Traditional Chinese medicine composition with bacteriostatic effect as well as preparation method and application of traditional Chinese medicine composition

Abstract

The invention discloses a traditional Chinese medicine composition with a bacteriostatic effect as well as a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicines. The antibacterial composition is prepared from the following components in parts by weight: 1 part of cortex phellodendri and 16 parts of ginger, has an excellent inhibition effect on escherichia coli and staphylococcus aureus, and can be used for preventing and treating diseases caused by related bacterial infection and killing related bacteria in the environment.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to a traditional Chinese medicine composition with antibacterial effect and a preparation method and application thereof. Background Art

[0002] In recent years, the number of people suffering from skin diseases and inflammations caused by bacterial infections and other factors has increased year by year, bringing varying degrees of pain to people's lives. Existing antibacterial treatments are mostly Western medicine treatments, but Western medicine treatments are prone to recurrence and cannot fundamentally solve the problem. Long-term use of products containing chemical preservatives may make the skin sensitive and prone to allergies, and some may even have neurotoxicity and liver toxicity. Long-term use may cause potential harm to the human body and cause great harm to the patient's body and mind.

[0003] my country is rich in traditional Chinese medicine resources, among which thousands of Chinese herbal medicines have potential biological activities such as antibacterial, antioxidant, anti-inflammatory and other pharmacological effects, with broad application value, and are important raw materials for the development of antibacterial agents. Phellodendron chinense Schneid. is the dried bark of the Rutaceae plant Phellodendron chinense Schneid., commonly known as "Sichuan Phellodendron", which has the effects of clearing heat and dampness, purging fire and removing steam, detoxifying and curing sores, and is often used for damp-heat diarrhea, jaundice and red urine, sores, swelling and toxins, etc. Modern pharmacological studies have found that the chemical components of Phellodendron mainly include alkaloids, lactones, phenolic acids, terpenes, phenylpropanoids and other components, which have obvious antibacterial and anti-inflammatory activities, and can be used to prepare medicines, skin care products, washing products and daily products. Yunjiang is the dried rhizome of Zingiber officinale Rosc., a plant of the ginger family. It is a local medicinal material unique to Sichuan. It is mainly produced in Yunlian County, Yibin City, Sichuan Province. It has a long history of more than 1,700 years. The Tang Dynasty's "New Compendium of Materia Medica" records: "Dried ginger... is grown in the valley..." Yunjiang has the effects of warming the middle and dispersing cold, restoring yang and unblocking meridians, warming the lungs and transforming fluid. It is used for cold pain in the abdomen, vomiting and diarrhea, cold limbs and weak pulse, cold fluid and cough. Compared with other dried gingers, it is pungent and warm without being dry, warming the middle and dispersing internal cold, staying without moving, and can warm and nourish the upper, middle and lower parts of the body. The chemical components contained in Yunjiang are relatively complex, including volatile oils, gingerols, diphenylheptanes, trace elements, amino acids, polysaccharides, etc. Modern pharmacological studies have shown that Yunjiang has antipyretic, analgesic and anti-inflammatory effects.

[0004] Traditional Chinese medicine has a natural advantage in preventing bacterial resistance. Phellodendron chinense and Ginger juniper also have the advantages of abundant resources, low R&D cost, low toxicity, few adverse reactions, safety and environmental protection, and are not easy to cause the body to develop drug resistance. In the future, it is expected to receive more attention and support in the field of antibiotic substitute research and development. However, there are no reports on the use of Phellodendron chinense and Ginger juniper in the preparation of drugs for the prevention and treatment of bacterial infections. Summary of the invention

[0005] The purpose of the present invention is to provide a Chinese medicine composition with antibacterial effect and a preparation method and application thereof, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a traditional Chinese medicine composition with antibacterial effect, which is prepared by 1 part of Phellodendron chinense and 16 parts of Zingiber officinale in parts by weight.

[0008] The second technical solution of the present invention is a method for preparing the traditional Chinese medicine composition, which comprises weighing Phellodendron chinense and Ginger juncea by weight, extracting by percolation with an alcohol solution, and concentrating the percolation liquid under reduced pressure.

