Tibetan medicine rhododendron flower water extract with bacteriostatic effect and preparation method thereof

Abstract

The application discloses a Tibetan medicine rhododendron flower water extract with bacteriostatic action and a preparation method thereof, and belongs to the technical field of medicines.The preparation method of the Tibetan medicine rhododendron flower water extract is as follows: Tibetan medicine rhododendron flower medicinal materials are cut into pieces to obtain material A; the material A is placed in a beaker, double distilled water is added, and the material A is soaked at normal temperature for 11-13 hours; then the material A is dispersed in an ultrasonic dispersion machine to obtain material B; the material B is filtered and evaporated to obtain concentrated extract; the concentrated extract is placed in a storage box and freeze-dried to obtain Tibetan medicine rhododendron flower water extract dry powder; the Tibetan medicine rhododendron flower water extract dry powder and distilled water are respectively placed in centrifugal tubes and subjected to dissolving and sterilization treatment, and are sealed, stored and reserved in reagent bottles to obtain the Tibetan medicine rhododendron flower water extract.The Tibetan medicine rhododendron flower water extract prepared by the application has no toxic side effects, multiple action targets, good inhibiting effect on multiple drug-resistant Escherichia coli and can effectively reduce the incidence of calf Escherichia coli diarrhea.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a Tibetan medicine rhododendron flower water extract with antibacterial properties and its preparation method. Background Technology

[0002] Calf diarrhea is a common disease in animal husbandry, with a global morbidity rate of 20% to 100% and a high mortality rate, severely restricting the development of the cattle industry. Years of research have confirmed that *Escherichia coli* is a major cause of diarrhea in young animals. Infected calves exhibit emaciation, diarrhea, dehydration, and even death. It is endemic and sporadic, most commonly affecting calves under 10 days old, and often occurs as a co-infection or secondary infection with other pathogens. *E. coli*-induced calf diarrhea is characterized by acute hemorrhagic enteritis, with feces possessing a distinctly foul odor. *E. coli* has numerous serotypes, and there is a lack of cross-immunity between different serotypes, making vaccine prevention less than ideal. The disease is usually treated with antibiotics; however, the irrational use and abuse of antibiotics have led to increasing bacterial resistance, resulting in increasingly poor treatment efficacy and the potential for drug resistance gene transfer, posing a threat to human health.

[0003] Currently, many scholars are beginning to focus on traditional medicines with advantages such as fewer toxic side effects, high safety, wide availability, and low price, such as Chinese herbal medicine and Tibetan medicine. Tibetan medicine has achieved good results in the treatment of epidemic diseases, indicating that it contains good antibacterial and antiviral drugs. Existing studies have shown that rhododendron has a variety of pharmacological activities, including expectorant, anti-inflammatory, analgesic, immunomodulatory, and insecticidal effects. Du Baozhong et al. found that rhododendron has a broad-spectrum antibacterial effect in their study on the in vitro antibacterial activity of extracts from 17 Tibetan medicinal materials against 13 common pathogens [J] (Plateau Science Research, 2021, No. 2). However, there are few reports on the application of Tibetan rhododendron in the treatment of Escherichia coli diarrhea in calves.

[0004] Therefore, developing a Tibetan medicine, rhododendron, that is highly safe and has a good inhibitory effect on multidrug-resistant Escherichia coli could provide a new option for treating Escherichia coli-induced diarrhea in calves. Summary of the Invention

[0005] In view of the above-mentioned problems in the prior art, the present invention provides a Tibetan medicine rhododendron water extract with antibacterial effect and its preparation method. The Tibetan medicine rhododendron water extract disclosed in this invention has high safety, multiple target sites, and good inhibitory effect on multidrug-resistant Escherichia coli, and can effectively reduce the incidence of Escherichia coli-induced diarrhea in calves.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for preparing a water extract of rhododendron flower with antibacterial properties, comprising the following steps:

[0008] (1) Cut the whole rhododendron plant into pieces to obtain material A;

[0009] (2) Place the material A obtained in step (1) into a beaker, add double-distilled water to the beaker, soak at room temperature for 11-13 hours, and then place the beaker in an ultrasonic disperser for ultrasonic dispersion to obtain a uniformly dispersed material B.

