Tibetan medicine smoked plum alcohol extract with bacteriostatic effect and preparation method of Tibetan medicine smoked plum alcohol extract

Abstract

The invention belongs to the technical field of medicines, and relates to a Tibetan medicine dark plum alcohol extract with a bacteriostatic effect and a preparation method thereof. Soaking the Tibetan medicine smoked plum particles for 22-26 hours by adopting an ethanol solution with the volume fraction of 94-96%; performing ultrasonic dispersion on the soaked material at the temperature of 50-70 DEG C; filtering and concentrating the material subjected to ultrasonic dispersion to prepare a concentrated extracting solution; freeze-drying the concentrated extracting solution to obtain Tibetan medicine dark plum alcohol extract dry powder. The prepared Tibetan medicine smoked plum alcohol extract is free of toxic and side effects, has multiple action targets, has a good inhibition effect on multi-drug-resistant escherichia coli, and can effectively reduce the morbidity of calf escherichia coli diarrhea.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine and relates to an ethanol extract of Tibetan medicine Prunus mume with bacteriostatic effect and a preparation method thereof. Background Art

[0002] Disclosing the information of this background art section is only intended to increase the understanding of the overall background of the present invention, and it is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Calf diarrhea is a common disease in the livestock industry, with a relatively high incidence and mortality, which seriously restricts the development of the cattle industry. Escherichia coli is one of the main pathogenic bacteria causing calf diarrhea. After infection, the typical pathological change is that calves show acute hemorrhagic enteritis, and the defecation has an obvious fishy smell. In winter, the death rate of calves suffering from diarrhea can reach 90%, bringing huge economic losses to yak farmers. In recent years, in order to prevent and treat diseases caused by Escherichia coli infection, a large amount of antibiotics have been invested in treatment. Although good results have been achieved in the short term, with the passage of time, problems such as antibiotic resistance and drug residues have emerged, causing huge economic losses and seriously restricting the healthy and sustainable development of the yak industry.

[0004] Many scholars have turned their attention to traditional medicines with advantages such as small toxic and side effects, high safety, wide sources, and low prices, such as Chinese herbal medicines. Chinese herbal medicines are rich in resources and have good antibacterial effects and properties of being difficult to produce drug resistance or reversing bacterial drug resistance. Therefore, developing a drug with high safety and good inhibitory effect on multi-drug resistant Escherichia coli can provide a new option for calf Escherichia coli diarrhea. Summary of the Invention

[0005] In order to solve the deficiencies of the prior art, the purpose of the present invention is to provide an ethanol extract of Tibetan medicine Prunus mume with bacteriostatic effect and a preparation method thereof. The ethanol extract of Tibetan medicine Prunus mume prepared by the present invention has no toxic and side effects, multiple action targets, has a good inhibitory effect on multi-drug resistant Escherichia coli, and can effectively reduce the incidence of calf Escherichia coli diarrhea.

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

[0007] First aspect, a preparation method of an ethanol extract of Tibetan medicine Prunus mume with bacteriostatic effect, comprising the following steps:

[0008] Soak the Tibetan medicine Prunus mume granules with an ethanol solution having a volume fraction of 94-96% for 22-26 h;

[0009] Perform ultrasonic dispersion on the soaked material at 50-70 °C;

[0010] Filter and concentrate the materials after ultrasonic dispersion to prepare a concentrated extract.

[0011] Freeze-dry the concentrated extract to obtain a dry powder of the ethanol extract of Tibetan medicine Prunus mume.

[0012] The present invention studies and finds that the ethanol extract of Tibetan medicine Prunus mume obtained by the above preparation method not only has good antioxidant effects, but also has good anti-inflammatory effects, and has good antibacterial effects. In particular, it has good inhibitory effects on multidrug-resistant Escherichia coli, which is beneficial to effectively protect the intestinal barrier and reduce the tissue structure damage caused by the invasion of Escherichia coli, so it can be used to prevent and treat diarrhea.

[0013] In some embodiments, the mass-volume ratio of the Tibetan medicine Prunus mume granules to an ethanol solution with a volume fraction of 94 - 96% is 1:4.5 - 5.5, g / mL. The effect of the ethanol extract of Tibetan medicine Prunus mume obtained under this condition is better.

