Ethanol phase extract of rumex madaio roots and application of ethanol phase extract

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses an ethanol phase extract of rumex madaio roots and application of the ethanol phase extract. An ethanol phase extract RmEE is extracted from rumex madaio roots through a 55-95% ethanol solvent extraction method, and the ethanol phase extract RmEE is further separated and purified to obtain a purified component RmEE-F2. The ethanol phase extract RmEE and the purified component RmEE-F2 of the ethanol phase extract RmEE can inhibit staphylococcus aureus and vibrio cholerae, and can be used as bacteriostatic agents. The ethanol phase extract of the rumex madaio root can inhibit the pollution of staphylococcus aureus and vibrio cholerae in meat of crucian and penaeus vannamei which are stored at low temperature (4 DEG C), and can be applied to aquatic products; the method can also be used for preparing food or medicines, and the prepared food or medicines also have an antibacterial effect.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to an ethanol phase extract of Rheum officinale root and application thereof. Background Art

[0002] Foodborne pathogens pose a serious threat to public health and the food industry. Among them, Vibrio cholerae is transmitted from environmental hosts to human hosts through contaminated water or food, causing the life-threatening disease "cholera". Staphylococcus aureus is transmitted through contact with fomites and person-to-person contact, causing skin infections, as well as fatal pneumonia and sepsis.

[0003] Antibiotics are the cornerstone of the treatment and control of infectious diseases and have been used for more than 70 years since their discovery. However, the inappropriate use of antibiotics leads to the emergence and spread of antibiotic-resistant pathogens. It is estimated that by 2050, the number of deaths caused by this may reach tens of millions. However, the development of new antibacterial compounds is slow and their clinical applications are scarce. Based on this, the development of new safe and effective antibacterial agents or innovative alternative prevention and control strategies to meet the severe challenge of pathogen resistance has become one of the key scientific issues that need to be urgently addressed.

[0004] Medicinal and edible plants have become a natural compound resource library for antimicrobial screening due to their outstanding advantages of safety, effectiveness and low cost. For example, Rumex madaio Makino has been used as a traditional Chinese medicinal material for more than a thousand years. According to the ancient Chinese book "Shennong's Herbal Classic" in 1828, Rumex madaio Makino can be used to relieve headaches, cure scabies, reduce fever, and treat gynecological diseases. So far, there is only one research report on the antibacterial effect of extracts from the roots of Rumex madaio Makino, and it was found that its methanol extract has the effect of inhibiting a variety of Gram-negative and Gram-positive pathogens. Ethanol is a food-grade and environmentally friendly solvent, and it shows good compatibility with many bioactive substances. Ethanol can also effectively reduce energy consumption related to solvent consumption. However, there are currently no literature reports on the antibacterial effect and application of the ethanol phase extract (Ethanol-Phase Extract from R.madaio, RmEE) of Rumex madaio Makino roots. Summary of the invention

[0005] The invention aims to provide an ethanol phase extract of Rheum officinale root, which can inhibit Staphylococcus aureus and Vibrio cholerae.

[0006] In order to achieve the above object, the specific technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides an ethanol-phase extract RmEE of rheum palmatum root, which is obtained by extracting from rheum palmatum root through an ethanol solvent extraction method. The specific steps are as follows:

[0008] S1. Rinse fresh rheum palmatum root thoroughly, cut it into small sections after drying, and pre-freeze it at -40 °C for 8 h; then freeze-dry it at -80 °C for 48 h; crush the freeze-dried sample and sieve it through a 300-mesh sieve;

[0009] S2. Add ethanol with a concentration of 55–95% to the sieved sample powder according to a solid-liquid ratio of 1:10 (m / v), magnetically stir and vortex-mix for 24 h; ultrasonically treat the mixture for 20 min, then filter it using a 20–25 μm microporous filter membrane, and collect the filtrate; evaporate and concentrate the filtrate using a rotary evaporator to obtain RmEE.

[0010] In a second aspect, the present invention provides a purified component RmEE-F2 of the ethanol-phase extract of rheum palmatum root, which is obtained by separating and purifying RmEE extracted with 75% ethanol as described above. The separation and purification method is as follows:

[0011] Dilute RmEE extracted with 75% ethanol with ultrapure water, centrifuge and collect the supernatant, then filter it through a sterile 0.22 μm filter membrane, and collect the filtrate; separate the filtrate sample using a Waters 2707 connected to a UPLC Sunfire C 18 chromatographic column; use a photodiode array detector to detect and collect single peaks in the wavelength range of 200–600 nm. At a wavelength of 211 nm, three obvious separated peaks are detected, and the substance corresponding to the separated peak eluted within 2.2–2.8 min (i.e., the second separated peak) is RmEE-F2.

[0012] In a third aspect, the present invention provides the use of the ethanol-phase extract RmEE of rheum palmatum root or its purified component RmEE-F2 as an antibacterial agent.

[0013] Furthermore, the antibacterial targets of the antibacterial agent are Staphylococcus aureus and Vibrio cholerae.

[0014] Furthermore, the antibacterial agent exerts antibacterial effects through the following aspects:

[0015] (a) Inhibiting the growth and proliferation of Staphylococcus aureus and Vibrio cholerae;

[0016] (b) Reducing the cell surface hydrophobicity of Staphylococcus aureus and Vibrio cholerae, increasing the cell membrane fluidity and the permeability of the inner cell membrane;

[0017] (c) Causing damage to the cell structure of Staphylococcus aureus and Vibrio cholerae, and leakage of intracellular nucleic acids and proteins;

[0018] (d) Inhibiting protein synthesis, cell wall synthesis, and β-lactam resistance pathways in Staphylococcus aureus;

[0019] (e) Inhibiting DNA replication, repair, and recombination in Vibrio cholerae, and biosynthetic pathways of cell wall and cell membrane.