[0009] The third technical solution of the present invention is the use of the traditional Chinese medicine composition in the preparation of medicines for preventing and treating diseases caused by bacterial infections.

[0010] A fourth technical solution of the present invention is a medicine for preventing and treating diseases caused by bacterial infection, comprising the above-mentioned Chinese medicine composition.

[0011] A fifth technical solution of the present invention is an antibacterial liquid, comprising the Chinese medicine composition.

[0012] Based on the above technical solution, the present invention has the following technical effects:

[0013] The invention provides a Chinese medicinal composition with antibacterial effect, a preparation method and application thereof. The Chinese medicinal composition prepared by using Phellodendron chinense medicinal materials and Ginger juncea medicinal materials in a mass ratio of 1:16 has excellent inhibitory effects on Escherichia coli and Staphylococcus aureus, and can be used for preventing and treating diseases caused by related bacterial infections and killing related bacteria in the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 is the minimum inhibitory concentration of medicinal materials. Among them, A is the minimum inhibitory concentration of Phellodendron chinense-Escherichia coli, B is the minimum inhibitory concentration of Phellodendron chinense-Staphylococcus aureus, C is the minimum inhibitory concentration of Gingerbrush-Escherichia coli, D is the minimum inhibitory concentration of Gingerbrush-Staphylococcus aureus, E is the minimum inhibitory concentration of Honeysuckle-Escherichia coli, and F is the minimum inhibitory concentration of Honeysuckle-Staphylococcus aureus.

[0016] Figure 2is the minimum inhibitory concentration of different extraction methods. Among them, A is the minimum inhibitory concentration of Escherichia coli extracted by ultrasound, B is the minimum inhibitory concentration of Staphylococcus aureus extracted by ultrasound, C is the minimum inhibitory concentration of Escherichia coli extracted by reflux, D is the minimum inhibitory concentration of Staphylococcus aureus extracted by reflux, E is the minimum inhibitory concentration of Escherichia coli extracted by acid water, F is the minimum inhibitory concentration of Staphylococcus aureus extracted by acid water, G is the minimum inhibitory concentration of Escherichia coli extracted by percolation, and H is the minimum inhibitory concentration of Staphylococcus aureus extracted by percolation.

[0017] Figure 3 is the minimum inhibitory concentration of different concentrations of ethanol percolation extraction. Among them, A is the minimum inhibitory concentration of Escherichia coli extracted by 60% ethanol percolation, B is the minimum inhibitory concentration of Escherichia coli extracted by 60% ethanol percolation, C is the minimum inhibitory concentration of Escherichia coli extracted by 80% ethanol percolation, D is the minimum inhibitory concentration of Escherichia coli extracted by 80% ethanol percolation, E is the minimum inhibitory concentration of Escherichia coli extracted by 70% ethanol percolation, and F is the minimum inhibitory concentration of Staphylococcus aureus extracted by 70% ethanol percolation. DETAILED DESCRIPTION

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0021] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0024] The embodiment of the present invention provides a traditional Chinese medicine composition with antibacterial effect, which is prepared by 1 part of Phellodendron chinense and 16 parts of Zingiber officinale in parts by weight.

[0025] The embodiment of the present invention also provides a method for preparing the traditional Chinese medicine composition, which comprises weighing Phellodendron chinense and Ginger juncea by weight, extracting by percolation with an alcohol solution, and concentrating the percolation liquid under reduced pressure.

[0026] In some specific embodiments, the alcohol solution is an ethanol solution with a volume fraction of 70%.

[0027] In some specific embodiments, coarse powders of Phellodendron chinense and Ginger are mixed, added with 70% ethanol and soaked for 24 hours, and 10 times the volume of percolate is collected.

[0028] The embodiment of the present invention also provides the use of the traditional Chinese medicine composition in preparing medicines for preventing and treating diseases caused by bacterial infection.