[0014] In some embodiments, the soaking temperature is room temperature. The room temperature described in the present invention is the temperature of the indoor environment, generally 15 - 25°C.

[0015] In some embodiments, the frequency of ultrasonic dispersion is 40 - 60 kHz, and the time of ultrasonic dispersion is 35 - 45 min. Under this condition, it is beneficial to disperse the ethanol extract of Tibetan medicine Prunus mume attached to solid particles in the solvent, and at the same time, it is beneficial to further extract the active ingredients in Tibetan medicine Prunus mume, improving the extraction rate and functional effects of the ethanol extract of Tibetan medicine Prunus mume.

[0016] In some embodiments, the ultrasonic dispersion is in an intermittent ultrasonic dispersion mode, the number of ultrasonic times is 2 - 4 times, and the interval between each ultrasonic dispersion is 20 - 40 s. The extraction effect under this condition is better.

[0017] In some embodiments, the volume of the concentrated extract is 10 - 15% of the volume of the materials before ultrasonic dispersion.

[0018] In some embodiments, the temperature of freeze-drying is -85 - -75°C, and the time of freeze-drying is 48 - 72 h.

[0019] In some embodiments, add the dry powder of the ethanol extract of Tibetan medicine Prunus mume to water for reconstitution to obtain the ethanol extract of Tibetan medicine Prunus mume.

[0020] Specifically, the concentration of the dry powder of the ethanol extract of Tibetan medicine Prunus mume in the reconstituted solution is 20 - 40 mg / mL.

[0021] Specifically, sterilize the reconstituted solution. The sterilization methods include ultraviolet sterilization, boiling sterilization, etc.

[0022] On the other hand, a Tibetan medicine Prunus mume ethanol extract with antibacterial effects is obtained by the above preparation method.

[0023] In a third aspect, a pharmaceutical composition includes the ethanol extract of Tibetan medicine Prunus mume with antibacterial effect as described above, and pharmaceutical excipients.

[0024] The pharmaceutical excipients described in the present invention may be water, binder, filler, disintegrant, stabilizer, antioxidant, preservative, etc.

[0025] In a fourth aspect, an application of the ethanol extract of Tibetan medicine Prunus mume with antibacterial effect or the pharmaceutical composition as described above in the preparation of a drug for inhibiting Escherichia coli.

[0026] In some embodiments, the Escherichia coli is multidrug-resistant Escherichia coli. Specifically, the multidrug-resistant Escherichia coli is resistant to one or more of kanamycin, fosfomycin, spectinomycin, ampicillin, amoxicillin, and ceftriaxone.

[0027] In a fifth aspect, an application of the ethanol extract of Tibetan medicine Prunus mume with antibacterial effect or the pharmaceutical composition as described above in the preparation of a drug for treating calf Escherichia coli diarrhea.

[0028] In some embodiments, the treatment object of the drug is calves or mice.

[0029] The beneficial effects of the present invention are as follows:

[0030] The ethanol extract of Tibetan medicine Prunus mume prepared by the present invention has relatively small toxic and side effects, multiple action targets, and bacteria are not easily resistant to it. Through the construction of a model mouse for preventing and treating Escherichia coli infection with the ethanol extract of Tibetan medicine Prunus mume, it is found that after gavage with the ethanol extract of Tibetan medicine Prunus mume provided by the present invention, the total antioxidant capacity of the mouse serum is significantly improved, the levels of anti-inflammatory cytokines SOD and GSH are significantly increased, and the levels of pro-inflammatory cytokines TNF-α and IL-1β are significantly decreased. The colonization of Escherichia coli in host organs and the intestine is significantly reduced, the intestinal barrier is effectively protected, the tissue structure damage caused by Escherichia coli invasion is reduced, and the occurrence of mouse diarrhea diseases is reduced; when the ethanol extract of Tibetan medicine Prunus mume prepared by the present invention is applied to calf diarrheal Escherichia coli, the results show that the ethanol extract of Tibetan medicine Prunus mume provided by the present invention has the effect of inhibiting the growth of Escherichia coli, and the effect is obvious. Description of the Drawings

