Smilax glabra leavening and preparation method and application thereof in hyperuricemia

Abstract

The invention discloses a rhizoma smilacis glabrae leavening and a preparation method and application thereof in hyperuricemia, and the scheme is as follows: inoculating a lactobacillus fermentation seed solution into a rhizoma smilacis glabrae traditional Chinese medicine stock solution, fermenting for 48-72 hours at 37 DEG C, and freeze-drying to obtain the rhizoma smilacis glabrae leavening; the lactobacillus brevis fermentation seed liquid is prepared by fermenting lactobacillus brevis YM 1301 and lactobacillus plantarum CICC24202, and the lactobacillus brevis YM 1301 and the lactobacillus plantarum CICC24202 are fermented; the lactobacillus fermentation seed liquid accounts for 6-10% of the volume of the rhizoma smilacis glabrae traditional Chinese medicine Results show that after the rhizoma smilacis glabrae is fermented, the contents of total flavone and total polyphenol are increased, and the polysaccharide content is reduced; the rhizoma smilacis glabrae leavening can significantly reduce serum uric acid, urea nitrogen and creatinine levels of HUA rats; the kidney injury caused by hyperuricemia is improved; the increase of inflammatory cytokines such as IL-1beta and TNF-alpha in serum is reduced, and the curative effect is superior to that of unfermented rhizoma smilacis glabrae; the mechanism of reducing uric acid may be by inhibiting the activity of xanthine oxidase in HUA rats and increasing the excretion of uric acid in excrement. The invention provides a solid theoretical reference basis for developing safe and effective uric acid-reducing traditional Chinese medicine large-scale health products.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine preparation, and particularly relates to a smilax glabra fermentation product and a preparation method thereof and an application thereof in treating hyperuricemia. Background Art

[0002] Hyperuricemia (HUA) is a disease with elevated uric acid levels caused by abnormal uric acid metabolism and purine metabolism disorders. The international definition of hyperuricemia is: clinical detection of serum uric acid levels under normal diet on different days exceeding 420μmo1 / L for men and 360μmo1 / L for women can be considered hyperuricemia. At present, hyperuricemia is divided into asymptomatic and symptomatic stages. The first stage of the asymptomatic period is the hyperuricemia stage, during which there is no deposition of sodium urate crystals; the second stage of the asymptomatic period is the sodium urate crystal deposition stage, during which no gouty arthritis occurs. The first stage of the symptomatic period is the occurrence and intermittent attacks of gouty arthritis, during which sodium urate crystals are deposited in the joints; the second stage of the symptomatic period is the chronic gouty arthritis stage, at which time tophi have formed in the joints, causing varying degrees of bone destruction. The main cause of hyperuricemia is the abnormal increase of uric acid content in serum. Uric acid is a weak acid with a high dissociation constant, so uric acid circulates in the blood mainly in the form of monovalent sodium salt (urate). Uric acid has both antioxidant and pro-oxidant properties, and its balance depends on genetic polymorphism, environmental conditions, the presence of other pro-oxidant or antioxidant molecules, cell type and the regulation of certain signaling pathways. The antioxidant properties of uric acid are more prominent outside cells. It can scavenge free radicals such as hydroxyl, peroxyl and singlet oxygen free radicals, providing beneficial support for cells and organs in coping with external oxidative stress. This role has been confirmed in neurological diseases such as stroke and Parkinson's disease. On the other hand, the pro-oxidant properties of uric acid are more prominent inside cells. The enhancement of XOD (xanthine oxidase) activity is directly related to the increase of intracellular uric acid levels. Excessive uric acid can cause oxidative stress in the body. Long-term high levels of uric acid may lead to serious diseases such as metabolic syndrome, arteriosclerosis and hypertension.