[0010] (3) After filtering the material B obtained in step (2) through the first filter screen and the second filter screen, the filtrate is evaporated and concentrated to obtain a concentrated extract.

[0011] (4) Place the concentrated extract obtained in step (3) in a storage box and freeze-dry it to obtain the dry powder of Tibetan medicine rhododendron water extract;

[0012] (5) Dissolve the dry powder of Tibetan rhododendron water extract obtained in step (4) and distilled water in centrifuge tubes. After dissolution, sterilize the centrifuge tube containing the solution by placing it in boiling water for 25-30 minutes. Then, seal the sterilized solution in a reagent bottle and store it at 4°C for later use. This is how Tibetan rhododendron water extract is obtained.

[0013] Preferably, in step (2), the mass-to-volume ratio of material A to double-distilled water is 1g:(3-4)mL.

[0014] Preferably, the parameters of the ultrasonic disperser in step (2) are set as follows: temperature 50-70℃, frequency 40-60kHz, and time 35-45min.

[0015] Preferably, in step (2), the ultrasonic dispersion is an intermittent ultrasonic dispersion method, with 3 ultrasonic dispersions and an interval of 30 seconds between each ultrasonic dispersion.

[0016] Preferably, in step (3), the first filter screen has a mesh size of 60 and the second filter screen has a mesh size of 100.

[0017] Preferably, the volume of the concentrated extract in step (3) is 10% to 15% of the volume of material B.

[0018] Preferably, the freeze-drying temperature in step (4) is -80°C and the freeze-drying time is 48 to 72 hours.

[0019] Preferably, the concentration of the Tibetan medicine rhododendron water extract obtained in step (5) is 25 mg / mL.

[0020] This invention also discloses a Tibetan medicine rhododendron water extract with antibacterial properties.

[0021] This invention also provides the application of a Tibetan medicine rhododendron water extract in the preparation of a drug for treating Escherichia coli-induced diarrhea in calves.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The Tibetan rhododendron water extract disclosed in this invention has high safety, multiple target sites, and is not easily resistant to bacterial growth. In a mouse model for the prevention and treatment of Escherichia coli infection, this invention demonstrated that gavage administration of the Tibetan rhododendron water extract significantly increased serum anti-inflammatory levels (interleukin-1β, interleukin-6, and interleukin-10), significantly reduced E. coli colonization in the host intestine, effectively protected the intestinal barrier, reduced tissue damage caused by E. coli invasion, and reduced the incidence of diarrhea in mice. Furthermore, application of the Tibetan rhododendron water extract to calf E. coli-induced diarrhea revealed that it has an inhibitory effect on E. coli, with significant efficacy (e.g., ...). Figure 7 ). Attached Figure Description

[0024] Figure 1 This is a colony diagram of Escherichia coli on MacConkey agar medium;

[0025] Figure 2 This is a colony diagram of Escherichia coli on eosin methylene blue agar.

[0026] Figure 3 A microscopic image of Gram-stained Escherichia coli;

[0027] Figure 4 The image shows the results of the Escherichia coli sugar fermentation experiment.