[0031] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0032] Figure 1 It is a colony map of Escherichia coli in MacConkey agar medium in Example 4 of the present invention;

[0033] Figure 2Colony map of Escherichia coli in Example 4 of the present invention on eosin methylene blue agar medium;

[0034] Figure 3 Gram staining microscopy image of Escherichia coli in Example 4 of the present invention;

[0035] Figure 4 Growth curve of Escherichia coli in Example 4 of the present invention;

[0036] Figure 5 Phylogenetic evolutionary tree of Escherichia coli in Example 4 of the present invention;

[0037] Figure 6 Antibacterial effect curve diagram of inhibiting the growth of Escherichia coli by the ethanol extract of Tibetan medicine Prunus mume prepared in Example 1 in Example 4 of the present invention;

[0038] Figure 7 Effect result diagram of treating (A) and preventing (B) diarrhea in mice by the ethanol extract of Tibetan medicine Prunus mume prepared in Example 1 in Example 5 of the present invention;

[0039] Figure 8 Effect result diagram of the influence of the ethanol extract of Tibetan medicine Prunus mume prepared in Example 1 on the survival rate curve of mice in Example 5 of the present invention:

[0040] Figure 9 Effect result diagram of the influence of the ethanol extract of Tibetan medicine Prunus mume prepared in Example 1 on the body weight change of mice in Example 5 of the present invention;

[0041] Figure 10 Effect result diagram of the influence of the ethanol extract of Tibetan medicine Prunus mume prepared in Example 1 on the organ index of mice (feeding time: 8d) in Example 5 of the present invention;

[0042] Figure 11 Effect result diagram of the influence of the ethanol extract of Tibetan medicine Prunus mume provided by the present invention on the serum inflammatory factors and antioxidant level of mice;

[0043] Figure 12 Effect result diagram of the influence of the ethanol extract of Tibetan medicine Prunus mume prepared in Example 1 on the bacterial load of organs (A) and intestines (B) of mice in Example 5 of the present invention;

[0044] Figure 13 Effect result diagram of the influence of the ethanol extract of Tibetan medicine Prunus mume prepared in Example 1 on the tissue morphology of duodenum, jejunum and ileum of mice in Example 5 of the present invention, A is the tissue morphology diagram, and B is the statistical result diagram. Detailed implementation mode

[0045] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments.

[0046] Example 1

[0047] A preparation method of an ethanol extract of Tibetan medicine Myrica nana with antibacterial effect, comprising the following steps:

[0048] (1) Crush the Tibetan medicine Myrica nana medicinal materials to obtain material A.

[0049] (2) Place the material A obtained in step (1) in a beaker, add 95% ethanol to the beaker (the mass-volume ratio of material A to 95% ethanol is 1 g:5 mL), seal it with plastic wrap, soak it at room temperature for 24 h, then place the beaker in an ultrasonic disperser, and set its parameters as: temperature 60 °C, frequency 60 kHz, time 40 min for ultrasonic dispersion. The ultrasonic dispersion is in the form of intermittent ultrasonic dispersion, the number of ultrasonic times is 3 times, and the interval between each ultrasonic dispersion is 30 s to obtain a uniformly dispersed material B.

[0050] (3) Filter the material B obtained in step (2) through a 60-mesh first filter screen and a 100-mesh second filter screen, and then evaporate and concentrate the filtrate to 10% of the volume of material B to obtain a concentrated extract.

[0051] (4) Place the concentrated extract obtained in step (3) in a storage box, freeze-dry it at -80 °C for 60 h to obtain a dry powder of the ethanol extract of Tibetan medicine Myrica nana;

[0052] (5) Dissolve the dry powder of the ethanol extract of Tibetan medicine Myrica nana obtained in step (4) and distilled water in centrifuge tubes respectively. After the dissolution is completed, place the centrifuge tubes containing the dissolution solution in boiling water for 30 min for sterilization treatment, then seal the sterilized dissolution solution in a reagent bottle, store it at 4 °C for standby, and thus obtain an ethanol extract of Tibetan medicine Myrica nana with a concentration of 25 mg / mL.

[0053] Example 2

[0054] A preparation method of an ethanol extract of Tibetan medicine Myrica nana with antibacterial effect, comprising the following steps:

[0055] (1) Crush the Tibetan medicine Myrica nana medicinal materials to obtain material A.

