Food Science and Engineering, Qilu University of Technology, Shandong Academy of Sciences, Jinan, 250353, People’s Republic of China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Bacterial biofilm Bacillus cereus Less polar ginsenosides Fusobacterium nucleatum Structure-activity

Ginsenoside has a wide range of pharmacological activities, but little research has been conducted on its microbial pathogen inhibition and the relationship between its antimicrobial activity and saponin structure. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) of a series of ginsenosides were used to investigate the antimicrobial activity of the ginsenosides. And the value of hydrophile–lipophile balance (HLB) was introduced as physicochemical factors to clarify the difference in bacteriostatic activity of saponins. The effects of active ginsenoside Rh2 on the Fusobacterium nucleatum and Bacillus cereus biofilm system were observed by calgary biofilm device (CBD) and fluorescence confocal microscopy (CLSM). The morphological characteristics were examined by using transmission electron microscope (TEM) to explore the cell damage. The antimicrobial effects were in the following order from high to low: saponins containing one glycosyl group > aglycones > two ligands > more than two ligands. Ginsenoside Rh2 and RK3 (hydrophile lipophilic balance value were both 1.15) showed high activity against the tested strains. Correlation analysis shows that HLB is associated with antimicrobial activity (p < 0.01). The antimicrobial activity increased with the HLB value of ginsenosides determined by changes in the length, the number, and the composition of sugar side chains. Specific polar ginsenosides cause bacterial death by destroying the membrane system. Based on their good broad-spectrum antibacterial activity, less polar ginsenosides have potential as bacteriostatic agents.

1. Introduction Ginsenosides are the main active substances in genus Panax L., such as American ginseng (Panax quinquefolius), Asian ginseng (Panax ginseng) and notoginseng (Panax notoginseng) (Baeg and So, 2013). Ginsenosides can be classified into polar ginsenosides and less polar saponins according to the number of sugars (Kwon et al., 2001). Polar saponins are mostly found in natural plants, whereas less polar saponins are extremely rare in nature and are therefore called rare ginseng saponins (Kwon et al., 2001). Experiments have confirmed that the less polar saponins in ginseng have higher pharmacological activity than the polar saponins (Duan et al., 2017; Lee et al., 2012). Therefore, many scholars have studied the transformation of ginsenosides, including physical methods (steaming, microwave, and sulfur fumigation), chemical methods (acid hydrolysis and alkaline hydrolysis),

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biotransformation (enzymatic hydrolysis, intestinal bacterial hydrolysis, endophytic bacterial transformation, edible fungal transformation, and soil microbial transformation) (Zheng et al., 2017). Biotransformation is a method of obtaining rare saponins with specificity, high efficiency, and low energy consumption (Cui et al., 2016; Quan et al., 2013). Moreover, physical and chemical methods can further convert rare saponins, which are easy to hydroxylate, hydrolyze, and isomerize by chemical methods, providing diversity in drug screening (Zhu et al., 2014; Xiao et al., 2017). Most rare saponins are prepared by transforming the roots of genus Panax L. (Xu et al., 2017; Du et al., 2014). Different types of ginsenosides are distributed in various parts of ginseng plants, with varying contents (Lee et al., 2017; Wang et al., 2014a, 2014b). Rare ginseng saponins can still be prepared by the conversion method (Hwang et al., 2014; Xue et al., 2016); however, ginseng roots can be harvested every

Corresponding authors at: School of Public Health and Management, Weifang Medical University, Weifang, 261053, People’s Republic of China. Corresponding author at: Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100081, People’s Republic of China. E-mail addresses: [email protected] (F. Zhang), [email protected] (G. Ren).

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https://doi.org/10.1016/j.indcrop.2019.111929 Received 13 August 2019; Received in revised form 16 October 2019; Accepted 31 October 2019 Available online 12 November 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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evaporation to obtain 0, 30%, 60%, 80%, and 95% eluted fractions and lyophilized to obtain HTS, HTS-1, HTS-2, HTS-3, and HTS-4, respectively. The Agilent 1200 HPLC preparation stage was equipped with a Grace Platinum EPS-C18 preparative column (22 mm × 250 mm, 5 μm) for further purification refer to the preliminary experimental method (Xue et al., 2017).

4–6 years (notoginseng 2–3 years), whereas the stem and leaf can be harvested every year. The latter has higher yield and ginsenoside content than the former. Thus, obtaining saponins from the stems and leaves of ginseng plants is more economical and provides higher yields than the roots (Yang et al., 2014). To date, more than 280 types of ginsenosides have been identified (Gu et al., 2013). Some have been proven to inhibit obesity, treat diabetes, promote hair growth, show neuroprotective effects, provide skin cell protection, and exhibit antifatigue, anticancer, antiapoptosis, antiinflammatory, and antiallergy properties (Tan et al., 2014a, b; Park et al., 2010; Shin et al., 2014; Chen et al., 2012; Lee et al., 2013; Samukawa et al., 2012). The microbial inhibitory activity of ginsenosides has been investigated. Polar ginsenosides from white ginseng do not inhibit the growth of Pseudomonas aeruginosa and show low antibacterial activity against Bacillus cereus and Staphylococcus aureus (Na et al., 2017; Battinelli et al., 2011; Wu et al., 2011) In a previous experiment, the less polar ginsenosides obtained from the stems and leaves of Panax quinquefolium by heat treatment had antimicrobial effects against Fusobacterium nucleatum, Clostridium perfringens, and Porphyromonas gingivalis, and the less polar ginsenosides from the roots of notoginseng had antifungal activities against Epidermophyton floccosum, Trichophyton rubrum, and Trichophyton mentagrophytes (Xue et al., 2016, 2017). Because of the limited availability of rare ginsenosides, the correlation of structures and mechanisms to antimicrobial activities is still not fully elucidated. In this study, the crude saponins from stem-leaf American ginseng were used to produce less polar ginsenosides by heat transformation. Nine rare ginsenosides (20(e)-Rh4, Rk3, 20(z)-Rh4, Rh2, 20S-Rg6, F4, 20S-Rg5, Rs3, and RK2) were obtained by silica gel, macroporous adsorption resin, and HPLC preparative chromatography and identified by NMR and HPLC-MS. Ten common polar compounds were obtained from stem and leaf saponins and identified by HPLC/MS and TLC verification with reference standard. We explored the antimicrobial effects of these 21 ginsenosides on three bacterial strains and three fungi to uncover possible structural-activity relationships. The effects of the less polar ginsenoside Rh2 on the bacterial biofilm system and acid production were investigated by transmission electron microscopy, fluorescence confocal microscopy and gas chromatography-mass spectrometry.