[0028] Figure 5 The growth curve of Escherichia coli;

[0029] Figure 6 A phylogenetic tree of Escherichia coli;

[0030] Figure 7 The antibacterial efficacy curve of the Tibetan medicine rhododendron water extract against the growth of Escherichia coli provided by the present invention;

[0031] Figure 8 The effect of the Tibetan medicine rhododendron flower water extract provided by this invention on changes in body weight in mice (groups C, M, and Z represent the control group, challenge group, and experimental group, respectively);

[0032] Figure 9The effects of the Tibetan medicine rhododendron water extract provided by this invention on the liver and spleen indices of mice (groups C, M, and Z represent the control group, challenge group, and experimental group, respectively);

[0033] Figure 10 The effect of the Tibetan medicine rhododendron water extract provided by this invention on serum inflammatory factors in mice (groups C, M, and Z represent the control group, challenge group, and experimental group, respectively);

[0034] Figure 11 The effect of the Tibetan medicine rhododendron flower water extract provided by the present invention on the bacterial load in mouse organs (groups C, M, and Z represent the control group, challenge group, and experimental group, respectively);

[0035] Figure 12 The effects of the Tibetan medicine rhododendron water extract provided by this invention on the morphology of mouse ileum and cecum tissue (groups C, M, and Z represent the control group, challenge group, and experimental group, respectively);

[0036] Figure 13 The results of STAMP differential analysis at the genus level for 16S amplicon sequencing (groups C, M, and Z represent the control group, challenge group, and experimental group, respectively);

[0037] Figure 14 To analyze the overlap of significantly different metabolites screened from each group in non-targeted metabolism analysis, Venn diagrams are used to illustrate the overlap (groups C, M, and Z represent the control group, challenge group, and experimental group, respectively).

[0038] Figure 15 The differential abundance score plots of all differentially metabolized pathways in each group were used for non-targeted metabolic analysis (groups C, M, and Z represent the control group, challenge group, and experimental group, respectively). Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] This invention provides a method for preparing a water extract of rhododendron flower with antibacterial properties, comprising the following steps:

[0041] (1) Cut the whole rhododendron plant into pieces to obtain material A.

[0042] (2) Place the material A obtained in step (1) into a beaker and add double-distilled water (the mass-volume ratio of material A to double-distilled water is 1g:(3-4)mL) into the beaker. Soak at room temperature for 11-13 hours, then place the beaker into an ultrasonic disperser and set its parameters as follows: temperature 50-70℃, frequency 40-60kHZ, time 35-45min for ultrasonic dispersion. The ultrasonic dispersion is an intermittent ultrasonic dispersion method, with 3 ultrasonic dispersions and an interval of 30s between each ultrasonic dispersion to obtain a uniformly dispersed material B.

[0043] (3) After filtering the material B obtained in step (2) through a 60-mesh first filter screen and a 100-mesh second filter screen, the filtrate is evaporated and concentrated to 10% to 15% of the volume of material B to obtain a concentrated extract.

[0044] (4) Place the concentrated extract obtained in step (3) in a storage box and freeze-dry at -80℃ for 48 to 72 hours to obtain the dry powder of Tibetan medicine rhododendron water extract.

[0045] (5) Dissolve the dry powder of Tibetan rhododendron water extract obtained in step (4) and distilled water in centrifuge tubes. After dissolution, place the centrifuge tube containing the solution in boiling water for 25-30 minutes for sterilization. Then, seal the sterilized solution in a reagent bottle and store it at 4°C for later use. This yields a Tibetan rhododendron water extract with a concentration of 25 mg / mL.

[0046] This invention also discloses a Tibetan medicine rhododendron water extract with antibacterial properties.

[0047] This invention also provides the application of a Tibetan medicine rhododendron water extract in the preparation of a drug for treating Escherichia coli-induced diarrhea in calves.

[0048] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments:

[0049] Example 1

[0050] 1. Isolation, screening and identification of Escherichia coli

[0051] 1.1 Isolation and purification of Escherichia coli: The applicant collected feces from calves of yaks in Tibet for the isolation of Escherichia coli.

