Preparation process of golden juice and application of golden juice in modern disease treatment

Abstract

The invention relates to the technical field of biomedicine, in particular to a preparation process of gold juice and application of the gold juice in modern disease treatment. The preparation process of the gold juice comprises the following steps: collecting and storing excrement; diluting and filtering; sealing in a jar; long-term curing; and digging the jar to take liquid. The invention discloses an application of Jinjiang in modern disease treatment. The application comprises the following disease treatment fields: the Jinjiang is used for treating septicopyemia; the compound is used for treating intestinal related diseases, including inflammatory bowel disease and irritable bowel syndrome; the traditional Chinese medicine composition is used for treating immune system diseases, including rheumatic arthritis and systemic lupus erythematosus; the pharmaceutical composition is used for treating metabolic syndromes including obesity, diabetes and hypertension. The preparation method disclosed by the invention achieves remarkable achievements in the aspects of a preparation process, modern medical application, pharmacological research, material component analysis, curative effect verification and the like of the traditional Chinese medicine Jinjin juice, and makes positive contributions to inheritance and development of the traditional Chinese medicine Jinjin juice.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a preparation process of gold juice and application thereof in the treatment of modern diseases. Background Art

[0002] In recent years, known or unknown infectious diseases such as severe acute respiratory syndrome, influenza, Ebola hemorrhagic fever, and novel coronavirus infection have become major public health issues. Modern medicine has achieved remarkable results in preventing and treating infectious diseases, but it still faces great difficulties in terms of rapid pathogen mutation, serious antibiotic resistance, increasing dysbiosis and superinfection, and direct intervention of new and emerging infectious diseases. Sepsis caused by infection is characterized by excessive response of the systemic inflammatory system. About 49 million people are diagnosed each year worldwide, with a mortality rate of nearly 21%. "Control infection and early rehydration" is the recognized treatment principle for sepsis. However, although antiviral and antibacterial drugs can reduce the replication of pathogens, they still cannot alleviate the damage to important organs such as the heart and kidneys caused by immune clearance during the infection process. With the increase of pathogen resistance and the emergence of new sources of infection, the diagnosis and treatment of sepsis is becoming more difficult, and we still need to find new treatment methods.

[0003] (I) The treatment methods for sepsis that have emerged in recent years have not yet fully met clinical needs.

[0004] (1) Immunosuppressants are used to control the inflammatory cytokine storm in sepsis, but their clinical efficacy is poor and difficult to promote.

[0005] Sepsis is an infection caused by pathogens invading the host. There are signs of "inflammatory storm" and immunosuppression in the immune system of patients with sepsis. Therefore, sepsis requires the removal of inflammatory inducers and the suppression of the body's persistent inflammatory response. Therefore, immunosuppression may be an effective strategy for the development of drugs for the treatment of sepsis. Activation of the cell surface receptor TLR4 has a decisive influence on sepsis and septic shock caused by LPS. Therefore, a large number of drugs have been developed and designed for TLR4-related target proteins, such as methylpiperidin pyrazole, IC14 antibody, and Eritoran. Increased levels of PD-1 and PD-L1 affect the prognosis of sepsis. Related inhibitory antibodies to PD-L1, such as the new short peptide (AntiPD-L1peptide) mycophenolate mofetil (MMF), can reverse T cell dysfunction and improve the survival rate of patients with sepsis. There are also related antibodies to IL-6 and INF-a, which need to be applied early, otherwise the clinical effect is poor. In clinical trials, these drugs have not been able to meet the expectations of anti-sepsis drugs, and the treatment effects are often too delayed to reverse the outcome of the disease.

[0006] (2) There are limited means to protect important organs in the treatment of sepsis.