2.3. Microbial cultures obtention and preparation The microbial cultures preparation was described by Xue and Ren (Xue et al., 2016; Ren et al., 2018). All standard bacterial and fungal strains were obtained from the Guangdong Microbiology Culture Center (Guangzhou, China). Fusobacterium nucleatum (ATCC 10953), Clostridium perfringens (ATCC 13124), and Porphyromonas gingivalis (ATCC 33277) were cultured in anaerobic blood agar (CDC anaerobic agar) base for 48 h at 37 °C in a YQX-II anaerobic incubator (Shanghai, China). The mixed gas consisted of 5% (v/v) CO2, 10% (v/v) H2, and 85% (v/v) N2. The final cell concentration was diluted with Gifu anaerobic medium (GAM) liquid medium and suspended to obtain 108 CFU/mL. Listeria ivanovii (ATCC 19119) was cultured in blood agar, Salmonella enteritidis (ATCC 14028), Pseudomonas aeruginosa, (ATCC27853), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (BNCC102555), and Bacillus cereus (ATCC14579) were cultured in nutrient agar (NA) for 24 h and at 37 °C. Fungal culture methods were used by previous report (Yang and Jiang, 2015). Epidermophyton floccosum (ATCC 52066), Trichophyton rubrum (ATCC 28188), and Trichophyton mentagrophyte (ATCC 9533) from Guangdong Microbiology Culture Center were inoculated into Sabouraud medium in a mould incubator and cultured at 37 °C for 72 h. After the fungus formed spores, the spores were gently washed with PBS and diluted to a spore concentration of 108 CFU/mL. 2.4. Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC)

American ginseng leaf and stem saponins (AGS) were purchased from Jilin Hongjiu Biotechnology Co., Ltd. (Jilin, China). Penicillin sodium, cefixime, crystal violet, miconazole nitrate and dimethyl sulfoxide (DMSO) were purchased from J & K (Shanghai, China). Ginsenoside standards (-Rg1, -Re, -Rb1, -Rc, -Rb2, -Rd, -20(R)-Rg2, -20(S)-Rg2, -20(R)-Rg3, -20(S)-Rg3, Rh2, PPD, PPT) with > 98% purity were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile and methanol were obtained from Merck (Darmstadt, Germany). Agar, blood agar, nutritious broth, Gifu anaerobic broth, anaerobic blood agar (CDC Anaerobic Agar) broth, and Sabouraud agar modified (SAM) were purchased from Beijing Solarbio Biotech Co., Ltd. (Beijing, China). The LIVE/DEAD™ BacLight™ Bacterial Viability Kit was purchased from Thermo Fisher Scientific (China) Co., Ltd. The other chemicals were of analytical or HPLC grade.

The MIC and MBC or MFC values were determined as described previously (Xue et al., 2017; Ren et al., 2018). Briefly, 100 μL dilutions (approximately 105 CFU/mL) of E. floccosum, T. rubrum, T. mentagrophytes, L. ivanovii, S. enteritidis, S. aureus, P. aeruginosa, S. epidermidis, B. cereus in nutrient broth and F. nucleatum, C. perfringens, and P. gingivalis in GAM broth were inoculated into microtiter plates. Then, 100 μL aliquots of the test sample solutions were added after a two-fold serial dilution with the corresponding medium broth (from 2 mg/mL to 3 μg/mL). Broths with 2% (v/v) DMSO were used as the solvent blank. The Petri dishes were incubated at 37 °C for 24 h with the exception of F. nucleatum, C. perfringens, and P. gingivalis, which were incubated at 37 °C for 48 h. Penicillin sodium and cefixime were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 10 mg/mL for the subsequent antibacterial experiments as positive control. Miconazole nitrate was diluted with DMSO to a concentration of 10 mg/mL as a positive control against fungi. The samples were also dissolved in DMSO at 10 mg/mL. The MIC was recorded as the lowest concentration of sample showing no detectable growth, as indicated by the absence of turbidity. Ten microliters of sub-inhibitory concentrations of the test compounds were placed in the corresponding solid medium for 48 h to determine the MBC or MFC values according to the growth of the microbial colonies.

2.2. Preparation of less polar ginsenosides

2.5. Biological film inhibition investigation by calgary biofilm device (CBD)

A total of 400 g of transformed American ginseng stem-leaf saponin was dissolved in as little water as possible, passed through HP-20 macroporous resin, and eluted with 3 column volumes of water and 30%, 60%, 80%, and 95% ethanol in water (Xue et al., 2017). Each eluted fraction was collected, and the solvent was removed by rotary

To determine the inhibition rate of the biological film, the film would attach to the calgary biofilm device and was stained with crystal violet solution after elution. Finally, the absorbance of crystal violet was determined by spectrophotometry. (Dana et al., 2018). The F. nucleatum and B. cereus were activated, then 100 μL of GAM liquid broth

2. Materials and methods 2.1. Chemicals

2

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and containing 106 CFU/mL F. nucleatum and 100 μL nutritious broth with 106 CFU/mL B. cereus were inoculated into calgary biofilm device (CBD), respectively. Ginsenoside Rh2 was added to CBD medium at concentrations of 1/2 MBC, 1/4 MBC, and 1/8 MBC, and 2% DMSO was used as a solvent control. After incubation in an anaerobic incubator at 37 °C for 24 h, the column cap was gently cleaned 3–5 times with ultrafiltered water. A total of 125 μL of 0.3% crystal violet was added to another clean CBD plate. The cleaned upper lid was closed and stained for 15 min, washed again 3–5 times with ultrafiltered water, and placed in another CBD plate to which 200 μL of 95% ethanol was added. The crystal violet on the biofilm was dissolved in 95% ethanol, and the absorbance (595 nm) was measured by a microplate reader. Each experiment was repeated three times.

