Application of stellera chamaejasme extract in preparation of medicine for inhibiting biological activity and biological membrane formation of superbacteria

Abstract

The invention belongs to the technical field of medicines and disease treatment, and particularly relates to application of a stellera chamaejasme extract in preparation of medicines for inhibiting the biological activity of superbacteria and the formation of a biological membrane of the superbacteria. Extracting the root of stellera chamaejasme with methanol, filtering, and collecting filtrate; concentrating the filtrate to obtain a methanol extract; the methanol extraction extract is extracted by ethyl acetate, and an obtained ethyl acetate extract is a stellera chamaejasme extract; wherein the material ratio of the overground part and root of stellera chamaejasme to methanol is 1: (2-5); the dosage ratio of the methanol extract to the ethyl acetate is 1: (2-6). Experiments prove that the stellera chamaejasme extract can significantly inhibit the biological activity of superbacteria, reduce the metabolic activity of biological membrane cells and inhibit the formation of biological membranes, a new direction is provided for research and development of drugs for resisting drug-resistant bacteria, the defects of existing antibiotics in the aspect of treating infection related to superbacterial biological membranes are overcome, and the preparation method is simple and low in cost.

Description

Technical Field

[0001] This invention belongs to the field of drug and disease treatment technology, specifically relating to the application of Daphne odora extract in the preparation of drugs that inhibit the bioactivity of superbugs and their biofilm formation. Background Technology

[0002] Daphne odora is a plant belonging to the genus Daphne in the family Thymelaeaceae. Stellera chamaejasme L The dried root of *Daphne odora*, a perennial herb, is also known as swallow flower or heartbreak grass. *Daphne odora* is harmful on grasslands. The above-ground parts are slightly toxic; ingestion by livestock can cause vomiting, diarrhea, and even death. Its robust root system has a high efficiency in converting soil nutrients, giving it a significant advantage in nutrient competition and a noticeable inhibitory effect on surrounding plants. It has strong reproductive capacity and can form patchy clusters after invading grasslands, seriously affecting the sustainable development of grassland animal husbandry and ecological balance. However, as a traditional Chinese medicine, *Daphne odora* has a long history of medicinal use, first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica). The root is used medicinally; it is pungent and warm in nature; it is toxic but has expectorant, digestive, and analgesic effects. Externally, it can treat scabies and kill parasites. The *Taiping Shenghui Fang* (Prescriptions for Universal Relief) of the Song Dynasty records its expectorant effects. The *Jingzhu Bencao* (Crystal Pearl Materia Medica) of the Qing Dynasty records its use in treating diarrhea and scabies. In modern clinical practice, Daphne odora, as a major ingredient in "Xiaozhong Jiuwei San" (a traditional Chinese medicine formula for reducing swelling), has analgesic, antipyretic, and anti-edema effects. Modern pharmacological studies have shown that Daphne odora possesses various activities, including anti-HIV, anti-tumor, anti-tuberculosis, anti-leukemia, and anti-epileptic activity.

[0003] Staphylococcus aureus ( Staphylococcus aureus As a common Gram-positive facultative anaerobic bacterium, it can cause serious diseases such as skin and soft tissue infections, pneumonia, and sepsis. Among them, methicillin-resistant Staphylococcus aureus (MRSA) is particularly problematic due to its high incidence of infection. mecA The gene encodes penicillin-binding protein-2, leading to dose-dependent resistance to antibiotics such as β-lactams, tetracyclines, and erythromycin. It has become a significant pathogen causing clinically fatal infections (such as bacteremia, osteomyelitis, and infections in diabetic patients). More seriously, MRSA can form a structured biofilm composed of microbial communities and their secreted extracellular polymeric substances (EPS). This biofilm dynamically develops through five stages: reversible attachment, irreversible adhesion, microcolony proliferation, maturation, and diffusion. It forms a three-dimensional physical barrier with a polysaccharide backbone, embedded proteins, and extracellular DNA, reducing antibiotic penetration by more than 80%. Furthermore, it upregulates the expression of resistance genes (such as...) through quorum sensing systems. icaA , fnbA), induces bacteria in the biofilm into metabolic dormancy and accelerates horizontal transfer of drug-resistant genes, causing the drug resistance of bacteria to increase by 10 times to 1000 times. Clinical studies have confirmed that 78% of chronic wound infections and 95% of catheter-related bloodstream infections are closely related to biofilms, and the MIC value of existing antibiotics to biofilm MRSA is often >512 μg / mL. That is, the existing antibiotics have poor inhibitory effect on biofilm MRSA and cannot effectively regulate biofilm formation-related genes. Therefore, a new strategy needs to be developed to solve the above problems. SUMMARY

[0004] In order to develop a new strategy for inhibiting or killing MRSA, solve the problem that the existing antibiotics have poor inhibitory effect on biofilm MRSA and cannot effectively regulate biofilm formation-related genes, the application provides a use of a Stellera chamaejasme extract in preparation of a medicine for inhibiting the biological activity of superbugs and the formation of biofilms thereof.

[0005] To achieve the above object, the application adopts the following technical scheme.

[0006] The application provides a use of a Stellera chamaejasme extract in preparation of a medicine for inhibiting the biological activity of superbugs and the formation of biofilms thereof. The Stellera chamaejasme extract is a methanol extract of a Stellera chamaejasme root, and the specific preparation method is as follows:

[0007] The Stellera chamaejasme root is extracted with methanol, filtered, and the filtrate is collected. The filtrate is concentrated to obtain a methanol extract. The methanol extract is redissolved with an organic cosolvent, and then mixed with diatomite at a mass ratio of 0.8-1.2:1 to obtain a mixed sample. The mixed sample is dried, and then extracted with petroleum ether and ethyl acetate in sequence. The ethyl acetate extract is the Stellera chamaejasme extract.