[0007] Infections with bacteria, viruses, rickettsiae and other microorganisms can lead to the release of inflammatory factors, endothelial dysfunction, apoptosis, etc. In addition to damaging target organs, they can also lead to dysfunction and even failure of other important organs. More and more clinical evidence shows that damage to important organs is closely related to death from infectious diseases. For example, myocardial injury seriously affects the prognosis of infectious diseases. Its incidence in sepsis caused by infection is as high as 60%, and deaths caused by it account for about 70% of the total mortality rate of sepsis. Although antibacterial and antiviral treatments can effectively control the replication and proliferation of pathogens and achieve good clinical efficacy in the early stages of infection, if damage to important organs occurs, these methods are very limited in reducing mortality. Therefore, actively seeking strategies to effectively control organ damage in sepsis is the key to further reducing the mortality rate of sepsis.

[0008] (II) Based on the view that “warm pathogens first attack the lungs and then spread to the pericardium”, “intense toxic heat” is the core pathogenesis of the inflammatory factor storm caused by sepsis and the damage to important organs. Therefore, traditional Chinese medicines that clear away heat and detoxify are the key drugs for treating sepsis.

[0009] Traditional Chinese medicine has achieved good results in understanding, preventing and treating infectious diseases based on the Wei Qi Ying Xue syndrome differentiation system. For example, Baihu Decoction is used to treat epidemic encephalitis B according to the Qi syndrome differentiation of Yangming heat, and Qingying Decoction is used in combination with Yinqiao Powder, Ma Xing Shi Gan Decoction, and Qianjin Weijing Decoction according to the Wei Qi Ying Xue syndrome differentiation to treat atypical pneumonia and influenza, both of which have achieved good results.

[0010] Clearing away heat and detoxifying is considered by TCM physicians as the key treatment for sepsis. The Treatise on Febrile Diseases states: "When heat toxins invade the heart... they cannot be resolved without... cooling medicine." This points out the extreme and acute nature of heat toxins in sepsis, which cannot be stopped without extremely cooling medicines.

[0011] (III) The clinical application of traditional Chinese medicines that are good at clearing away heat and detoxifying is limited. Jinzhi is very cooling and has strong heat-clearing and detoxifying effects, so it has important development significance.

[0012] Many traditional Chinese medicines that are good at clearing heat and detoxifying, such as natural bezoar, are greatly limited in clinical application. The physical and chemical properties and efficacy of substitutes such as artificial bezoar are not ideal and cannot meet clinical needs. "Ben Cao Qiu Zhen" records that Jin Jue can "greatly detoxify heat. When damp heat is prevalent, the toxicity is overwhelming, and the situation is dangerous and cannot be controlled. Use this to pour it, and swallow it down a little. Because its smell is similar. Therefore, it can go straight into the nest and break the poison." It can be seen that the traditional Chinese medicine Jin Jue is very cool and has a strong ability to clear heat and detoxify. Literature excavation found that Jin Jue can be used in the Qi Ying syndrome of febrile diseases to clear Ying and detoxify. There are 18 articles involving Qi syndrome in the literature, with symptoms of abdominal distension and constipation, thirst and drinking, and sweating; there are 58 articles involving Ying syndrome, with symptoms of high fever, coma and delirium, and insomnia at night. These symptoms are equivalent to critical cases caused by infection in modern medicine. Therefore, actively developing Jin Jue has important clinical significance. Summary of the invention

[0013] The present invention provides a preparation process of golden juice and its application in modern disease treatment, and has achieved remarkable results in the preparation process of traditional Chinese medicine golden juice, modern medical application, pharmacological research, material component analysis and efficacy verification, and has made positive contributions to the inheritance and development of traditional Chinese medicine golden juice.