[0003] Hyperuricemia (HUA) is mainly manifested as an imbalance in the homeostasis of serum uric acid levels. Modern medicine shows that hyperuricemia is mainly caused by increased production or reduced excretion of uric acid, accompanied by inflammatory response and oxidative stress of the body. About 20% of uric acid in the human body is exogenous, mainly produced by the body's catabolism of ingested food. About 80% of uric acid in the human body is endogenously generated, mainly in the catabolism of nucleic acids, which produces a large amount of purine. Purine is usually converted into hypoxanthine and xanthine in the liver. At this time, hypoxanthine and xanthine can be oxidized to uric acid by xanthine oxidase in the liver and enter the blood circulation. Due to the degradation of human uricase, uric acid cannot be further catalyzed into allantoin and can only exist in the blood in the form of urate. It can be seen from the two sources of uric acid that there are many factors that control the production of uric acid in the body, and the production of uric acid is subject to multiple regulation. In terms of uric acid production, the increase in products during nucleic acid metabolism and changes in the activity of key enzymes can cause excessive increase in uric acid. Xanthine dehydrogenase (XDH) can be converted into xanthine oxidase (XOD) through sulfhydryl residue oxidation or proteolysis. Adenosine deaminase (ADA) is the key rate-limiting enzyme in the metabolism of purine to uric acid. Its main function is to catalyze the irreversible conversion of adenosine to inosine. Increased ADA expression or activity will lead to increased uric acid production. Excessive intake of high-purine foods such as soy products, seafood, offal, sugary soft drinks, beer, etc., increases exogenous purines, thereby increasing uric acid production. If not intervened in time, the continuous accumulation of uric acid can easily lead to HUA. Primary HUA is related to heredity and gene mutation, and is mainly manifested by abnormal activity of certain key enzymes in the purine metabolism process, such as decreased activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT), excessive activity of phosphoribosyl pyrophosphate synthetase (PRPP) or XOD, which may cause increased synthesis of purine nucleotides, enhanced metabolism, and excessive production of uric acid. In addition, cell division or renewal can also cause abnormal increases in uric acid levels, which are mainly related to diseases such as lymphoproliferative diseases, myeloproliferative diseases, erythrocytic hyperplasia, and psoriasis. In terms of uric acid excretion, about 30% of uric acid in the human body is metabolized through the intestines and excreted from the body with feces, and about 70% of uric acid is excreted through the kidneys with urine. Intestinal dysfunction, decreased glomerular filtration, reduced tubular secretion, and enhanced tubular reabsorption can all lead to insufficient uric acid excretion, which in turn causes HUA. Uric acid excretion is mainly completed through uric acid transporters, which can be divided into two types: uric acid reabsorption transporters and uric acid excretion transporters. There are two main types of urate reabsorption transporters: uric acid-anion exchange transporter 1 (URAT1) and glucose transporter 9 (GLUT9).URAT1 is the main transporter of uric acid reabsorption in the kidney. It is a membrane transporter protein encoded by the SLC22A12 gene and is only expressed on the brush border side of the proximal tubular epithelium. It transports uric acid from the kidney to the blood, thereby mediating the reabsorption of uric acid. The GLUT9 protein is from the GLU family and is encoded by the SLC2A9 gene. Unlike other members of the GLU family, the transport rate of uric acid by GLUT9 is much higher than that of glucose or other monosaccharides. Therefore, it is believed that the primary function of GLUT9 is to transport uric acid. It is distributed in the basolateral membrane of the proximal tubule and is mainly responsible for the reabsorption of uric acid. The proximal tubular reabsorption of uric acid is controlled by URAT1, which absorbs uric acid to the basolateral membrane, and then GLUT9 transfers uric acid to the renal interstitium for reabsorption. Renal excretion transporters include organic anion transporters (OATs) of the SLC22 gene family and adenosine triphosphate-binding transporter G superfamily member 2 (ABCG2). Anion transporter 1 (OAT1) is an important transporter for urate secretion. It is mainly located on the outside of the basement membrane of epithelial cells and transports uric acid from the renal interstitium to the epithelial cells of the renal tubules. Studies have shown that in hyperuricemia, the expression of OAT1 is reduced, which means that the kidney's ability to excrete uric acid is also reduced. In addition, ABCG2 is required for the excretion of uric acid from renal tubular cells to their lumen. ABCG2 is expressed on the brush border side of the renal proximal tubular epithelial cells and in the intestine, and its abnormal function will also reduce the kidney's ability to excrete uric acid.

[0004] At present, there is no clear treatment guideline for asymptomatic hyperuricemia in the world. The current treatment is mainly to reduce uric acid through drugs, so that the serum uric acid level returns to normal. According to their treatment mechanism, the common uric acid-lowering drugs in clinical practice are divided into two categories: drugs that inhibit uric acid production and promote uric acid excretion. Allopurinol is the first-line treatment drug for HUA. It is a competitive XOD inhibitor that directly blocks the synthesis of uric acid in the liver and other organs, thereby reducing the patient's serum uric acid level and reducing the occurrence of complications. Drugs targeting urate transporters mainly include benzbromarone, probenecid, etc. Benzbromarone and probenecid are both typical URAT1 inhibitors. By inhibiting the reabsorption of uric acid by the proximal tubules of the kidney, uric acid excretion is increased and plasma uric acid levels are reduced. However, Western medicine has many problems in lowering uric acid, such as poor compliance and adverse reactions. For example, allopurinol can cause a series of serious adverse reactions, such as hematological abnormalities, diarrhea, and drug fever. Benzbromarone can cause liver damage, skin reactions, etc. Therefore, finding highly effective and safe uric acid-lowering drugs has become an urgent problem to be solved.

[0005] Traditional Chinese medicine has pharmacodynamic characteristics such as diversity, selectivity, complexity, nonlinearity, and bidirectionality. Due to the characteristics of chronic hyperuricemia, traditional Chinese medicine has been widely used in its treatment. At present, the uric acid-lowering prescriptions used in clinical practice by traditional Chinese medicine include: Ermiao San, Simiao San, Danggui Niantong Tang, Xiexin Tang, etc. Studies have shown that the active ingredients of traditional Chinese medicine also have a good uric acid-lowering effect, such as: rutin, berberine, chlorogenic acid, and huangqi glycosides. Tusmilax is the dried rhizome of Smilax glabra Roxb., a plant of the Liliaceae family, and more than 200 compounds have been isolated. So far, more than 170 chemical components have been identified, including flavonoids and flavonoid glycosides, organic acids and phenolic acids, polysaccharides, sterols, phenylpropanoids, phenols and phenolic glycosides, other glycosides and volatile oils. Astilbin, total saponins, alkaloids and other ingredients in Smilax glabra have the effects of lowering uric acid, protecting kidney and liver, relieving pain, anti-inflammatory and benefiting joints, anti-tumor, immunomodulatory, antibacterial and antifungal, and improving arrhythmia. Studies have shown that Smilax glabra is often used in compound prescriptions for lowering uric acid, such as Compound Smilax glabra Granules, Dioscorea Plantago and Poria Decoction, and Huoxue Simiao Decoction, which shows that Smilax glabra is unique in lowering uric acid. At the same time, Smilax glabra is also a health product with both medicinal and edible properties. Smilax glabra is often used to make soup to remove dampness and detoxify the body.