HLB= 7 +

∑ Hi–

n × 0.475

i=1

m: the number of hydrophilic groups in the molecule H: the value of the i hydrophilic group n: the number of lipophilic groups in the molecule 2.9. Acute toxicity studies in mice For the acute toxicity study, male and female SPF grade Wistar rats had an average weight of 200 ± 50 g. All were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., which production license number is SCXK (Lu) 20140007. Upon arrival at the laboratory, the animals were acclimatized in cages under standard environmental conditions of light/dark cycles (12 h/12 h), temperature (23 ± 1 °C), with 40 ± 5% of humidity and air changes. Animals had free access to tap water and standard pellet diet. The air is exchanged by the automatic air circulation system to ensure the normal breathing of the rats, and the disinfection is carried out regularly for 7 days. Adapt to the environment of the house. Rats were randomly divided into five groups (10 mice/group). The HTS were administered to rats by gavage (n = 10 in each group) at doses of 1000, 2150, 4640, 10,000 mg/kg, while the control group received the water only. The acute oral toxicity test was performed in accordance with the OECD code of practice (TG 423) and national food safety standards of China (GB 15193.3-2014).

2.6. Biological film inhibition investigation by confocal laser scanning microscopy (CLSM) The effect of the samples on bacterial activity was observed by CLSM through SYTO-9 and propidium iodide (PI) fluorescent staining of live and dead cells (Wu et al., 2011). The SYTO-9 and PI tube dyes were separately dissolved, diluted to 0.1% with sterile distilled water and mixed in equal proportions. Suspensions of F. nucleatum and B. cereus were diluted with PBS to a concentration of 106 CFU/mL. Then, 2.6 mL of GAM containing 100 μl of F. nucleatum and NA liquid medium containing 100 μl of B. cereus was added to a 24-well plate, respectively. The test compound was added to the well and brought up to 3 mL with the corresponding liquid medium. Finally, test compound concentration of 1/2MIC, MIC, and MBC were applied. Subsequently, 10 mm × 10 mm slides were placed in the wells after adding all liquids and cultured at 37 °C for 72 h in an anaerobic incubator for F. nucleatum and cultured at 37 °C for 48 h in an incubator for B. cereus. The medium was replaced with fresh medium every day. The medium was then washed 3–5 times with sterile distilled water. After incubation of the stains in equal proportions (SYTO-9 : PI = 1 : 1), the cells were incubated for 15 min in the dark, and the film formation and cell survival were observed under a CLSM (Carl Zeiss AG, LMS 700, Heidenheim, Germany).

2.10. Data analysis Data are presented as the mean of three replicates ± standard deviation. One-way ANOVA with Duncan’s multiple range test and bivariate correlation with spearman were used to analyze the results with SPSS 13.0 and Sigma Plot 10.0, respectively. A p value of < 0.01 was determined to be statistically significant. 3. Results and discussion 3.1. Extraction and isolation

2.7. Visualization of cell damage by transmission electron microscopy (TEM)

A total of 8.5 g of HTS-2, 120.9 g of HTS-3 and 200.5 g of HTS-4 were obtained. In early experiments, we found that the content of less polar ginsenosides (-F4, Rg3, Rg6, Rk3, Rg5, Rh2) was only 0.10 ± 0.01 mg/mg in AGS, while the contents of less polar ginsenosides in HTS 0.76 ± 0.02 mg/mg were significantly increased after heated. After purification by HP-20, the content of less polar ginsenosides increased to 0.91 ± 0.03 mg/mg in HTS-4 and the HPLC chromatogram of American ginseng saponins (A), HTS (B), HTS-3 (C) and HTS-4 (D) (Xue et al., 2016). Therefore, HTS was chosen to isolate rare ginsenosides. In short, 75.0 g of HTS-4 was dissolved in anhydrous methanol solution, and 380 g of 160–200 mesh silica gel was added, mixed and loaded using a dichloromethane-methanol system of 10:0; 9.5:0.5; 9:1; 8:2; 7:3; 5:5; 2:8 ratio elution, one fraction per 1000 mL, where 6000 mL was one column volume, and 2–3 column volumes of each ratio were used for elution. The fractions were combined according to thin layer chromatography, and a total of 7 groups, crude 1 (Frs 1–9; 3.4 g), crude 2 (Frs 10–16; 12.6 g), crude 3 (Frs 17–21; 30.7 g), crude 4 (Frs 21–28; 47.2), crude 5 (Frs 29–38; 26.2 g), crude 6 (Frs 39–44, 7.8 g), and crude 7 (Frs 45–54, 11.2 g), were selected. The yield was 79.2%. Crude-2 (8.7 g) and crude-3 (10.4 g) were loaded on an LH-20 column and eluted with 80:20 and 75:25 MeOH/H2O, respectively. Three major fractions, HTS-24, HTS-38, and HTS-W, were obtained. These fractions were subjected to isocratic elution on analytical NP HPLC with gradient elution to afford pure 1–14. Crude-4 (15.21 g) was loaded on an LH-20 column and eluted with MeOH/H2O 50 : 50 and 60 : 40 to obtain HTS-dis and HTS-40. These

The integrity of cells and organelles can be observed by TEM, according to a previously reported method (Lee et al., 2014). F. nucleatum, and B. cereus (approximately 1 × 107 CFU/ml) were combined with different concentrations of saponins (control, 1/2MIC, MIC, and MBC) and incubated for 24 h at 37 °C in incubator, respectively. The suspensions were harvested by centrifugation at 3000 ×g for 10 min. The cell-agar blocks were fixed in 2.5% (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 90 min and washed three times in the same buffer for 10 min. The cell-agar blocks were then post-fixed for 2 h in 1% (w/v) osmium tetroxide, washed three times with sterile distilled water, and dehydrated using a series of concentrations of ethanol (50%, 60%, 70%, 80%, 90%, 95% and 100%) for 30 min each. After infiltration into a mixture (propylene oxide: resin = 1: 3), the samples were embedded using the EPON-12 kit and polymerized at 60 °C for 72 h. The polymerized cell-agar blocks were sliced with an ultramicrotome (Reichert SuperNova; Leica, Wetzlar, Germany) to obtain thin sections (50–60 nm). The sections were mounted on grids and stained with uranyl acetate and Reynold’s lead citrate to enhance contrast. 2.8. Hydrophile–lipophile balance (HLB) value The HLB value of ginsenosides were calculated theoretically by the method of Ou and Davies (Ou et al., 2009; Davies, 1957). In short, the HLB value of a surfactant can be calculated from its molecular structure. The Davies’ calculation formula is: 3

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Fig. 1. The main molecular structure of ginsenoside; protopanaxadiol type (A); protopanaxatriol type(B); other type (C).