[0014] The technical solution adopted by the present invention is: a preparation process of gold juice, comprising the following steps:

[0015] Step 1: Feces collection and storage: Collect fresh feces, put them into containers, and store them at -20℃ to ensure the basic efficacy;

[0016] Step 2: dilution and filtration: dilute the feces with distilled water and filter with gauze to remove the residue and retain the pure feces dilution;

[0017] Step 3: Sealing the jar: Pour the filtered fecal dilution into a clean porcelain jar, cover the jar mouth with a porcelain bowl, and then seal the jar mouth with yellow clay stirred with salt water to ensure sealing;

[0018] Step 4: Long-term aging: bury the porcelain jar 1 meter underground for at least 1 year, during which time it will ferment naturally;

[0019] Step 5, dig out the jar and take the liquid: dig out the porcelain jar, remove the seal, and take the yellowish transparent liquid on the upper layer, which is the gold juice.

[0020] As a further improvement of the present invention, the inclusion criteria for the selection of stool donors include: healthy children aged 6-14 years; no history of using antibiotics in the last 3 months; no history of using intestinal microecological preparations in the last 3 months.

[0021] As a further improvement of the present invention, the exclusion criteria for selecting stool donors include: having digestive system diseases and digestive system symptoms such as gastrointestinal tumors, Crohn's disease, ulcerative colitis, and constipation; having a history of immune system diseases and a history of using any immunosuppressants; traveling to areas with endemic diarrhea in the past 6 months; and having a history of infectious diseases in the past 3 months.

[0022] As a further improvement of the present invention, the ratio of feces to distilled water is 1:6.

[0023] The application of a kind of golden juice in modern disease treatment includes the following disease treatment fields:

[0024] Used to treat sepsis, by clearing away heat and detoxifying, reducing inflammatory factor storm, protecting important organ functions, and reducing mortality;

[0025] Used to treat intestinal diseases, including inflammatory bowel disease and irritable bowel syndrome, by regulating the balance of intestinal flora, improving the intestinal microenvironment, and alleviating symptoms;

[0026] Used to treat immune system diseases, including rheumatoid arthritis and systemic lupus erythematosus, by regulating immune function, reducing inflammatory response and improving the condition;

[0027] Used to treat metabolic syndrome, including obesity, diabetes, and hypertension, by improving the structure of intestinal flora, regulating metabolic pathways, and improving metabolic indicators.

[0028] As a further improvement of the present invention, the analysis of the gold juice components uses 16SrDNA combined with non-targeted metabolomics technology to fully understand its active ingredients and mechanism of action.

[0029] As a further improvement of the present invention, the 16SrDNA analysis technology is used to identify and quantify the microbial community structure in the fecal dilution solution, revealing the changing patterns of different microorganisms during the preparation process of golden juice; the non-targeted metabolomics technology is used to analyze the small molecule metabolites in golden juice, exploring its potential action pathway in disease treatment.

[0030] As a further improvement of the present invention, the gold juice can be combined with other treatment methods of modern medicine during use, including antibiotics, antiviral drugs, and immunosuppressants, to improve the treatment effect.

[0031] The beneficial effects of the present invention are as follows: (1) The present invention realizes the preparation method of the traditional Chinese medicine Jin Juice through a scientific and rigorous process, thereby ensuring the preparation quality and stability of the efficacy of the Jin Juice; on this basis, the application of the Jin Juice in modern medicine is confirmed, including its application potential in the fields of sepsis, intestinal-related diseases, immune system diseases, metabolic syndrome treatment, etc.; at the same time, by screening the effective drug ingredients in the Jin Juice (such as intestinal flora and specific compounds), combined with animal experiments, it is confirmed that the Jin Juice has a significant therapeutic effect in the treatment of diseases such as sepsis, which effectively makes up for the shortcomings of current treatment methods in controlling organ damage and reducing mortality.