[0020] Furthermore, the bacteriostatic agent can be used for preventing and controlling bacterial contamination in food, drugs, or aquatic products. Specifically, it can be used as an internal additive to prepare food, drugs, or aquatic products, or as an external storage agent for food, drugs, or aquatic products.

[0021] Furthermore, the food is instant, fresh, refrigerated, or frozen food.

[0022] Furthermore, the dosage forms of the drugs are liquid, powder, tablet, capsule, etc.

[0023] Furthermore, the aquatic products include crucian carp and white shrimp. The bacteriostatic agent has the effect of inhibiting the contamination of Staphylococcus aureus and Vibrio cholerae in the meat of crucian carp and white shrimp stored at low temperature (4°C).

[0024] The present invention has the following beneficial effects:

[0025] 1. The present invention provides an ethanol-phase extract RmEE of Rheum palmatum root and its purified component RmEE-F2, which have the effect of inhibiting Staphylococcus aureus and Vibrio cholerae.

[0026] 2. The present invention also conducts research on the bacteriostatic mechanism of the ethanol-phase extract of Rheum palmatum root, and proves that it can inhibit the growth and proliferation of Staphylococcus aureus and Vibrio cholerae; reduce their cell surface hydrophobicity, increase cell membrane fluidity and intracellular membrane permeability, resulting in leakage of intracellular nucleic acids and proteins and damage to cell structure; inhibit protein synthesis, cell wall synthesis, and β-lactam resistance pathways in Staphylococcus aureus; inhibit DNA replication, repair, and recombination in Vibrio cholerae, and biosynthetic pathways of cell wall and cell membrane; thus leading to cell death.

[0027] 3. The ethanol-phase extract of Rheum palmatum root of the present invention can be used for the preparation of food or drugs, and the prepared food or drugs also have bacteriostatic effects.

[0028] 4. The ethanol-phase extract of Rheum palmatum root of the present invention can inhibit the contamination of Staphylococcus aureus and Vibrio cholerae in the meat of crucian carp and white shrimp stored at low temperature, reflecting its bacteriostatic effect in aquatic products. Description of the Drawings

[0029] Figure 1Antibacterial activity experimental results of RmEE and RmEE-F2 in the present invention. A: Prep-HPLC separation and purification of RmEE; B: Experimental results of agar disc diffusion method, B1 and B2 are Staphylococcus aureus ATCC 25923 and Vibrio cholerae GIM 1.449 respectively; C: Experimental results of growth curve; C1 and C2 are Staphylococcus aureus ATCC 25923 and Vibrio cholerae GIM 1.449 respectively.

[0030] Figure 2 Effects of RmEE-F2 in the present invention on CSH (A), CMF (B), ICMP (C and D, being Staphylococcus aureus ATCC 25923 and Vibrio cholerae GIM 1.449 respectively), intracellular nucleic acid (E) and protein (F) of Staphylococcus aureus ATCC 25923 and Vibrio cholerae GIM 1.449. ***: p < 0.001.

[0031] Figure 3 Changes in cell surface structure of Staphylococcus aureus ATCC 25923 (A and B) and Vibrio cholerae GIM 1.449 (C and D) before and after treatment with RmEE-F2 in the present invention.

[0032] Figure 4 Main metabolic pathways changed by RmEE-F2 in the present invention in Staphylococcus aureus ATCC 259239 and Vibrio cholerae GIM 1.449. They are volcano plots (A and B) of DGEs in Staphylococcus aureus ATCC 25923 and Vibrio cholerae GIM 1.449 respectively, and their enriched main metabolic pathways (C and D).

[0033] Figure 5 Experimental results of RmEE-F2 in the present invention inhibiting the contamination of Staphylococcus aureus ATCC 25923 (A and C), Vibrio cholerae GIM 1.449 (B and D) in crucian carp and white shrimp. Detailed implementation mode

[0034] The technical solution of the present invention is further described below through examples. Obviously, the described examples are only a part of the examples of the present invention, rather than all the examples. All other implementation manners obtained by those skilled in the art based on the examples of the present invention without creative efforts belong to the scope protected by the present invention.

[0035] Note: The methods used in the examples are all conventional methods unless otherwise specified, and the reagents are all conventional reagents unless otherwise specified.

[0036] Example 1 Preparation and antibacterial test of ethanol extract RmEE from roots of Rumex crispus

[0037] (1) Preparation of Ethanol-Phase Extract of Rumex japonicus Houtt. Root

[0038] Ethanol solvent extraction method: Wash the fresh Rumex japonicus Houtt. root samples with tap water, cut them into small pieces about 1 cm long after air-drying, and pre-freeze them in a -40 °C refrigerator for 8 h. Then, place them in a freeze dryer and freeze-dry them at -80 °C for 48 h. Crush the freeze-dried samples using a multi-functional crusher, and sieve them through a 300-mesh sieve for standby use.

[0039] Weigh 10 g of the above sample powder, and add 100 mL of ethanol with different concentrations (analytical grade, 55%, 75%, 95%, diluted with sterile ultrapure water) at a solid-liquid ratio of 1:10 (m / v). Use a magnetic stirrer to vortex-mix for 24 h. Then, ultrasonically treat the above mixture using an ultrasonic cell disruptor with the following parameters: power: 300 W; ultrasonic on-time: 1 s; ultrasonic off-time: 1 s; probe diameter: 6 mm; working time: 20 min. Filter the ultrasonically treated sample solution through a 20–25 μm microporous filter membrane, collect the filtrate, and evaporate and concentrate it using a rotary evaporator to obtain RmEE, which is stored in the dark at 4 °C for standby use.