(Zhou et al., 2016). Compound 5, white powder (methanol), brown spot under UV lamp, 10% sulphuric acid in sm., HR ESI MS m/z: 621.4 [M−H]−, 13C NMR 151 MHz, (MeOD) data As shown in Table 2. Compared with the literature, the compound was identified as ginsenoside Rh2 (Zhou et al., 2016). Compound 7, white powder (methanol), brown spot under UV lamp, 10% sulphuric acid sm., HR ESI MS m/z: 765.4 [M−H]−, 1H NMR (600 MHz, MeOD), 3.51 (1H, dd, m, H-3α); 1.64 (1H, m, H-5α); 4.62 (1H, dd, J = 9.5, 3. 0 Hz, H-6β); 3.92 (1H, m, H-12α); 1.36 (3H, s, H29); 1. 15 (3H, s, H-19); 5.14 (1H, br s, Ha-21); 4.17 (1H, br s, Hb-21); 5.22 (1H, m, H-24); 1.75 (3H, s, H-26); 1.70 (3H, s, H-27); 2.14 (3H, s, H-28); 1. 26 (3H, s, H-29); 5.33 (1H, d, J =6.6 Hz, H-1′). And 13C NMR 151 MHz, (MeOD) data are shown in Table 2. Compared with the literature, the determination of the compound is ginsenoside 20 (z)-Rg6 (Zhou et al., 2016). Compound 8, white powder (methanol), brown spot under UV lamp, 10% sulphuric acid, sm., HR ESI MS m/z: 765.4 [M−H]−, 1H NMR (600 MHz, MeOD), 3.31 (1H, dd, J = 10.8, 4.5 Hz, H-3α); 1.29 (1H, d, J = 10.4 Hz, H-5α); 4.45 (1H, m, 3.0 Hz, H-6β); 3.77 (1H, m, H-12α); 1.36 (3H, s, H-18); 0.95 (3H, s, H-19); 2.25 (3H, s, H-21); 5. 33 (1H, m, H-22); 4.67 (1H, m, H-24); 1. 75 (3H, s, H-26); 1.60 (3H, s, H-27); 1.97 (3H, s, H-28); 1.19 (3H, s, H-29); 0.90 (3H, s, H-30); 4.90 (1H, m, J =6.4 Hz, H-1′). And 13C NMR 151 MHz, MeOD) data are shown in Table 2. Compared with the literature, the identified compound is ginsenoside F4 (Zhou et al., 2016). Compound 10, white powder (methanol), brown spot under UV lamp, 10% sulphuric acid, sm., HR ESI MS m/z: 765.4 [M−H]−, 1H NMR (600 MHz, MeOD), 3.18 (1H, dd, J = 11.6, 4.4 Hz, H-3α); 3.98 (1H, m, H-12α); 1.09 (3H, s, H-18); 1.02 (3H, s, H-19); 1.89 (3H, s, H21); 5.11 (1H, m, H-22); 5.01 (1H, m, H-24); 1.62 (3H, s, H-26); 1.57

fractions were subjected to isocratic elution on an analytical NP HPLC with gradient elution to afford pure 15-19. 3.2. Compound identification Twenty one compounds were isolated and identified from HTS-4 (Fig. 1). Most of the isolated compounds were less polar ginsenosides. Some 13C NMR spectroscopic data of less polar ginsenosides 20(e)-Rh4, Rk3, 20(z)-Rh4, Rh2, 20S-Rg6, F4, 20S-Rg5, Rs3, and RK2 are shown in Table 1. The identification of other compounds was compared with the corresponding references by HPLC/MS and TLC. The data are not shown. Compound 1, white powder (methanol), brown spot under UV lamp, 10% sulphuric acid sm., HR ESI MS m/z: 619.4 [M−H]−, 1H NMR (600 MHz, MeOD), 3.59 (1H, m, H-3α); 4.37 (1H, d, J =6 Hz, H-6β); 3.87 (1H, m, H-12α); 5.44 (1H, m, H-22); 5.18 (1H, m, H-24); 1.70(3H, s, H-26); 1.69 (3H, s, H-27); 2. 07 (3H, s, H-28); 1.60 (3H, s, H-29); 0.88 (3H, s, H-30); 5.10 (1H, d, J = 7.8 Hz, H-1′). And 13C NMR 151 MHz, MeOD) data are shown in Table 2, the data were compared with the literature and the identified compound is ginsenoside 20 (E)-Rh4 (Zhou et al., 2016). Compound 3, white powder (methanol), brown spot under UV lamp, 10% sulphuric acid sm., HR ESI MS m/z: 619.4 [M−H]−, 1H NMR (600 MHz, MeOD) 4.13 (1H, dd, J = 11.2, 3.9 Hz, H-3α); 1.18 (1H, d, J = 10.4 Hz, H-5α); 4.57 (1H, m, H-6β); 4.13 (1H, m, H-12α); 1.25 (3H, s, H-29); 1.07 (3H, s, H-29); 5.07 (1H, br s, Ha-21); 4.81(1H, br s, Hb21); 5.19 (1H, t, J =6.8 Hz, H-24); 1.64 (3H, s, H-26); 1.57 (3H, s, H27); 2.05 (3H, s, H-28); 1.56 (3H, s, H-29); 0.83 (3H, s, H-30); 4.95 (1H, d, J = 7.8 Hz, H-1′). And 13C NMR 151 MHz, MeOD) data are shown in Table 2. The identified compound is ginsenoside Rk3 compared with the literature 4

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Table 1 13 C NMR spectroscopic data for less polar ginsenoside. pos

20(E)-Rh4

Rk3

20(z)-Rh4

Rh2

20(z)-Rg6

F4

20S-Rg5

Rs3

RK2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 6-glc 1' 2' 3' 4' 5' 6' 2'-rha 1" 2" 3" 4" 5" 6" Ac-CH3

