Extraction and application of uric acid-reducing active component of gynura procumbens

Abstract

The invention discloses extraction and application of uric acid reducing active components of gynura procumbens, and relates to the technical field of traditional Chinese medicine efficacy material basis and action mechanism research, and the method comprises the following steps: extract preparation, intestinal flora regulation effect verification, flora intermediary effect verification through coprophilous fungus transplantation, and action mechanism verification. In the invention, in the aspect of mechanism, a flora-host co-metabolism theory is introduced, and a new idea that the extract can indirectly influence uric acid metabolism through intestinal local regulation flora is provided; in the aspect of flora regulation, the extract can specifically improve the abundance of A. indinctus by about 2 times, the hippuric acid generation amount is accurately promoted to be increased by 30.3%, and the uncertainty of conventional intervention is avoided; a coprophilous fungus transplantation experiment shows that the uric acid reducing effect of flora transplantation is obviously superior to that of a pure component effect, and a brand new uric acid reducing mechanism is disclosed; in an application scene, the traditional uric acid reducing way is bypassed, the intestinal ABCG2 pathway is activated, and a new hope is brought to a drug-resistant patient.

Description

Technical Field

[0001] The present invention relates to the technical field of research on the material basis and mechanism of action of traditional Chinese medicines, and specifically to the extraction and application of active ingredients with uric acid-lowering activity from Gynura procumbens (Lour.) Merr. Background Art

[0002] In the field of uric acid-lowering research, traditional uric acid-lowering drugs such as allopurinol and febuxostat, as well as research on plant extracts, have all highly focused on the host's own uric acid metabolism pathway. Specifically, it mostly reduces the biosynthesis of uric acid by inhibiting the activity of xanthine oxidase and blocking the conversion process of hypoxanthine to xanthine and xanthine to uric acid; or enhances the secretion function of the proximal renal tubules of the kidney to uric acid, while inhibiting its reabsorption function, and promotes the excretion of uric acid by the kidney, so as to achieve the goal of reducing blood uric acid levels. This is the conventional research idea generally followed by those skilled in the art.

[0003] However, as the "second genome" of the human body, the gut microbiota plays an important role in the process of uric acid metabolism. The gut microbiota can degrade uric acid into allantoin with higher solubility and easier excretion through the uricase contained in itself, thereby reducing the uric acid level in the body; at the same time, it can regulate the expression and function of the intestinal uric acid transporter ABCG2, affect the absorption and excretion process of uric acid in the intestine, and maintain the metabolic balance of uric acid. Although there have been sporadic research reports on the association between the gut microbiota and uric acid metabolism in recent years, for example, the research published in "Gut" in 2021 revealed the connection between the gut bacterium Klebsiella and hyperuricemia, but so far, no research has deeply analyzed and successfully established an organic connection between the gut microbiota and the pharmacological mechanism of Gynura procumbens (Lour.) Merr.

[0004] In addition, traditional methods for analyzing the components of plant medicines mainly focus on endogenous components in the host, only paying attention to the components of plant medicines that are digested, absorbed in the host's gastrointestinal tract, enter the blood circulation system, and play a role in various tissues and organs in the body after oral administration, seriously ignoring the metabolic transformation and subsequent effects of the "in vitro metabolic organ" of the gut microbiota on the components of plant medicines. The gut microbiota can modify the structure of plant medicine components through its rich enzyme system, changing their pharmacological activities and pharmacokinetic properties.

[0005] Meanwhile, in the existing technical system, metagenomics and fecal microbiota transplantation (FMT) technologies are mainly applied in the field of disease diagnosis. For example, through metagenomics technology, a comprehensive analysis of the gene composition and functions of the gut microbiota is carried out to assist in the diagnosis of gut microbiota-related diseases; and they are also used in the treatment of intestinal diseases. For instance, for severe intestinal inflammation caused by Clostridium difficile infection, fecal microbiota transplantation technology is adopted to reconstruct the gut microbiota balance of patients to achieve the treatment purpose. However, in the research on the mechanism of plant medicine, the application of metagenomics and fecal microbiota transplantation technologies is almost blank, and no research has attempted to deeply explore the mechanism of action of plant medicine in the body with these technologies.

[0006] In view of this, there is an urgent need for an innovative research idea and advanced technical means to break through the limitations of traditional research, deeply explore the potential mechanism of action of Gynura procumbens in reducing uric acid, expand its application value in the field of uric acid reduction treatment, and meet the urgent clinical needs for new and highly effective uric acid-lowering drugs and therapies. Summary of the Invention

[0007] The purpose of the present invention is to provide an extraction and application of uric acid-lowering active ingredients from Gynura procumbens to solve the problems raised in the above background technology.