[0006] The Chinese herbal medicine Smilax glabra is often used to remove dampness and detoxify, and it also has a relatively clear uric acid-lowering effect. However, like other Chinese herbal medicines, the uric acid-lowering effect of Smilax glabra is not yet significant and rapid enough; in addition, the currently known anti-hyperuricemia drugs mainly achieve the purpose of reducing blood uric acid concentration by inhibiting uric acid production in the body (such as allopurinol, which inhibits xanthine oxidase, thereby inhibiting uric acid production) and promoting uric acid excretion from urine (such as benzbromarone and probenecid as URAT1 inhibitors, which inhibit uric acid reabsorption and increase uric acid excretion in urine). Both have clear toxic and side effects, especially URAT1 inhibitors, which greatly increase the burden on the kidneys and cause renal damage. In fact, patients with hyperuricemia generally have a high renal load, and most patients with long-term hyperuricemia are accompanied by renal damage. The source of uric acid is food and production in the body, and the excretion of uric acid is mainly urine and feces. Previous drugs rarely pay attention to the excretion of uric acid in feces. About 30% of the uric acid in the human body is metabolized through the intestines and excreted with feces, so the excretion of uric acid through the intestines is also very important for the relief of hyperuricemia, but the detailed molecular mechanism of uric acid excretion through the intestines is still unclear. However, there is clear experimental evidence that changes in intestinal flora and certain intestinal bacteria have the function of alleviating hyperuricemia. Summary of the invention

[0007] The purpose of the present invention is to provide a smilax glabra fermented product and a preparation method thereof and application thereof in treating hyperuricemia.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] The invention discloses a method for preparing a smilax glabra fermented product. The method comprises the steps of inoculating lactobacillus fermented seed liquid into a smilax glabra traditional Chinese medicine stock liquid, fermenting the product at 37°C for 48 to 72 hours, and freeze-drying the product to obtain the smilax glabra fermented product.

[0010] Wherein, the lactobacillus fermented seed liquid is obtained by fermenting Lactobacillus brevis YM 1301 and Lactobacillus plantarum CICC24202;

[0011] The lactobacillus fermented seed liquid accounts for 6% to 10% of the volume of the Chinese smilax glabra original liquid.

[0012] Preferably, the lactobacillus fermented seed liquid is a mixture of Lactobacillus brevis YM 1301 seed liquid and Lactobacillus plantarum CICC24202 seed liquid in a volume ratio of 1:1.

[0013] Preferably, the OD value of the lactobacillus fermentation seed liquid is adjusted to 1.0.

[0014] Preferably, the Chinese smilax glabra traditional Chinese medicine stock solution is prepared according to the following method:

[0015] After drying, crush and sieve the Rhizoma Smilacis Glabrae to obtain Rhizoma Smilacis Glabrae powder for later use;

[0016] Water is added to the smilax glabra powder, and then α-amylase is added for enzymolysis for 4-5 hours, and then the pH value is adjusted to neutral, sterilized, and the smilax glabra traditional Chinese medicine stock solution is prepared.

[0017] More preferably, the usage ratio of Smilax glabra powder to water is 1 g: (4-8) mL.

[0018] More preferably, the added amount of α-amylase is 0.2% of the volume of the Smilax glabra traditional Chinese medicine stock solution, the enzymatic treatment temperature is 60° C., and the pH value is 6.5.

[0019] The present invention also discloses a fermented smilax glabra obtained by the above-mentioned preparation method. In the fermented smilax glabra, the total flavonoids content is 2.59±1.34% (about 1.6 times the total flavonoids content of unfermented smilax glabra), the total polyphenol content is 1.93±0.24% (about 1.45 times the total polyphenol content of unfermented smilax glabra), and the astilbin content is reduced, which is about 52% of that of unfermented smilax glabra.

[0020] The invention also discloses the use of the above-mentioned Smilax glabra fermented product in preparing food or health food for improving the blood uric acid level of non-hyperuricemia patients.

[0021] Preferably, the food or health food is a food or health food that reduces serum uric acid, urea nitrogen and creatinine levels; or,

[0022] Food or health food for improving kidney damage caused by high uric acid; or,

[0023] Food or health care that reduces the increase of inflammatory factors in serum; or,

[0024] Foods or health foods that increase uric acid excretion in feces.