39.2 26.9 88.8 39.8 56.5 18.5 35.8 37.2 50.5 39.7 32.1 71 48 51.8 31.4 26.9 54.9 16.7 16.4 73 27 36 23.1 126.1 130.8 25.9 17.3 28.2 16 17.4

34.02 18.41 77.15 36.84 56.03 88.27 39.97 39.17 50.23 35.04 32.01 71.59 50.68 50.54 28.6 26.22 50 16.73 16.73 148.34 110.38 32.31 18.18 120.32 141.12 17.03 16.52 27.99 13.51 15.85

34.65 27.99 77.13 36.83 56.03 88.29 39.37 39.19 50.22 35.05 32.32 71.63 50.18 50.34 32.08 18.41 50.62 16.72 17.04 148.25 13.31 110.12 26.21 120.93 141.38 18.17 17.04 28.2 15.85 16.52

39.2 26.9 88.8 39.8 56.5 18.5 35.8 37.2 44 39.7 32.1 71 48 51.8 31.4 26.9 54.9 16.7 16.4 73 27 36 23.1 126.1 130.8 25.9 17.3 28.2 16 17.4

33.41 29.41 77.68 38.9 60.03 73.46 40.72 39.01 44.91 38.82 32.01 70.49 51.22 50.8 31.28 26.38 49.74 16.59 16.59 154.67 107.13 32.01 30.18 124.31 130.78 26.18 15.97 30.51 16.46 15.66

38.85 26.97 77.11 39.19 61.72 76.95 49.96 39.58 53.73 36.54 34.52 73 56.13 61.44 34.91 25.96 51.17 17.84 16.3 144.2 14.77 124.2 25.86 124.78 130.58 25.11 15.32 30.6 15.71 15.37

38.86 16.54 79.52 39.06 50.03 16.44 31.82 39.11 44.12 31.92 26.4 70.3 40.6 50 29.95 26.21 40.68 15.65 16.12 138.77 14.68 123.58 24.47 124.36 130.31 16.51 16.39 26.14 15.57 16.13

36.6 17.87 89.87 38.95 56.1 15.9 31.27 39.23 48.14 34.75 27.52 70.17 39.8 50.46 26.99 24.51 50.82 14.71 15.32 72.68 25.82 32.1 15.99 123.57 130.3 16.46 15.69 26.11 15.44 15.79

40.64 24.44 79.49 44.16 61.5 16.4 38.89 49.7 50.01 39.15 30.96 74.2 60.47 50.51 30.02 28.03 49.92 15.54 11.53 148.96 107.79 31.93 26.14 123.23 130.72 16.54 16.1 26.57 14.7 15.59

106.8 75.8 78.4 71.7 78.3 63

105.91 74.27 77.35 70.65 74.46 61.69

105.81 74.42 77.36 70.66 74.44 61.69

106.8 75.8 78.4 71.7 78.3 63

100.15 78.33 77.77 72.4 76.64 61.7

103.12 89.91 79.67 76.31 21.89 70.21

104.16 78.44 76.29 61.5 72.58 50.76

103.08 79.64 76.93 61.69 76.46 56.14

104.21 76.36 78.49 72.57 77.76 70.35

100.2 70.83 71.06 72.54 68.31 24.5

104.01 70.74 74.91 76.49 70.52 24.51

107.14 72.44 77.7 60.42 74.11 50.79

103.99 76.28 77.09 70.5 74.89 61.4 168.01

identified compound is ginsenoside Rk2 (Zhou et al., 2016) After high-temperature conversion, the less polar ginsenosides first obtained from HTS are consistent with the main species of red ginseng (Quan et al., 2015; Liu et al., 2011). The feasibility of obtaining less polar ginsenosides from the stems and leaves of Panax species is thereby proven. The composition indicates that the transformed stem and leaf saponins and the components after ginseng root saponin transformation are consistent. The compound structure is altered by temperature conversion: dammarane ginsenoside C-17 will change, as will Rg5, Rk1, Rk3, Rh4, etc.; and the dehydration of C-20 and C-22 produces different saponins with type E and Z double bonds. In raw ginseng and fresh ginseng, the original ginseng diol and the original ginseng triol saponin are mostly 20 (S) types, and 20 isomerization was achieved by heating (In et al., 2016). Human intestinal bacteria can subsequently convert the saponins Rg1 and Rh4 into CK, indicating that low-polarity saponins may be better absorbed by the human body (Kalai et al., 2015). Among these compounds, 20(S)-Rg3 and 20(R)-Rg3 are anticancer drugs in clinical use (Guo et al., 2018; Lu et al., 2018). During the transformation process, the polar saponin undergoes not only desugarization and dehydration but is also the conversion of the C17 side chain and the isomerization of C-20. A similar situation occurs in the biotransformation process (Wan et al., 2013; Wang et al., 2014a,

(3H, s, H-27); 1.23 (3H, s, H-28); 0.89 (3H, s, H-30); 4.87 (1H, d, J = 7.6 Hz, H-1′); 5.05 (1H, d, J = 7.6,Hz, H-1″).And 13C NMR 151 MHz, MeOD) data are shown in Table 2. Compared with the literature, the identified compound is ginsenoside 20S-Rg5 (Zhou et al., 2016). Compound 11, white powder (methanol), brown spot under UV lamp, 10% sulphuric acid, sm., R ESI MS m/z：919.4 [M−H]−, 1H NMR (600 MHz, MeOD), 3.51 (1H, dd, J = 11.4, 4. 2 Hz, H-3α); 4.21 (1H, m, H-12α); 1.07 (3H, s, H-18); 1.19 (3H, s, H-19);1.55 (3H, s, H21); 5.25 (1H, m, H-24); 1.66 (3H, s, H-26); 1. 64 (3H, s, H-27); 1.41 (3H, s, H-28); 0.91 (3H, s, H3-29); 1.04 (3H, s, H-30); 5.01 (1H, d, J = 7.6 Hz, H-1′); 5.44 (1H, m, H-1″); 4.97 (1H, m, Ha-6″); 4.76 (1H, m, Hb-6″); 2.06 (3H, s, Ac−CH3). And 13C NMR 151 MHz, MeOD) data are shown in Table 2. Compared with the literature, the identified compound is ginsenoside Rs3 (Zhou et al., 2016). Compound 12, white powder (methanol), brown spot under UV lamp, 10% sulphuric acid, sm., HR ESI MS m/z: 7: 919.4 [M−H]−, 1H NMR (600 MHz, MeOD), 3.53 (1H,m,H-3α);4.38 (1H,d,J =6 Hz,H-6β); 2.57 (1H,dd,J = 12.64,2. 83 Hz,H-7); 4.02 (1H,m,H-12α); 1.01 (3H,s,H-19); 1.87 (3H,s,H-21); 5.44 (1H,m,H-22); 5.19 (1H,brt,H-24); 1.57 (3H,s,H-26); 1.56 (3H,s,H-27); 2.07(3H,s,H-28); 1.54 (3H,s,H-29); 0.88 (3H,s,H-30); 4.99 (1H,d,J = 7.8 Hz,H-1′). And 13C NMR 151 MHz, MeOD) data are shown in Table 2. Compared with the literature, the 5