[0008] The Stellera chamaejasme root and the methanol are mixed at a solid-liquid ratio of 1:2-5.

[0009] The mass ratio of the mixed sample and the petroleum ether is 1:20-50.

[0010] The application researches and finds that the Stellera chamaejasme extract has a significant inhibitory effect on MRSA, VRE and biofilms thereof, can effectively regulate biofilm formation-related genes, and has no drug resistance. Therefore, the application proves that the Stellera chamaejasme extract can be used to inhibit or kill MRSA and biofilms thereof, and can be used in the preparation of a new medicine for inhibiting or killing MRSA or an anti-drug-resistant bacteria medicine, which provides a new strategy for developing an antibiotic substitute with antibacterial and antibiofilm functions, and helps to expand the high-value application of Stellera chamaejasme medicinal materials in the field of anti-infection.

[0011] Preferably, the Stellera chamaejasme extract has antibacterial and inhibiting biofilm formation activities on any one or more of the following superbugs: Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Enterococcus (VRE), resistant Helicobacter pylori (RHP) and Penicillin-resistant Streptococcus pneumoniae (PRSP).

[0012] Preferably, after the mixed sample is dried, the sample is extracted with petroleum ether to obtain a petroleum ether extract fraction (petroleum ether extract) and a residual aqueous phase; and the residual aqueous phase is extracted with ethyl acetate to obtain an ethyl acetate extract fraction (ethyl acetate extract).

[0013] The volume ratio of the residual aqueous phase to the ethyl acetate is 1:20-100.

[0014] Preferably, the organic cosolvent is methanol.

[0015] Preferably, when the Stellera chamaejasme roots are extracted with methanol, a heating reflux extraction method is used.

[0016] The conditions of the heating reflux extraction method are heating and condensation reflux at 78-82℃.

[0017] Preferably, the medicine uses the Stellera chamaejasme extract as an active ingredient and is supplemented with a pharmaceutically acceptable excipient or carrier.

[0018] Preferably, the excipient includes any one or more of starch, dextrin, lactose, sodium carboxymethyl cellulose, sodium carboxymethyl starch, mannitol and sucrose.

[0019] Preferably, the carrier includes any one or more of liposomes, microemulsion carriers and nanocarriers.

[0020] The application also provides an effective component of the Stellera chamaejasme extract, which is a substance obtained by separation and purification of the Stellera chamaejasme extract.

[0021] The substance includes four compounds: compound I-compound IV, and specifically includes two flavonoids and two diterpenoids. Compound I and compound II are flavonoids, and compound III and compound IV are terpenoids. The structural formula of the compound I is shown as formula I, the structural formula of the compound II is shown as formula II, the structural formula of the compound III is shown as formula III, and the structural formula of the compound IV is shown as formula IV:

[0022] .

[0023] In the formula I, R1 is selected from methoxy (-OCH3), ethoxy (-OC2H5) or isopropoxy (-OCH (CH3)2), and R2-R6 are all selected from hydroxyl, methyl (-CH3) or acetyl.

[0024] In the formula II, R1 is selected from methoxy (-OCH3), ethoxy (-OC2H5), isopropoxy (-OCH (CH3)2) or tert-butoxy (-OC (CH3)3), R2 is selected from hydroxyl, methyl (-CH3) or acetyl, R3 is selected from methoxy (-OCH3), ethoxy (-OC2H5), isopropoxy (-OCH (CH3)2) or tert-butoxy (-OC (CH3)3), and R4 is selected from hydroxyl, methyl (-CH3) or acetyl.

[0025] In the formula III, R1 is selected from hydrogen atom or vinyl, R2 is selected from hydrogen atom or hydroxyl, R3 is selected from hydrogen atom or methyl, R4 is selected from hydrogen atom or carbonyl, and R5 is selected from hydrogen atom or hydroxyl.

[0026] In the formula IV, R1 is selected from hydrogen atom or methyl, R2 is selected from carbonyl or hydroxyl, R3 is selected from hydrogen atom, hydroxyl or acetyl, R4 is selected from hydrogen atom or hydroxyl, and R5 is selected from hydrogen atom, hydroxyl or acetyloxy.

[0027] The application also provides application of the effective component of the Stellera chamaejasme extract in preparation of an antibiotic drug with antibacterial and antibiofilm functions.

[0028] Preferably, the drug for resisting drug-resistant bacteria takes the effective component of the Stellera chamaejasme extract as an active component, and is supplemented with a pharmaceutically acceptable auxiliary material or carrier.

[0029] Preferably, the auxiliary material includes any one or more of starch, dextrin, lactose, sodium carboxymethyl cellulose, sodium carboxymethyl starch, mannitol and sucrose.

[0030] Preferably, the carrier includes any one or more of liposomes, microemulsion carriers and nanocarriers.

[0031] Compared with the prior art, the application has the following beneficial effects: 1. This invention provides the application of Daphne odora extract in the preparation of antibiotic drugs with both antibacterial and anti-biofilm functions, offering a novel method for combating drug-resistant bacteria. The invention's research found that the Daphne odora extract significantly inhibits MRSA and VRE and their biofilms, effectively regulating biofilm formation-related genes without inducing drug resistance. Therefore, this invention demonstrates that the Daphne odora extract can be used to inhibit or kill MRSA and its biofilms, and subsequently in the preparation of new drugs to inhibit or kill MRSA or drugs against drug-resistant bacteria. This will provide a new strategy for developing antibiotic alternatives with both antibacterial and anti-biofilm functions, and will also help expand the high-value application of Daphne odora in the field of anti-infection.