[0040] Under the condition of -80 °C, after freeze-drying for 48 h, the water loss rate of fresh Rumex japonicus Houtt. root is 74.84%. The extraction rate of 75% ethanol > 55% ethanol > 95% ethanol, which are 33.8%, 31.10%, and 25.57% respectively.

[0041] (2) Antibacterial Activity Test of Ethanol-Phase Extract of Rumex japonicus Houtt. Root

[0042] 1. Agar Disk Diffusion Method

[0043] Test strains and culture conditions: Inoculate Staphylococcus aureus ATCC 29213 strain into sterilized Tryptic Soy Broth (TSB, pH 7.0–7.2, 1.0% NaCl) medium at a volume fraction of 1%, and culture it at 37 °C, 180 rpm for 16–18 h. After two subcultures and activation, streak it on a TSB agar plate and culture it at 37 °C for 16–18 h. Pick a single colony and inoculate it into fresh TSB liquid medium, and culture it until the mid-logarithmic growth phase (mid-LGP, OD 600 = 0.6–0.8), and determine the bacterial concentration using the conventional plate counting method for standby use.

[0044] Similarly, Vibrio cholerae strain GIM1.449 was inoculated into sterilized TSB (pH 8.4–8.5, 3.0% NaCl) medium; the quality control strain Escherichia coli ATCC 25922 was inoculated into Luria-Bertani (LB) medium (pH 7.0–7.2) and cultured at 37 °C until mid-LGP (Table 1).

[0045] Table 1 Test strains and their media

[0046]

[0047] ATCC: American Type Culture Collection, USA; GCCC: Guangdong Culture Collection Center, China

[0048] Cultured broths (1×10 8 CFU / mL) of the above-mentioned test strains were respectively and evenly spread on sterilized Mueller-Hinton Agar (M-HA) agar plates (100 μL / plate). After the bacterial broth was absorbed, sterile filter paper disks (10 μL / disk, diameter: 6 mm) impregnated with RmEE (100 mg / mL) were evenly placed on them. They were cultured at 37 °C for 12 h, and the diameter of the inhibition zone (DIZ) was measured. Sterile water was used as the negative control group (NC); a gentamicin (CN, 10 μg) test paper disk was used as the positive control group (CN); Escherichia coli ATCC 25922 was used as the quality control strain. Each group of experiments was repeated three times. The results are shown in Table 2 below.

[0049] 2. Minimum inhibitory concentration method

[0050] In a sterilized 96-well bacterial culture plate, using an eight-channel pipette, sterilized M-H Broth (M-HB) liquid medium (100 μL / well) was added. In the wells of the first column, RmEE (100 μL / well, final concentration 100 mg / mL) was added. After mixing with the M-HB medium therein, 100 μL / well was aspirated and added to the wells of the second column, and so on, until the wells of the 11th column. In the wells of the 12th column, sterile water (100 μL / well) was added as a control. Using an eight-channel pipette, the cultured broths of the above-mentioned test strains (10 μL / well, 1.5×10 6(CFU / mL), add it to each well of a 96-well bacterial culture plate, and culture it at 37 °C for 18–24 h. The lowest drug concentration that completely inhibits bacterial growth in the culture well is its minimum inhibitory concentration (MIC). The growth control wells must show acceptable growth (≥2 mm bottom covered or distinct turbidity). Each experiment is repeated three times. The results are shown in Table 2.

[0051] As can be seen from Table 2, RmEE extracted with different concentrations of ethanol can all inhibit Staphylococcus aureus ATCC 29213. Among them, the inhibitory effect of RmEE extracted with 95% ethanol > RmEE extracted with 75% ethanol > RmEE extracted with 55% ethanol. The observed DIZ values are 17.00 ± 0.5, 16.50 ± 0.37, and 14.00 ± 0.42 respectively. The DIZ value of the positive control CN is 18.0 ± 1.41 mm.

[0052] For Vibrio cholerae GIM1.449, the inhibitory effect of RmEE extracted with 75% ethanol > RmEE extracted with 95% ethanol > RmEE extracted with 55% ethanol. The observed DIZ values are 11.50 ± 0.32, 10.00 ± 0.34, and 9.00 ± 0.29 respectively. The DIZ value of the positive control is 23.00 ± 0.25.

[0053] In addition, there is no inhibition zone in the blank control; RmEE extracted with different concentrations of ethanol has no inhibitory effect on Escherichia coli ATCC25922, while the DIZ value of the positive control for inhibiting this bacterium is 17 ± 0.50. These experimental results show that for Staphylococcus aureus ATCC 29213, RmEE extracted with 95%–55% ethanol has a similar antibacterial effect to the positive control antibiotic CN; for Vibrio cholerae GIM1.449, the inhibitory effect of the positive control CN is stronger than that of RmEE.

[0054] As can be seen from Table 2, the MIC values of RmEE extracted with 95% ethanol, 75% ethanol, and 55% ethanol for inhibiting Staphylococcus aureus ATCC25923 are 98 μg / mL, 98 μg / mL, and 195 μg / mL respectively; the MIC values for inhibiting Vibrio cholerae GIM1.449 are 391 μg / mL, 391 μg / mL, and 781 μg / mL respectively.

