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CN-122012665-A - Three-fruit Shang Kangyan component high-screening method based on intestinal flora metabolic model

CN122012665ACN 122012665 ACN122012665 ACN 122012665ACN-122012665-A

Abstract

The invention discloses a three-fruit Shang Kangyan component high-screening method based on an intestinal flora metabolic model, and belongs to the technical field of traditional Chinese medicine active component screening. The method comprises the steps of constructing a special intestinal flora metabolism model (comprising an in-vitro SHIME dynamic simulation system and an in-vivo flora humanized mouse model) of the three-fruit decoction, preparing a three-fruit decoction total extract, different polarity parts and single prototype components, co-culturing the three-fruit decoction with the in-vitro flora to obtain metabolites, locking the high-activity prototype components and the metabolites through a three-level anti-inflammatory activity screening system, and performing structural identification, anti-inflammatory mechanism verification, content and metabolic efficiency detection and in-vivo effectiveness confirmation, and repeating optimization to form a standardized method. The invention solves the problems that the traditional screening does not consider pain points with intestinal metabolism and incomplete results, takes direct and indirect anti-inflammatory activities into account, has accurate fit of screening results to clinic, controllable operation and good repeatability, and can be used for quality control of three fruit soup, formulation improvement and anti-inflammatory innovative drug development.

Inventors

  • LI BIN
  • CUI MINGXUE
  • NIU YANFEI

Assignees

  • 青岛科技大学

Dates

Publication Date
20260512
Application Date
20260302

Claims (9)

  1. 1. The three-fruit Shang Kangyan component high-screening method based on the intestinal flora metabolic model is characterized by comprising the following steps of: s1, constructing a special intestinal flora metabolic model of the three-fruit soup, wherein the special intestinal flora metabolic model comprises an in-vitro intestinal flora metabolic model and an in-vivo intestinal flora metabolic model; s2, preparing a three-fruit soup total extract, effective parts with different polarities and single prototype components, co-culturing the three-fruit soup total extract and the single prototype components with an in-vitro intestinal flora metabolism model, and separating to obtain a metabolite group and a non-metabolizing prototype component; S3, adopting a three-level anti-inflammatory activity screening system to sequentially perform anti-inflammatory activity screening, single activity screening of prototype components and metabolites, and intestinal flora regulation activity screening, and locking high-activity components; s4, carrying out structural identification on the high-activity component, and verifying an anti-inflammatory action mechanism of the high-activity component; S5, detecting the content and metabolic efficiency of the high-activity component, verifying the in-vivo effectiveness of the high-activity component through an in-vivo intestinal flora metabolic model, and finally locking the high-activity core component of the three fruits Shang Kangyan; s6, repeated screening verification, optimizing screening parameters and forming a standardized screening method.
  2. 2. The three-fruit Shang Kangyan-component high-screening method based on an intestinal flora metabolism model according to claim 1, wherein in the step S1, an in-vitro intestinal flora metabolism model adopts an SHIME dynamic intestinal simulation system, a standardized mixed intestinal flora is inoculated, the standardized mixed intestinal flora is obtained from fresh feces of 3-5 healthy volunteers through anaerobic gradient dilution and centrifugal purification, the flora abundance is more than or equal to 10 9 CFU/mL, the Shannon index is more than or equal to 3.5, the similarity with the human colon flora is more than or equal to 85%, and the metabolic conversion of ellagic acid to urolithin A can be realized.
  3. 3. The three-fruit Shang Kangyan component high-screening method based on an intestinal flora metabolic model according to claim 1, wherein in the step S1, the in-vivo intestinal flora metabolic model is a flora humanized mouse model, and the three-fruit Shang Kangyan component high-screening method is obtained by performing gastric lavage inoculation on an SPF-grade sterile mouse and standardized mixed intestinal flora, wherein the similarity of the intestinal flora of the mouse and the colonic flora of a human body is more than or equal to 85%.
  4. 4. The method for high screening of three-fruit Shang Kangyan components based on an intestinal flora metabolic model according to claim 1 is characterized in that in the step S2, three-fruit soup samples are prepared by mixing myrobalan, fructus Terminaliae Billericae and fructus phyllanthi according to a mass ratio of 1:1:1, crushing, reflux-extracting with 70% ethanol, concentrating under reduced pressure to obtain total extract, extracting with organic solvent gradient to obtain effective parts with different polarities, and separating with column chromatography to obtain single prototype component with purity of more than or equal to 98%.
  5. 5. The method for high selection of the three-fruit Shang Kangyan components based on the intestinal flora metabolic model according to claim 1, wherein in step S3, the specific method for three-level anti-inflammatory activity selection is as follows: S31, a level 1, namely, acting the co-culture supernatant on an RAW264.7 macrophage model induced by LPS, and screening a high-activity culture system with an inflammatory factor inhibition rate of more than or equal to 50% and an NO inhibition rate of more than or equal to 45%; S32, separating and purifying a metabolite group and an unmetabolized prototype component, calculating IC 50 value of each component, wherein the IC 50 value is less than or equal to 50 mu mol/L, namely the high-activity component, and locking the components of type A (prototype is inactive and high in activity of the metabolite) and type B (prototype is active and is enhanced in activity after metabolism); S33, detecting the influence of a sample on the intestinal flora structure, and screening components which can obviously increase the abundance of anti-inflammatory bacteria by more than or equal to 30%, reduce the abundance of pro-inflammatory bacteria by more than or equal to 25% and improve the expression quantity of intestinal barrier tight junction protein by more than or equal to 20%.
  6. 6. The method for high screening of the three-fruit Shang Kangyan components based on the intestinal flora metabolic model according to claim 1, wherein in the step S4, UPLC-MS/MS and NMR technology are adopted to carry out structural identification on the high-activity components, the high-activity components comprise prototype components and metabolites, the prototype components are gallic acid, corilagin, ellagic acid and myrobalan acid, and the metabolites are gallate lactone, urolithin A, urolithin B and gallic acid-glucuronic acid conjugate.
  7. 7. The method for screening three-fruit Shang Kangyan components based on an intestinal flora metabolic model according to claim 1, wherein in step S5, the content of the high-activity prototype component in the three-fruit soup compound is not less than 0.1%, the metabolite production amount is not less than 20% of the initial amount of the prototype component, the intestinal flora metabolic conversion rate of the prototype component is not less than 30%, and the in vivo inflammation inhibition rate is not less than 60%.
  8. 8. The method for high-screening the three-fruit Shang Kangyan components based on the intestinal flora metabolic model according to claim 1, wherein in the step S2, the co-culture condition is that anaerobic culture is carried out at 37 ℃ for 24-72 h, the concentration of a sample is 100-500 mug/mL, and 0.1% ascorbic acid is added into the sample after the co-culture for antioxidation treatment, and the sample is stored at a low temperature of 4 ℃.
  9. 9. The method for high-screening the three-fruit Shang Kangyan components based on the metabolic model of intestinal flora according to claim 1, wherein in the step S4, the inhibition of the high-activity component on the inflammatory pathway of NF- κ B, MAPK and the inhibition of the endotoxin LPS of intestinal tract are verified by using Western Blot method and double luciferase reporter gene method.