[0032] 2. This invention is the first to report that Daphne odora extract has inhibitory and killing effects on superbugs, proving that it can be used to prepare drugs against drug-resistant bacteria.

[0033] 3. This invention has found that the purified extracts and active ingredients of Daphne odora have significant inhibitory effects on MRSA and VRE. Furthermore, the active substances were isolated, purified, and their structures identified, and their anti-superbug activity and mechanism of action were studied. The diterpenoid compounds also exhibit antiviral and antibacterial effects, and when combined with dihydroflavonoids, they showed good antibacterial synergistic effects. This will provide new ideas for the development of novel antibiotics, turning Daphne odora from waste into a valuable resource, and also helping the active ingredients in Daphne odora to become antibiotic alternatives. Attached Figure Description

[0034] Figure 1 This is a flowchart illustrating the separation process of methanol extract from the roots of Daphne odora in this invention.

[0035] Figure 2 The results of the detection of the antibacterial zone of the Daphne odora extract in this invention are shown.

[0036] Figure 3 This is the separation and purification process for the ethyl acetate fraction in this invention.

[0037] Figure 4 The results show the inhibition zone detection results of the effective components Fr.EA-1 to 5 of Daphne odora in this invention; wherein, Figure 4 Figure A in the middle Figure 4 Figure B in the figure shows the detection results of the inhibition zone of the effective component Fr.EA-1 of Daphne odora. Figure 4 Figure C in the middle Figure 4 Figure D in the figure shows the detection results of the inhibition zone of the effective component Fr.EA-2+3 of Daphne odora. Figure 4 E diagram in Figure 4 Figure F in the figure shows the detection results of the inhibition zone of the effective component Fr.EA-4 of Daphne odora. Figure 4 G-graph inFigure 4 The H-figure in the figure shows the detection results of the inhibition zone of the effective component Fr.EA-5 of Daphne odora.

[0038] Figure 5 This is a flowchart illustrating the separation process of the effective components Fr.EA-1 to 5 of Daphne odora in this invention.

[0039] Figure 6 The flowchart shows the further separation and purification process of the effective component Fr.EA-4-7 from Daphne odora in this invention.

[0040] Figure 7 These are the MIC values ​​of compounds 1 to 4, the effective monomers of the effective component Fr.EA-4-7 in Daphne odora from this invention, against MRSA; wherein, Figure 7 Figure A shows the MIC values ​​of Fr.EA-4-7 effective monomers, compounds 1 and 2, against MRSA. The three parallel samples in the same row are parallel samples. Figure 7 Figure B in the figure shows the MIC values ​​of Fr.EA-4-7 effective monomer compounds 3 and 4 against MRSA. The three parallel samples in the same row are parallel samples.

[0041] Figure 8 The experimental results show the effect of the effective monomer compound 1 in this invention on biofilm formation: Figure 8 Figure A shows crystal violet images of biofilms after co-incubation of different concentrations of compound 1 with bacterial culture, indicating its inhibitory effect. Figure 8 Figure B shows the crystal violet-stained biofilms dissolved in ethanol after co-incubation of different concentrations of compound 1 with bacterial culture. The six samples from left to right are Control group, 1 / 2MIC group, MIC group, 2×MIC group, 4×MIC group and 8×MIC group, respectively. Figure 8 Figure C in the figure shows the inhibition rate of MRSA biofilm formation in each group; Figure 8 Figure D in the image shows the three-dimensional structure of immature MRSA biofilms after drug inhibition treatment under laser confocal microscopy (left, top view; right, side view), where from top to bottom are the Control group, MIC group, 2×MIC group, 4×MIC group, and 8×MIC group.

[0042] Figure 9 The results of detecting the anti-SA and MRSA inhibition zones of compound 1 in this invention are as follows: Figure 9 Figure A in the figure shows the detection results of the anti-SA inhibition zone of compound 1; Figure 9 Figure B in the figure shows the detection results of the anti-MRSA inhibition zone of compound 1; Figure 9 Figure C in the diagram shows the specific measured values ​​of the inhibition zones of compound 1 against SA and MRSA.

[0043] Figure 10The results of the biofilm influence of the combination of the diterpenoid compound and the bis-dihydroflavonoid, i.e., the combination of compound 3 and compound 4, are shown in the following table: Figure 10 A in the table is a biofilm crystal violet picture of the combination of different concentrations of compound 3 and compound 4, i.e., the combination of the diterpenoid compound and the bis-dihydroflavonoid, after incubation with the bacterial liquid, for detecting the inhibitory effect; Figure 10 B in the table is a biofilm stained with crystal violet dissolved in ethanol after incubation of the combination of different concentrations of compound 3 and compound 4, i.e., the combination of the diterpenoid compound and the bis-dihydroflavonoid, with the bacterial liquid, wherein the six samples from left to right are Control group, 1 / 2 MIC group, MIC group, 2x MIC group, 4x MIC group and 8x MIC group; Figure 10 C in the table is the inhibition rate of MRSA biofilm formation in each group; Figure 10 D in the table is the three-dimensional structure of the immature MRSA biofilm under the laser confocal microscope after drug inhibition treatment (left, top view; right, side view), wherein the groups from top to bottom are Control group, MIC group, 2x MIC group, 4x MIC group and 8x MIC group. DETAILED DESCRIPTION

[0044] The application will be described in detail below with reference to the drawings and specific examples, but should not be understood as limiting the application. If not specifically stated, the technical means used in the following examples are conventional means familiar to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specifically stated, can be obtained from commercial channels.