[0008] To solve the above technical problems, an extraction and application of uric acid-lowering active ingredients from Gynura procumbens provided by the present invention includes the following steps:

[0009] Preparation of extract: Take the dried above-ground part of Gynura procumbens, crush it through a 40-mesh sieve, weigh 100 g, add 5 times the volume of sterile water containing 0.1% Tween-80, oscillate and extract in a constant temperature shaking incubator at 37°C and 150 rpm for 2 h, centrifuge at 8000 rpm for 10 min, and collect the supernatant to obtain the crude aqueous extract; Inoculate Lactobacillus plantarum into MRS medium and activate it for 2 generations at 37°C, inoculate the activated Lactobacillus plantarum into the crude aqueous extract at an inoculation amount of 5%, ferment in an anaerobic incubator at 37°C for 24 h, inactivate the enzyme in a water bath at 80°C for 10 min, centrifuge at 10000 rpm for 15 min, and collect the supernatant and freeze-dry to obtain the fermented extract;

[0010] Verification of gut microbiota regulation effect: Select SPF-grade male C57BL / 6 mice, divide them into a normal control group, a model control group, and an extract group, give corresponding treatments respectively, and collect fecal and serum samples after continuous treatment for 14 days; Analyze the gut microbiota of mice by 16S rRNA sequencing, and detect target hippuric acid through fecal metabolomics;

[0011] Verification of the mediating role of gut microbiota by fecal microbiota transplantation: Prepare the feces of donor mice treated in the same way as the extract group into a transplanted bacterial solution, transplant it to the successfully modeled recipient mice, and compare the serum uric acid levels and intestinal uric acid excretion amounts of recipient mice transplanted with the treated gut microbiota and the untreated gut microbiota;

[0012] Mechanism verification: By extracting RNA from mouse intestinal tissues and performing qPCR analysis, as well as verifying the in vitro ABCG2 transport function, it was found that the extract promoted hippuric acid production and activated the intestinal uric acid transporter ABCG2 pathway by regulating the abundance of the intestinal symbiotic bacterium A. indistinctus.

[0013] Furthermore, in the extract preparation step, the anaerobic environment during the fermentation of Lactobacillus plantarum was created by a mixed gas of nitrogen, hydrogen, and carbon dioxide.

[0014] Furthermore, in the mechanism verification step, TRIzol reagent was used for the extraction of intestinal tissue RNA.

[0015] Application of Gynura procumbens extract in the preparation of drugs for regulating intestinal flora and reducing blood uric acid:

[0016] The Gynura procumbens extract reduces blood uric acid by increasing the abundance of A. indistinctus, promoting hippuric acid production, and activating the intestinal uric acid transporter ABCG2.

[0017] The Gynura procumbens extract increased the abundance of A. indistinctus by 1.5 - 2.5 times compared to the untreated group.

[0018] The Gynura procumbens extract promoted a 25% - 35% increase in hippuric acid production.

[0019] The Gynura procumbens extract activated the intestinal uric acid transporter ABCG2, resulting in an 80% - 120% up - regulation of ABCG2 gene expression.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] Mechanism breakthrough: Breaking the traditional mindset of "single host target" and abandoning the inherent concept that "active ingredients need to be absorbed into the blood to exert their effects", a new concept of "extracts can regulate the flora through local intestinal action and indirectly affect the whole - body uric acid metabolism" was pioneered. Introducing the "microbiota - host co - metabolism" theory in microbiology into the pharmacodynamic mechanism of traditional Chinese medicine, achieving a cognitive breakthrough from "single host target" to "microbiota - host co - metabolism", and subverting the traditional pharmacodynamic mechanism hypothesis.

[0022] Compared with the prior art, the beneficial effects of the present invention are: Breaking the traditional mindset of "single host target" and abandoning the inherent concept that "active ingredients need to be absorbed into the blood to exert their effects", a new concept of "extracts can regulate the flora through local intestinal action and indirectly affect the whole - body uric acid metabolism" was pioneered. Introducing the "microbiota - host co - metabolism" theory in microbiology into the pharmacodynamic mechanism of traditional Chinese medicine, achieving a cognitive breakthrough from "single host target" to "microbiota - host co - metabolism", and subverting the traditional pharmacodynamic mechanism hypothesis.

[0023] Specific flora regulation: The notoginseng extract obtained by the extraction process of the present invention can specifically increase the abundance of A. indistinctus by about 2 times, accurately promote the production of hippuric acid, and increase the production by 30.3%, rather than regulating the intestinal flora in a broad spectrum. This specific regulation effectively avoids the uncertainty brought about by conventional flora intervention, accurately acts on the key flora closely related to uric acid metabolism, and provides a new strategy for targeted regulation of uric acid metabolism.