[0025] The invention also discloses application of the above-mentioned Smilax glabra fermented product in preparing medicine for treating hyperuricemia.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention uses mixed fermentation of Lactobacillus plantarum CICC 24202 (Lp) and Lactobacillus breris YM 1301 (Lb) to ferment Smilax glabra, and inoculates the mixture into a Chinese medicine stock solution containing Smilax glabra to obtain Smilax glabra fermented product. Then, hypoxanthine and potassium oxonate are used to establish a HUA rat model, and allopurinol is used as a positive drug. By detecting key indicators such as serum uric acid, creatinine, urea nitrogen, cytokines, and xanthine oxidase of HUA rats, the therapeutic effect of Smilax glabra on HUA before and after fermentation is explored, so as to lay a solid theoretical foundation for the development and application of uric acid-lowering products. The results of the present invention show that after fermentation, the content of total flavonoids and total polyphenols increases, and the content of polysaccharides decreases; the fermented product of Rhizoma Smilacis Glabrae (FRSG) can significantly reduce the levels of serum uric acid, urea nitrogen and creatinine in HUA rats; improve kidney damage caused by high uric acid; reduce the increase of cytokines such as IL-1β and TNF-α in serum, and its therapeutic effect is better than that of unfermented Rhizoma Smilacis Glabrae (RSG); the mechanism of FRSG in lowering uric acid may be through inhibiting the activity of xanthine oxidase (XOD) in HUA rats and increasing the excretion of uric acid in feces. This study provides a solid theoretical reference for the development of safe and effective uric acid-lowering Chinese medicine health products.

[0028] Furthermore, the present invention obtained the optimal fermentation process of Smilax glabra through experiments, that is, the material-liquid ratio is 1:6, the fermentation time is 66h, and the inoculation amount is 8%. Experimental verification under this process condition found that the total acidity of the Smilax glabra fermentation liquid is 22.5g / L. There is no significant difference between the theoretical value and the actual value of the independent t test, indicating that the prediction conditions of the model are consistent with the actual situation. The live bacteria in the fermentation liquid were counted under the optimal fermentation conditions. After counting by the dilution plate method, the number of live bacteria in the fermentation liquid was finally counted to be 8.6*10 8 CFU / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the interactive effect of each factor on the total acidity of the fermentation broth; among them, A is the interactive effect of fermentation time and inoculation amount on total acidity; B is the interactive effect of fermentation time and liquid ratio on total acidity; C is the interactive effect of liquid ratio and inoculation amount on total acidity;

[0030] Figure 2 is an HPLC chromatogram; wherein A is astilbin reference substance, B is Smilax glabra; C is fermented Smilax glabra 1: astilbin;

[0031] Figure 3 The effects of RSG and FRSG on serum uric acid in HUA rats;

[0032] Figure 4 The effect of RSG and FRSG on serum creatinine and urea nitrogen in HUA rats; A is creatinine; B is urea nitrogen;

[0033] Figure 5 The effects of RSG and FRSG on urinary uric acid in HUA rats;

[0034] Figure 6 The effects of RSG and FRSG on fecal uric acid in HUA rats;

[0035] Figure 7 The rat kidney pathological sections and scores; A represents the rat kidney pathological sections-HE staining; B represents the rat kidney pathological sections scoring results;

[0036] Figure 8 The effects of RSG and FRSG on serum IL-1β and TNF-α in HUA rats; A is IL-1β; B is TNF-α;

[0037] Fig. 9 The effects of RSG and FRSG on serum XOD in HUA rats;

[0038] Fig.10 For PCA analysis. DETAILED DESCRIPTION

[0039] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings 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 should fall within the scope of protection of the present invention.

[0040] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0041] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0042] 1. Materials and methods

[0043] 1.1 Traditional Chinese medicine and strains

[0044] Smilax glabra Roxb. is provided by the Northwest National Engineering Laboratory for Endangered Medicinal Materials; the strains are Lactobacillus breris YM 1301 (Lactobacillus breris YM 1301) and Lactobacillus plantarum CICC 24202 (Lactobacillus plantarum CICC 24202). The present invention is not particularly limited to the sources of Lactobacillus plantarum CICC 24202 and Lactobacillus breris YM 1301, and conventional commercial products in the art can be used. In a specific embodiment of the present invention, Lactobacillus plantarum CICC 24202 is purchased from the Strain Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, and Lactobacillus breris YM 1301 is purchased from the Strain Collection Center of the Institute of Microbiology, Kunming.

[0045] 1.2 Reagents

[0046] MRS culture medium (Beijing Aoboxing Biotechnology Co., Ltd.); potassium oxonate (Sigma Biological Reagent Co., Ltd.); hypoxanthine (Sigma Biological Reagent Co., Ltd.); allopurinol tablets (Hefei Jiulian Pharmaceutical Co., Ltd.); uric acid (UA) detection kit (Nanjing Jiancheng Bioengineering Institute); rat IL-1β enzyme-linked immunosorbent assay kit (Shanghai ELISA Co., Ltd.).

[0047] 1.3 Experimental instruments

[0048] High-pressure steam autoclave (Hefei Huatai Medical Equipment Co., Ltd.); intelligent constant temperature and humidity incubator (Ningbo Jiangnan Instrument Factory); clean bench (Shanghai Yuejin Medical Equipment Factory); electric constant temperature water bath (Beijing Kewei Yongxing Instrument Co., Ltd.); vacuum freeze dryer (Bio Tek, USA); microplate reader (Thermo Fisher Scientific Inc.).