[0032] (2) The present invention innovatively adopts a method combining 16SrDNA with non-targeted metabolomics technology to conduct a comprehensive and accurate analysis of the microbial community structure and small molecule metabolites in golden juice; 16SrDNA technology can identify and quantify the microbial community in golden juice and reveal its changing patterns during the preparation process; while non-targeted metabolomics technology can deeply analyze the small molecule metabolites in golden juice and explore their potential action pathways in disease treatment; it not only improves the accuracy and comprehensiveness of golden juice component analysis, but also provides a solid scientific basis for subsequent pharmacological research and clinical application. DETAILED DESCRIPTION

[0033] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0034] The present invention provides a process for preparing gold juice, comprising the following steps:

[0035] Step 1: Feces collection and storage: Collect fresh feces, put them into containers, and store them at -20℃ to ensure the basic efficacy;

[0036] Step 2: dilution and filtration: dilute the feces with distilled water and filter with gauze to remove the residue and retain the pure feces dilution;

[0037] Step 3: Sealing the jar: Pour the filtered fecal dilution into a clean porcelain jar, cover the jar mouth with a porcelain bowl, and then seal the jar mouth with yellow clay stirred with salt water to ensure sealing;

[0038] Step 4: Long-term aging: bury the porcelain jar 1 meter underground for at least 1 year, during which time it will ferment naturally;

[0039] Step 5, dig out the jar and take the liquid: dig out the porcelain jar, remove the seal, and take the yellowish transparent liquid on the upper layer, which is the gold juice.

[0040] The inclusion criteria for the selection of stool donors of the present invention include: healthy children aged 6-14 years; no history of antibiotic use in the last 3 months; no history of intestinal microecological preparation use in the last 3 months. The exclusion criteria for the selection of stool donors include: digestive system diseases and digestive system symptoms such as gastrointestinal tumors, Crohn's disease, ulcerative colon, and constipation; history of immune system diseases and use of any immunosuppressants; travel to endemic diarrhea areas in the last 6 months; history of infectious diseases in the last 3 months.

[0041] The application of a kind of golden juice in modern disease treatment includes the following disease treatment fields: used for treating sepsis, by clearing away heat and detoxifying, reducing inflammatory factor storm, protecting weight organ function and reducing mortality; used for treating intestinal related diseases, including inflammatory bowel disease and irritable bowel syndrome, by regulating the balance of intestinal flora, improving intestinal microenvironment and alleviating symptoms; used for treating immune system diseases, including rheumatoid arthritis and systemic lupus erythematosus, by regulating immune function, reducing inflammatory response and improving the condition; used for treating metabolic syndrome, including obesity, diabetes and hypertension, by improving intestinal flora structure, regulating metabolic pathways and improving metabolic indicators.

[0042] The gold juice component analysis of the present invention combines 16SrDNA with non-targeted metabolomics technology to fully understand its active ingredients and mechanism of action. 16SrDNA analysis technology is used to identify and quantify the microbial community structure in fecal dilutions, revealing the changing patterns of different microorganisms during the preparation of gold juice; non-targeted metabolomics technology is used to analyze small molecule metabolites in gold juice to explore its potential pathways of action in disease treatment.

[0043] During use, the golden juice of the present invention can be combined with other treatment methods of modern medicine, including antibiotics, antiviral drugs, and immunosuppressants, to improve the treatment effect.

[0044] Embodiment 1 (preparation process of gold juice)

[0045] (1) Selection of fecal donors

[0046] Inclusion criteria: ① healthy children aged 6-14 years; ② no history of antibiotic use in the last 3 months; ③ no history of intestinal probiotic use in the last 3 months.

[0047] Exclusion criteria: ① having digestive diseases and digestive system symptoms such as gastrointestinal tumors, Crohn's disease, ulcerative colitis, constipation, etc.; ② having a history of immune system diseases and a history of using any immunosuppressants; ③ not having traveled to areas with endemic diarrhea in the past 6 months; ④ having a history of infectious diseases in the past 3 months.

[0048] (2) Feces collection

[0049] Five healthy children were screened, including two males and three females with an average age of 7.52 years. Their fresh feces were collected and stored in a 4°C refrigerator for later use.