[0055] Table 2 Antibacterial activity experimental results of different concentration ethanol extracts of Rheum palmatum roots

[0056]

[0057] Note: DIZ, diameter of inhibition zone, including the diameter of the test strip (6 mm); MIC, minimum inhibitory concentration; CN: Gentamicin positive control (10 μg / mL). The values are expressed as the mean ± standard deviation (S.D.) of three parallel determinations.

[0058] Example 2 Separation, purification of the ethanol extract of Rumex patientia roots and antibacterial experiment of RmEE-F2

[0059] Dilute the RmEE extracted with 75% ethanol to a concentration of 10 mg / mL with ultrapure water (analytical grade), centrifuge at 12,000 rpm for 20 min, and collect the supernatant; then, filter through a sterile 0.22 μm filter membrane and collect the filtrate. Use Waters 2707 (Waters, Milford, MA, USA) connected to a UPLC Sunfire C 18 chromatographic column (5 μm, 10 × 250 mm, Waters, USA) to separate the filtrate sample. Column temperature: 40 °C, injection volume: 100 μL. Mobile phase A is ultrapure water (analytical grade), mobile phase B is methanol (analytical grade), and the flow rate is 4 mL / min (isocratic elution: 0–15 min, 50% A, 50% B). Detect and collect single peaks within the wavelength range of 200–600 nm using a photodiode array detector.

[0060] As Figure 1 (A) shows that by scanning for 14 min at a wavelength of 211 nm, three distinct separated peaks, namely components 1–3 (Fraction 3–Fraction 3, F1–F2), were detected, and components 1–3 were collected separately. Using the above method, the antibacterial activities of each component were analyzed.

[0061] The DIZ values of component 2 of the RmEE extracted with 75% ethanol (hereinafter referred to as: RmEE-F2) against Staphylococcus aureus ATCC25923 and Vibrio cholerae GIM 1.449 were 11.50 ± 0.50 mm and 10.50 ± 0.35 mm ( Figure 1 , B); the MIC values were both 391 μg / mL. Components 1 and 2 showed weak antibacterial activities.

[0062] Based on the above test results, in the following examples, the 1×MIC values of RmEE-F2 against Staphylococcus aureus ATCC 25923 and Vibrio cholerae GIM 1.449 were both 391 μg / mL.

[0063] Example 3 Growth and proliferation inhibition tests of RmEE-F2 against Staphylococcus aureus ATCC 25923 and Vibrio cholerae GIM 1.449

[0064] Staphylococcus aureus ATCC 25923 and Vibrio cholerae GIM1.449 strains cultured to mid-LGP were inoculated into TSB medium supplemented with RmEE-F2 (1×MIC or 1 / 2×MIC) at 1% volume fraction respectively, and cultured at 37°C and 180 rpm for 24 h. An automatic growth curve analyzer was used to measure their growth curves. TSB medium without RmEE-F2 was used as a control, and the following analysis was similar.

[0065] As Figure 1 (C-1) shows that under the treatment conditions of 1×MIC (391 μg / mL) and 1 / 2×MIC (195.5 μg / mL) of RmEE-F2, the maximum biomass (OD 600 = 1.03, OD 600 = 1.31) of Staphylococcus aureus ATCC 25923 was reduced by 0.30 times and 0.12 times respectively compared with the control group (OD 600 = 1.46) (p<0.05).

[0066] Similarly, compared with the maximum biomass of the control group (OD 600 = 0.91), under the treatment conditions of 1×MIC (391 μg / mL) and 1 / 2×MIC (195.5 μg / mL) of RmEE-F2, the maximum biomass (OD 600 = 0.57, OD 600 = 0.67) of Vibrio cholerae GIM 1.449 decreased by 0.37 times and 0.26 times respectively (p<0.05) ( Figure 1 , C-2).

[0067] These experimental results indicate that RmEE-F2 significantly inhibits the growth and proliferation of Staphylococcus aureus ATCC 25923 and Vibrio cholerae GIM1.449.

[0068] Example 4 RmEE-F2 changes the key cell biophysical parameters of Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923

[0069] Bacterial cell surface hydrophobicity (CSH), cell membrane fluidity (CMF), and inner cell membrane permeability (ICMP) are key biophysical parameters for maintaining their cell structure and function, and are also the main targets of antibacterial drugs.

[0070] CSH assay: From the culture suspensions of bacteria treated with RmEE-F2 at the concentration of 1×MIC (391 μg / mL) for 2 h, 4 h, and 6 h, 1 mL was taken respectively, and then 1 mL of hexadecane was added. After vortex mixing for 5 min and standing at room temperature for 30 min, the supernatant was discarded. The OD 415 absorbance value was measured using a multifunctional microplate reader. Each test was repeated three times.

[0071] As Figure 2 (A) shows, compared with the control group with 0% RmEE-F2 concentration, after treatment with RmEE-F2 for 2 h, 4 h, and 6 h, the CSH of Vibrio cholerae GIM 1.449 decreased significantly by 1.54-fold, 3.43-fold, and 9.17-fold respectively (p < 0.05), and showed a decrease dependent on the treatment time. Similarly, the CSH of Staphylococcus aureus ATCC 25923 decreased by 1.18-fold, 1.42-fold, and 2.47-fold respectively (p < 0.01).