Description

Three-fruit Shang Kangyan component high-screening method based on intestinal flora metabolic model Technical Field The invention relates to the field of intestinal flora metabolic models, in particular to a three-fruit Shang Kangyan component high-screening method based on an intestinal flora metabolic model. Background The Sanguo decoction is a classical heat-clearing, detoxifying and anti-inflammatory prescription in Tibetan medicine, is composed of three core medicinal materials of myrobalan, fructus terminaliae bellae and phyllanthus emblica, is widely applied to the treatment of various inflammation related diseases such as lung heat, laryngalgia, pestilence and the like in clinic, and has long-term clinical practice verification of anti-inflammatory efficacy. Modern pharmaceutical researches show that the anti-inflammatory activity of the three-fruit soup mainly comes from natural chemical components such as polyphenols, tannins and the like contained in the three-fruit soup, such as gallic acid, corilagin, ellagic acid, myrobalan acid and the like, but the components are only part of complex chemical components of the three-fruit soup, and how to accurately screen out high-activity components which have strong anti-inflammatory activity, definite in-vivo efficacy and development value from a plurality of components is a core problem of modern researches of the three-fruit soup. At present, the existing screening method of the three-fruit Shang Kangyan component mainly adopts a traditional screening system of combining an in-vitro cell model (such as RAW264.7 macrophage model) with an in-vivo animal model, and the core thought is to separate prototype components in three-fruit soup, and screen out high-activity prototype components by detecting the inhibition of components on indexes such as inflammatory factors (TNF-alpha, IL-6, IL-1 beta, NO and the like). However, the screening method has obvious limitations and cannot be fit with the actual pharmacokinetic process of a human body, wherein the core anti-inflammatory component in the three-fruit soup is polyphenol and tannin compounds, the components have larger molecular weight and limited water solubility/fat solubility, only a small amount of the components can be directly absorbed by small intestine after being orally taken, more than about 70% of the components can enter colon, and the components are converted into small molecule metabolites through the metabolic actions of hydrolysis, decarboxylation, reduction and the like of intestinal flora of the human body, and the metabolites are often the main forms of the anti-inflammatory action in the human body. The existing screening method does not consider metabolic conversion links of intestinal flora, and has two key defects that firstly, the original components with no activity and high activity of metabolic products are easy to miss, so that the screening result is incomplete, secondly, the original components with high activity in vitro and degradation and inactivation in vivo by the intestinal flora are mistakenly used as core development targets, the waste of research and development resources is caused, and the in-vivo efficacy and in-vitro activity of the screened components are disjointed, so that the clinical application requirements are difficult to meet. In addition, the existing screening method only focuses on the direct anti-inflammatory activity of the components, does not consider the effect characteristics of the three-fruit soup compound for realizing indirect anti-inflammatory by adjusting the balance of intestinal flora and repairing intestinal barriers, cannot capture the synergistic activity of the components 'direct anti-inflammatory and indirect anti-inflammatory', and is not matched with the anti-inflammatory characteristics of the three-fruit soup 'multi-component, multi-target and multi-way'. Meanwhile, in the prior art, although a small amount of researches pay attention to the relevance of intestinal flora and traditional Chinese medicine anti-inflammatory (such as the research of regulating the intestinal flora by part of traditional Chinese medicine compositions), a set of special screening technical system aiming at three fruit soups is not formed, the accurate adaptation of an intestinal flora metabolism model and anti-inflammatory activity screening is not realized, a closed loop screening flow of metabolic conversion, layering activity screening, mechanism verification and in vivo confirmation is not established, and the core pain point of 'in vitro activity and in vivo efficacy disjointing and incomplete screening result' in three fruit Shang Kangyan component screening cannot be solved. Therefore, a high screening method of three-fruit Shang Kangyan components is needed, which can simulate the metabolic process of human intestinal canal, give consideration to direct and indirect anti-inflammatory activity and accurately fit the screening result to the clini