[0050] (3) Preparation of Gold Juice

[0051] Place feces in a container and store it at minus 20 degrees Celsius to ensure the basic efficacy; dilute the feces with distilled water (the ratio of feces to distilled water is 1:6), and filter it with gauze to remove the residue and retain the pure feces dilution; pour the filtered feces dilution into a clean porcelain jar, cover the jar mouth with a porcelain bowl, and then seal it with yellow clay stirred with salt water to ensure it is airtight; bury the porcelain jar 1 meter underground for at least 1 year, during which time it will ferment naturally; dig out the porcelain jar, remove the seal, and take the upper layer of slightly yellowish transparent liquid, which is the gold juice.

[0052] Example 2 (Animal Experimental Method for Evaluating the Efficacy of Gold Juice)

[0053] 1. Experimental animals

[0054] Healthy male C57BL / 6 mice weighing 18-22 g were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., license number: SCXK (Beijing) 2019-0008. The temperature of the breeding environment was maintained at 25±2℃, the light-dark cycle was replaced by 12h, and the mice were adaptively fed under conventional experimental conditions with free drinking water and food for 1 week.

[0055] (II) Main reagents

[0056]

[0057]

[0058]

[0059] (III) Main instruments

[0060]

[0061]

[0062] 4. Research Methods

[0063] (1) Experimental groups

[0064] ①Sixty C57BL / 6 mice were randomly divided into a normal group (Control), a model group (Model), and a Jinzhi group (Jinzhi), with 20 mice in each group; the effect of Jinzhi on the 72h survival rate and MSS score of septic mice was observed

[0065] ②Forty-eight C57BL / 6 mice were randomly divided into a normal group (Control), a model group (Model), a Jinzhi group (Jinzhi) and a dexamethasone-positive drug group (DXMS), with 12 mice in each group; the protective effect of Jinzhi on cardiac damage in septic mice was detected.

[0066] (2) Dosage

[0067] Preliminary experiments were conducted on three doses (concentrations) of 50mL (0.13mL / 20g), 100mL (0.26mL / 20g), and 200mL (0.52mL / 20g). It was found that 100mL (0.26mL / 20g) was the best dose (concentration). The dose (concentration) of gold juice in this experiment was 100mL (0.26mL / 20g). The dose of dexamethasone acetate tablets was 2.25mg / 70kg / d, which was equivalent to a dose of 5.46μg / 20g for mice.

[0068] Note: According to the body surface area method in "Pharmacological Experimental Methodology" edited by Professor Xu Shuyun, the dosage for a 20g mouse is 9.1 times that for a 70kg adult.

[0069] (3) Dosage regimen

[0070] ① Each mouse in the normal group and model group was gavaged with 13 mL / kg of saline; each mouse in the gold juice group was gavaged with 13 mL / kg of gold juice. Two hours later, mice in the normal group were intraperitoneally injected with 15 mg / kg of saline, and mice in the model group and gold juice group were intraperitoneally injected with 15 mg / kg of LPS to establish a sepsis infection model.

[0071] ② Each mouse in the normal group and model group was gavaged with 13 mL / kg of saline; each mouse in the gold juice group was gavaged with 13 mL / kg of gold juice; each mouse in the positive drug group was gavaged with 273 μg / kg of dexamethasone. Two hours later, mice in the normal group were intraperitoneally injected with 15 mg / kg of saline, and mice in the other three groups were intraperitoneally injected with 15 mg / kg of LPS to establish a sepsis infection model.

[0072] (4) Observation of efficacy indicators

[0073] ① Survival time of septic mice: After the golden juice gavage intervention and LPS establishment of the septic infection model, the number of mice deaths in each group was recorded every 12 hours from 0 to 72 hours. Finally, the 72-hour survival rate of mice in each group was calculated.

[0074] ② MSS score of septic mice: After intragastric administration of gold juice and establishment of sepsis infection model with LPS, the MSS statistical score of mice in each group was performed at 72 hours. The scoring content included six aspects: appearance, activity, response to stimulation, consciousness, eye movement and breathing. The six aspects and the total score were statistically analyzed and the average score was calculated.