[0045] Example 1: Stipa bungeana crude extraction steps and minimum inhibitory concentration detection Experimental method: micro-broth dilution method The Stipa bungeana collected in the field in the Ningxia grassland is divided into dry aboveground parts and dry roots. 25 kg of the dry aboveground parts of Stipa bungeana are added with 3 times the amount of methanol, heated to reflux at 80°C for extraction for 3 times, and then the 3 times of extraction liquids are combined and concentrated to recover the organic solvent to prepare the methanol extract of the aboveground parts of Stipa bungeana, which is also called the methanol extraction part of the aboveground parts of Stipa bungeana.

[0046] The dry roots of Stipa bungeana are 25 kg, which are added with 3 times the amount of methanol, heated to reflux at 80°C for extraction for 3 times, and then the 3 times of extraction liquids are combined and concentrated to recover the organic solvent to prepare the methanol extract of the roots of Stipa bungeana, which is also called the methanol extraction part of the roots of Stipa bungeana.

[0047] The methanol extract of the aboveground parts of Stellera chamaejasme and the methanol extract of the roots of Stellera chamaejasme were respectively configured into solutions with concentration gradients of 1000 g / mL, 500 g / mL, 250 g / mL, 125 g / mL and 62.5 g / mL using double distilled water, and the minimum inhibitory concentrations of the extract of the aboveground parts of Stellera chamaejasme and the extract of the roots of Stellera chamaejasme against the four drug-resistant bacteria were detected by the micro-broth dilution method.

[0048] The Staphylococcus aureus (ATCC 25923) and the four drug-resistant bacteria were methicillin-resistant Staphylococcus aureus (ATCC 43300), vancomycin-resistant Enterococcus (ATCC 51299), drug-resistant Helicobacter pylori (ATCC 43504) and penicillin-resistant Streptococcus pneumoniae (ATCC 51915).

[0049] The strains were all from the Microbial Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences.

[0050] The minimum inhibitory concentration is also referred to as the MIC value.

[0051] The steps for detecting the minimum inhibitory concentrations of the extract of the aboveground parts of Stellera chamaejasme and the extract of the roots of Stellera chamaejasme against the four drug-resistant bacteria by the micro-broth dilution method are as follows: the strains stored in an ultra-low temperature refrigerator at -80°C were activated on LB solid medium and cultured at 37°C overnight. The next day, the methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, drug-resistant Helicobacter pylori and penicillin-resistant Streptococcus pneumoniae colonies were inoculated into 5 mL of LB liquid medium respectively, and cultured at 37°C, 220 rpm overnight. When the OD 600 of the bacterial liquid was 1.0, it was diluted to 1×10 6 CFU / mL for standby. 100 µL of LB liquid medium was first added to each well, and 100 µL of the extract of the aboveground parts of Stellera chamaejasme and the extract of the roots of Stellera chamaejasme with a concentration of 1 mg / mL were added to the first well, so that the drug concentration in the first column was 500 g / mL. Then, 100 µL was sucked out from the first well into the second well, and the drug concentration was diluted by 2 times in turn. Then, 100 µL of the bacterial liquid with a concentration of 1×10 6 CFU / mL was added to each well. The positive control group was 100 µL of MH liquid medium and 100 µL of the methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, drug-resistant Helicobacter pylori and penicillin-resistant Streptococcus pneumoniae bacterial liquid with a concentration of 1×10 6 CFU / mL, and the negative control group was 200 µL of MH liquid medium. After incubation in a 37°C constant temperature incubator for 24 hours, the wells were clear, sterile and grew, the wells were turbid, and the bacteria survived. The MIC value was determined.

[0052] Overnight refers to a culture time ≥ 12 h.

[0053] The results are: the methanol extract of the roots of Stellera chamaejasme is clear in the well at a concentration of 500 g / mL, and turbid at 250 g / mL. The methanol extract of the aerial parts of Stellera chamaejasme is turbid in the well.

[0054] The above results show that the methanol extract of the roots of Stellera chamaejasme has a significant inhibitory effect on methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and penicillin-resistant Streptococcus pneumoniae at 500 g / mL, while the methanol extract of the aerial parts of Stellera chamaejasme has no significant inhibitory effect.

[0055] Example 2: Extraction and extraction process of methanol extract of roots of Stellera chamaejasme The dry roots of Stellera chamaejasme 25 kg, add 3 times the amount of methanol, heat reflux extraction at 80℃ for 3 times, combine the 3 times of extraction liquid, and after concentrating and recovering the solvent, obtain 8.6 kg of methanol extract. The methanol extract is redissolved with methanol, mixed with 1:1 of diatomite, dried in an oven at 45℃, and then extracted with petroleum ether, dichloromethane, ethyl acetate and methanol in turn, to obtain 210 g of petroleum ether extract, 630 g of dichloromethane extract, and 1.5 kg of ethyl acetate extract. The extraction and extraction process is shown in Figure 1 .

[0056] The petroleum ether extract is also called PE extract, the dichloromethane extract is also called DCM extract, and the ethyl acetate extract is also called Stellera chamaejasme EA extract.