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Table 2 The antimicrobial activity of ginsenosides (μg/mL). 　

E. floccosum

Samples

MIC

MFC

MIC

Rg1 Re Rb1 Rb2 Rc Rd 20R-Rg2 20S-Rg2 20z-Rg6 F4 20(z)-Rh4 20(E)-Rh4 Rg3(S) Rg3(R) Rk3 Rg5 Rh2 Rk2 Rs3 PPD PPT Penicillin sodium Cefixime Miconazole nitrate

125 250 250 250 250 250 62.5 62.5 31.3 31.3 16 16 31.3 31.3 16 16 16 16 > 500 31.3 62.5 —— —— 4

250 > 500 > 500 > 500 > 500 > 500 125 125 125 125 62.5 62.5 125 125 62.5 62.5 31.3 31.3 > 500 250 125 —— —— 16

62.5 250 250 250 250 250 62.5 62.5 31.3 31.3 16 16 31.3 31.3 16 16 16 16 > 500 31.3 62.5 —— —— 4

T. rubrum

T. mentagrophyte

C. perfringens

F. nucleatum

P. gingivalis

MFC

MIC

MFC

MIC

MBC

MIC

MBC

MIC

MBC

250 > 500 > 500 > 500 > 500 > 500 125 125 125 62.5 62.5 62.5 125 250 62.5 62.5 31.3 31.3 > 500 250 250 —— —— 16

125 250 250 250 250 250 62.5 62.5 31.3 31.3 16 16 31.3 62.5 16 16 16 16 > 500 31.3 62.5 —— —— 4

250 > 500 > 500 > 500 > 500 > 500 125 125 62.5 32.5 62.5 62.5 125 250 62.5 62.5 31.3 31.3 > 500 250 250 —— —— 16

> 500 > 500 > 500 > 500 > 500 > 500 62.5 62.5 125 125 16 16 31.3 31.3 31.3 16 31.3 31.3 > 500 62.5 125 8 62.5 ——

> 500 > 500 > 500 > 500 > 500 > 500 125 125 250 250 62.5 62.5 125 125 125 62.5 125 125 > 500 250 250 16 125 ——

> 500 > 500 > 500 > 500 > 500 > 500 62.5 62.5 31.3 31.3 16 16 31.3 31.3 16 16 16 16 > 500 62.5 31.3 > 500 8 ——

> 500 > 500 > 500 > 500 > 500 > 500 125 125 250 250 62.5 62.5 125 250 125 62.5 125 125 > 500 250 250 > 500 31.3 ——

> 500 > 500 > 500 > 500 > 500 > 500 62.5 62.5 125 125 31.3 31.3 31.3 31.3 62.5 16 16 16 > 500 62.5 31.3 > 500 16 ——

> 500 > 500 > 500 > 500 > 500 > 500 125 125 250 250 62.5 62.5 250 125 125 62.5 62.5 62.5 > 500 250 250 > 500 62.5 ——

　

L. ivanovii

Samples

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

Rg1 Re Rb1 Rb2 Rc Rd 20R-Rg2 20S-Rg2 20z-Rg6 F4 20(z)-Rh4 20(E)-Rh4 Rg3(S) Rg3(R) Rk3 Rg5 Rh2 Rk2 Rs3 PPD PPT Penicillin sodium Cefixime Miconazole nitrate

> 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 125 62.5 125 125 > 500 > 500 > 500 > 500 62.5 ——

> 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 500 > 500 > 500 > 500 > 500 125 ——

> 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 62.5 62.5 62.5 > 500 16 > 500 125 125 62.5 > 500 ——

> 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 > 500 125 125 125 > 500 62.5 > 500 500 500 125 > 500 ——

62.5 > 500 > 500 > 500 > 500 > 500 62.5 62.5 62.5 62.5 31.3 31.3 31.3 31.3 31.3 16 16 16 > 500 62.5 125 62.5 > 500 ——

250 > 500 > 500 > 500 > 500 > 500 125 125 125 125 62.5 62.5 62.5 62.5 62.5 62.5 62.5 62.5 > 500 250 250 125 > 500 ——

> 500 > 500 > 500 > 500 > 500 > 500 125 125 125 125 125 125 125 125 125 62.5 62.5 62.5 > 500 125 125 > 500 > 500 ——

> 500 > 500 > 500 > 500 > 500 > 500 250 250 250 250 250 > 500 > 500 > 500 500 125 125 125 > 500 > 500 > 500 > 500 > 500 ——

62.5 250 > 500 > 500 > 500 > 500 62.5 62.5 31.3 31.3 16 16 31.3 31.3 16 16 8 8 > 500 62.5 31.3 8 8 ——

250 > 500 > 500 > 500 > 500 > 500 125 125 62.5 62.5 62.5 62.5 125 125 31.3 31.3 16 16 > 500 250 250 16 16 ——

62.5 250 > 500 > 500 > 500 > 500 62.5 62.5 31.3 31.3 16 16 16 16 16 16 8 8 > 500 31.3 62.5 125 125 ——

125 > 500 > 500 > 500 > 500 > 500 125 125 62.5 62.5 62.5 62.5 125 125 125 31.3 16 16 > 500 62.5 125 500 > 500 ——