[0075] ③ Detection of inflammatory factor levels: 24 hours after the LPS sepsis infection model was established, mice were anesthetized with chloral hydrate to collect blood from the abdominal aorta. The arterial blood of each mouse was collected into a sodium citrate blood collection tube, and the blood was placed at 4°C for 20 min after slight up and down shaking. After the anticoagulant effect was produced, the blood was centrifuged at a speed of 3000 r / min-1. The supernatant was collected after centrifugation for 15 min, and the content of key inflammatory factors in the serum of each group of mice was detected by ELISA: IL-6, IL-10, TGF-β1, TNF-α, and MCP-1.

[0076] Example 3 (Technology of using 16sDNA to analyze bacterial flora in gold juice)

[0077] (1) Main reagents and instruments

[0078] CTAB (prepared by Shanghai Ouyi Biomedical Technology Co., Ltd.), Qubit dsDNA Assay Kit (Life Technologies, Cat. No. Q328520), HiFi Hot Start Ready Mix high-fidelity enzyme (Kapa Biosystems, Cat. No. KK2501), desktop high-speed centrifuge (Eppendorf Centrifuge, model 5418), PCR instrument (Bio-rad, model 580BR10905), electrophoresis instrument (Tanon, model HE-120), gel imager (Tanon, model 2500), pipette (Eppendorf), Agilent 2100 Bioanalyzer chip analysis system, Illumina Miseq high-throughput sequencing platform.

[0079] (2) DNA extraction and PCR amplification

[0080] Thaw the samples stored at -80℃ at room temperature. The genomic DNA of the samples was extracted by CTAB method, and then the purity and concentration of DNA were detected by agarose gel electrophoresis. Then take an appropriate amount of sample in a centrifuge tube and dilute the sample to 1ng / μl with sterile water. Using the diluted genomic DNA as a template, according to the selection of the sequencing region, use specific primers with Barcode, front primer: 338F-5'-ACTCCTACGGGAGGCAGCA3'; back primer: 806R-5'-GGACTACHVGGGTWTCTAAT3', and use HiFi Hot Start Ready Mix high-fidelity enzyme (Kapa Biosystems, Cat. No. KK2501) for PCR.

[0081] (3) Purification and sequencing of PCR products

[0082] The PCR product was detected by electrophoresis, purified by magnetic beads, used as a template for the second round of PCR, and amplified by the second round of PCR, and detected by electrophoresis again, purified by magnetic beads, and the 16SrRNA V3-4 region PCR product was quantified by Qubit. Equal amounts of samples were mixed according to the concentration of PCR products and sequenced.

[0083] (4) Biological analysis

[0084] Trimmomatic software was used to accurately remove the original double-end sequences, and sequences containing ambiguous bases (ambiguous), single-base high-repetitive regions (homologous), and sequences that were too short. The parameters for accurate removal of impurities are: remove sequences containing N bases, and retain sequences with a base quality score Q20 of at least 75%. At the same time, usearch was used to detect and remove chimeric sequences in the sequences. After the sequencing data was preprocessed to generate high-quality sequences, the UPARSE software was used to classify the sequences into multiple OTUs based on the similarity of the sequences. The parameter is that sequences with a similarity greater than or equal to 97% are classified as one OTU unit. The representative sequences of each OTU were selected using the QIIME software package, and all representative sequences were compared and annotated with the Greengenes database. The species comparison and annotation used the RDP classifier software, and the annotation results with a confidence interval greater than 0.7 were retained.