[0057] Example 3: Detection of antibacterial effect by inhibition zone Experimental method: filter paper method The filter paper method was used to measure the size of the inhibition zone of MRSA. A puncher with a diameter of 6 mm was used to punch out several filter paper pieces, which were sterilized by high-pressure steam for 1 hour and then dried in an oven at 37℃ for standby. The cultured MRSA liquid was taken and placed in a 96-well plate. The OD value was measured at a wavelength of 600 nm. The MRSA liquid was diluted to 2.0×10 6 CFU / mL with medical 0.9% saline. The diluted bacterial liquid was taken with a sterile cotton swab, and the excess bacterial liquid was scraped off the wall of the tube. The bacterial liquid was evenly applied to the meat broth (MH) solid culture medium. Then two sterile dry filter paper pieces were placed on the medium, and 10 µL of the prepared drug (ethyl acetate extract 500 mg / mL, 500 g / mL, 250 g / mL, 125 g / mL, 62.5 g / mL and 31.25 g / mL) and two control groups (DMSO solution, MH liquid medium) were added to the corresponding filter paper pieces.

[0058] The culture method is the same as that in Example 1. MRSA is cultured in LB liquid medium to the logarithmic growth phase. When the OD 600 =1.0 of the bacterial solution, it is diluted to 1×10 6 CFU / mL. The LB solid medium is divided into four parts to make six concentration gradients (500 mg / mL, 500 g / mL, 250 g / mL, 125 g / mL, 62.5 g / mL, 31.25 g / mL of ethyl acetate extraction fraction, and two control groups (DMSO solution, MH liquid medium). After marking on the bottom of the culture dish, it is inverted and cultured in a 37°C constant temperature incubator for 12 hours, and then the size of the inhibition zone is measured.

[0059] The results are shown in Table 1. Figure 2 At a concentration of 500 mg / mL, a clear inhibition zone appears around it.

[0060] From the above results, it can be seen that at a concentration of 500 mg / mL, the drug has good antibacterial effect on MRSA strain.

[0061] Example 4: Evaluation of the antibacterial effect of MIC value Experimental method: micro-broth dilution method The culture method is the same as that in Example 1. MRSA is cultured in LB liquid medium to the logarithmic growth phase. When the OD 600 =1.0 of the bacterial solution, it is diluted to 1×10 6 CFU / mL, and then diluted in a 96-well plate to obtain five concentration gradients (250 g / mL, 125 g / mL, 62.5 g / mL, 31.25 g / mL and 15.625 g / mL) of the drug to be tested. The bacterial solution and the control group are added and cultured at 37°C for 24 hours. 20 µL of CCK-8 is added to each well in the dark, and incubated in a 37°C incubator for 1 hour. The absorbance (OD value) of each well of the 96-well plate is measured at 600 nm wavelength using a microplate reader, and the MIC value is determined.

[0062] The results of the determination are shown in Table 1.

[0063] Table 1: MIC values of each extraction fraction Note: PE refers to the petroleum ether fraction; EA refers to the ethyl acetate fraction; DCM refers to the dichloromethane fraction; MeOH refers to the methanol fraction.

[0064] The MIC value shows that the EA part of Stellera chamaejasme has good bacteriostatic effect, and the other three parts do not have obvious bacteriostatic effect. Therefore, it can be determined that the ethyl acetate part of Stellera chamaejasme has obvious effect on inhibiting MRSA. Among them, the MIC of the EA part of Stellera chamaejasme is 62.5 g / mL.

[0065] Example 5: Ethyl acetate part separation and purification steps and subordinate component MIC detection results Experimental method: micro-broth dilution method The ethyl acetate extraction part is dissolved in methanol, then 200 mesh silica gel is added in a beaker according to the weight ratio of ethyl acetate part sample to silica gel 1:1.5, and the sample is stirred, and then the sample is separated by normal phase silica gel column chromatography, and the eluent is ethyl acetate:methanol=30:1, 20:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1:1 and pure methanol elution, and the same components are combined by thin layer plate detection, and 13 components Fr.EA-1~Fr.EA-13 are obtained from the ethyl acetate extraction part, and the MIC value of each component is detected. The specific detection method is the same as that of Example 1.

[0066] Among them, the ethyl acetate part separation and purification process and MIC detection results are shown in Figure 3 and Table 2.

[0067] Table 2: MIC detection results of components Fr.EA-1~Fr.EA-13 The results show that the EA-1~EA-5 components of the ethyl acetate extraction part have significant inhibitory effect on MRSA, and other components can also inhibit the growth of MRSA. Among them, the MIC of Fr.EA-1 and Fr.EA-5 is 15.6 g / mL.

[0068] Example 6: Bacteriostatic determination of effective components Fr.EA-1~5 Experimental method: filter paper method Select components Fr.EA-1, components Fr.EA-2+3, components Fr.EA-4, components Fr.EA-5 for bacteriostatic circle experiment, prepare 500 g / mL of components, dilute gradiently to prepare 250 g / mL, 125 g / mL, 62.5 g / mL, 31.25 g / mL, 15.625 g / mL, six concentration gradients, three groups of repeated experiments, and MH liquid medium as negative control, observe the size of bacteriostatic circle, and the specific detection method is the same as that of Example 3.

[0069] Among them, the bacteriostatic circle results are shown in Figure 4 .

[0070] As Figure 5The separation flow chart of effective components Fr.EA-1-5 of Stellera chamaejasme L. is shown.

[0071] The results show that the inhibition zone experiment results are consistent with the MIC results, and the components Fr.EA-1, Fr.EA-2+3, Fr.EA-4 and Fr.EA-5 all have good antibacterial effect. Further separation and purification of the components Fr.EA-1, Fr.EA-2+3, Fr.EA-4 and Fr.EA-5.