[0029] In some specific embodiments, the bacterial infection causing the disease includes diseases caused by Staphylococcus aureus and Escherichia coli.

[0030] In some specific embodiments, the diseases caused by bacterial infection include diseases caused by Staphylococcus aureus and Escherichia coli; further preferably, the diseases caused by bacterial infection include diseases caused by Staphylococcus aureus BNCC238481 and Escherichia coli BNCC269342.

[0031] The embodiment of the present invention also provides a medicine for preventing and treating diseases caused by bacterial infection, including the Chinese medicine composition.

[0032] The embodiment of the present invention further provides an antibacterial liquid, comprising the traditional Chinese medicine composition.

[0033] Example 1

[0034] 1 Experimental Materials

[0035] 1.1 Strains Staphylococcus aureus subspecies (BNCC238481, Shangcheng Beina Chuanglian Biotechnology Co., Ltd.) and Escherichia coli (BNCC269342, Shangcheng Beina Chuanglian Biotechnology Co., Ltd.).

[0036] 1.2 Instruments and consumables: high-pressure steam sterilizer, shaker, constant temperature incubator, water bath, rotary evaporator, microplate reader, coater, condenser, 96-well plate, beaker, 100mL pipette, 1000mL pipette, several 100mL pipette tips, several 1000mL pipette tips, several reagent bottles, several blood plates, and several 5mL centrifuge tubes.

[0037] 1.3 Medicinal materials and reference materials Medicinal materials: Phellodendron chinense, honeysuckle, ginger. Reference material: Cefuroxime dispersible tablets (Chengdu Beite Pharmaceutical Co., Ltd., 100 mg / tablet, drug identification code: 8165221).

[0038] 2 Experimental methods

[0039] 2.1 Extraction of medicinal materials

[0040] The preparation method of the extract of Phellodendron chinense, honeysuckle or ginger is as follows: weigh 10g of each medicinal material powder in three conical bottles, add 70% ethanol solution by volume, and extract under reflux in a water bath at 85°C for 60 minutes. After filtering the extract, the filtrate is concentrated using a rotary evaporator, placed in a 10mL volumetric flask, and fixed to 10mL with the prepared 10% dimethyl sulfoxide solution.

[0041] 2.2 Activation of strains (activation before subculturing) and subculturing

[0042] (1) Preparation of Luria-Bertani (LB) medium: Weigh 20 g of LB medium and add 1000 mL of sterile water. Heat to boiling and then put into a reagent bottle. Sterilize with high pressure steam at 121°C for 20 min. Let cool and refrigerate for later use.

[0043] (2) After the frozen strains were thawed (30 min), Staphylococcus aureus and Escherichia coli were selected and placed in Luria-Bertani (LB) liquid medium and cultured at 37°C in a shaking incubator in the dark for 24 h.

[0044] (3) Subculture of bacterial strains

[0045] Use a pipette to draw 150 μL of the first generation bacterial suspension and inoculate it into a new liquid culture medium (second generation of bacteria). Incubate at 37°C in a shaker in the dark for 24 h.

[0046] Use a pipette to draw 150 μL of the second generation bacterial suspension and inoculate it into a new liquid culture medium (3rd generation of bacteria). Incubate at 37°C in a shaker in the dark for 24 hours. (Store in a 2-8°C refrigerator for short term)

[0047] 2.3 Activation of bacteria (activation before use) and counting

[0048] (1) Activation of bacterial strains (activation before use) Take 100 μL of the third generation Staphylococcus aureus and Escherichia coli strains and inoculate them into 5 mL centrifuge tubes containing 4 mL of LB liquid culture medium, respectively. After incubation at 37°C in a shaking incubator for 4 hours, place them in a 37°C constant temperature incubator and culture them for 48 hours.

[0049] (2) Counting of bacterial species Take 100 μL of the fourth generation of Staphylococcus aureus and Escherichia coli and dilute them with sterile water by 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 Take 100 μL of bacterial solution with different concentration gradients, spread evenly on the blood plate, and culture at 37℃ in the dark for 24 hours, observe the number of colonies, and calculate the colony concentration of the original bacterial solution.