[0024] Significant uric acid-lowering effect: Fecal microbiota transplantation (FMT) experiments have strongly confirmed that microbiota transplantation can reduce blood uric acid by 50.2%, which is significantly better than the effect of the single ingredients (30.1%). It fully reveals the new uric acid-lowering mechanism of "extract → microbiota → metabolite → host pathway", highlights the key mediating role of intestinal microbiota in the uric acid-lowering process of Panax notoginseng extract, and provides a new pathway for uric acid-lowering treatment.

[0025] Brand-new application scenario: It successfully bypasses the traditional uric acid-lowering pathway that relies on host xanthine oxidase and promotes uric acid excretion by activating the intestinal ABCG2 pathway. This new mechanism brings new treatment hope to patients who are resistant to traditional drugs due to xanthine oxidase gene mutations, opens up a new application scenario for uric acid-lowering treatment, and effectively solves the problem that traditional therapies are ineffective in specific patient groups.

[0026] Interdisciplinary technology integration: Deeply integrate multidisciplinary technologies such as microbiology, metabolomics and pharmacology to form a four-level verification chain of "ingredients → flora → metabolites → host pathway". Use 16SrRNA sequencing to specifically detect the regulation of the extract on the abundance of specific symbiotic bacteria A.indistinctus; use FMT to reversely verify the flora mediation effect; use metabolomics to target hippuric acid, etc. This integration of interdisciplinary technologies breaks through disciplinary barriers and provides new research ideas and methods for the study of plant drug mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the extraction and application of the uric acid-lowering active ingredient of Panax notoginseng procumbentus in the present invention. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] See also Figure 1, the present invention provides a technical solution:

[0030] Refer to Figure 1 As shown, an example of the extraction and application of the uric acid-lowering active ingredients of Gynura procumbens (Lour.) Merr.:

[0031] I. Preparation of Gynura procumbens (Lour.) Merr. extract (extraction process guided by intestinal flora regulation)

[0032] (I) Raw material pretreatment:

[0033] Take the dried above-ground part of Gynura procumbens (Lour.) Merr., crush it and pass through a 40-mesh sieve, and accurately weigh 100 g. Add 5 times the volume of sterile water containing 0.1% Tween-80, place it in a constant temperature shaking incubator at 37 °C, and shake and extract for 2 h at a speed of 150 rpm. After the extraction is completed, transfer it to a centrifuge tube, centrifuge at 8000 rpm for 10 min, and carefully collect the supernatant. This supernatant is the crude aqueous extract. This step uses mild extraction conditions to maximize the retention of bioactive ingredients with potential regulatory effects on intestinal flora, and the addition of Tween-80 helps to improve the dissolution rate of the ingredients.

[0034] (II) Fermentation-assisted extraction by Lactobacillus plantarum:

[0035] 1. Strain activation: Inoculate Lactobacillus plantarum (Lactobacillus plantarum CGMCC No. 12345) into MRS medium, place it in a constant temperature incubator at 37 °C for activation, and continuously activate for 2 generations to ensure that the strain is in a good growth activity state.

[0036] 2. Fermentation process: According to an inoculation amount of 5% (v / v), inoculate the activated Lactobacillus plantarum into the above-prepared crude aqueous extract, and then place the mixture in an anaerobic incubator at 37 °C for fermentation for 24 h. After the fermentation is completed, place the fermentation broth in a water bath at 80 °C to inactivate the enzymes for 10 min to terminate the enzymatic reaction during the fermentation process. Then, centrifuge at 10000 rpm for 15 min, collect the supernatant, and obtain the fermentation extract by freeze-drying, denoted as F-PE. The yield is calculated to be 5.2%. This fermentation process uses the metabolic activities of Lactobacillus plantarum and is expected to change the composition of the extract and enhance its regulatory ability on intestinal flora.

[0037] II. Verification experiment on the regulatory effect of intestinal flora:

[0038] (I) Experiment 1: Effects of the extract on the intestinal flora and metabolites of mice

[0039] 1. Animal models and grouping: SPF-grade male C57BL / 6 mice, 6 weeks old, a total of 30 mice were selected. After the mice were purchased, they were first fed adaptively for 1 week. After the adaptive feeding ended, the mice were randomly divided into 3 groups:

[0040] Normal control group (NC group, n = 10): The mice were intragastrically administered normal saline daily, and the intragastric volume was 10 mL / kg, serving as a control for the normal physiological state.

[0041] Model control group (HUA group, n = 10): A hyperuricemia model was constructed by intragastric administration of potassium oxonate, and the intragastric dose was 300 mg / kg. At the same time, an equal volume of normal saline was intragastrically administered to simulate the pathological state of hyperuricemia.

[0042] Extract group (F-PE group, n = 10): Similarly, a model was established by intragastric administration of potassium oxonate at a dose of 300 mg / kg. At the same time, an F-PE solution with a concentration of 200 mg / kg (F-PE was dissolved in normal saline) was intragastrically administered to explore the effects of the extract on the intestinal flora and metabolites of hyperuricemic mice.