[0049] 1.4 Experimental methods

[0050] 1.4.1 Activation of strains

[0051] Lactobacillus breris YM 1301 (Lb) and Lactobacillus plantarum CICC 24202 (Lp) were inoculated into MRS liquid culture medium, and activated by static culture in a 37°C constant temperature incubator for 12 hours, and activated for two generations. After Lp and Lb were mixed at a ratio of 1:1, a fermentation seed liquid was obtained.

[0052] 1.4.2 Traditional Chinese Medicine Fermentation

[0053] After drying, crush the smilax glabra and pass it through a 100-mesh sieve for later use. Take 60g of smilax glabra powder and put it into a fermentation bottle. Add 300mL of distilled water and then add α-amylase for enzymatic hydrolysis (enzyme addition: 0.2%, temperature: 60°C, pH: 6.5). Use iodine colorimetry to determine whether the enzymatic hydrolysis is complete. After the enzymatic hydrolysis is completed, adjust the pH to neutral and sterilize with high-pressure steam (121°C, 101KPa, 15min) to obtain the smilax glabra Chinese medicine stock solution. Mix the two seed solutions of Lb and Lp in a ratio of 1:1 and inoculate them into the Chinese medicine stock solution at an inoculation rate of 8%. After fermentation at 37°C for 48h, freeze-dry the sample to obtain the fermented smilax glabra.

[0054] 1.4.3 Optimization of fermentation process

[0055] Design Expert software was used to optimize the fermentation process of Smilax glabra by taking fermentation time (X1), inoculation amount (X2) and material-liquid ratio (X3) as variables and total acidity in the fermentation broth (Y) as the response value. The factor level coding of the response surface experiment is shown in Table 1.

[0056] Table 1

[0057]

[0058] 1.4.3 Comparison of substances before and after fermentation of Smilax glabra

[0059] The contents of total flavonoids, total polyphenols, polysaccharides and the changes of main active substances in Smilax glabra before and after fermentation were compared.

[0060] 1.4.4 Experimental evaluation of the effect of fermented Smilax glabra on lowering uric acid

[0061] 1.4.3.1 Animals

[0062] The experimental animals used in this experiment were all SD male rats, 6-8 weeks old, with an initial weight of 150-160 g, purchased from the Animal Experiment Center of Shaanxi Normal University, with an animal production license number of SCXK(Shaanxi)2021-402. The animal experimental operation process was carried out in accordance with the Guidelines for Laboratory Animal Care and approved by the Animal Care and Use Committee of Shaanxi Normal University, with the approval number ECES-2019-0322.

[0063] 1.4.3.2 Animal grouping and treatment

[0064] 50 male SD rats, weighing 150-160 g, were kept in a clean animal room at a constant temperature of 24±1℃.

[0065] Relative humidity was 50%-70%, with 12h light and 12h dark per day, and the rats were adapted for one week, during which time they were fed with basic feed and had free access to water. One week later, the SD rats were divided into a normal group (Normal Control, NC) and a modeling group. The modeling group was intraperitoneally injected with potassium oxonate (PO, 250mg / kg) and gavage with hypoxanthine (HX, 300mg / kg) every morning for modeling. The normal control group (10 rats) was intraperitoneally injected with saline and gavage with 0.5% CMC-Na solution (1mL / 100g) for one week. One week later, the serum uric acid value of the rats was measured, and the serum uric acid value was significantly higher than that of normal rats, which was considered to be a successful modeling. The HUA model rats were evenly divided into four groups, namely the model group (MOD), the Smilax glabra group (RSG), the fermented Smilax glabra group (FRSG), and the allopurinol group (ALLOP), with 10 rats in each group. The dosage of each group is shown in Table 2 below. Three weeks later, the 24-hour feces and urine of the rats were collected using metabolic cages. After the last dose, the rats were fasted for 12 hours, and 1.5 mL of blood samples were collected from the orbital venous plexus after the rats were anesthetized with sodium pentobarbital. Subsequently, the rats were killed by cervical dislocation, and tissues such as the kidney and liver were removed. Part of the kidney tissue was fixed in 4% paraformaldehyde for the preparation of histopathological sections; the remaining kidney tissue and liver tissue were packaged and stored in a -80°C refrigerator. After the blood sample was allowed to stand at room temperature for 1 hour, it was centrifuged at 7000g for 10 minutes at 4°C, and the upper serum was taken for subsequent detection of biochemical indicators.

[0066] Table 2

[0067]

[0068] 1.4.3.3 Serum biochemical index detection

[0069] The corresponding biochemical kits were used to detect the levels of serum uric acid (UA), creatinine (CRE) and blood urea nitrogen (BUN), and the specific operations were carried out according to the instructions of the kits.

[0070] 1.4.3.4 Detection of serum cytokines and XOD

[0071] The levels of TNF-α and IL-1β in serum samples were detected according to the operating instructions of the ELISA kit, and the level of XOD in serum was detected according to the instructions of the kit.