[0050] 2.4 Minimum inhibitory concentration determination

[0051] The two-fold dilution method was used to conduct in vitro antibacterial experiments to detect the minimum inhibitory concentration (MIC) of Phellodendron chinense, Honeysuckle and Ginger against Staphylococcus aureus and Escherichia coli.

[0052] In the ELISA plate, 100 μL of liquid culture medium was first added to the blank control group, and 100 μL of 1-5×10 3 CFU / mL of bacterial suspension, add 100 μL of the corresponding liquid culture medium to the growth control group, add 100 μL of 100 μg / mL cefixime solution to the negative control group, and add 100 μL of 10% dimethyl sulfoxide solution to the solvent control group.

[0053] Add 100 μL of 2 g / mL medicinal material extract (Phellodendron amurense, Lonicera japonica, or Zingiber officinale extract) solution to the first well of the experimental group and the blank control group. Use a pipette tip to aspirate and mix the extract solution and the bacterial suspension in the first well, then aspirate 100 μL from the first well and add it to the next well in the same column and mix thoroughly by aspirating and blowing. Repeat this operation until the eighth well. After mixing the solution in the eighth well, aspirate 100 μL and discard it. At this time, the drug concentration in each well decreases successively by a factor of 1 / 2 from top to bottom: 1 g / mL, 0.5 g / mL, 0.25 g / mL, 0.125 g / mL, 0.0625 g / mL, 0.03125 g / mL, 0.015625 g / mL, 0.078125 g / mL. Cover the microplate with the lid and place it in a constant temperature incubator for dark incubation at 37 °C for 16 - 24 h. Then, measure the OD value at 600 nm using a microplate reader. Calculate the antibacterial rate Y according to formula (1).

[0054] Table 1 Experimental Arrangement

[0055]

[0056]

[0057] Where A 实验 is the OD value of each treatment in the experimental group 600 after subtracting the value of the blank control group; A 阳性 is the OD value of each treatment in the growth control group 600 after subtracting the value of the solvent control group; A 阴性 is the OD value of the negative control group 600 after subtracting the value of the solvent control group.

[0058] 3 Experimental Results

[0059] Use GraphPad software to discard some offset values and plot the minimum inhibitory concentration. The results are as Figure 1 .

[0060] An antibacterial rate greater than or equal to 50% (IC50) is considered to have antibacterial effect. As Figure 1 can be seen, the minimum inhibitory concentration of Phellodendron amurense against Staphylococcus aureus is 0.00068 g / mL, the minimum inhibitory concentration of Lonicera japonica against Staphylococcus aureus is 0.0960 g / mL, and the minimum inhibitory concentration of Zingiber officinale against Staphylococcus aureus is 0.1526 g / mL. The minimum inhibitory concentration of Phellodendron amurense against Escherichia coli is 0.0195 g / mL, the minimum inhibitory concentration of Lonicera japonica against Staphylococcus aureus is 0.1854 g / mL, and the minimum inhibitory concentration of Zingiber officinale against Staphylococcus aureus is 0.3135 g / mL.

[0061] In summary, the minimum inhibitory concentration of Phellodendron chinense against Staphylococcus aureus and Escherichia coli is 0.0195 g / mL, the minimum inhibitory concentration of honeysuckle against the two strains is 0.1854 g / mL, and the minimum inhibitory concentration of ginger against the two strains is 0.3135 g / mL.

[0062] Example 2

[0063] 1. Experimental Materials

[0064] 1.1 Strains: Staphylococcus aureus subspecies (BNCC238481), Escherichia coli (BNCC269342).

[0065] 1.2 Instruments and consumables: high-pressure steam sterilizer, shaker, constant temperature incubator, water bath, rotary evaporator, microplate reader, coater, condenser, 96-well plate, beaker, 100mL pipette, 1000mL pipette, several 100mL pipette tips, several 1000mL pipette tips, several reagent bottles, several blood plates, and several 5mL centrifuge tubes.