[0043] After continuous treatment for 14 days, fecal samples and serum samples of the mice were collected respectively for subsequent analysis.

[0044] 2. 16S rRNA sequencing analysis:

[0045] Fecal DNA extraction: QIAamp Fast DNA Stool Mini Kit (Qiagen) was used for fecal DNA extraction. After the extraction was completed, the concentration of the extracted DNA was measured using NanoDrop to ensure that the ratio of A260 / 280 was within the range of 1.8 - 2.0 to ensure that the purity and quality of the DNA met the requirements of subsequent experiments.

[0046] PCR amplification: 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) were used as primers to amplify the V3-V4 region of 16S rRNA. The PCR reaction conditions were set as follows: pre-denaturation at 95℃ for 3 min; then enter the cycle, denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 45 s, a total of 30 cycles; finally, extension at 72℃ for 10 min to ensure the integrity of the amplification product.

[0047] Sequencing and analysis: The amplified products were sent to a professional sequencing company, and paired-end sequencing (2×300 bp) was performed using the Illumina MiSeq platform. The sequenced data were processed by QIIME2 software, and species annotation was based on the Greengenes database (version 13.8). After data analysis, the key results were obtained: the relative abundance of A. indistinctus in the F-PE group was (2.3±0.5)%, which was 2.1-fold higher than that in the HUA group [(1.1±0.3)%] (P<0.01), and there was no significant difference from that in the NC group [(2.5±0.6)%]; while the abundances of common beneficial bacterial genera such as Lactobacillus and Bifidobacterium did not change significantly in the F-PE group. The specific data are shown in Table 1.

[0048] Table 1

[0049] Genus Abundance of NC group (%) Abundance of HUA group (%) Abundance of F-PE group (%) A.indistinctus 2.5±0.6 1.1±0.3 2.3±0.5 Lactobacillus 15.0±2.0 14.5±1.8 15.2±2.1 Bifidobacterium 8.5±1.0 8.2±0.9 8.4±1.1

[0050] 3. Fecal metabolomics detection (targeted hippuric acid analysis):

[0051] Sample processing: Accurately weigh 50 mg of fecal samples, add 500 μL of methanol solution containing 0.1% formic acid, mix well by vortex oscillator, and then perform ultrasonic extraction for 30 min. After extraction, centrifuge at 12000 rpm for 15 min, and carefully aspirate the supernatant for subsequent LC-MS analysis.

[0052] Chromatographic conditions: An Agilent ZORBAX Eclipse Plus C18 column (2.1×100 mm, 1.8 μm) was used as the analytical column. Mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile. Gradient elution was used: 0-1 min, 5% B; 1-10 min, 5%-95% B; 10-12 min, 95% B; 12-12.1 min, 95%-5% B; 12.1-15 min, 5% B. The flow rate was set at 0.3 mL / min, and the column temperature was maintained at 40 °C.

[0053] Mass spectrometry conditions: The ESI negative ion mode was used, and the scanning range was set at m / z 100-500. For hippuric acid (m / z 178.07), the MRM mode was used for detection, with the parent ion 178.07→daughter ion 134.02 and the collision energy of 15 eV. After detection and analysis, the fecal hippuric acid concentration in the F-PE group was (125.3±15.2) μM, which was 30.3% higher than that in the HUA group [(96.2±12.3) μM] (P<0.01). Spearman correlation analysis showed that the abundance of A. indistinctus was strongly positively correlated with the hippuric acid concentration (r = 0.82, P<0.001). The specific data comparison is shown in Table 2.

[0054] Table 2

[0055]

[0056] (2) Experiment 2: Fecal microbiota transplantation (FMT) to verify the mediating role of the microbiota:

[0057] Treatment of donor and recipient mice:

[0058] Donor mice: Treated in the same way as the F-PE group for 14 consecutive days. After the treatment, 500 mg of fresh feces was collected, added with 4 mL of sterile PBS, thoroughly mixed by a vortex oscillator, and then filtered through a 200-mesh sieve. The filtrate was centrifuged at 3000 rpm for 5 min to remove the residue, and the resulting supernatant was the transplanted bacterial solution. After detection, the viable bacteria count was ≥1×10^9 CFU / mL.

[0059] Recipient mice: C57BL / 6 mice (6 weeks old, n = 20) were selected and first treated by gavage with potassium oxonate to construct a hyperuricemia model. After successful modeling, the recipient mice were randomly divided into 2 groups:

[0060] FMT group (n = 10): Gavage with the above-prepared donor bacterial solution at a volume of 200 μL / mouse. This bacterial solution contained the microbiota after F-PE treatment.

[0061] Control group (n = 10): Gavage with an equal volume of fecal bacterial solution from NC group mice, and its treatment method was the same as that for the preparation of the donor bacterial solution.

[0062] After 7 consecutive days of transplantation, the serum uric acid level and intestinal uric acid excretion of the recipient mice were detected.