[0052] The specific procedure is as follows: Collected feces from diarrheal calves and yaks are aseptically inoculated into a nutrient broth medium. The broth is then placed in a constant-temperature shaker at 37°C and 180 rpm for 2 hours for amplification. 50 μL of the culture is transferred to 3 mL of LB medium and enriched by shaking at 37°C and 180 rpm for 2 hours. The enriched culture is then aseptically streaked onto MacConkey agar medium and incubated at 37°C for 18–24 hours. Once red single colonies (e.g., [missing information]) grow on the MacConkey solid medium... Figure 1 Using a sterile inoculation loop, pick a single red colony and streak it in three zones onto an eosin methylene blue agar plate. Incubate at 37°C for 24 hours and observe colony growth (e.g., ...). Figure 2 Single colonies with a black color and a greenish metallic sheen were selected and inoculated onto LB nutrient agar medium. After purification and incubation at 37°C for 24 hours, single colonies from the purified culture were smeared and Gram-stained (e.g., ...). Figure 3 ), sugar fermentation test (e.g.) Figure 4 ).

[0053] 1.2 Determination of bacterial growth curve: Thirty-three sterilized test tubes were filled with 3 mL of MRS liquid medium, and then 30 μL of overnight bacterial culture was added to each tube. The tubes were incubated at 37°C in a shaker. Three tubes were removed from each group at 0 h, 2 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h, 7 h, 9 h, 12 h, and 14 h, and the OD600 value was measured after shaking. The average value of the three parallel experiments at each time point was calculated to plot the growth curve. Figure 5 As shown in the figure, the growth curve shows a linear increase in viable bacteria from 4 to 8 hours. During this period, the bacteria grow rapidly at a stable geometric progression. The bacteria exhibit typical morphology, staining, and biological activity, and are sensitive to external environmental factors.

[0054] 1.3 Taxonomic identification of *E. coli*: Genomic DNA was extracted from single colonies using a DNA extraction kit. PCR amplification of the genomic DNA was performed using universal 16S rDNA primers, followed by sequencing to obtain the 16S rDNA gene sequence. The sequence was then entered into NCBI for BLAST alignment to construct a phylogenetic tree, such as... Figure 6 As shown in the figure, the isolated strain has a homology of 99.65% to 99.79% or higher with Escherichia coli (GenBank accession number: CP042982.1, CP055251.1), indicating extremely high similarity. The isolated strain was identified as Escherichia coli.

[0055] 2. In vitro antibacterial test of the Tibetan medicine rhododendron water extract of this invention

[0056] The *Escherichia coli* from Tibetan calves described in this embodiment was used as the pathogen indicator bacterium to test the antibacterial ability of the rhododendron water extract of the present invention.

[0057] LB broth and Tibetan rhododendron water extract were mixed to achieve concentrations of 12.5 mg / mL, 6.25 mg / mL, and 3.125 mg / mL in the culture medium, respectively. The concentration of E. coli was adjusted to 10... 6 After adding CFU / mL, the extract was added to the prepared culture medium containing the Tibetan herb Rhododendron flower water extract. All groups were cultured in a constant temperature shaker at 37℃ and 180 r / min. Equal volumes of bacterial suspension were taken from each group at 0h, 2h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 7h, and 9h, and the absorbance was measured at 600nm using a spectrophotometer. A growth curve was plotted with each time point on the x-axis and OD600nm on the y-axis. The average value of three parallel experiments at each time point was calculated to plot the inhibition curve. Figure 7 As shown in the figure, the growth curve of Escherichia coli in the culture medium containing Tibetan rhododendron water extract is significantly lower than that in the pure bacterial solution. Moreover, the Tibetan rhododendron water extract with a concentration of 12.5 mg / mL has the most significant antibacterial effect on Escherichia coli. This indicates that the Tibetan rhododendron water extract disclosed in this invention has an inhibitory effect on Escherichia coli, and the effect is obvious.

[0058] Example 2

[0059] This embodiment is an experiment demonstrating the application of the Tibetan medicine rhododendron water extract of the present invention in the prevention of Escherichia coli-induced diarrhea in mice.