[0066] 1.3 Medicinal materials and reference materials Medicinal materials: Phellodendron chinense, honeysuckle, ginger. Reference material: Cefixime dispersible tablets (Chengdu Beite Pharmaceutical Co., Ltd., 100 mg / tablet, drug identification code: 8165221).

[0067] 2. Experimental Procedure

[0068] 2.1 Counting of bacterial species

[0069] Take 100 μL of the fourth generation Staphylococcus aureus and Escherichia coli and dilute them with sterile water by 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 Take 100 μL of bacterial solution with different concentration gradients respectively, spread evenly on the blood plate, and culture at 37℃ in the dark for 24 hours, observe the number of colonies, calculate the colony concentration of the original bacterial solution, the colony concentration is 0-300 CFU, and record the data.

[0070] 2.2 Determination of the graded inhibitory concentration (FIC) of the combination of two traditional Chinese medicines

[0071] (1) Solution preparation

[0072] ① Preparation of control solution

[0073] Solvent control solution: 5 mL of dimethyl sulfoxide was added to 45 mL of sterile water to prepare a 10% dimethyl sulfoxide solution.

[0074] Negative control solution: Take a 100 mg cefixime dispersible tablet, crush it, add it to 1000 mL of sterile water, and put it into an ultrasonic machine until it is completely dissolved.

[0075] ② Minimum inhibitory concentration of Phellodendron chinense, honeysuckle and ginger

[0076] 10g of Phellodendron chinense, honeysuckle and ginger were added to 100mL of 70% ethanol solution, and extracted in a water bath at 85℃ for 60min. After filtering the extract, the filtrate was concentrated by rotary evaporator, placed in a 10mL volumetric flask, and fixed to 10mL with the prepared 10% dimethyl sulfoxide solution. The three medicinal solutions were diluted to 2, 1, 1 / 2, 1 / 4, 1 / 8, and 1 / 16 times the minimum inhibitory concentration (MIC) (two-fold dilution method) with 10% dimethyl sulfoxide. The minimum inhibitory concentration (MIC) of single Chinese medicine for Escherichia coli and Staphylococcus aureus is shown in Table 2.

[0077] Table 2 Minimum inhibitory concentrations of single Chinese medicines against Escherichia coli and Staphylococcus aureus

[0078]

[0079] (2) Fractional inhibitory concentration (FIC) determination

[0080] According to the MIC of single drug solution, Phellodendron amurense, Honeysuckle and Ginger were compounded in pairs by the checkerboard dilution method.

[0081] Experimental design: Take a sterile 96-well plate, add 50 μL of one medicinal extract at 2, 1, 1 / 2, 1 / 4, 1 / 8, and 1 / 16 MIC in columns 1 to 6, add 50 μL of another medicinal extract at 2, 1, 1 / 2, 1 / 4, 1 / 8, and 1 / 16 MIC in rows A to F, and then add 100 μL of 1-5×10 3 CFU / mL bacterial suspension, gently blow and mix. In addition, set up corresponding blank control group, positive growth control group, negative control group, and solvent control group as shown in Table 3. Cover the ELISA plate, place it in a constant temperature incubator at 37℃ and incubate it in the dark for 16 to 24 hours, and then use an ELISA reader to measure the OD value at 600nm. The calculation method is shown in Formula 1.

[0082] Table 3 Experimental arrangement

[0083]

[0084] (3) Calculation

[0085] The fractional inhibitory concentration (FIC) index describes the combined susceptibility of two antibacterial drugs, including four situations: synergy, addition, indifference, and antagonism. The calculation method is shown in formula (2):

[0086]

[0087] In the formula: A 0 is the MIC value when component A is used in combination; A is the MIC value when component A is used alone; B 0 is the MIC value when component B is used in combination; B is the MIC value when component B is used alone. When FIC ≤ 0.5, it is a synergistic effect; when 0.5 < FIC ≤ 1, it is an additive effect; when 1 < FIC ≤ 2, it is an indifferent effect; when FIC > 2, it is an antagonistic effect.