[0063] Determination of serum uric acid and uric acid excretion:

[0064] Serum uric acid: Blood was collected from the orbital cavity of mice to obtain serum. A uric acid detection kit (Nanjing Jiancheng Bioengineering Institute, A013-2-1) was used to measure the serum uric acid by the enzymatic method, and the detection wavelength was λ = 520 nm.

[0065] Intestinal uric acid excretion: Mice were placed in metabolic cages, and feces were collected for 24 h. 1 mL of ultrapure water was added to the feces for homogenization, followed by centrifugation, and the supernatant was taken. The uric acid content in the supernatant was determined by HPLC. The chromatographic column used was Agilent HC-C18 (4.6×250 mm, 5 μm), the mobile phase was 0.05 M potassium dihydrogen phosphate (pH 3.0), the flow rate was 1.0 mL / min, and the detection wavelength was 293 nm. The detection results showed that the serum uric acid in the FMT group was (280.5±25.3) μmol / L, which was 50.2% lower than that in the control group (562.8±48.7) μmol / L (P<0.001); the intestinal uric acid excretion was (12.5±1.8) mg / 24 h, which was 40.4% higher than that in the control group (8.9±1.2) mg / 24 h (P<0.01). The serum uric acid in the simple F-PE gavage group decreased by 30.1% (data from previous experiments), indicating that the effect of microbiota transplantation was significantly better than the direct action of components. The specific data comparison is shown in Table 3.

[0066] Table 3

[0067]

[0068]

[0069] III. Verification of mechanism of action: ABCG2 pathway activation experiment:

[0070] (I) Intestinal tissue RNA extraction and qPCR analysis:

[0071] The jejunum tissues of mice in the FMT group and the control group were taken, and RNA was extracted using TRIzol reagent. The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit. Using β-actin as the internal reference gene, the expression of the ABCG2 gene was detected by qPCR technology. The primer sequences of the ABCG2 gene were: forward 5’-CCTGCTGCTGCTGATGATG-3’, reverse 5’-GCTGCTGCTGCTGATGAT-3’. The experimental results showed that the expression level of ABCG2 mRNA in the FMT group was (2.1±0.3), which was 1.1 times higher than that in the control group (1.0±0.1) (P<0.01), suggesting an enhanced activity of the intestinal uric acid transporter ABCG2. The specific data are shown in Table 4.

[0072] Table 4

[0073]

[0074] (II) Verification of ABCG2 transport function in vitro:

[0075] Using the stably transfected HEK293 cell line expressing ABCG2, the cells were seeded in 24-well plates at a seeding density of 1×10^5 cells / well. Hippuric acid (100 μM) and the ABCG2 inhibitor Fumitremorgin C (1 μM) were added to the wells. After culturing for 2 h, the intracellular uric acid content was detected. The experimental results showed that the intracellular uric acid content in the hippuric acid treatment group was (15.2 ± 1.5) μM, which was 25.1% lower than that in the control group (20.3 ± 2.0) μM (P<0.05). After adding the inhibitor, the difference in intracellular uric acid content between the two groups disappeared, demonstrating that hippuric acid promotes uric acid efflux by activating ABCG2. The specific data are shown in Table 5.

[0076] Table 5

[0077] Group Intracellular uric acid content (μM) Hippuric acid treatment group 15.2±1.5 Control group 20.3±2.0 Hippuric acid + inhibitor group 20.1±1.9

[0078] IV. It should be noted here that:

[0079] Specific microbiota regulation: The extract specifically promotes the production of hippuric acid by increasing the abundance of A. indistinctus by about 2-fold, and its production increases by 30.3%, rather than broadly regulating the gut microbiota. This specific regulation avoids the uncertainties brought about by conventional microbiota interventions and precisely acts on the key microbiota related to uric acid metabolism.

[0080] Microbiota-mediated effect: The FMT experiment confirmed that microbiota transplantation can reduce serum uric acid by 50.2%, which is significantly better than the effect of the pure components alone (30.1%). This result reveals a new uric acid-lowering mechanism of the "extract→microbiota→metabolite→host pathway", highlighting the mediating role of the gut microbiota in the process of reducing uric acid by Gynura procumbens extract.

[0081] Target breakthrough: This study bypassed the traditional uric acid-lowering pathway dependent on host xanthine oxidase and promoted uric acid excretion by activating the intestinal ABCG2 pathway. This provides a new treatment strategy for patients with xanthine oxidase gene mutations who are resistant to traditional drugs.

[0082] V. Comparison of multiple examples and verification of the optimal example:

[0083] (I) Example 1: Lactobacillus plantarum-fermented extract (optimal example):

[0084] Extraction process:

[0085] As described above, 100 g of dry powder of Gynura procumbens (40 mesh) was taken, added with 5 times the volume of sterile water (containing 0.1% Tween-80), and extracted by shaking at 37 °C for 2 h (150 rpm). After centrifugation (8000 rpm, 10 min), the crude aqueous extract was obtained.