[0060] Sixty mice weighing 23g±2g were randomly divided into three groups: a control group (n=20, group C) fed a basal diet and administered 0.2mL / (mice·day) of sterile saline by gavage; a challenge group (n=20, group M) fed a basal diet and administered 0.2mL / (mice·day) of sterile saline by gavage; and an experimental group (n=20, group Z) fed a basal diet and administered 0.2mL / (mice·day) of the Tibetan herbal rhododendron water extract disclosed in this invention by gavage. Starting from day 13, mice in groups M and Z were challenged by intraperitoneal injection of Escherichia coli bacterial solution (OD600=1.5). Group C received no treatment. After challenge, all groups continued to be fed according to the above feeding method for 24 hours. Blood was then collected from the eyes of mice in groups C, M, and Z. The internal organs of the mice were dissected and observed for lesions. The liver and spleen were removed, fascia and adipose tissue were removed, and the mice were weighed. The liver and spleen indices were calculated. Figure 9As shown in the figure, the liver and spleen indices of groups M and Z were higher than those of group C. Compared with group M, group Z had a lower liver and spleen index, indicating that feeding the Tibetan medicine rhododendron water extract disclosed in this invention can, to some extent, inhibit the effect of Escherichia coli on the liver and spleen of mice. From the start of the experiment, the mice in groups C, M, and Z were weighed daily. Figure 8 As shown in the figure, in the 13 days before the challenge, the body weight of mice in each group (C, M, and Z) showed a linear increase, and there was no significant difference in body weight among the groups. This indicates that the Tibetan medicine rhododendron water extract disclosed in this invention does not affect the normal growth of mice and has high safety. Starting from the 13th day, groups M and Z were challenged, while group C received no treatment. Mice in group M developed diarrhea after challenge, and their body weight decreased more significantly than that of mice in groups Z and C. Mice in groups Z and C both had mild diarrhea, but there was no significant difference in body weight between the two groups. This demonstrates that the Tibetan medicine rhododendron water extract disclosed in this invention has an inhibitory effect on Escherichia coli.

[0061] Blood collected from the eyeballs of mice in groups C, M, and Z of this embodiment was allowed to stand at room temperature for 2 hours, then centrifuged at 4°C and 4000 rpm for 10 minutes. The supernatant was collected as mouse serum and stored at 4°C for later use. Excess serum was frozen at -20°C. Serum interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10) were measured using an enzyme-linked immunosorbent assay (ELISA) kit. Figure 10 As shown in the figure, compared with group M, group Z significantly reduced the levels of IL-1β, IL-6, and IL-10 in mouse serum (P<0.05); and there was no significant difference in the levels of IL-1β, IL-6, and IL-10 in the serum of mice in groups Z and C (P<0.05). This indicates that the Tibetan medicine rhododendron water extract disclosed in this invention helps to alleviate the inflammatory response in mice caused by Escherichia coli infection.

[0062] Three mice were randomly selected from each of groups C, M, and Z in this embodiment. After blood was collected from the eyeballs, approximately 0.1g of tissue from the heart, liver, spleen, lung, kidney, duodenum, jejunum, ileum, cecum, colon, and rectum was weighed and placed in a sterile EP tube. 2mL of physiological saline and two grinding beads were added, and the mixture was thoroughly homogenized to obtain the stock solution. The stock solution was then serially diluted 10-fold with sterile physiological saline to a final concentration of 10. -6 Escherichia coli were screened and counted using MacConkey agar medium, and 20 μL of the medium was diluted 10⁻⁶ times. -1 10 -2 10 -3 10 -4 10 -5 10 -6Mix the contents thoroughly and drop them onto MacConkey sterile cell counting plates. Allow the bacterial culture to flow down naturally, then invert the plates and incubate at 37°C for 10–12 hours. Perform cell counting. Organ bacterial load is as follows: Figure 11 As shown in the figure, compared with group M, group Z showed a significant reduction in the colonization of Escherichia coli in various organs and intestinal segments (except rectum) of mice (P<0.05). This indicates that the Tibetan medicine rhododendron water extract disclosed in this invention can greatly reduce the colonization of Escherichia coli in various organs and intestinal segments (except rectum) of mice, and has a protective effect on mice infected with Escherichia coli.