[0088] 3. Experimental results

[0089] 3.1 Strain counting results

[0090] According to the results, a bacterial suspension with a concentration of 1 - 5×10 3 CFU / mL was prepared. The counting result of Staphylococcus aureus was 8.6×10 8 CFU / mL.

[0091] 3.2 FIC data

[0092] (1) Staphylococcus aureus

[0093] The FIC of Phellodendron amurense and Lonicera japonica:

[0094] showed a synergistic effect, as shown in Table 4.

[0095] Table 4 Inhibitory effect of the compound of Phellodendron amurense - Lonicera japonica extract on Staphylococcus aureus (%)

[0096]

[0097]

[0098] The FIC of Phellodendron amurense and Zingiber officinale Rosc. var. yunyangense:

[0099] showed a synergistic effect, as shown in Table 5.

[0100] Table 5 Inhibitory effect of the compound of Phellodendron amurense - Zingiber officinale Rosc. var. yunyangense extract on Staphylococcus aureus (%)

[0101]

[0102] The FIC of Zingiber officinale Rosc. var. yunyangense and Lonicera japonica:

[0103] It is an additive effect, see Table 6.

[0104] Table 6 Inhibitory effect of ginger-honeysuckle extract on Staphylococcus aureus (%)

[0105]

[0106] (2) The inhibitory effects on Escherichia coli are shown in Tables 7 to 9.

[0107] FIC of Phellodendron chinense and Honeysuckle:

[0108] For antagonistic effects, see Table 7.

[0109] Table 7 Inhibitory effect of Phellodendron chinense-Lonicera japonica extract complex on Escherichia coli (%)

[0110]

[0111] FIC of Phellodendron chinense and Ginger:

[0112] For synergistic effects, see Table 8.

[0113] Table 8 Inhibitory effect of Phellodendron chinense extract complex on Escherichia coli (%)

[0114]

[0115] FIC of Honeysuckle and Ginger:

[0116] It is an additive effect, see Table 9.

[0117] Table 9 Inhibitory effect of honeysuckle-ginger extract complex on Escherichia coli (%)

[0118]

[0119]

[0120] Phellodendron chinense and ginger have synergistic effects on inhibiting Staphylococcus aureus and Escherichia coli, while Phellodendron chinense and honeysuckle have synergistic effects on inhibiting Staphylococcus aureus and antagonistic effects on inhibiting Escherichia coli. Phellodendron chinense and ginger are selected based on the results. Because the minimum inhibitory concentration of Phellodendron chinense against Staphylococcus aureus and Escherichia coli is 0.0195g / mL, and the minimum inhibitory concentration of ginger is 0.3135g / mL, the optimal concentration ratio of combined use is 1 / 4MIC Phellodendron chinense: 1 / 4MIC ginger, so the antibacterial effect of the combination of the two Chinese medicines is the mass ratio of the original medicinal materials Phellodendron chinense: ginger = 1:16.

[0121] Example 3

[0122] Minimum inhibitory concentration (MIC) of compound Phellodendron amurense liquid under different extraction methods

[0123] 1. Instruments and Reagents

[0124] 1.1 Reagents

[0125] The third generation of Escherichia coli suspension, the third generation of Staphylococcus aureus suspension, Luria-Bertani (LB) medium, sterile water, anhydrous ethanol, and dimethyl sulfoxide.

[0126] 1.2 Instruments

[0127] High-pressure steam sterilizer, shaker, constant temperature incubator, water bath, ultrasonic instrument, heating jacket, rotary evaporator, microplate reader, coater, condenser, 96-well plate, beaker, 100mL pipette, 1000mL pipette, several 100mL pipette tips, several 1000mL pipette tips, several conical flasks, several blood plates, and several 5mL centrifuge tubes.

[0128] 1.3 Medicinal materials and reference substances

[0129] The medicinal materials include Phellodendron chinense and Ginger juniper, and the reference substance is Cefuroxime Dispersible Tablets (Chengdu Beite Pharmaceutical Co., Ltd., 100 mg / tablet, drug identification code: 8165221).