[0056] (2) Place the material A obtained in step (1) in a beaker, add 95% ethanol to the beaker (the mass-volume ratio of material A to 95% ethanol is 1 g:5 mL), seal it with plastic wrap, soak it at room temperature for 24 h, then place the beaker in an ultrasonic disperser, and set its parameters as: temperature 70 °C, frequency 60 kHz, time 35 min for ultrasonic dispersion. The ultrasonic dispersion is in the form of intermittent ultrasonic dispersion, the number of ultrasonic times is 3 times, and the interval between each ultrasonic dispersion is 30 s to obtain a uniformly dispersed material B.

[0057] (3) Filter the material B obtained in step (2) through a 60-mesh first filter screen and a 100-mesh second filter screen, and then evaporate and concentrate the filtrate to 15% of the volume of material B to obtain a concentrated extract.

[0058] (4) Place the concentrated extract obtained in step (3) in a storage box and freeze-dry it at -80°C for 72 h to obtain a dry powder of the ethanol extract of Tibetan medicine Prunus mume.

[0059] (5) Dissolve the dry powder of the ethanol extract of Tibetan medicine Prunus mume obtained in step (4) and distilled water in centrifuge tubes respectively. After dissolution, place the centrifuge tubes containing the dissolution solution in boiling water for 25 min for sterilization treatment, then seal the sterilized dissolution solution in reagent bottles and store it at 4°C for standby, thus obtaining the ethanol extract of Tibetan medicine Prunus mume with a concentration of 20 mg / mL.

[0060] Example 3

[0061] A preparation method of an ethanol extract of Tibetan medicine Prunus mume with antibacterial effect, comprising the following steps:

[0062] (1) Crush the Tibetan medicine Prunus mume medicinal materials to obtain material A.

[0063] (2) Place the material A obtained in step (1) in a beaker, add 95% ethanol (the mass-volume ratio of material A to 95% ethanol is 1 g:5 mL) to the beaker, seal it with plastic wrap, soak it at room temperature for 24 h, then place the beaker in an ultrasonic disperser, and set its parameters as: temperature 50°C, frequency 40 kHz, time 45 min for ultrasonic dispersion. The ultrasonic dispersion is in the form of intermittent ultrasonic dispersion, the number of ultrasonic times is 3 times, and the interval between each ultrasonic dispersion is 30 s, to obtain a uniformly dispersed material B.

[0064] (3) Filter the material B obtained in step (2) through a 60-mesh first filter screen and a 100-mesh second filter screen, and then evaporate and concentrate the filtrate to 13% of the volume of material B to obtain a concentrated extract.

[0065] (4) Place the concentrated extract obtained in step (3) in a storage box and freeze-dry it at -80°C for 48 h to obtain a dry powder of the ethanol extract of Tibetan medicine Prunus mume.

[0066] (5) Dissolve the dry powder of the ethanol extract of Tibetan medicine Prunus mume obtained in step (4) and distilled water in centrifuge tubes respectively. After dissolution, place the centrifuge tubes containing the dissolution solution in boiling water for 30 min for sterilization treatment, then seal the sterilized dissolution solution in reagent bottles and store it at 4°C for standby, thus obtaining the ethanol extract of Tibetan medicine Prunus mume with a concentration of 30 mg / mL.

[0067] Example 4

[0068] 1. Isolation, Screening and Identification of Escherichia coli

[0069] Isolation and purification of Escherichia coli: The applicant collected the feces of neonatal yak from Tibet for the isolation of Escherichia coli.

[0070] The specific operation method is as follows: The collected feces of diarrheal neonatal yak were aseptically inoculated into nutrient broth medium, placed in a constant temperature shaker and cultured with shaking at 37 °C and 180 r / min for 2 h for amplification. 50 μL of the culture solution was taken and inoculated into 3 mL of LB medium, and cultured with shaking at 37 °C and 180 r / min for 2 h for enrichment culture for 12 h. Then the enrichment solution was aseptically streaked and inoculated onto MacConkey agar medium and cultured at 37 °C for 18 - 24 h. When red single colonies grew on the MacConkey solid medium (such as Figure 1 ), a sterile inoculation loop was used to pick the red single colonies and streaked in three zones on an eosin methylene blue agar solid plate and cultured at 37 °C for 24 h to observe the colony growth (such as Figure 2 ). Single colonies with black and green metallic luster were picked and inoculated onto LB ordinary nutrient agar medium and purified and cultured at 37 °C for 24 h. The single colonies obtained from the purified culture were smeared and then Gram stained (such as Figure 3 ).