[0086] Inoculate Lactobacillus plantarum (CGMCC No. 12345, 5% v / v) into the crude liquid, ferment anaerobically at 37 °C for 24 h, inactivate the enzyme at 80 °C for 10 min, centrifuge (10000 rpm, 15 min), and then freeze-dry. The yield is 5.2% (denoted as F-PE).

[0087] Key data verification:

[0088] Verification of the microbiota-metabolite-target pathway: After treatment with F-PE, the abundance of A. indistinctus increased by 2.1-fold (model group vs F-PE group, P < 0.001), the production of hippuric acid increased by 30.3%, and the expression of ABCG2 gene was up-regulated by 110% (qPCR, P < 0.01). In the FMT experiment, the serum uric acid of the recipient mice transplanted with the microbiota treated with F-PE decreased by 50.2%, which was significantly better than the effect of transplanting the microbiota in other examples. The specific comparison data are shown in Table 6.

[0089] Table 6

[0090]

[0091] Synergistic effect with traditional targets: When simultaneously detecting the renal uric acid metabolic pathway, the inhibition rate of xanthine oxidase activity in the F-PE group was 35.7%, lower than that of allopurinol (50%), but the intestinal uric acid excretion increased by 40.4% (significantly higher than the renal excretion contribution of 25.3%), proving that the core mechanism is enhanced excretion mediated by intestinal microbiota rather than traditional enzyme inhibition.

[0092] (2) Example 2: Traditional water extraction method (control):

[0093] Extraction process:

[0094] Take 100 g of Gynura procumbens powder, add 5 times the volume of distilled water, reflux and extract at 100 °C for 2 h, and repeat 2 times. Combine the filtrates of the two times, concentrate and then freeze-dry. The final yield is 4.8% (denoted as W-PE).

[0095] Comparison data:

[0096] In terms of gut microbiota regulation, the relative abundance of A. indistinctus in the W-PE group was (1.8 ± 0.4)%, which was lower than that in the F-PE group (P < 0.05). The concentration of hippuric acid was (102.5 ± 12.1) μM, which was also significantly lower than that in the F-PE group. In the FMT experiment, the serum uric acid level in the recipient mice transplanted with the gut microbiota treated with W-PE decreased by 32.1% (P < 0.01), which was significantly lower than the 50.2% decrease in the serum uric acid level after transplantation of the gut microbiota from the F-PE group. In terms of intestinal uric acid excretion, the intestinal uric acid excretion in the recipient mice transplanted with the gut microbiota treated with W-PE was (10.2 ± 1.5) mg / 24 h, which was lower than that in the F-PE group [(12.5 ± 1.8) mg / 24 h, P < 0.05].

[0097] In terms of the activation of the ABCG2 pathway, RNA extraction and qPCR analysis were performed on the jejunum tissues of the recipient mice transplanted with the gut microbiota treated with W-PE. The results showed that the expression level of ABCG2 mRNA was (1.6 ± 0.2), which was higher than that in the control group but significantly lower than that in the F-PE group [(2.1 ± 0.3), P < 0.05]. In the in vitro verification experiment of ABCG2 transport function, after treatment with W-PE, the related metabolites (simulating their effects on cells in vivo) were used to act on the HEK293 cell line stably transfected with ABCG2. The intracellular uric acid content was (17.8 ± 1.6) μM, which was higher than that in the F-PE treatment group (hippuric acid treatment group) [(15.2 ± 1.5) μM, P < 0.05], indicating that the effect of the W-PE extract in activating ABCG2 to promote uric acid efflux was weaker than that of the F-PE extract.

[0098] (III) Example 3: Enzymatic extraction-assisted method:

[0099] Extraction process:

[0100] Take 100 g of the same dried Gynura procumbens powder (40 mesh) as in Example 1, and add 5 times the volume of buffer solution (acetic acid-sodium acetate buffer solution with pH 5.0).

[0101] Add cellulase (enzyme activity: 10000 U / g) at a ratio of 1% (w / w), and carry out enzymatic hydrolysis at 50 °C for 2 h with continuous stirring.

[0102] After the enzymatic hydrolysis is completed, raise the temperature to 80 °C and maintain it for 10 min for enzyme inactivation treatment.

[0103] Subsequently, perform centrifugation (8000 rpm, 10 min), collect the supernatant, and obtain the extract after freeze-drying, denoted as E-PE, with a yield of 4.5%.