[0072] Example 7: MIC value determination of effective components Fr.EA-1-5 Experimental method: micro-broth dilution method The MIC values of the components Fr.EA-1, Fr.EA-2+3, Fr.EA-4 and Fr.EA-5 are shown in Table 3.

[0073] Table 3 MIC values of four components under Fr.EA-1 The MIC values of the components Fr.EA-2+3 are shown in Table 4.

[0074] Table 4 MIC values of one component under Fr.EA-2 The MIC values of the components Fr.EA-3 are shown in Table 5.

[0075] Table 5 MIC values of five components under Fr.EA-4 The MIC values of the components Fr.EA-5 are shown in Table 6.

[0076] Table 6 MIC values of ten components under Fr.EA-5 From the above experimental results, it can be concluded that the components Fr.EA-1-5 have the best antibacterial effect, and the remaining components also have good antibacterial effect.

[0077] Example 8: separation and purification results of components Fr.EA-4-7 and their MIC values Experimental method: micro-broth dilution method The component Fr.EA-4-7 was separated and purified by a chromatographic column. The Fr.EA-4-7 was mixed and sampled, 300 mesh silica gel was added to the sample to be separated and stirred uniformly to a fine powder for standby. A clean and dried chromatographic column was vertically installed on an iron stand, and a developing agent was added to about 80% of the column height, 300 mesh silica gel was added as a filler, and it was left overnight. After the chromatographic column was settled and balanced, the sample was added. A series of chemical reagents with decreasing polarity were added for gradient elution (dichloromethane:methanol=50:1~1:1, methanol), and the obtained substances were distilled by a rotary evaporator and identified by TCL thin layer chromatography. A total of 11 substances were obtained, numbered as component Fr.EA-4-7-1, component Fr.EA-4-7-2, component Fr.EA-4-7-3, component Fr.EA-4-7-4, component Fr.EA-4-7-5, component Fr.EA-4-7-6, component Fr.EA-4-7-7, component Fr.EA-4-7-8, component Fr.EA-4-7-9, component Fr.EA-4-7-10, and component Fr.EA-4-7-11.

[0078] The separation and purification process of Fr.EA-4-7 is shown in the following diagram. Figure 6

[0079] The component Fr.EA-4-7 was separated and purified by a chromatographic column. The Fr.EA-4-7 was mixed and sampled, 300 mesh silica gel was added to the sample to be separated and stirred uniformly to a fine powder for standby. A clean and dried chromatographic column was vertically installed on an iron stand, and a developing agent was added to about 80% of the column height, 300 mesh silica gel was added as a filler, and it was left overnight. After the chromatographic column was settled and balanced, the sample was added. A series of chemical reagents with decreasing polarity were added for gradient elution (dichloromethane:methanol=50:1~1:1, methanol), and the obtained substances were distilled by a rotary evaporator and identified by TCL thin layer chromatography. A total of 11 substances were obtained, numbered as component Fr.EA-4-7-1, component Fr.EA-4-7-2, component Fr.EA-4-7-3, component Fr.EA-4-7-4, component Fr.EA-4-7-5, component Fr.EA-4-7-6, component Fr.EA-4-7-7, component Fr.EA-4-7-8, component Fr.EA-4-7-9, component Fr.EA-4-7-10, and component Fr.EA-4-7-11.

[0080] Table 7 Weight of the components under component Fr.EA-4-7 The component Fr.EA-4-7 was separated and purified by a chromatographic column. The Fr.EA-4-7 was mixed and sampled, 300 mesh silica gel was added to the sample to be separated and stirred uniformly to a fine powder for standby. A clean and dried chromatographic column was vertically installed on an iron stand, and a developing agent was added to about 80% of the column height, 300 mesh silica gel was added as a filler, and it was left overnight. After the chromatographic column was settled and balanced, the sample was added. A series of chemical reagents with decreasing polarity were added for gradient elution (dichloromethane:methanol=50:1~1:1, methanol), and the obtained substances were distilled by a rotary evaporator and identified by TCL thin layer chromatography. A total of 11 substances were obtained, numbered as component Fr.EA-4-7-1, component Fr.EA-4-7-2, component Fr.EA-4-7-3, component Fr.EA-4-7-4, component Fr.EA-4-7-5, component Fr.EA-4-7-6, component Fr.EA-4-7-7, component Fr.EA-4-7-8, component Fr.EA-4-7-9, component Fr.EA-4-7-10, and component Fr.EA-4-7-11.

[0081] Table 8 MIC values of the 5 fractions under component Fr.EA-4-7 ​From a series of antibacterial experiments, it can be concluded that the ethyl acetate extraction part has obvious antibacterial effect, and the MIC = 61.25 g / mL. The antibacterial activity of the 12 components obtained by separating and purifying the ethyl acetate extraction part is the best for the components Fr.EA-1 and Fr.EA-5, and the MIC = 15.625 g / mL. The components Fr.EA-2+3 and Fr.EA-4 also have good antibacterial effect. Further separation and purification of the components Fr.EA-4-7 obtains 11 components, and the best components are the components Fr.EA-4-7-5, Fr.EA-4-7-6, Fr.EA-4-7-7 and Fr.EA-4-7-8, and the MIC values are all MIC = 31.25 g / mL.