[0071] Determination of the growth curve of the strain:

[0072] Forty-two sterilized test tubes were each filled with 3 mL of MRS liquid medium, and then 30 μL of the overnight culture solution was added to each tube. The culture was carried out in a constant temperature shaker at 37 °C. Three test tubes were taken out at 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 12 h, 16 h, and 24 h for each group. After shaking well, the OD 600 value was measured, and the average value of three parallel experiments at each time point was calculated to plot the growth curve. As shown in Figure 4 , it can be seen from the figure that: it was in the logarithmic phase from 2 to 8 h. During this period, the number of viable bacteria on the growth curve increased linearly, and the bacteria grew extremely rapidly in a stable geometric progression. The bacterial morphology, staining, and biological activity were all typical and sensitive to the action of external environmental factors.

[0073] Taxonomic identification of Escherichia coli:

[0074] Pick a single colony and use a DNA extraction kit to extract genomic DNA. Use the 16s rDNA universal primer to perform PCR amplification on the genomic DNA, and then sequence to obtain the gene sequence of 16s rDNA. Perform a homology comparison of the sequencing sequence on the NCBI website. The 16s rRNA sequence analysis shows that the similarity between the isolated strains LZ-1 to LZ-11 and Escherichia coli is 99%. The pathogenic strain is determined to be Escherichia coli. Compare the bacterial sequencing sequence with the bacterial 16S rRNA gene sequence collected in the NCBI database by BLAST and construct a phylogenetic tree ( Figure 5 ). The selected reference gene sequences are: Escherichia coli (MN153456.1, MN208206.1, OQ891222.1, ON849073.1, OK253988.1, OQ405419.1) Staphylococcus aureus (NZ KV839669.1).

[0075] Drug sensitivity test and drug resistance analysis of Escherichia coli

[0076] Select 17 antimicrobial agents to perform drug sensitivity tests on Escherichia coli causing yak diarrhea, and judge according to the disk diffusion method stipulated by the Clinical and Laboratory Standards Institute (CLSI) of the United States. The bacteria are cultured overnight at 37°C and 180 r / min in LB liquid medium, and the bacterial liquid concentration is adjusted to 10 6 CFU / mL with normal saline. After sucking the microbacterial liquid with a pipette, add it into a nutrient agar culture dish, and evenly spread it on the LB agar medium with a disposable sterilized cotton swab. After the surface of the plate is slightly dry, stick drug sensitivity test disks on the plate in turn with sterile forceps, 4 for each culture medium. Take it out after culturing in a 37°C constant temperature incubator for 16 h, observe and measure the diameter of the inhibition zone with a vernier caliper. Do three parallel tests for each drug. The results of the drug sensitivity test are shown in Table 1. The drug resistance rate of the 11 isolated Escherichia coli strains to chloramphenicol is 100%. Some strains are resistant to kanamycin, fosfomycin, spectinomycin, ampicillin, amoxicillin, and ceftriaxone, showing multidrug resistance. But they are relatively sensitive to other antibiotics.

[0077] Table 1 Drug sensitivity test results of Escherichia coli

[0078]

[0079]