S. enteritidis

S. aureus

P. aeruginosa

S. epidermidis

B. cereus

Re showed stronger antimicrobial activity (MIC < 1 μg/mL) against Candida albicans and Staphylococcus capitis (Bo et al., 2012). However, in subsequent studies, it was found that the antibacterial activity of polar saponins is weak. Lower MICs (31.3 μg/mL) and MFCs (250 μg/mL) were observed when the same strain of fungi was treated with PPD than with PPT (MICs of 62.5 μg/mL). Interestingly, the inhibitory bacterial activity of PPT (MIC of 31.3 μg/mL) was higher than that of PPD (MIC of 62.5 μg/ mL). Saponins with two sugar chains have moderate bacteriostatic activity; the MIC values were 31.3 μg/mL against the tested bacterial strains, and the MBCs against the same strains were 250 μg/mL. The sugar-based less polar ginsenosides exhibit good bacteriostatic effects. Consistent with previous research, the sugar moiety is a key factor in moderating the antitumor activity of ginsenosides (Lee et al.,

2014b). The practical application has a wide range of effects, and how to improve the conversion rate of the target less polar ginsenosides needs further study. 3.3. Antimicrobial activity of ginsenosides To further explore the material basis of the antimicrobial activity after transformation, the MICs and MBC or MFCs of each ginsenoside are reported in Table 2. Bacteria and fungi were assayed, but no activity (MICs > 500 μg/mL) was detected for saponins containing three glycosyl groups (ginsenoside Rg1, Re, Rb1, Rb2, Rc, and Rd) at the higher concentration used. The selected microorganisms showed high growth inhibition when treated with compounds containing one molecule of glycoside (Rk3, Rh4, Rh2, Rg5). MICs of 16 μg/mL (Rk3, Rg5, Rh2, Rh4) were recorded with F. nucleatum. Protopanaxatriol ginsenosides 6

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Table 3 Specifications of chemical structure and HLB value of ginsenosides. Composition of sugar chains saponins

Saponin typea

3-C/6-C

20-C

-OH(free)

-OH (sorbitan)

-COO- (Sorbitan)

-O-

-CHn(n > 0)

HLB value

Rg1 Re Rb1 Rb2 Rc Rd 20R-Rg2 20S-Rg2 20z-Rg6 F4 20(z)-Rh4 20(E)-Rh4 Rg3(S) Rg3(R) Rk3 Rg5 Rh2 Rk2 Rs3 PPD PPT

Protopanaxatriol Protopanaxatriol Protopanaxadiol Protopanaxadiol Protopanaxadiol Protopanaxadiol Protopanaxatriol Protopanaxatriol Protopanaxadiol Protopanaxadiol Protopanaxatriol Protopanaxatriol Protopanaxadiol Protopanaxadiol Protopanaxatriol Protopanaxatriol Protopanaxadiol Protopanaxatriol Protopanaxatriol Protopanaxadiol Protopanaxatriol

-glc -glc(2-1)glc -glc(2-1)glc -glc(2-1)glc -glc(2-1)glc -glc(2-1)glc -glc(2-1)rha -glc(2-1)rha -glc(2-1)rha -glc(2-1)rha -glc(2-1)rha -glc(2-1)rha -glc(2-1)glc -glc(2-1)glc -glc -glc(2-1)glc -glc -glc glc(2,1)glc-6-Ac —— ——

-Oglc -Oglc -Oglc(6-1)glc -Oglc(6-1)arap -Oglc(6-1)araf -Oglc —— —— —— —— —— —— —— —— —— —— —— —— —— —— ——

2 2 1 1 1 1 3 3 2 2 2 2 2 2 2 2 2 1 1 3 4

8 11 14 13 13 11 6 6 6 6 6 6 7 7 4 7 4 4 6 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0

4 4 8 8 8 6 4 4 4 4 4 4 4 4 2 4 2 2 4 0 0

36 42 48 47 47 42 36 36 36 36 36 36 36 36 30 36 30 30 37 24 24

2.9 4.15 3.5 3.475 3.475 2.25 3.8 3.8 1.9 1.9 1.9 1.9 2.4 2.4 1.15 2.4 1.15 −0.75 6.325 1.3 3.2

2014). The antimicrobial effects, from high to low, were in the order of saponins containing one glycosyl group > aglycone > two ligands > three or more. In addition, the configuration and the active group sites of ginsenosides also affect the bacteriostatic activity of saponins (Kalai et al., 2015).

3.4. Relationship between antimicrobial and HLB of ginsenosides HLB values of ginsenosides are listed in Table 3, together with chemical specifications. The aglycone of ginsenosides PPD and PPT showed low HLB values of 1.3 and 1.2, respectively. Interestingly, the HLB value was reduced when there was only one sugar bond, because the loss of free hydroxyl groups and the increase in sorbitol contributed more lipophilicity to the calculation. Rk3, Rh2, had HLB values of 1.15, 1.15, -0.75, respectively. Then, as the number of sugar chains increases, the HLB value also becomes higher. Re, Rb1, Rb2 had HLB values of 4.15, 3.475, 3.475, respectively. Correlation analysis shows that HLB is associated with antimicrobial activity (p < 0.01). The relationship was illustrated with two typical microorganisms panels on Fig. 2. Curve estimation (cubic) used for the relation were collected from the data of twenty one ginseng saponins (n = 21). The best fitting for regression line formulas were calculated as follows; the MIC of F. nucleatum R2 = 0.747, the MIC of B. cereus R2 = 0.544. This result is superior to the model in which the HLB value of soyasaponins affects the adjuvant effect (Oda et al., 2003). The saponin compound possesses both a hydrophilic group and a lipophilic group, and thus exhibits surfactant-like characteristics. Based on this, the saponin can reduce the surface tension in the aqueous solution, and forms micelles when the critical micelle concentration is reached, so that the biological macromolecule undergoes certain structural changes (Bo et al., 2012; Chavarha et al., 2010). For eukaryotic cell membranes (cells, fungi), saponins can be linked to sterols on their surface, causing perforation and rupture of its cell membrane, eventually leading to the collapse of the biofilm system (Bo and Melzig, 2013). This may be why the HLB value affects the inhibition of microbial activity by ginsenosides. The less polar ginsenosides have a certain inhibitory effect on fungi, Gram-negative bacteria and Grampositive bacteria. According to the antimicrobial activity results and the relationship between HLB, these two kinds of bacteria (F. nucleatum and B. cereus) were further selected as a mechanism to explore.