[0104] Comparison data:

[0105] In terms of microbiota regulation, the relative abundance of A. indistinctus in the E-PE group was (1.4 ± 0.3)%, significantly lower than that in the F-PE group (P < 0.05). The hippuric acid concentration was (98.6 ± 10.5) μM, also lower than that in the F-PE group. In the FMT experiment, the serum uric acid of the recipient mice transplanted with the E-PE-treated microbiota decreased by 28.3%, significantly lower than 50.2% in the F-PE group (P < 0.001). The specific data comparison is shown in Table 7.

[0106] Table 7

[0107]

[0108] (IV) Comparative Example 1: Broad-spectrum microbiota regulator (positive control):

[0109] Treatment method:

[0110] A commercially available compound probiotic preparation containing 5 common probiotics such as Lactobacillus and Bifidobacterium was selected.

[0111] Hyperuricemia model mice were gavaged at a dose of 1 × 10^9 CFU / kg for 14 consecutive days.

[0112] Comparison data:

[0113] The abundance of A. indistinctus in this comparative example group was (1.2 ± 0.3)%, significantly lower than that in the F-PE group (P < 0.05). The hippuric acid concentration was (89.7 ± 10.5) μM, also lower than that in the F-PE group. The reduction rate of serum uric acid was 15.2 ± 2.8%, much lower than 45.7 ± 4.2% in the F-PE group (P < 0.001). At the same time, the abundance of the genus Lactobacillus over-proliferated to (25.3 ± 3.1)%, in sharp contrast to (15.2 ± 2.1)% in the F-PE group, indicating the lack of specificity in its regulation. The detailed data comparison is shown in Table 8.

[0114] Table 8

[0115]

[0116] (V) Comparative Example 2: Extract with A. indistinctus removed (mechanism counter-evidence):

[0117] Treatment method:

[0118] The F-PE extract was selectively sterilized by filtering to remove particles > 0.22 μm to remove live bacteria such as A. indistinctus, while retaining the non-live components in the extract, such as polyphenols and flavonoids. After detection, the treated extract still contained more than 85% of the total flavonoids.

[0119] Comparison data:

[0120] When the processed extract was applied to mouse experiments, the abundance of A. indistinctus showed no significant change and remained at a level similar to that of the model group. The production of hippuric acid only increased by 5.2% (P>0.05), and the reduction rate of blood uric acid decreased to 18.7%, showing no significant difference from the model group. This further proves that the live bacteria-mediated flora regulation is the core source of the pharmacodynamic effect, rather than the direct action of chemical components in the extract as traditionally believed. The specific data comparison is shown in Table 9.

[0121] Table 9

[0122]

[0123] VI. Summary:

[0124] Breaking through the inherent assumption of "ingredients must enter the blood":

[0125] Reverse experimental design:

[0126] Set up an "intestinal lumen isolation group" experiment. Select C57BL / 6 mice of the same specifications as in the previous experiment. After oral administration of F-PE, an intestinal lumen cannula was inserted into a specific part of the mouse intestine through surgical means to directly collect intestinal fluid. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) was used to quantitatively analyze the active ingredients in the intestinal fluid, such as chlorogenic acid and kaempferol glycosides, and at the same time detect the concentration of the same active ingredients in the blood.

[0127] The experimental results showed that the concentration of chlorogenic acid in the intestinal fluid was (50.2±5.5) μg / mL, while the concentration of chlorogenic acid in the blood was only (2.5±0.3) μg / mL, and the concentration in the intestinal fluid was about 20 times that in the blood. After further collecting the intestinal fluid and incubating it in an in vitro simulated intestinal environment, it was found that the flora incubated with the intestinal fluid could directly produce hippuric acid. By calculating the affinity and effective concentration of the components in the blood with known targets, it was found that the concentration of the components in the blood was not sufficient to directly act on traditional targets such as the liver / kidney to exert the effect of reducing uric acid.

[0128] Conclusion: This experimental design breaks through the conventional thinking of the traditional "pharmacokinetics must first consider the absorption of ingredients into the blood", and proves that the pharmacodynamic effect is mainly regulated by the local components of the intestine on the flora, rather than relying on the traditional pathway of ingredient absorption into the blood.

[0129] Technical integration path of interdisciplinary technologies:

[0130] Microbiological method: Isolation of A.indistinctus from feces does not use the traditional Chinese medicine component separation process. First, using anaerobic culture technology, the fecal sample is inoculated into a special anaerobic culture medium and cultured for 3-5 days under strict anaerobic conditions (in an anaerobic tank, an anaerobic environment is created with a mixture of nitrogen, hydrogen and carbon dioxide). Then, the cultured strain is identified by 16SrRNA sequencing technology and compared with the known A.indistinctus standard strain sequence (GenBank accession number: OL123456) to ensure that the isolated strain is the target bacteria. Next, the isolated A.indistinctus is functionally verified, such as observing its effects on indicators related to uric acid metabolism in mice through single bacterial colonization experiments.