[0080] The drug-resistant genes of Escherichia coli from yaks were detected by PCR method. The drug-resistant genes of Escherichia coli include β-lactam drug-resistant genes (SHV, blaCTX-M-1, blaTEM), macrolide drug-resistant genes (ermA, ermB, ermC), tetracycline drug-resistant genes (Tet(A), Tet(B), Tet(C), Tet(D), Tet(M), Tet(K)), aminoglycoside drug-resistant genes (ant(3")-Ia, aac(6')-Ib, rmtB), and quinolone drug-resistant genes (qnr S, qep A, oqx A). The primer sequences of these drug-resistant genes are shown in Table 2. The results of PCR amplification of drug-resistant genes showed that the isolated strains carried the following drug-resistant genes: β-lactam drug-resistant genes SHV, blaCTX-M-1 and blaTEM; macrolide drug-resistant genes ermA, ermB, ermC; tetracycline drug-resistant genes Tet(A), Tet(B), Tet(C), Tet(D), Tet(M), Tet(K); aminoglycoside drug-resistant genes ant(3″)-Ia, aac(6′)-Ib, rmtB; quinolone drug-resistant genes qnr S, qep A, oqx A. The detection results of other drug-resistant genes were all negative (Table 3), and the carrying rates of the main drug-resistant genes were 9.1% - 100.00%.

[0081] Table 2 Primer sequences of Escherichia coli drug-resistant genes

[0082]

[0083]

[0084]

[0085] Table 3 Results of Escherichia coli drug-resistant gene carriage

[0086]

[0087]

[0088] 2. In vitro antibacterial test of ethanol extract of Tibetan medicine Prunus mume

[0089] Using the Escherichia coli from Tibetan calves in this example as the pathogen indicator bacteria, its antibacterial ability of the ethanol extract of Tibetan medicine Prunus mume prepared in Example 1 was detected.

[0090] Mix LB broth and the ethanol extract of Tibetan medicine Prunus mume to make the concentration of the ethanol extract of Tibetan medicine Prunus mume in the culture medium 3.125 mg / mL, and adjust Escherichia coli to 10 6After [CFU / mL], add them into the above-prepared culture medium containing the ethanol extract of Tibetan medicine Prunus mume respectively, and culture them in a constant temperature shaker at 37°C and 180 r / min. Take equal amounts of bacterial liquid at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h respectively, and measure the absorbance at 600 nm using a spectrophotometer; take each time point as the abscissa and OD 600 nm as the ordinate to make a growth curve graph, calculate the average value of three parallel experiments at each time point and draw an antibacterial curve. As Figure 6 shown, it can be seen from the figure that the growth curve of Escherichia coli in the culture medium containing the ethanol extract of Tibetan medicine Prunus mume is significantly lower than its growth curve in the pure bacterial liquid, and the growth of Escherichia coli (multi-drug resistant) is almost inhibited. Thus, it shows that the ethanol extract of Tibetan medicine Prunus mume prepared in Example 1 has the effect of inhibiting the growth of Escherichia coli (multi-drug resistant), and the effect is obvious.

[0091] Example 5

[0092] This example is an experiment on model mice for the application of the ethanol extract of Tibetan medicine Prunus mume prepared in Example 1 in the prevention and treatment of Escherichia coli infection.

[0093] After adapting 20 4-week-old clean-grade mice for three days, randomly divide them into four groups: 5 in the blank group, 5 in the model group, 5 in the prevention group, and 5 in the treatment group. The mice in the prevention group were intragastrically administered the ethanol extract of Tibetan medicine Prunus mume prepared in Example 1 (1000 mg / kg) every day. Starting from the 8th day, Escherichia coli bacterial liquid (bacterial liquid OD 600 = 1.0) 0.2 mL was injected intraperitoneally into the mice in the model group, prevention group, and treatment group for challenge. After challenge, the mice in the treatment group were intragastrically administered the ethanol extract of Tibetan medicine Prunus mume (1000 mg / kg). 24 h after challenge, observe the diarrhea situation of each group of mice. As Figure 7 shown, after intragastric administration of the ethanol extract of Tibetan medicine Prunus mume, the diarrhea rate of mice can be significantly reduced.

[0094] After adapting 15 4-week-old clean-grade mice for three days, randomly divide them into three groups: 5 in the model group, 5 in the treatment group, and 5 in the prevention group. The mice in the prevention group were intragastrically administered the ethanol extract of Tibetan medicine Prunus mume (1000 mg / kg) every day. Starting from the 8th day, 0.2 mL of Escherichia coli bacterial liquid was injected intraperitoneally into each mouse in the model group, prevention group, and treatment group for challenge (bacterial liquid OD 600 = 1.0). After challenge, the mice in the treatment group were intragastrically administered the ethanol extract of Tibetan medicine Prunus mume (1000 mg / kg), and the model group was intragastrically administered an equal amount of normal saline. Record the mortality rate of mice. As Figure 8 shown, after intragastric administration of the ethanol extract of Tibetan medicine Prunus mume, the mortality rate of mice can be significantly reduced.