[0072] 1.4.3.5 Observation and evaluation of renal tissue pathology

[0073] After the kidney tissue was fixed with paraformaldehyde, it was dehydrated, transparent, and embedded. A 5 μm tissue wax slice was cut, dewaxed and hydrated, and then stained with hematoxylin-eosin (H&E), dehydrated and transparent, and the slice was sealed with gum. Finally, the structure and morphology of the kidney tissue were observed under a microscope. Three people who were not aware of the experimental protocol were randomly assigned to perform the renal tissue pathology scoring.

[0074] 1.4.3.6 Comprehensive evaluation of drug efficacy by PCA analysis

[0075] IBM SPSS Statistics 26 software was used to reduce the dimension of the measured efficacy indicators and comprehensively evaluate the treatment effects of each group.

[0076] 1.5 Data Analysis

[0077] The experimental data were processed by IBM SPSS Statistics 26 and GraphPad Prism 5 software and expressed as mean ± standard error (Mean ± SEM). The difference between the groups was analyzed by t test, when p < 0.05, it was considered to be significantly different, and when p < 0.01, it was considered to be extremely significantly different.

[0078] 2. Results and Analysis

[0079] 2.1 Response surface methodology to investigate the fermentation process of Smilax glabra

[0080] Table 3 Response surface significance test and variance analysis

[0081]

[0082] See Table 3. Based on the single factor results, the Design Expert software was used to conduct a three-factor three-level response surface analysis experiment on the fermentation process of Smilax glabra. The experiment determined the total acidity of the Smilax glabra fermentation liquid, where A, B, and C represent the fermentation time, inoculation amount, and material-liquid ratio. Taking the total acidity as the response value, according to the regression model, the linear relationship between the total acidity variables is obtained as follows:

[0083] Total acidity = -144.2520 + 3.0810*A + 8.8605*B + 11.9340*C - 0.0187*AB - 0.0487*AC

[0084] -0.1575*BC-0.0201*A 2 -0.4545*B 2 -0.7020*C 2

[0085] As can be seen from Table 3, statistical analysis shows that high F values ​​and low P values ​​represent that the model is highly significant. The smaller the P value, the better the fit between the equation and the actual situation, and the model is suitable for use. The F value and P value of the lack of fit item indicate that the fit is not significant, and the test value is in good agreement with the predicted value. The correlation coefficient R2 is 0.968079, indicating that the model has good credibility, and the interaction coefficient AC is significant (P < 0.05).

[0086] See also Figure 1 , the optimal fermentation conditions of Smilax glabra were analyzed by Design-Export software: the material-liquid ratio was 1:6, the fermentation time was 66h, and the inoculation amount was 8%. Experimental verification was carried out under this condition, and the total acidity of the Smilax glabra fermentation liquid was 22.5g / L. There was no significant difference between the theoretical value and the actual value of the independent t-test, indicating that the prediction conditions of the model were consistent with the actual situation. The live bacteria in the fermentation liquid were counted under the optimal fermentation conditions. After counting by the dilution coating plate method, the number of live bacteria in the fermentation liquid was finally counted to be 8.6*10 8 CFU / mL.

[0087] 2.2 Changes in the content of total flavonoids, polyphenols, and polysaccharides before and after fermentation of Smilax glabra

[0088] Table 4 Comparison of substances before and after fermentation of Smilax glabra

[0089]

[0090] As shown in Table 4, the total flavonoids content of Smilax glabra after fermentation reached 2.592±1.34%, which is 1.6 times the total flavonoids content of unfermented Smilax glabra; the total polyphenol content of Smilax glabra after fermentation was 1.937±0.24%, and the content of total polyphenols in fermented Smilax glabra was significantly increased compared with that in unfermented Smilax glabra; the dry matter of Smilax glabra polysaccharide was obtained by water extraction and alcohol precipitation, and compared with the sugar content in unfermented Smilax glabra polysaccharide, the content of fermented Smilax glabra polysaccharide was significantly reduced, which may be caused by the consumption of probiotics during the growth process.

[0091] Table 5 Determination of astilbin content before and after fermentation of Smilax glabra

[0092]

[0093] Studies have shown that most flavonoids in plants exist in the form of glycosides, which are not easily absorbed in the human body and have low utilization rates. After fermentation, flavonoids can be converted into aglycones and exist in a free form. Astilbin, also known as 3-O rhamnosyl methyl ester of yew leaves, is the quality control active ingredient of the traditional Chinese medicine Smilax glabra. As shown in Table 5, after fermentation of Smilax glabra, the content of astilbin (0.46%) decreased by 52%, but it can be seen from the HPLC chromatogram that the content of aglycones in other forms increased. This may be due to the fact that during the fermentation process, the enzyme environment provided by the probiotics hydrolyzed astilbin, causing it to exist in the form of aglycones with a smaller molecular weight.