[0063] Four mice were randomly selected from each of groups C, M, and Z in this embodiment. A 2cm sample of ileum and cecum was taken from each mouse, and after removing the contents, the samples were fixed in 4% paraformaldehyde fixative for 2 days. HE-stained sections were prepared, and after dehydration, soaking in soft and hard wax, paraffin embedding, sectioning (5μm), spreading, copying, drying, dewaxing, and hematoxylin-eosin staining, the prepared sections were observed under an optical microscope to examine the morphology of the ileum and colon tissue. Figure 12 As shown in the figure, the Tibetan medicine rhododendron water extract disclosed in this invention can effectively protect the intestinal barrier and reduce tissue damage caused by Escherichia coli invasion.

[0064] Example 3

[0065] This embodiment is an analysis of the detection of mouse feces in groups C, M and Z of embodiment 2 above.

[0066] (1) Sequencing analysis of mouse fecal microbial diversity

[0067] The V3-V4 variable region was amplified and sequenced using the Illumina novaseq 6000 sequencing platform. Six fecal samples were collected from each of groups C, M, and Z. DNA was extracted from the samples, tested, and then amplified by PCR. The amplified products were purified and quantitatively analyzed using fluorescence. Data quality assessment (QC) was performed at each stage from DNA extraction to sequencing. The raw sequencing data underwent quality control analysis using Qiime2 default parameters, including quality filtering, noise reduction, splicing, and dechimerism. Sequences with an abundance less than 10 (summed from all samples) were filtered out to obtain ASVs. α and β diversity index analysis and sequence depth detection were performed based on ASVs. Based on taxonomic information, community structure analysis and species difference analysis were conducted at each taxonomic level. At the genus level, significant differences between groups were identified, such as... Figure 13 As shown in the figure, the Escherichia coli-Shigella detected in group M was significantly higher than that in groups Z and C. This indicates that the Tibetan medicine rhododendron water extract disclosed in this invention can effectively reduce the colonization of Escherichia coli-Shigella in the intestines of mice.