[0085] Example 4 (Non-targeted metabolomics analysis of gold juice components)

[0086] (1) Main reagents and instruments

[0087] Methanol, formic acid, water, and acetonitrile were purchased from CNW Company, L-2-chlorophenylalanine was purchased from Shanghai Hengchuang Biotechnology Co., Ltd., Lyso PC17:0. Fully automatic sample rapid grinding instrument (Shanghai Jingxin Industrial Development Co., Ltd., JXFSTPRP-24 / 32), ultrasonic cleaning machine (Ningbo Xinzhi Biotechnology Co., Ltd., SB-5200DT), vortex oscillator (Shanghai Hannuo Instrument Co., Ltd., TYXH-I), desktop high-speed refrigerated centrifuge (Shanghai Luxiangyi Centrifuge Instrument Co., Ltd., TGL-16MS), high-resolution mass spectrometer (Waters, Xevo G2-XS QTof), high-performance liquid chromatograph (Waters, ACQUITY UPLC), chromatographic column [Waters, ACQUITY UPLC BEH C18 (100*2.1mm, 1.7μm)].

[0088] (2) Sample pretreatment

[0089] All extraction reagents were precooled at -20℃ before use. Take 200μL of sample and put it into a 1.5mL centrifuge tube, centrifuge for 15min (13000rpm, 4℃), take 100ul supernatant; then add 20μL of internal standard (L-2-chlorophenylalanine, 0.3mg / mL; C-17, 0.01mg / mL, both in methanol), add 400ul methanol-acetonitrile (V:V=2:1), vortex oscillation for 1min, ultrasonic extraction in an ice water bath for 5min, and stand at -20℃ for 30min; centrifuge for 10min (13000rpm, 4℃), take 200μL of supernatant and put it into an LC-MS injection vial with an inner liner; quality control samples (QC) were prepared by mixing equal volumes of the extracts of all samples, and the volume of each QC was the same as that of the sample.

[0090] (3) Liquid chromatography-mass spectrometry analysis conditions

[0091] The study used an ultra-high performance liquid chromatography-mass spectrometry system coupled with a Xevo G2-XS QTof high-resolution mass spectrometer for data collection and analysis.

[0092] Chromatographic conditions:

[0093] ①Chromatographic column: ACQUITY UPLC BEH C18 (100mm×2.1mm, 1.7um); ②Column temperature: 45℃; ③Mobile phase: A-water (containing 0.1% formic acid), B-acetonitrile (containing 0.1% formic acid); ④Flow rate: 0.4mL / min; ⑤Injection volume: 2μL.

[0094] The elution gradient is shown in the following table.

[0095]

[0096]

[0097] Mass spectrometry conditions:

[0098] ① Ion source: ESI; sample mass spectrometry signal acquisition uses positive and negative ion scanning modes respectively.

[0099] The mass spectrometry parameters are listed in the following table.

[0100] Parameter Positive / Negative Ion Capillary Voltages, kV +3，-2 Sampling Cone, V 40 Source Offset, V 80 Source Temperature, °C 120 Desolvation Temperature, °C 450 Desolvation Gas Flow, L / h 800 Cone Gas Flow, L / h 50 Mass Range, amu 50-1000 Scan Time, s 0.1 Collision Energy (Low Energy) 6 Ramp Collision Energy (High Energy) 20-35 Scan Type <![CDATA[MS E centroid]]>

[0101] (4) Data processing

[0102] UNIFI 1.8.1 software was used to collect raw data, and the precursor tolerance, product tolerance, and production threshold of Progenesis QI v2.3 software (NonlinearDynamics, Newcastle, UK) were set to 5 ppm, 10 ppm, and 5%, respectively, for baseline filtering, peak identification, integration, retention time correction, peak alignment, and normalization. The Human Metabolome Database (HMDB), Lipidmaps (v2.3), and METLIN databases were used to identify and characterize compounds based on accurate mass, secondary fragmentation, and isotope distribution.

[0103] Delete the ion peaks with missing values ​​(0 value) > 50% in the extracted data group, and screen the compounds according to the qualitative results of the compounds with a score of ≥ 30 points (out of 60 points). The qualitative results < 30 points are considered inaccurate and deleted. The data obtained after screening are standardized, and the obtained ion intensities are LOG transformed, medianized, and auto-scaling standardized before data analysis.