[0094] The medical definition of hyperuricemia is: the fasting blood uric acid level on two different days is higher than 420μmol / L for men and higher than 360μmol / L for women. The level of uric acid in serum is a key indicator of hyperuricemia. At the same time, the body will also suffer certain kidney damage under the condition of high uric acid for a long time. Therefore, it is of great significance to detect the levels of uric acid, creatinine and urea nitrogen in serum. This experiment used drug intervention, and after four weeks of potassium oxonate combined with hypoxanthine modeling. Figure 2 , 3 As shown in Figure 2, compared with normal rats, the serum uric acid level of the model group rats was significantly increased (p<0.01), which was 1.83 times that of the normal rats. Figure 4 , the renal function indicators CRE and BUN of the model group rats increased significantly (p<0.05), indicating that long-term high serum uric acid levels can cause kidney damage, which is consistent with the characteristics of hyperuricemia and also indicates that the HUA rat model was successfully established. RSG and FRSG intervention in HUA rats can alleviate the increase in serum UA, CRE and BUN in HUA rats, indicating that both drugs have good uric acid-lowering and renal protective effects. The effect of FRSG is particularly prominent, which can significantly reduce the levels of serum UA and BUN (p<0.05), and there is also a trend to reduce CRE. It is worth noting that the serum uric acid level of HUA rats almost returned to normal levels after treatment with allopurinol (p<0.01), but the renal protection is unsatisfactory.

[0095] Effect of FRSG on uric acid excretion in HUA rats

[0096] There are two forms of uric acid excretion in the human body, two-thirds of which are excreted through the kidneys and one-third through the intestines. In this experiment, 24-hour urine of HUA rats was collected and the uric acid content was detected. Figure 5As shown in the figure, the uric acid content in the urine of HUA rats was 435.2 mg / L, which was significantly higher than the urinary uric acid level of normal rats (p<0.05), indicating that a large amount of uric acid was produced in the HUA rats and part of the uric acid was excreted from the body in the form of urine. After drug intervention, the urinary uric acid levels of rats in each drug group were significantly lower than those of HUA rats (p<0.05). This result suggests that RSG and FRSG may inhibit the synthesis of uric acid in HUA rats, thereby reducing the level of uric acid.

[0097] The intestine is not only responsible for purine absorption, but also an important organ for uric acid excretion. Figure 6 As shown in the fecal uric acid content determination, it was found that there was no difference in the FEUA level between normal rats and HUA rats. However, after intervention with FRSG, the level of FEUA was significantly increased (p<0.05), which may be due to the fermentation characteristics of FRSG.

[0098] When the animals were treated, it was found that compared with normal rats, the kidneys of HUA rats were enlarged, yellow-brown, and had uneven stripes on the surface. The apparent state of the kidneys in the drug-treated groups was improved, but it is worth noting that the appearance of the kidneys of the rats in the allopurinol group was roughly the same as that of the model group. Figure 7 Pathological sections showed that compared with normal rats, the renal tubules of HUA rats were significantly dilated, epithelial vacuolation occurred, brush borders fell off, and casts formed inside; the glomerular basement membrane was thickened and edematous; inflammatory cells infiltrated the renal interstitium, showing a serious pathological state (p<0.01). RSG and FRSG significantly improved the renal pathological state of HUA rats, and FRSG had a particularly prominent protective effect on the kidneys of HUA rats (p<0.01). It is worth noting that allopurinol did not improve the existing renal damage in rats.

[0099] Effect of FRSG on serum cytokine levels in HUA rats

[0100] Uric acid has been identified as an endogenous adjuvant that can drive immune responses without microbial stimulation, and long-term high levels of uric acid can have adverse effects on the body’s health. Figure 8 As shown in the results, compared with normal rats, the levels of IL-β and TNF-α in the serum of HUA rats were significantly increased (p<0.05), indicating that long-term high levels of serum uric acid can lead to severe inflammation in the body. RSG and FRSG can improve the increase of serum IL-β to varying degrees, among which FRSG has a more prominent improvement effect (P<0.01). At the same time, the increase of serum TNF-α can be alleviated after administration of RSG and FRSG, among which FRSG has a better effect (p<0.05).

[0101] Effect of FRSG on serum XOD levels in HUA rats

[0102] Xanthine oxidase (XOD) is a key enzyme in the uric acid metabolic pathway. Its activity directly affects the amount of uric acid produced and is often used as a target for uric acid-lowering drugs. Fig. 9 As shown in the results, the serum XOD level of HUA rats was significantly higher than that of normal rats (p<0.05), indicating that the XOD activity in HUA rats was high and a large amount of uric acid was produced. After drug treatment, RSG had a tendency to reduce the serum XOD level of HUA rats, but there was no statistical difference. Compared with the model group, FRSG could significantly reduce the serum XOD level of HUA rats (p<0.05); allopurinol is a commonly used uric acid-lowering drug targeting XOD in clinical practice, and its inhibition of XOD activity was particularly significant (p<0.01).

[0103] 2.8 Comparison of the therapeutic effects of each medication group using PCA analysis

[0104] Table 6

[0105]

[0106] Principal component analysis is to transform the original multiple indicators into a few representative comprehensive indicators. These indicators can reflect most of the information of the original indicators, and each indicator remains independent to avoid overlapping information. PCA analysis plays an important role in the analysis of natural medicines. We selected 7 core indicators such as UA, CRE, and BUN for PCA analysis, and obtained two principal components PC1 and PC2, whose expressions are y1 = -0.380zx1-0.331zx2-0.452zx3-0.252zx4-0.334zx5-0.396zx6-0.457zx7; y2 = -0.229zx1+0.486zx2+0.131zx3-0.717zx4-0.331zx5+0.213zx6+0.161zx7. Then the Y value is calculated according to the proportion of each group on different principal components, and the size of the Y value can represent the effect of the drug. As shown in Table 6, the Y value of normal rats was the highest, and the Y value of model rats was the lowest. After drug treatment, the comprehensive therapeutic effect of FRSG was better than RSG and ALLOP, and the Y value ranked second. PCA analysis was used to more objectively and comprehensively evaluate the therapeutic effect of FRSG on HUA rats. (See Fig.10 )