[0072] CMF assay: The culture suspensions of bacteria treated with RmEE-F2 at the concentration of 1×MIC for 2 h, 4 h, and 6 h were added into sterilized 96-well bacterial culture plates (200 μL / well) respectively, and then 10 mM 1,6-diphenyl-1,3,5-hexatriene (DPH) solution (2.0 μL / well) was added. Using a multifunctional microplate reader, with an excitation filter of 360 / 40, a detection filter of 460 / 40, and a dichroic mirror of 400 nm, the horizontal polarization (Ivh) and vertical polarization (Ivv) fluorescence of each well were detected respectively. Calculated according to the following formula: rDPH = (Ivv - G×Ivh) / (Ivv + 2×G×Ivh), where G = 0.85. Each test was repeated three times.

[0073] As Figure 2 (B) shows, compared with the control group with 0% RmEE-F2 concentration, after treatment with RmEE-F2 at the concentration of 1×MIC for 2 h, 4 h, and 6 h, the CMF of Staphylococcus aureus ATCC 25923 increased significantly by 1.63-fold, 1.86-fold, and 3.71-fold respectively (p < 0.001). Similarly, the CMF of Vibrio cholerae GIM 1.449 also showed an increase dependent on the treatment time (1.05 - 1.14-fold).

[0074] ICMP assay: The cultured bacterial suspension treated with RmEE-F2 at the above 1×MIC concentration was added into a sterilized 96-well bacterial culture plate (200 μL / well) respectively, and then 10 mM o-nitrophenyl-β-D-galactopyranoside (ONPG, 2.5 μL / well) was added. Incubate at 37 °C. Every 30 min, use a multifunctional microplate reader to measure the OD 415 absorbance value of each well. Each experiment was repeated three times.

[0075] As Figure 2 (C and D) show that compared with the control group with an RmEE-F2 concentration of 0%, after treatment with 1×MIC concentration of RmEE-F2 for 6 h, the ICMP of Staphylococcus aureus ATCC 25923 and Vibrio cholerae GIM 1.449 increased significantly by 1.11 times and 1.15 times respectively (p < 0.05).

[0076] These experimental results indicate that RmEE-F2 (1×MIC) can reduce the CSH of Staphylococcus aureus ATCC 25923 and Vibrio cholerae GIM 1.449; while increasing its ICMP and CMF.

[0077] Example 5 RmEE-F2 causes leakage of intracellular nucleic acids and proteins in Staphylococcus aureus ATCC 25923 and Vibrio cholerae GIM 1.449

[0078] In the cultured bacterial suspensions treated with RmEE-F2 at the above 1×MIC concentration for 2 h, 4 h, and 6 h, take 1.5 mL respectively, centrifuge at 3500 rpm for 5 min at 4 °C, collect the supernatant, and use a multifunctional microplate reader to measure its OD 260 absorbance value. After treatment for 24 h, use a Bradford method protein concentration assay kit to measure the concentration of extracellular proteins in the supernatant samples according to the kit instructions. Each experiment was repeated three times.

[0079] As Figure 2 (E) shows that compared with the control group with an RmEE-F2 concentration of 0%, after treatment with RmEE-F2 for 2 h, 4 h, and 6 h, the extracellular nucleic acid leakage of Staphylococcus aureus ATCC 25923 increased by 1.12 times, 1.22 times, and 1.24 times respectively (p < 0.001). Similarly, the extracellular nucleic acid leakage of Vibrio cholerae GIM 1.449 increased by 1.73 - 2.05 times (p < 0.001).

[0080] As Figure 2As shown in (F), after treatment with RmEE-F2 at 24 h, compared with the control group, the extracellular protein contents of Staphylococcus aureus ATCC25923 and Vibrio cholerae GIM 1.449 increased by 1.43-fold and 1.71-fold, respectively (p<0.001).

[0081] Example 6 Scanning Electron Microscope Observation of the Destruction of the Cell Structures of Staphylococcus aureus ATCC 25923 and Vibrio cholerae GIM 1.449 by RmEE-F2

[0082] The culture bacterial suspensions treated with RmEE-F2 at the above 1×MIC concentration for 2 h, 4 h, and 6 h were centrifuged at 4000 rpm for 10 min at 4°C, the supernatant was discarded, and the bacterial cell precipitates were collected. The bacterial cells were washed twice with sterile 1×PBS buffer (pH 7.2–7.4). Then, the cells were fixed with 2.5% glutaraldehyde at 4°C for 12 h and dehydrated with gradient ethanol (30%, 50%, 80%, 90%, 100%) for 15 min. After that, 20 μL of the sample was dropped onto a coverslip and dried at room temperature. The cell morphology of the test strains was observed using a Scanning Electron Microscope (SEM, 5.0 kV, 30,000×). Each test was repeated three times.

[0083] As Figure 3 shown in (A and B), in the control group with an RmEE-F2 concentration of 0%, the cells of Staphylococcus aureus ATCC 25923 were spherical, with a plump shape, a flat surface, and a complete structure. In contrast, after treatment with 1×MIC of RmEE-F2 for 2 h, slight depressions appeared on the cell surface; after treatment for 4 h, severe depressions appeared; after treatment for 6 h, the cells ruptured and the contents leaked.

[0084] Similarly, in the control group with an RmEE-F2 concentration of 0%, the cells of Vibrio cholerae GIM 1.449 were rod-shaped and curved, with a plump shape, a flat surface, and a complete structure. In contrast, after treatment with 1×MIC of RmEE-F2 for 2 h, the cell surface shrank; after treatment for 4 h, the cell structure was severely damaged; after treatment for 6 h, the cells ruptured ( Figure 3 , C and D).