Fig. 2. Relation between antimicrobial responses on F. nucleatum (a); B. cereus (b) (the MIC value μg/mL, Y-axis) and HLB values (X-axis) of ginsenosides.

3.5. Biofilm inhibition by CBD A biofilm of the bacteria has certain adhesive properties, and the upper cover of the CBD has a columnar structure so that the biofilm produced by the bacteria covers the column, thereby facilitating 7

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nucleatum cell morphology with intact cell walls and uniform intracellular components. However, bacteria treated with Rh2 at 1/2MIC showed great morphological alterations (Fig. 5 B). The cell wall was broken and separated from the cell membrane. Cellular contents accumulated and coagulated near the cell membrane. Fig. 5C shows the bacterial morphology after 8 h of treatment at the minimum inhibitory bacteria concentration (MIC). Compared with Fig. 5B, the cell wall was basically broken. F. nucleatum treated with Rh2 at the MBC exhibited a deformed shape, showing rupture of the membranes with nucleic acid and protein leaching out of the cell (Fig. 5D). Compared with F. nucleatum, the effect of saponin on B. cereus wall is more obvious. The cell wall of Gram-positive bacteria B. cereus without ginsenosides have certain thickness, smooth and intact the edges (Fig.5E). As the drugs at different concentrations continue to stimulate, the cell walls become thinner and thinner until they rupture (Fig. 5F–H). The morphological alterations could intuitively reflect the principle of action of less polar saponins. The results of this experiment confirmed that less polar ginsenosides cause bacterial death by destroying the membrane system (Xue et al., 2016). Previous laboratory studies found no significant differences between the drug-treated group and the negative control at the transcriptome level, further demonstrating that saponin acts directly on the membrane system of bacteria. The reaction mechanism of less polar ginseng saponins on bacteria should be further studied. 3.8. Acute toxicity Administration of HTS at dose of 4.64 g/kg, 10.00 g/kg body weight caused slight diarrhea at day 2 in two groups of rats. In addition, the non-observed adverse reaction level (NOAEL) of the mouse steroidal HTS in this study was 10 g/kg, which is equivalent to 100 times the normal human dose in the clinical prescription. At present, most of the toxicity studies on ginseng are greater than 5 g/kg, furthermore, no increases in the incidence of cancer or non-neoplastic lesions were detected (Mancuso and Santangelo, 2017).

Fig. 3. The inhibition rate of less polar ginsenosides against bacterial biofilm of F. nucleatum (a) and B. cereus (b).

subsequent crystal violet staining and reading. Ginsenoside Rh2 was added to the culture medium of F. nucleatum and B. cereus at concentrations of 1/2 MBC, 1/4 MBC, and MIC. The results are shown in Fig. 3 (A–B). Fig. 3. (A) shows that as the concentration of less polar ginseng saponin increased, the inhibition rate of biofilm formation increased gradually. When the concentration of ginsenoside in the bacterial solution reached the 1/2 MBC level, the inhibition rate reached 94.10%. The effect of saponin on the two biofilms of bacteria is consistent. CBD is a high-throughput screening assay used to check the viability of biofilm (Kalai et al., 2015). To verify the experimental results, we also used a fluorescence confocal microscope to directly observe colony formation stained by SYTO-9 and PI.

4. Conclusion The structure-activity relationship of the antibacterial activity of ginsenosides indicates that the antibacterial activity of saponins is closely related to the polarity of compounds. One of the main reasons is that polar ginsenosides reduce the polarity by de-saccharification and dehydration, and the affinity of less polar saponins is stronger with microbial cell membranes. Certain less polar ginsenosides can destroy the stability of the bacterial membrane system by exerting optimal surfactant effects, further causing cell collapse. At low concentrations, bacteria can be inhibited from forming cell envelopes, while at the MBC level, less polar saponins can penetrate biofilms to kill bacteria. However, as the saponin is further hydrolyzed, the bacteriostatic activity also decreases as the surface activity of the saponin decreases. Based on the good broad-spectrum antibacterial activity of specific polar ginsenosides, the application range of American ginseng stem and leaf saponins has been expanded.

3.6. Biofilm inhibition by CLSM These effects of biofilm inhibition became more pronounced after treatment for four days. The cultured F. nucleatum and B. cereus biofilm were observed under a 600x laser confocal microscope (Fig. 4A–H). The ginsenoside Rh2 inhibited F. nucleatum biofilm formation in a concentration-dependent manner. F. nucleatum without any added reagent formed a thick biofilm on the slide, and the green field contained live bacteria, as shown in Fig. 4 (A); the F. nucleatum was dead and biofilm structure fragmented on the slide with the MBC level of ginsenosideRh2 (D). When the 1/2MIC or MIC level of Rh2 was added, the visual field was dark yellow due to the superposition of the dead and living bacteria. The brightness of the different fields of view also changed accordingly (B, C). In contrast to polar ginsenosides, less polar ginsenosides can not only kill cells but also dissociate cells in biofilms (Kalai et al., 2015).

Funding This study was funded by project No. ZR2019PH043 supported by Shandong Provincial Natural Science Foundation. Informed consent

3.7. Morphological alterations All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

As shown in Fig. 5A, cells in the control group had typical F. 8

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Fig. 4. Co-focus images for F. nucleatum and B. cereus (A–H). A, E, control; B, F treated with concentration of 1/2MIC of less polar ginsenoside Rh2; C, G treated with concentration of MIC of less polar ginsenoside Rh2; D, H treated with concentration of MBC of less polar ginsenoside Rh2.

9

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Fig. 5. The morphological alteration of F. nucleatum and B. cereus cells treated with different concentration of less polar ginsenosides (A–H). A, E, control; B, F treated with concentration of 1/2MIC of less polar ginsenoside Rh2; C,G treated with concentration of MIC of less polar ginsenoside Rh2; D,H treated with concentration of MBC of less polar ginsenoside Rh2.

Declaration of Competing Interest

Weifang Medical College and project No. ZR2019PH043supported by Shandong Provincial Natural Science Foundation.

The authors declare that they have no conflict of interest performed by any of the authors.

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