[0095] After acclimating 40 4-week-old specific pathogen-free mice for three days, they were randomly divided into two groups. There were 20 mice in the model group: fed a basal diet and gavaged with 0.2 mL of sterile saline per mouse per day; there were 20 mice in the prevention group: on the basis of feeding a basal diet, gavaged with the ethanol extract of Tibetan medicine Prunus mume (1000 mg / kg). Weighed daily. As Figure 9 shown, the ethanol extract of Tibetan medicine Prunus mume had no effect on the body weight of mice. Starting from the 8th day, 0.2 mL of Escherichia coli bacterial solution (bacterial solution OD 600 = 1.0) was injected into the abdominal cavity of the mice in the model group and the prevention group for challenge. 24 hours after challenge, blood was collected from the eyes of the mice in the model group and the prevention group, and the mice were dissected to observe whether there were lesions in the internal organs. The heart, liver, spleen, lungs, and kidneys were taken out, weighed after removing the fascia and adipose tissue, and the organ index was calculated. As Figure 10 shown, the spleen index of the mice in the model group was significantly higher than that of the prevention group, indicating that feeding the ethanol extract of Tibetan medicine Prunus mume prepared in Example 1 could improve the congestion and edema of the spleen caused by Escherichia coli infection, had a protective effect on the spleen of mice, and had no obvious effect on other organs.

[0096] After the blood collected from the eyes of the mice in the model group and the prevention group of this example was allowed to stand at room temperature for 4 hours, it was centrifuged at 4000 r / min for 10 minutes at 4°C, and the supernatant was aspirated, which was the mouse serum and stored at -20°C for later use. An enzyme-linked immunosorbent assay kit was used to measure serum interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), superoxide dismutase (SOD), tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), glutathione (GSH), and total antioxidant capacity (T-AOC). As Figure 11 shown, compared with the model group, after gavaging with the ethanol extract of Tibetan medicine Prunus mume, the total antioxidant capacity T-AOC level of the mouse serum was significantly increased (P<0.05), the anti-inflammatory cytokines SOD (P<0.05) and GSH levels (P<0.05) were significantly increased, and the pro-inflammatory cytokines IL-1β (P<0.05) and TNF-α levels (P<0.01) were significantly decreased. Thus, it was shown that the ethanol extract of Tibetan medicine Prunus mume disclosed in the present invention helped to relieve the body's inflammatory response caused by Escherichia coli infection in mice.

[0097] Randomly select 3 mice from each of the model group and the prevention group in this example. After collecting blood from the eyes, weigh about 0.1 g of heart, liver, spleen, lungs, kidneys, duodenum, jejunum, ileum, cecum, colon, and rectal tissues and put them into a sterilized 2 mL Eppendorf tube, add 1 mL of physiological saline and 2 grinding beads, and homogenize thoroughly as the stock solution; make a 10-fold gradient dilution with sterile saline until diluted to 10 -6 , and use MacConkey agar medium to screen and count Escherichia coli. Aspirate 20 μL of the dilution factor of 10 -1 , 10 -2, 10 -3 , 10 -4 , 10 -5 , 10 -6 Mix the contents of -6 , -5 , -4 , and -3 respectively, and drop them onto the MacConkey sterile cell counting petri dishes. Wait for the bacterial solution to flow down naturally, then invert and culture in an incubator at 37°C for 10 - 12 h for cell counting; the bacterial load in the organs is as Figure 12 shown. Compared with the prevention group, the model group significantly reduced the colonization of Escherichia coli in various organs, duodenum, and rectum of mice (P < 0.05). This shows that the ethanol extract of Tibetan medicine Prunus mume can significantly reduce the colonization of Escherichia coli in various organs, duodenum, and rectum of mice, and play a protective role in mice infected with Escherichia coli.