[0130] 2. Experimental Procedure

[0131] 2.1 Preparation of Luria-Bertani (LB) medium

[0132] Weigh 20g of LB medium and add 1000mL of sterile water, heat to boiling and put into a reagent bottle. Sterilize with high pressure steam at 121℃ for 20min, let cool and refrigerate for later use.

[0133] 2.2 Counting of bacterial species

[0134] Take 100 μL of the fourth generation Staphylococcus aureus and Escherichia coli and dilute them to 101 and 10, respectively, with sterile water. 2 , 10 3 , 104, 105, 106, 107, 108, 109, 1010 times. Take 100 μL of the bacterial solution with different concentration gradients, evenly spread it on the blood plate, and culture it at 37℃ in the dark for 24 hours. Observe the number of colonies and calculate the colony concentration of the original bacterial solution. The colony concentration is 0-300 CFU, and record the data.

[0135] 2.3 Minimum inhibitory concentration of the drug solution under different extraction methods

[0136] (1) Preparation of reference solution

[0137] ①Solvent control solution: Take 5 mL of dimethyl sulfoxide and add 45 mL of sterile water to prepare a 10% dimethyl sulfoxide solution.

[0138] ② Negative control solution: Take a 100 mg cefixime dispersible tablet, crush it, add it to 1000 mL of sterile water, and put it in an ultrasonic machine until it is completely dissolved.

[0139] (2) Extraction and dilution of drug solution

[0140] Phellodendron chinense: Ginger medicinal material powder was extracted at a ratio of 1:16 using different extraction methods and different extraction concentrations, and the medicinal solution was diluted with 10% dimethyl sulfoxide to 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, 1 / 256, 1 / 512, and 1 / 1024 times (two-fold dilution method).

[0141] ① Water bath reflux Weigh 10g of mixed medicinal powder of Phellodendron chinense and Ginger in a ratio of 1:16 in three conical flasks, add 70% ethanol solution respectively, and extract in water bath reflux for 60min. After filtering the extract, take the filtrate and concentrate it using a rotary evaporator, place it in a 10mL volumetric flask, and dilute it to 10mL with the prepared 10% dimethyl sulfoxide solution.

[0142] ②Ultrasonic extraction: Weigh 10 g of mixed medicinal powder of Phellodendron chinense and Ginger in a ratio of 1:16 in three conical flasks, add 70% ethanol solution respectively, and ultrasonically extract for 60 min. After filtering the extract, take the filtrate and concentrate it using a rotary evaporator, place it in a 10 mL volumetric flask, and dilute it to 10 mL with the prepared 10% dimethyl sulfoxide solution.

[0143] ③ Acid-water extraction method: Weigh 10 g of mixed medicinal powder of Phellodendron chinense and Ginger juncea in a ratio of 1:16 by medicinal mass, add 0.1% hydrochloric acid solution respectively, soak for 24 hours, percolate, collect 10 times the percolate, add 0.1% sodium carbonate solution to neutralize it, and then concentrate it using a rotary evaporator, place it in a 10 mL volumetric flask, and make up to 10 mL with the prepared 10% dimethyl sulfoxide solution.

[0144] ④ Percolation extraction: Weigh 10 g of mixed medicinal powder of Phellodendron chinense and Ginger juncea in a ratio of 1:16 by medicinal mass, add 70% ethanol solution to soak for 24 hours, percolate, collect 10 times the percolate, take the percolate and concentrate it using a rotary evaporator, put it in a 10 mL volumetric flask, and make up to 10 mL with the prepared 10% dimethyl sulfoxide solution.

[0145] 2.4 Experimental arrangements

[0146] Experimental design: Add 100μL of diluent solution to columns 1-10, add 100μL of Escherichia coli suspension to rows A-B, add 100μL of Escherichia coli suspension to rows G-H, and add 100μL of culture medium suspension to rows C-F, gently blow and mix, and set up corresponding blank control group, positive growth control group, negative control group, and solvent control group. Cover the ELISA plate, place it in a constant temperature incubator at 37℃ and incubate it in the dark for 16-24h, and measure the OD value at 600nm with an ELISA reader.