[0104] In summary, the preparation process of the gold juice of the present invention and its application in the treatment of modern diseases realize the preparation process of the traditional Chinese medicine gold juice through scientific methods, and confirm its therapeutic potential in modern medicine for sepsis, intestinal diseases, immune diseases, metabolic syndrome and adjuvant treatment of cancer; at the same time, combined with 16SrDNA and non-targeted metabolomics technology, the microbial community and small molecule metabolites in the gold juice are comprehensively analyzed, the accuracy and comprehensiveness of the component analysis are improved, and the foundation is laid for the pharmacological research and clinical application of the gold juice.

[0105] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A process for preparing gold juice, characterized in that: The following steps are involved: Step 1: Feces collection and storage: Collect fresh feces, put them into containers, and store them at -20℃ to ensure the basic efficacy; Step 2: dilution and filtration: dilute the feces with distilled water and filter with gauze to remove the residue and retain the pure feces dilution; Step 3: Sealing the jar: Pour the filtered fecal dilution into a clean porcelain jar, cover the jar mouth with a porcelain bowl, and then seal the jar mouth with yellow clay stirred with salt water to ensure sealing; Step 4: Long-term aging: bury the porcelain jar 1 meter underground for at least 1 year, during which time it will ferment naturally; Step 5, dig out the jar and take the liquid: dig out the porcelain jar, remove the seal, and take the yellowish transparent liquid on the upper layer, which is the gold juice.

2. The process for preparing gold juice according to claim 1, characterized in that: The inclusion criteria for the stool donor selection include: healthy children aged 6-14 years; no history of antibiotic use in the past 3 months; no history of intestinal probiotic use in the past 3 months.

3. The process for preparing gold juice according to claim 1, characterized in that: The exclusion criteria for the selection of stool donors include: digestive system diseases and digestive system symptoms such as gastrointestinal tumors, Crohn's disease, ulcerative colitis, and constipation; a history of immune system diseases and a history of using any immunosuppressants; travel to areas with endemic diarrhea in the past 6 months; and a history of infectious diseases in the past 3 months.

4. The process for preparing gold juice according to claim 1, characterized in that: The ratio of feces to distilled water was 1:

6.

5. An application of gold juice in the treatment of modern diseases, characterized in that: The following disease treatment areas are included: Used to treat sepsis, by clearing away heat and detoxifying, reducing inflammatory factor storm, protecting important organ functions, and reducing mortality; Used to treat intestinal diseases, including inflammatory bowel disease and irritable bowel syndrome, by regulating the balance of intestinal flora, improving the intestinal microenvironment, and alleviating symptoms; Used to treat immune system diseases, including rheumatoid arthritis and systemic lupus erythematosus, by regulating immune function, reducing inflammatory response and improving the condition; Used to treat metabolic syndrome, including obesity, diabetes, and hypertension, by improving the structure of intestinal flora, regulating metabolic pathways, and improving metabolic indicators.

6. The use of a golden juice in the treatment of modern diseases according to claim 5, characterized in that: The gold juice component analysis uses 16SrDNA combined with non-targeted metabolomics technology to fully understand its active ingredients and mechanism of action.

7. The use of a golden juice in the treatment of modern diseases according to claim 6, characterized in that: The 16SrDNA analysis technology is used to identify and quantify the microbial community structure in the fecal dilution solution, revealing the changing patterns of different microorganisms during the preparation of golden juice; the non-targeted metabolomics technology is used to analyze the small molecule metabolites in golden juice and explore its potential action pathway in disease treatment.

8. The use of a golden juice in the treatment of modern diseases according to claim 5, characterized in that: During use, the golden juice can be combined with other treatment methods of modern medicine, including antibiotics, antiviral drugs, and immunosuppressants, to improve the treatment effect.

Images