[0082] Example 9: Further separation and purification of component Fr.EA-4-7-7 Further purification of the component Fr.EA-4-7-7 was carried out by using a semi-preparative liquid chromatography system of NS4205 model of Jiangsu Hanbang Science and Technology Co., Ltd. The specific operation is as follows: 10 mg of the component Fr.EA-4-7-7 was weighed and dissolved in 50 mL of methanol to prepare a liquid chromatography sample with a concentration of 0.2 mg / mL. A 30% methanol solution by volume was used as the mobile phase. After the instrument was turned on, baseline running was first carried out. After the baseline was stable, the sample was injected using a sample needle, and the sample control was within 200 µL. During the process, the spectrum was observed and the peak value compound was collected.

[0083] The effective monomer substance (monomer compound) obtained is 4, which are compound 1~compound 4 in turn.

[0084] Among them, compound 1 and compound 2 belong to flavonoids, and compound 3 and compound 4 belong to terpenes.

[0085] The structural formula of compound 1 is shown as formula 1, the structural formula of compound 2 is shown as formula 2, the structural formula of compound 3 is shown as formula 3, and the structural formula of compound 4 is shown as formula 4: .

[0086] Example 10: MIC value detection of compound 1 and compound 2 against MRSA Experimental method: micro-broth dilution method The specific detection method is the same as in Example 1. 100µL of compound 1 and compound 2 at a concentration of 1920g / mL are added to the first 3 wells of the first row, and so on, until ten concentration gradients of compound 1 and compound 2 are obtained. Then, bacterial culture or MH liquid culture medium is added to each well and incubated in a constant temperature incubator at 37°C for 24 hours. After that, 10µL of MTT is added to each well and incubated in a dark incubator at 37°C for 2 hours to determine the MIC value.

[0087] Table 9 MIC values ​​of compounds 1-4 The results are as follows Figure 7 As shown: Compounds 1 to 4 have a significant inhibitory effect on MRSA.

[0088] The results above indicate that compounds 1 to 4 of the effective monomers Fr.EA-4-7 from Daphne odora significantly inhibit MRSA. Specifically, the MIC values ​​for compounds 1, 2, 3, and 4 are all 1.8725 g / mL.

[0089] Example 11: Compound 1 significantly inhibits the formation of MRSA biofilm. Experimental method: Crystal violet staining method Add 2 mL of bacterial suspension (100-fold dilution of overnight-shaken bacteria) and different concentrations of Compound 1 (1 / 2 MIC, MIC, 2 MIC, 4 MIC, 8 MIC) or 2 mL of bacterial suspension as a control to a 6-well plate. Incubate at 37°C for 48 h. After biofilm formation, aspirate the bacteria with an insulin needle, wash gently 2-3 times with PBS, and after aspirating all PBS, add crystal violet dye and stain for 20-30 minutes. Aspirate the crystal violet and wash 3 times with PBS. Dissolve in 95% ethanol for 5 min, and then measure the OD value at 570 nm using a microplate reader to calculate the biofilm disruption rate. Disruption rate % = (OD control group / OD experimental group) / OD control group × 100%. Figure 8 As shown.

[0090] It should be noted that when using an insulin needle to aspirate the bacterial solution, try to avoid aspirating the biofilm.

[0091] The PBS was sourced from Sangon Biotech Co., Ltd., with a concentration of 0.01 mol / L and a pH of 7.4.

[0092] The results are as follows Figure 8 As shown, the intensity of crystal violet staining decreases with increasing concentration of compound 1.

[0093] From the above results, it can be seen that compound 1 significantly inhibits the formation of MRSA biofilm.

[0094] Example 12: Detection of MRSA antibacterial inhibition zone of compound 1 Experimental method: filter paper method The specific detection method is the same as that of Example 3. 10 μL of prepared compound 1 and two groups of control (DMSO solution, MH liquid medium) were dropped on the corresponding filter paper, and the size of the inhibition zone was measured after 12 hours of culture in a 37°C constant temperature incubator. The results are shown in Figure 9

[0095] From the above results, it can be seen that obvious antibacterial inhibition zones appear around the filter paper on SA and MRSA strains. From the above results, it can be seen that compound 1 has good antibacterial effect on SA and MRSA strains.

[0096] Example 13: FIC value detection of MRSA of compound 3 and compound 4 in combination Experimental method: micro-broth dilution method The specific detection method is the same as that of Example 1. 100 μL of compound 1 and compound 2 with a concentration of 1920 g / mL were added to the first three holes in the first row, and the same was done in the subsequent rows. Finally, ten concentration gradients of compound 1 and compound 2 were obtained. Bacterial solution or MH liquid medium was added to each hole, and it was incubated in a 37°C constant temperature incubator for 24 hours. Then 10 μL of MTT was added to each hole in the dark, and it was incubated in a 37°C incubator in the dark for 2 hours, so as to determine the MIC value. FIC (Fractional Inhibitory Concentration) is a method for evaluating the interaction of antibacterial drugs. It can determine the inhibitory or killing effect of different drugs or drug combinations on microorganisms, and help to judge the synergistic effect, additive effect or antagonistic effect between drugs. The principle of FIC determination is based on MIC, i.e. the minimum concentration at which a drug can inhibit the growth of microorganisms. By testing the MIC values of drugs used alone and in combination, the FIC index is calculated, and the nature of drug interaction is analyzed. For the joint drug sensitivity experiment, on the basis of the detection of the MIC value of compound 3 in Example 1, compound 4 was added to each hole for FIC value determination. The results show that the combination of diterpenoid and bis-dihydroflavonoid, i.e. compound 3 and compound 4, shows obvious synergistic antibacterial effect, and the FIC value is <1 g / mL.