[0107] In summary, the present invention uses the powdery and edible properties of Chinese medicine Smilax glabra to ferment lactic acid bacteria and explore the uric acid-lowering effect of its fermentation products. The results show that the optimal fermentation process of Smilax glabra is: material-liquid ratio 1:6, fermentation time 66h, and inoculation amount 8%; the total flavonoid content of Smilax glabra after fermentation is 1.6 times that of unfermented, the polyphenol content increases, and the Smilax glabra polysaccharide content decreases; the content of astilbin decreases after fermentation, and the content of other aglycones increases; in the treatment of HUA rats, FRSG can reduce the levels of serum uric acid, creatinine and urea nitrogen in HUA rats, increase the excretion of uric acid in feces, improve kidney damage caused by high uric acid, reduce the increase of IL-1β and TNF-α in serum, and significantly inhibit the activity of XOD; PCA was used to comprehensively analyze the efficacy of FRSG, and the results showed that the comprehensive therapeutic effect of FRSG was better than allopurinol and RSG. Studies have shown that Smilax glabra has better HUA treatment effect after lactic acid bacteria fermentation, which provides a reliable theoretical experimental basis for the research and development of Chinese medicine fermentation-related products that have the effect of alleviating hyperuricemia and have the advantages of high safety, easy production and preservation, and high quality stability.

[0108] Based on the above reasons, the present invention conducts probiotic fermentation on Smilax glabra, detects the anti-hyperuricemia effect of the fermented product, and observes whether the fermented Smilax glabra has better anti-hyperuricemia effect. The experimental results of the present invention confirm that the Smilax glabra fermented by probiotics has better anti-hyperuricemia effect, can significantly increase the excretion of uric acid in feces, and significantly reduce the renal function damage caused by hyperuricemia, providing a new technical strategy and product manufacturing solution for alleviating hyperuricemia.

[0109] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing a fermented product of Smilax glabra, characterized in that: Inoculating Lactobacillus fermented seed liquid into the Chinese herbal medicine stock solution of Smilax glabra, fermenting at 37°C for 48-72 hours, and freeze-drying to obtain the Smilax glabra fermented product; The lactobacillus fermented seed liquid is obtained by fermenting Lactobacillus brevis YM 1301 and Lactobacillus plantarum CICC24202; the lactobacillus fermented seed liquid accounts for 6% to 10% of the volume of the Chinese smilax glabra stock liquid.

2. The method for preparing the fermented smilax glabra according to claim 1, characterized in that: The lactobacillus fermented seed liquid is prepared by mixing Lactobacillus brevis YM 1301 seed liquid and Lactobacillus plantarum CICC24202 seed liquid in a volume ratio of 1:

1.

3. The method for preparing the fermented product of Smilax glabra according to claim 1, characterized in that: The OD value of the Lactobacillus fermentation seed solution was adjusted to 1.

0.

4. The method for preparing the fermented product of Smilax glabra according to claim 1, characterized in that: The Chinese herbal medicine stock solution of Smilax glabra is prepared according to the following method: After drying, crush and sieve the Rhizoma Smilacis Glabrae to obtain Rhizoma Smilacis Glabrae powder for later use; Water is added to the smilax glabra powder, and then α-amylase is added for enzymolysis for 4-5 hours, and then the pH value is adjusted to neutral, sterilized, and the smilax glabra traditional Chinese medicine stock solution is prepared.

5. The method for preparing the fermented product of Smilax glabra according to claim 4, characterized in that: The dosage ratio of Smilax glabra powder to water is 1g: (4-8)mL.

6. The method for preparing the fermented product of Smilax glabra according to claim 4, characterized in that: The added amount of α-amylase is 0.2% of the volume of the Smilax glabra traditional Chinese medicine stock solution, the enzymatic treatment temperature is 60° C., and the pH value is 6.

5.

7. The fermented product of Smilax glabra obtained by the preparation method according to any one of claims 1 to 6, characterized in that: The total flavonoids content of the fermented Smilax glabra is 2.59±1.34%, which is 1.6 times that of the unfermented Smilax glabra; the total polyphenols content is 1.93±0.24%, which is 1.45 times that of the unfermented Smilax glabra; and the astilbin content is reduced to 52% of that of the unfermented Smilax glabra.

8. Use of the Smilax glabra fermented product according to claim 7 in the preparation of food or health food for improving the blood uric acid level in patients without hyperuricemia.

9. The use according to claim 8, characterized in that The food or health food is a food or health food that reduces serum uric acid, urea nitrogen and creatinine levels; or, Food or health food for improving kidney damage caused by high uric acid; or, Food or health care that reduces the increase of inflammatory factors in serum; or, Foods or health foods that increase uric acid excretion in feces.

10. Use of the Smilax glabra fermented product according to claim 7 in the preparation of a medicament for treating hyperuricemia.

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