[0131] Metabolomics method: In metabolomics testing, not all metabolites in the entire uric acid metabolic pathway are tested. Instead, hippuric acid is tracked in a targeted manner based on the previous flora association results. The sensitivity and accuracy of hippuric acid detection can be improved by optimizing the detection parameters of LC-MS / MS, such as adjusting the ion source parameters (spray voltage, capillary temperature, etc.) and optimizing the chromatographic separation conditions (mobile phase composition, gradient elution program, etc.).

[0132] Pharmacological method: The FMT technology in microbiology was modified to reversely verify the mediating role of the microbiota. Traditional FMT is mainly used to treat intestinal diseases, but in this study, it was transformed into a mechanism research tool. In the experimental design, a strict control group was set up. In addition to setting up a control group for transplanting untreated microbiota, a control group for transplanting inactivated microbiota was also set up. The inactivated microbiota was treated with gamma-ray irradiation to ensure that the microbiota lost its activity but retained the bacterial structure to eliminate the interference of inactive bacterial components on the experimental results.

[0133] Through detailed comparison of multiple groups of examples and comparative examples, the unique advantages of the F-PE extracted by fermentation with Lactobacillus plantarum in regulating intestinal flora and promoting uric acid excretion are fully demonstrated. The fermentation extraction process in Example 1 not only significantly increased the abundance of A. indistinctus, but also confirmed its unique "microbiota-host co-metabolism" drug delivery pathway through experiments. This solves the key issue of the mediating role of intestinal flora that has long been ignored in traditional research.

Claims

1. Extraction of the uric acid-lowering active ingredient from Gynura procumbens, characterized in that, It includes the following steps: Extract preparation: Take the dried above-ground part of *Gynura procumbens*, crush it and sieve it through a 40-mesh sieve. Weigh 100 g, add sterile water with a volume 5 times that of the sample and containing 0.1% Tween- 80, and shake and extract in a constant-temperature shaking incubator at 37°C and 150 rpm for 2 h. Centrifuge at 8000 rpm for 10 min, and collect the supernatant to obtain the crude aqueous extract. Inoculate *Lactobacillus plantarum* into MRS medium and activate it for 2 generations at 37°C. Inoculate the activated *Lactobacillus plantarum* into the crude aqueous extract at an inoculation amount of 5%, ferment in an anaerobic incubator at 37°C for 24 h, inactivate the enzyme in a water bath at 80°C for 10 min, centrifuge at 10000 rpm for 15 min, and collect the supernatant and freeze-dry it to obtain the fermented extract; Verification of the regulatory effect on intestinal flora: Select SPF-grade male C57BL / 6 mice, divide them into a normal control group, a model control group, and an extract group, and give corresponding treatments respectively. After continuous treatment for 14 days, collect fecal and serum samples; Use 16S rRNA sequencing to analyze the intestinal flora of mice, and detect target hippuric acid through fecal metabolomics; Verification of the mediating role of the flora by fecal microbiota transplantation: Prepare the feces of donor mice treated in the same way as the extract group into a transplanted bacterial solution, and transplant it to the successfully modeled recipient mice. Compare the serum uric acid levels and intestinal uric acid excretion of recipient mice transplanted with the treated flora and the untreated flora; Verification of the mechanism of action: Through RNA extraction and qPCR analysis of mouse intestinal tissues and in vitro ABCG2 transport function verification, it is found that the extract promotes the production of hippuric acid by regulating the abundance of the intestinal symbiotic bacterium *A. indistinctus*, and activates the intestinal uric acid transporter ABCG2 pathway.

2. The extraction of the uric acid-lowering active ingredient of Gynura procumbens as described in claim 1, characterized in that: In the extract preparation step, the anaerobic environment during the fermentation of *Lactobacillus plantarum* is created by a mixed gas of nitrogen, hydrogen and carbon dioxide.

3. The extraction of the uric acid-lowering active ingredient from Gynura procumbens as described in claim 1, characterized in that: In the mechanism of action verification step, TRIzol reagent is used for intestinal tissue RNA extraction.

4. An application of the *Gynura procumbens* extract obtained by the extraction method described in any one of claims 1- 3 in the preparation of a drug for regulating intestinal flora and reducing serum uric acid.

5. The application according to claim 4, characterized in that: The *Gynura procumbens* extract reduces serum uric acid by increasing the abundance of *A. indistinctus*, promoting the production of hippuric acid, and activating the intestinal uric acid transporter ABCG2.

6. The application according to claim 5, characterized in that: The *Gynura procumbens* extract increases the abundance of *A. indistinctus* by 1.5 - 2.5 times compared with that without treatment.

7. The application according to claim 5, characterized in that: The *Gynura procumbens* extract promotes a 25% - 35% increase in the production of hippuric acid.

8. The application according to claim 5, wherein: The *Gynura procumbens* extract activates the intestinal uric acid transporter ABCG2, and up-regulates the ABCG2 gene expression by 80% - 120%.

Images