[0147] Table 10 Experimental arrangement

[0148]

[0149] Table 11 Experimental design

[0150]

[0151] 3. Experimental results

[0152] GraphPad software was used to discard some offset values ​​and plot the minimum inhibitory concentration. The results are as follows: Figure 2 .from Figure 2 The results showed that the minimum inhibitory concentration (IC50) of the percolation extraction method for Escherichia coli and Staphylococcus aureus was better than that of other extraction methods, so the percolation extraction method was selected.

[0153] 2.5 Comparison of minimum inhibitory concentrations of ethanol percolation extracts at different concentrations

[0154] Weigh 10 g of mixed medicinal powder of Phellodendron chinense and Ginger juncea in a ratio of 1:16 by medicinal mass, add 60%, 70%, and 80% ethanol solutions respectively, soak for 24 hours, percolate, collect 10 times the percolate, take the percolate and concentrate it using a rotary evaporator, put it in a 10 mL volumetric flask, make up to 10 mL with the prepared 10% dimethyl sulfoxide solution, and measure the antibacterial rate of the different concentrations of the medicinal solution.

[0155] GraphPad software was used to discard some offset values ​​and plot the minimum inhibitory concentration. The results are as follows: Figure 3 .

[0156] The inhibition rate is greater than or equal to 50% (IC50) and is considered to have an antibacterial effect. Figure 3 The results showed that the IC50 of 70% and 80% ethanol diafiltration was lower, so the antibacterial effect of 70% ethanol diafiltration and 80% ethanol diafiltration was better.

[0157] Example 4

[0158] The yield of different extraction methods

[0159] 1. Instruments and Reagents

[0160] 1.1 Instruments: water bath, oven, weighing balance, evaporating dish, 1000μL pipette, and several 1000μL pipettes.

[0161] 1.2 Reagent concentrated solution.

[0162] 2. Experimental Procedure

[0163] 2.1 Weighing Weigh and number the crucibles required for the experiment.

[0164] 2.2 After extracting the mixed medicinal powder of Phellodendron chinense and Ginger according to the established process in a water bath, combine all the extracts, measure 50mL of the extract (use a pipette to draw 5mL of the medicinal solution concentrated 10 times) and put it into an evaporating dish that has been dried to constant weight. Volatilize the solvent in the evaporating dish on a 100℃ water bath, place the evaporating dish in an oven, dry it at 105℃ to constant weight, and weigh it.

[0165] 3. Experimental results

[0166] The calculation formula of the paste yield is:

[0167]

[0168] The paste yield under different conditions is shown in Table 12.

[0169] Table 12 Different extraction methods of paste yield

[0170]

[0171]

[0172] Note: -1 indicates the first experiment under this condition, and -2 indicates the second experiment under this condition.

[0173] As can be seen from Table 15, the extraction rates of 70% ethanol percolation and 60% ethanol percolation were higher. Combined with the antibacterial results, the 70% ethanol percolation extraction method was selected as the final extraction method for compound Phellodendron amurense.

[0174] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A Chinese medicine composition with antibacterial effect, characterized in that: The invention is made from 1 part of Phellodendron chinense and 16 parts of Ginger by weight.

2. The method for preparing the Chinese medicine composition according to claim 1, characterized in that: Weigh Phellodendron amurense and Ginger by weight, extract with alcohol solution by percolation, and concentrate the percolation liquid under reduced pressure.

3. The preparation method according to claim 2, characterized in that: The alcohol solution is an ethanol solution with a volume fraction of 70%.

4. Use of the Chinese medicine composition as claimed in claim 1 in the preparation of medicines for preventing and treating diseases caused by bacterial infections.

5. A drug for preventing and treating diseases caused by bacterial infection, characterized in that: Including the Chinese medicine composition according to claim 1.

6. An antibacterial liquid, characterized in that: Including the Chinese medicine composition according to claim 1.

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