[0085] Example 7 RmEE-F2 Alters Multiple Metabolic Pathways of Staphylococcus aureus ATCC 25923 and Vibrio cholerae GIM 1.449

[0086] Illumina RNA sequencing experiment: In the cultured bacterial suspension treated with RmEE-F2 at 1×MIC concentration for 6 h, RNase inhibitors were added respectively, and the bacteria were collected by centrifugation. Total RNA of the bacterial samples was extracted using an RNA extraction kit according to the kit instructions. Purification, analysis, and Illumina RNA sequencing of the RNA samples were completed by Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China), using the Illumina HiSeq2500 platform (Illumina, USA). Each experiment was repeated three times. Compared with the control group, genes with Fold change (FC) ≥ 2.0 or ≤ 0.5, and Benjamini / Hochberg (BH-adjusted FDR) p < 0.05 were defined as Differentially Expressed Genes (DEGs). Gene Set Enrichment Analysis (GSEA) was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http: / / www.genome.jp / KEGG / ).

[0087] (I) Changes in metabolic pathways of Staphylococcus aureus ATCC 25923

[0088] Comparative transcriptome analysis results showed that compared with the control group, the DEGs identified in the RmEE-F2 treatment group accounted for 41.79% (959 / 2295) of the total genes of Staphylococcus aureus ATCC 25923. Among them, 389 DEGs were significantly downregulated at the transcriptional level (FC ≤ 0.5), while 570 DEGs were significantly upregulated (FC ≥ 2.0). These DEGs were mainly enriched in 9 metabolic pathways of Staphylococcus aureus ATCC 25923, such as ribosome metabolism; valine, leucine, and isoleucine biosynthesis; lysine biosynthesis; arginine biosynthesis; β-lactam resistance, etc. (p < 0.05)( Figure 4 , A and C).

[0089] For example, in ribosome metabolism, approximately 9 DEGs were significantly downregulated at the transcriptional level (0.216–0.489-fold, p < 0.05). For example, the 50S ribosomal protein L7 / L12 (rplL, PQQ26_02565) was significantly downregulated (0.240-fold, p < 0.05). This protein plays an important role in peptide bond formation at the peptidyl transferase center and ensures the smooth export of newly synthesized polypeptide chains. The 50S ribosomal protein L14 (rplN, PQQ26_11285) was also significantly downregulated (0.216-fold, p < 0.05), and this protein has been shown to have significant antibacterial activity and anti-biofilm formation ability against multidrug-resistant Staphylococcus aureus in vitro and in vivo.

[0090] In lysine and arginine biosynthesis, approximately 11 DGEs were significantly downregulated in Staphylococcus aureus ATCC25923 (0.094–0.492-fold, p < 0.05). For example, aspartate-semialdehyde dehydrogenase (PQQ26_06670), which is involved in microbial amino acid and cell wall biosynthesis processes, was significantly downregulated (0.453-fold, p < 0.05). Argininosuccinate lyase (argH, PQQ26_04235) was strongly inhibited (0.094-fold, p < 0.05), and this enzyme catalyzes the conversion of argininosuccinate to L-arginine and fumarate.

[0091] In valine, leucine, and isoleucine biosynthesis, approximately 10 DEGs were significantly downregulated (0.060–0.460-fold, p < 0.05). For example, 3-isopropylmalate dehydrogenase (leuB, PQQ26_10305; leuD, PQQ26_10315) was significantly inhibited (0.268-fold; 0.230-fold, p < 0.05). This enzyme plays an important role in the biosynthesis of leucine and complex lipids, as well as in oxidative stress and virulence responses. Ketol-acid reductoisomerase (ilvC, PQQ26_10295) was also significantly inhibited (0.334-fold, p < 0.05), and this enzyme is the second enzyme in valine, leucine, and isoleucine biosynthesis and is also a new target for fungicides.

[0092] In β-lactam resistance, approximately 12 DEGs were significantly inhibited (0.323–0.495-fold, p < 0.05). For example, penicillin-binding proteins (PBPs, PQQ26_05490) were significantly downregulated (0.329-fold, p < 0.05). This protein is the main target of β-lactam antibiotics and plays a key role in cell wall construction and bacterial replication. The ATP-binding cassette (PQQ26_04380) was also significantly downregulated (0.323-fold, p < 0.05), thereby inhibiting the active transport of substances on the biofilm driven by ATP.

[0093] In summary, RmEE-F2 significantly altered nine metabolic pathways of Staphylococcus aureus ATCC 25923, mainly inhibiting protein synthesis, cell wall synthesis, β-lactam resistance, etc., leading to cell lysis and decreased antibiotic tolerance.

[0094] (II) Metabolic pathway changes of Vibrio cholerae GIM1.449

[0095] The results of comparative transcriptome analysis showed that compared with the control group, the DEGs identified in the RmEE-F2 treatment group accounted for 61.3% (2301 / 3753) of the total genes of Vibrio cholerae GIM1.449. Among them, 2213 DEGs were significantly downregulated at the transcriptional level (FC ≤ 0.5), while 88 DEGs were significantly upregulated (FC ≥ 2.0). These DEGs were mainly enriched in 10 metabolic pathways of Vibrio cholerae GIM1.449, such as DNA replication, mismatch repair, homologous recombination, nucleotide excision repair, peptidoglycan biosynthesis, fatty acid biosynthesis, aminoacyl-tRNA biosynthesis, etc. (p < 0.05)( Figure 4 , B and D).