[0097] Example 14: Compound 3 and compound 4 in combination significantly inhibit the formation of MRSA biofilm Experimental method: crystal violet staining method ​The specific detection method is the same as that in Example 11, and different concentrations of compound 3 and compound 4 (1 / 2 MIC, MIC, 2 MIC, 4 MIC, 8 MIC) or 2 mL of bacterial solution are added as controls. The results are shown in Figure 10 .

[0098] The results are shown in Figure 10 . As the concentration of compound 3 and compound 4 increases, the crystal violet staining intensity decreases.

[0099] From the above results, it can be seen that the combination of compound 3 and compound 4 has good synergistic effect and significantly inhibits the formation of MRSA biofilm.

[0100] The application provides application of Stellera chamaejasme extract and effective components in preparation of anti-drug-resistant bacteria medicines, and provides a new method for resisting drug-resistant bacteria. It is found that the ethyl acetate extraction part of the Stellera chamaejasme methanol extract has the functions of resisting drug-resistant bacteria and resisting biofilm, and the effective substances are separated, purified, and identified, and the anti-'super bacteria' activity and mechanism are researched, wherein the diterpenoid compounds also have the functions of resisting viruses and resisting bacteria, and the diterpenoid compounds combined with bis-dihydroflavonoids show good antibacterial synergistic effect, which provides a research direction and a thought for limited clinical drug-resistant bacteria treatment means. It is proved that the Stellera chamaejasme extract can be used for inhibiting or killing MRSA, and then used in preparation of new medicines for inhibiting or killing MRSA or anti-drug-resistant bacteria medicines.

[0101] It should be noted that when numerical ranges are involved in the present application, both endpoints of each numerical range and any number between the two endpoints can be selected, and in order to prevent redundancy, preferred embodiments are described in the present application.

[0102] Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to the embodiments once they know the basic inventive concept, and these changes and modifications all fall within the scope of all changes and modifications of the present application.

Claims

1. The application of Daphne odora extract in the preparation of drugs that inhibit the bioactivity of superbugs and their biofilm formation, characterized in that, The roots of Daphne odora were extracted with methanol, filtered, and the filtrate was collected. The filtrate was concentrated to obtain a methanol extract. The methanol extract was redissolved and then mixed with diatomaceous earth at a mass ratio of 0.8-1.2:1 to obtain a mixed sample. The mixed sample was dried and then extracted with petroleum ether and ethyl acetate in sequence. The ethyl acetate extract obtained was the Daphne odora extract. The ratio of the root of Daphne odora to the methanol is 1:2~5. The mass ratio of the mixed sample to the petroleum ether is 1:20~50.

2. The application according to claim 1, characterized in that, The Daphne odora extract exhibits antibacterial activity and inhibits biofilm formation against any one or more of the following superbugs: Methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, drug-resistant Helicobacter pylori strains, and penicillin-resistant Streptococcus pneumoniae.

3. The application according to claim 1, characterized in that, After the mixed sample is dried, it is first extracted with petroleum ether to obtain the petroleum ether extract and the remaining aqueous phase; the remaining aqueous phase is then extracted with ethyl acetate to obtain the ethyl acetate extract. The volume ratio of the remaining aqueous phase to the ethyl acetate is 1:20~100.

4. The application according to claim 1, characterized in that, When extracting the roots of Daphne odora with methanol, the extraction method used was heating and reflux. The extraction method using heating and reflux is performed under the conditions of heating and reflux at 78℃~82℃.

5. The application according to claim 1, characterized in that, The drug uses the Daphne odora extract as the active ingredient, supplemented with pharmaceutically acceptable excipients or carriers.

6. The application according to claim 5, characterized in that, The excipients include any one or more of starch, dextrin, lactose, sodium carboxymethyl cellulose, sodium carboxymethyl starch, mannitol, and sucrose.

7. The application according to claim 5, characterized in that, The carrier includes any one or more of liposomes, microemulsion carriers, and nanocarriers.

8. An effective component of a Daphne odora extract, characterized in that, The effective component of the Daphne odora extract is the substance obtained by separation and purification of the Daphne odora extract as described in claim 1. The substance comprises compounds I through IV; the structural formulas of compounds I through IV are shown in formulas I through IV respectively: ； In Formula I, R1 is selected from methoxy, ethoxy, or isopropoxy, and R2 to R6 are all selected from hydroxy, methyl, or acetyl groups; In Formula II, R1 is selected from methoxy, ethoxy, isopropoxy or tert-butoxy, R2 is selected from hydroxy, methyl or acetyl, R3 is selected from methoxy, ethoxy, isopropoxy or tert-butoxy, and R4 is selected from hydroxy, methyl or acetyl. In Formula III, R1 is selected from hydrogen atom or vinyl group, R2 is selected from hydrogen atom or hydroxyl group, R3 is selected from hydrogen atom or methyl group, R4 is selected from hydrogen atom or carbonyl group, and R5 is selected from hydrogen atom or hydroxyl group. In Formula IV, R1 is selected from hydrogen atom or methyl, R2 is selected from carbonyl or hydroxyl, R3 is selected from hydrogen atom, hydroxyl or acetyl, R4 is selected from hydrogen atom or hydroxyl, and R5 is selected from hydrogen atom, hydroxyl or acetoxy.

9. The use of the active ingredient of the Daphne odora extract according to claim 8 in the preparation of drugs against drug-resistant bacteria.

10. The application according to claim 9, characterized in that, The anti-drug-resistant bacterial drug uses the active ingredient of the Daphne odora extract as the active ingredient, supplemented with pharmaceutically acceptable excipients or carriers.

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