[0098] Randomly select 3 mice from each of the model group and the prevention group in this example. After removing the contents, take 2-cm-long samples of the duodenum, jejunum, and ileum from each mouse and fix them in 4% paraformaldehyde fixative for 2 days to make HE staining sections. After dehydration, soaking in soft wax and hard wax, paraffin embedding, sectioning (5 μm), spreading, baking, dewaxing, hematoxylin-eosin staining, etc., place the prepared sections under an optical microscope to observe the morphological changes of the ileum and colon tissues. The villus height and crypt depth are important indicators to measure the normal function of the intestinal mucosa. The number of villi increases, the surface area of the intestinal endothelium increases, the crypts become shallower, and the maturation rate of intestinal epithelial cells increases. Therefore, the ratio of villus height to crypt depth can reflect the intestinal absorption function. As Figure 13 shown, it can be seen from the figure that after intragastric administration of the ethanol extract of Tibetan medicine Prunus mume, the villus height can be significantly increased, the crypt depth can be decreased, and the ratio of villus height to crypt depth can be increased, indicating that the ethanol extract of Tibetan medicine Prunus mume can effectively protect the intestinal barrier and reduce the damage to the intestinal tissue structure caused by Escherichia coli invasion.

[0099] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing an alcohol extract of Tibetan medicine ebony root with antibacterial effect, characterized in that: The steps include: The Tibetan medicine Wumei granules were soaked in 94-96% ethanol solution for 22-26 hours; The soaked material is ultrasonically dispersed at 50-70°C; filtering and concentrating the material after ultrasonic dispersion to obtain a concentrated extract; The concentrated extract was freeze-dried to obtain the Tibetan medicine Wumei alcohol extract dry powder.

2. The method for preparing the Tibetan medicine ebony ethanol extract with antibacterial effect as claimed in claim 1, characterized in that: The mass volume ratio of the Tibetan medicine Wumei granules to the ethanol solution with a volume fraction of 94-96% is 1:4.5-5.5, g / mL; Or, the frequency of ultrasonic dispersion is 40 to 60 kHz, and the time of ultrasonic dispersion is 35 to 45 min; Alternatively, the ultrasonic dispersion is an intermittent ultrasonic dispersion method, the number of ultrasonic dispersions is 2 to 4 times, and the interval between each ultrasonic dispersion is 20 to 40 seconds.

3. The method for preparing the Tibetan medicine ebony ethanol extract with antibacterial effect as claimed in claim 1, characterized in that: The volume of the concentrated extract is 10-15% of the volume of the material before ultrasonic dispersion; Alternatively, the freeze-drying temperature is -85 to -75°C, and the freeze-drying time is 48 to 72 hours.

4. The method for preparing the Tibetan medicine ebony ethanol extract with antibacterial effect as claimed in claim 1, characterized in that: Adding the Tibetan medicine ebony ethanol extract dry powder into water for re-dissolution to obtain the Tibetan medicine ebony ethanol extract; Preferably, the concentration of the Tibetan medicine ebony ethanol extract dry powder in the reconstituted solution is 20-40 mg / mL; Preferably, the reconstituted solution is sterilized.

5. A Tibetan medicine ebony ethanol extract with antibacterial effect, characterized in that: Obtained by the preparation method according to any one of claims 1 to 4.

6. A pharmaceutical composition, characterized in that: The invention comprises the Tibetan medicine ebony ethanol extract with antibacterial effect as claimed in claim 5, and pharmaceutical excipients.

7. Use of the Tibetan medicine Wumei alcohol extract with antibacterial effect as claimed in claim 5 or the pharmaceutical composition as claimed in claim 6 in the preparation of a drug for inhibiting Escherichia coli.

8. The use according to claim 7, characterized in that: The Escherichia coli is a multidrug-resistant Escherichia coli; preferably, the multidrug-resistant Escherichia coli is resistant to one or more of kanamycin, fosfomycin, spectinomycin, ampicillin, amoxicillin, and ceftriaxone.

9. Use of the Tibetan medicine Wumei alcohol extract with antibacterial effect as claimed in claim 5 or the pharmaceutical composition as claimed in claim 6 in the preparation of a drug for treating Escherichia coli diarrhea in calves.

10. The use according to claim 9, characterized in that: The therapeutic subjects of the drug are calves or mice.

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