[0096] For example, in mismatch repair, about 20 DEGs were significantly downregulated (0.183–0.469-fold, p < 0.05). For example, the DNA mismatch repair protein MutS (mutS, GTH07_11350) and the DNA mismatch repair endonuclease MutL (mutL, GTH07_12235) were significantly downregulated by 0.257-fold and 0.316-fold, respectively (p < 0.05). The single-stranded DNA-binding protein (GTH07_11935) was also significantly inhibited (0.225-fold, p < 0.05). The core components of DNA polymerase III (dnaE, GTH07_03160; dnaQ, GTH07_03215; dnaN, GTH07_00010; dnaX, GTH07_08890; holA, GTH07_09345), which are essential for bacterial replication and growth, were all significantly inhibited (0.312–0.375-fold, p < 0.05).

[0097] In fatty acid biosynthesis, about 23 DEGs were significantly downregulated in Vibrio cholerae GIM 1.449 (0.090–0.428-fold, p < 0.05). For example, β-ketoacyl-ACP synthase II (fabF, GTH07_04290) was significantly inhibited (0.166-fold, p < 0.05). This enzyme catalyzes the key step in the elongation of fatty acid chains in FAS-II, and the fatty acids produced by FAS-II are important components for constructing the bacterial membrane.

[0098] In peptidoglycan biosynthesis, approximately 19 DEGs were significantly inhibited (0.166–0.431-fold, p<0.05). Peptidoglycan is an important component of the cell wall, providing a rigid cell structure for bacteria. Enzymes related to peptidoglycan synthesis (murA, GTH07_01875; murB, GTH07_12430; murC, GTH07_02455; murE, GTH07_02425; murG, GTH07_02450; mraY, GTH07_02435) were all significantly inhibited (0.321–0.431-fold, p<0.05). Among them, MurA catalyzes the first reaction in peptidoglycan synthesis; MurC and MurE catalyze the ligation reaction of L-Ala, D-Glu, m-DAP or L-Lys, D-Ala; MraY catalyzes the transfer of phosphorylated N-acetylmuramyl-pentapeptide motif to undecaprenyl phosphate carrier lipid; MurG catalyzes the last step in peptidoglycan synthesis. In addition, penicillin-binding protein 1 (GTH07_01290; mrcB, GTH07_11015), penicillin-binding protein 2 (mrdA, GTH07_09360), and penicillin-binding protein 3 (GTH07_02420) were all significantly downregulated (0.231–0.388-fold, p<0.05), and these proteins are all targets of antibiotic action.

[0099] In summary, RmEE-F2 significantly altered 10 metabolic pathways in Vibrio cholerae GIM 1.449, mainly inhibiting DNA replication, repair, and homologous recombination, and repressing the biosynthesis of cell wall and cell membrane, thus leading to cell lysis and death of Vibrio cholerae GIM 1.449.

[0100] Example 8: Test on the inhibition of Vibrio cholerae GIM 1.449 and Staphylococcus aureus ATCC 25923 contamination in crucian carp and white shrimp (Litopenaeus vannamei) by RmEE-F2

[0101] Collection and analysis of crucian carp and white shrimp (Litopenaeus vannamei) samples: Fresh crucian carp and white shrimp (Litopenaeus vannamei) samples were collected from a certain aquatic product market in Shanghai, China, and quickly transported to the laboratory in an ice box. The collected fish and shrimp samples were washed with running tap water. Under sterile conditions, the fish skin and shrimp shell were removed, and the fish and shrimp meat were cut into small pieces (about 1×1×1 cm) with a sterilized scalpel, then rinsed three times with sterile water and sterilized by ultraviolet light for 20 min. In each 1 g of fish and shrimp meat samples, 9 mL of sterilized 1×PBS buffer was added, and homogenized for 3 min. The homogenates were respectively spread on agar plates (100 μL / plate) and cultured overnight at 37°C, and the number of colonies was counted. Each test was repeated three times. If no colony growth was observed, the samples could be used for subsequent tests.

[0102] Preparation and grouping tests of artificially contaminated samples: As described above, Staphylococcus aureus ATCC25923 and Vibrio cholerae GIM 1.449 were separately inoculated into TSB medium and cultured at 37 °C until mid-LGP. Then, they were diluted to 1×10 6 CFU / mL with 1×PBS and added to the homogenized meat samples of the above-mentioned crucian carp and white shrimp, Litopenaeus vannamei, respectively. A negative control group was set up in the experiment, in which the test strains were inoculated without adding RmEE-F2; a treatment group, in which the test strains were inoculated and 1×MIC concentration of RmEE-F2 (391 μg / mL) was added; a positive control group, for Staphylococcus aureus, vancomycin (10 μg / mL) was added; for Vibrio cholerae, doxycycline (10 μg / mL) was added. The three groups of test samples were stored at 4 °C for 0 h, 6 h, 12 h, and 24 h, respectively. Then, the test samples of each group were transferred to a sterile homogenization bag, 9 mL of 1×PBS was added, and homogenized for 3 min. The homogenized samples were serially diluted, 100 μL / plate was taken and evenly spread on agar plates, and cultured at 37 °C for 12 h, and the number of colonies was counted. Each test was repeated three times. Bacteriostatic rate (%) = (Ba–Bt) / Ba×100%. Among them, Ba is the viable count (CFU / mL) of the test strain in the negative control group; Bt is the viable count (CFU / mL) of the test strain in the treatment group.

[0103] As Figure 5 (A and B) shown, compared with the control group without adding RmEE-F2, after treatment at 4 °C for 6 h, 12 h, and 24 h with 1×MIC concentration of RmEE-F2 added, the viable count of Staphylococcus aureus ATCC 25923 in the crucian carp samples decreased significantly by 0.26, 0.83, and 1.01 Log CFU / g (p<0.01); the bactericidal rates were 44.4%, 83.57%, and 91.17%, respectively. Similarly, the viable count of Vibrio cholerae 1.449 in the crucian carp samples decreased significantly by 0.49, 0.63, and 0.99 Log CFU / g (p<0.01); the bactericidal rates were 67.32%, 76.79%, and 89.65%, respectively.