[0068] (2) High-resolution non-targeted metabolomics analysis of mouse feces

[0069] Built on a high-resolution mass spectrometer (triple TOF), relying on its powerful high-resolution mass analyzer, this study performs unbiased, large-scale, and systematic detection of various metabolites in samples. Six fecal samples were collected from each of groups C, M, and Z. After extracting the supernatant, chromatographic-mass spectrometry (GC-MS) analysis was performed. The raw data were converted to mzXML format using ProteoWizard, and then peak alignment, retention time correction, and peak area extraction were performed using XCMS software. The data extracted by XCMS were first used for metabolite structure identification. Experimental preprocessing and data processing each required experimental quality evaluation, and finally, data analysis was conducted. Data analysis included statistical analysis of the identification results based on the number of identified metabolites and their chemical classification. Figure 14As shown, from top to bottom, the plots display the differential abundance scores for all differentially expressed metabolic pathways in C vs M, C vs Z, and Z vs M. The Y-axis represents the name of the differentially expressed pathway, and the X-axis represents the differential abundance score (DA score). The DA score represents the overall change in the expression of all metabolites in the pathway. A score of 1 indicates an upregulation trend for all identified metabolites in that pathway, while a score of -1 indicates a downregulation trend. The length of the line segment represents the absolute value of the DA score, and the size of the dot at the endpoint of the line segment indicates the number of metabolites in that pathway; a larger dot indicates a greater number of metabolites. The intensity of the line segment and dot color corresponds to the DA score. The score values ​​are proportional; the darker the red (right side of the figure), the more likely the overall expression of the pathway is upregulated, and the darker the blue (left side of the figure), the more likely the overall expression of the pathway is downregulated. The figure shows that group C identified 224 potential differentially expressed metabolites compared to group M, of which 112 were upregulated (primarily α-ionone, (2-oxo-2,3-dihydro-1h-indole-3-ylidene)-acetic acid, 1-(1z-octadecenyl)-2-(4z,7z,10z,13z,16z,19z-docosahexadecyl)-acetic acid). The group C group was mainly composed of (-)-epigallocatechin, (2-oxo-2,3-dihydro-1h-indole-3-ylidene)-acetic acid, and (1-(1z-octadecenyl)-2-(4z,7z,10z,13z,16z,19z-2-oxoyl)-sn-glycero-3-phosphoethanolamine). Group C showed 171 differentially regulated metabolites compared to Group Z, with 110 upregulated (mainly γ-aminobutyric acid, (2-oxo-2,3-dihydro-1h-indole-3-ylidene)-acetic acid, 1-(1z-octadecenyl)-2-(4z,7z,10z,13z,16z,19z-2-oxo ... The group Z showed 61 downregulated metabolites compared to the group M, with 17 upregulated metabolites (mainly 1-(1z-octadecenyl)-2-(5z,8z,11z,14z-eicosatetraenyl)-sn-glycerol-3-phosphate choline, and 1-hexadecyl)-sn-glycerol-3-phosphate choline. -2-(5z,8z,11z,14z-eicosatetraenoyl)-sn-glycerol-3-phosphocholine was the main component, with 8 downregulated components (mainly 1-hydroxyanthraquinone, 7-methylguanine, and nitrobutyl ester); this suggests that the differential metabolism between group Z and group M may be the main reason for the antibacterial effect of the Tibetan medicine rhododendron water extract disclosed in this invention on Escherichia coli; all the significantly enriched metabolic pathways mentioned above are classified and visualized according to their next higher level of pathway-hierarchy, such as... Figure 15 As shown.

[0070] The present invention provides a detailed description of a Tibetan rhododendron flower water extract with antibacterial properties and its preparation method. Specific examples have been used to illustrate the principles and implementation methods of the invention. The descriptions of these examples are merely for the purpose of helping to understand the method and core ideas of the invention. It should be noted that those skilled in the art can make various improvements and modifications to the invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. The use of a Tibetan herbal rhododendron flower water extract in the preparation of a drug for treating Escherichia coli-induced diarrhea in calves caused by Escherichia coli and Shigella, characterized in that... The drug works by reducing the colonization level of Escherichia coli-Shigella in the intestine; the concentration of the Tibetan medicine rhododendron water extract is 25 mg / mL; the preparation method of the Tibetan medicine rhododendron water extract is as follows: (1) Cut the whole rhododendron plant into small pieces to obtain material A; (2) Place the material A obtained in step (1) into a beaker and add double-distilled water to the beaker, wherein the mass-volume ratio of material A to double-distilled water is 1g:(3~4)mL; after soaking at room temperature for 11~13h, place the beaker in an ultrasonic disperser for intermittent ultrasonic dispersion, the ultrasonic frequency is 3 times, the interval between each ultrasonic dispersion is 30s, the ultrasonic parameters are set to temperature 50~70℃, frequency 40~60kHz, and total time 35~45min, to obtain uniformly dispersed material B; (3) After filtering the material B obtained in step (2) through a 60-mesh first filter screen and a 100-mesh second filter screen, the filtrate is evaporated and concentrated to 10%~15% of the volume of material B to obtain a concentrated extract. (4) Place the concentrated extract obtained in step (3) in a storage box and freeze-dry it at -80℃ for 48~72h to obtain the dry powder of Tibetan medicine rhododendron water extract; (5) Dissolve the dry powder of Tibetan rhododendron water extract obtained in step (4) and distilled water in centrifuge tubes. After dissolution, sterilize the centrifuge tube containing the solution in boiling water for 25-30 minutes. Then seal the sterilized solution in a reagent bottle and store it at 4°C for later use. This is how Tibetan rhododendron water extract is obtained.

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