[0104] As Figure 5As shown in (C and D), compared with the control group without RmEE-F2, after treatment with 1×MIC concentration of RmEE-F2 for 6 h, 12 h, and 24 h, the viable counts of Staphylococcus aureus ATCC 25923 in the Litopenaeus vannamei samples decreased significantly by 0.38, 0.67, and 0.93 Log CFU / g (p<0.01), respectively; the bactericidal rates were 58.51%, 78.82%, and 88.21%, respectively. Similarly, the viable counts of Vibrio cholerae GIM 1.449 in the Litopenaeus vannamei samples decreased significantly by 0.89, 0.93, and 1.0 Log CFU / g (p<0.05), respectively; the bactericidal rates were 87.18%, 88.11%, and 90.01%, respectively.

[0105] These experimental results indicate that under low-temperature (4°C) storage conditions, RmEE-F2 can effectively inhibit the contamination of Staphylococcus aureus ATCC 25923 and Vibrio cholerae GIM 1.449 in Crucian carp and Litopenaeus vannamei meat, and its antibacterial effect is comparable to that of the positive control antibiotics vancomycin and doxycycline ( Figure 5 ).

[0106] In summary, the ethanol-phase extract of Rumex obtusifolius roots provided by the present invention can inhibit the growth and proliferation of Staphylococcus aureus and Vibrio cholerae; reduce their cell surface hydrophobicity, increase cell membrane fluidity and intracellular membrane permeability, resulting in damaged cell structure and leakage of intracellular nucleic acids and proteins; inhibit the protein synthesis, cell wall synthesis, and β-lactam resistance pathway of Staphylococcus aureus; inhibit the DNA replication, repair, and recombination, and the biosynthesis pathways of cell wall and cell membrane of Vibrio cholerae; and also has the effect of inhibiting the contamination of Staphylococcus aureus and Vibrio cholerae in Crucian carp and Litopenaeus vannamei meat stored at low temperature (4°C). The ethanol-phase extract of Rumex obtusifolius roots of the present invention can also be used in the preparation of food or drugs, and the prepared food or drugs also have antibacterial effects.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. An ethanol extract RmEE of Rheum officinale root, characterized in that: It is extracted from the root of Rheum officinale by ethanol solvent extraction method, and the specific steps are as follows: S1. Rinse the fresh Rheum officinale roots, dry them, cut them into small pieces, pre-freeze them at -40℃ for 8h, and then freeze-dry them at -80℃ for 48h. Crush the freeze-dried samples and sieve them through a 300-mesh sieve. S2. Add 55–95% ethanol to the sieved sample powder at a solid-liquid ratio of 1:10, and mix by magnetic stirring for 24 h. Ultrasonicate the mixture for 20 min, filter it with a 20–25 μm microporous filter membrane, and collect the filtrate. Use a rotary evaporator to evaporate and concentrate the filtrate to obtain RmEE.

2. A purified fraction RmEE-F2 of an ethanol extract of Rheum officinale root, characterized in that: The RmEE is separated and purified according to claim 1, and the separation and purification method is as follows: The RmEE extracted with 75% ethanol was diluted with ultrapure water, the supernatant was collected after centrifugation, and then filtered through a sterile 0.22 μm filter membrane to collect the filtrate; a Waters 2707 was connected to a UPLC Sunfire C 18 The filtrate sample was separated by a chromatographic column; a photodiode array detector was used to detect and collect single peaks in the wavelength range of 200-600 nm. At a wavelength of 211 nm, three obvious separation peaks were detected. The substance corresponding to the separation peak within 2.2-2.8 min of elution was RmEE-F2.

3. Use of the ethanol extract RmEE of the root of Rheum officinale according to claim 1 or the purified fraction RmEE-F2 of the ethanol extract of the root of Rheum officinale according to claim 2 as an antibacterial agent.

4. The use according to claim 3, characterized in that: The antibacterial agent inhibits Staphylococcus aureus and Vibrio cholerae.

5. The use according to claim 4, characterized in that: The antibacterial agent exerts an antibacterial effect through the following aspects: (a) Inhibit the growth and proliferation of Staphylococcus aureus and Vibrio cholerae; (b) Reduce the cell surface hydrophobicity, increase the cell membrane fluidity and intracellular membrane permeability of Staphylococcus aureus and Vibrio cholerae; (c) Causes damage to the cell structure of Staphylococcus aureus and Vibrio cholerae, and leakage of intracellular nucleic acids and proteins; (d) Inhibit protein synthesis, cell wall synthesis, and β-lactam resistance pathways of Staphylococcus aureus; (e) Inhibits DNA replication, repair and recombination, and the biosynthesis pathways of cell wall and cell membrane of Vibrio cholerae.

6. The use according to claim 4, characterized in that: The antibacterial agent can be used for the prevention and control of bacterial contamination in food, medicine or aquatic products.

7. The use according to claim 6, characterized in that: The food is ready-to-eat, fresh, refrigerated or frozen food.

8. The use according to claim 6, characterized in that: The dosage form of the medicine is liquid, powder, tablet or capsule.

9. The use according to claim 6, characterized in that: The aquatic products include crucian carp and whiteleg shrimp, and the antibacterial agent has the effect of inhibiting the contamination of Staphylococcus aureus and Vibrio cholerae in the crucian carp and whiteleg shrimp meat stored at low temperature.

Images