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CN-121970751-A - Aloesin composition for inhibiting growth of vibrio desulphurisation and generation of hydrogen sulfide, and screening method and application thereof

CN121970751ACN 121970751 ACN121970751 ACN 121970751ACN-121970751-A

Abstract

The invention belongs to the technical field of pharmaceutical compositions, and particularly relates to an aloin composition for inhibiting growth of vibrio desulphurisation and generation of hydrogen sulfide, and a screening method and application thereof. The invention prepares the raw material mixture by uniformly mixing aloin, flavone, sea buckthorn extract, sappan wood extract and erigeron breviscapus extract, and the obtained composition is light yellow powder, has good effects of inhibiting growth of sulfate reducing bacteria and vibrio desulphurisation and reducing generation of hydrogen sulfide, and is particularly suitable for solving the problem of hydrogen sulfide emission caused by sulfate reducing bacteria such as vibrio desulphurisation and the like. The method for inhibiting the growth of sulfate reducing bacteria and vibrio desulphurisation and reducing the generation of hydrogen sulfide by virtual screening has the advantages of novel screening method, simple operation of the preparation method of the composition, easily available raw materials, low cost and easy repetition, and has important significance for preparing products for inhibiting the growth of vibrio desulphurisation and the generation of hydrogen sulfide.

Inventors

  • DENG DUN
  • Weng Guangying
  • LIU ZHICHANG
  • MA XIANYONG
  • SONG MIN
  • YU MIAO
  • RONG TING

Assignees

  • 广东省农业科学院动物科学研究所

Dates

Publication Date
20260505
Application Date
20251217

Claims (9)

  1. 1. An aloin composition for inhibiting the growth of vibrio desulphurisation and the generation of hydrogen sulfide, which is characterized by comprising aloin, flavone, sea buckthorn extract, sappan wood extract and erigeron breviscapus extract.
  2. 2. The aloin composition according to claim 1, wherein the aloin is 200-250 parts, flavone is 100-120 parts, sea buckthorn extract is 30-40 parts, sappan wood extract is 20-30 parts, erigeron breviscapus extract is 10-20 parts by weight.
  3. 3. A screening method of natural compounds for inhibiting growth of vibrio desulphurisation and generation of hydrogen sulfide, comprising the steps of: 1) Homologous protein phylogenetic analysis and sequence alignment for confirming protein target-target protein catabolite sulfite reductase 1.1 The sequence and crystal structure of the target enzyme Dsr are obtained from the Uniprot database and the PDB database, and the amino acid sequence and the protein crystal structure of the dissimilated sulfite reductase produced by Desulfovibrio vulgaris are downloaded from the Uniprot database and the PDB database respectively; 1.2 Phylogenetic analysis and sequence multiplex alignment): Generating a Dsr phylogenetic tree by adopting MEGA X (adjacent method); Alignment of the crystal protein structure using Pairwise Structure Alignment in RCSB PDB; 1.3 Using structural analysis software to determine the active pocket and key amino acid residues of the enzyme: Determining amino acid residues involved in a substrate binding pocket using different active sites recommended by the analysis MOE v2015.10011 software SITE FINDER module, performing multiple alignment on Dsr homologous protein sequences using Clustal Omega, editing, visualizing and analyzing the sequence alignment using Jalview; 2) Virtual screening is carried out based on Dsr enzyme structure, and potential compounds are screened out 2.1 A preliminary screening compound library is established, similarity screening is carried out with known inhibitors, 32 ten thousand molecules are randomly selected from a ChemBridge compound database to be a virtual screening database, and the 32 ten thousand molecules are compared with the inhibitor molecules with known Dsr structures to calculate similarity values; 2.2 Selecting 10,000 molecules with the similarity value ordered at the front, filtering by opera' slead-likefilter in MOE, and removing non-conforming molecules; Filtering to obtain molecules, and further processing by using a Wash module in MOE; 2.3 Molecular docking calculations were performed with Dsr protein as receptor: then adopting molecular docking and taking Dsr as a receptor; The binding pocket in the protein is determined by the SITE FINDER module in the MOE; Before docking, selecting an AMBER10, namely an EHT force field and an R-field implicit solvent model, and docking by adopting an reduced fit mode; After the receptor is introduced into the MOE software, the protonation state of the protein and the position of the hydrogen atom are determined by the LigX module in the MOE software; 2.4 According to the butt scoring and cluster analysis, finally selecting 20 diversified candidate compounds, namely sorting the binding modes of the ligand and the receptor through a London dG scoring function, and then evaluating the first 10 conformations again through a GBVI/WSA dG method to obtain a final conformation; after the butt joint is completed, selecting the first 100 compounds with the lowest butt joint scoring, performing cluster analysis of molecular fingerprints, and finally identifying a diversified subset with 20 hit compounds; 5 compounds of flavone, aloin, isorhamnetin, rutin and scutellarin are screened out, and the best potential compound screened out is aloin; 3) Preliminary screening of sulfate reducing bacteria enriched flora by using compound 3.1 Enrichment and culture of flora SRB Mixed flora from fecal samples Culturing livestock manure, and separating vibrio desulphurisation after black precipitation occurs in a culture system and odor of the smelly eggs escapes, which indicates that sulfate reducing bacteria in the manure are enriched; The enriched SRB bacterial liquid is subjected to serial gradient dilution by using sterile normal saline, and is coated on a Postgate solid culture medium, a proper gradient coated flat plate is selected, and different bacterial colonies are selected for flat plate streaking according to different bacterial colony morphology, size and other indexes until the bacterial colony morphology and size are uniform; finally, selecting monoclonal colonies with different indexes such as morphology, size and the like, and inoculating the monoclonal colonies into a Postgate culture medium for anaerobic culture; 3.2 Primary activity test) Co-culturing the candidate compound with the enriched SRB flora; 3.3 Evaluation of Effect The inhibition effect of the compound on the activity of the whole SRB flora is primarily estimated by measuring the pH value, sulfate consumption and the yield of metabolic products such as hydrogen sulfide, methyl mercaptan and the like after culture; 4) Compound desulfur vibrio single bacteria re-screening The single colony D. Vulgaris of the purified desulfurated vibrio is separated from the enriched bacterial colony, and the growth curve is measured to perform the specific activity test and effect evaluation.
  4. 4. A screening method according to claim 3, wherein the similarity value is at least 0.53.
  5. 5. A screening method according to claim 3, wherein the non-conforming molecules are to be removed under the conditions of a) N or O atoms as hydrogen bond donors not more than 5;b) number of N and O atoms as hydrogen bond acceptors not more than 8;c), molecular weight not more than 450, d) log P value in the range of [ -3.5-4.5], e) number of tricyclic to octacyclic rings not more than 4, f) number of rotatable bonds not more than 10.
  6. 6. A screening method according to claim 3, wherein the LigX module is set at a ph=7 and at a temperature of 300K.
  7. 7. A screening method according to claim 3, wherein 5 natural compounds of flavone, aloin, isorhamnetin, rutin and scutellarin are finally co-screened.
  8. 8. The method of claim 7, wherein the final natural compound selected is aloin.
  9. 9. Use of an aloin composition according to claim 1 or 2, or a natural compound according to claim 7 or 9, for the preparation of a product for inhibiting the growth of vibrio desulphurisation and the production of hydrogen sulphide.

Description

Aloesin composition for inhibiting growth of vibrio desulphurisation and generation of hydrogen sulfide, and screening method and application thereof Technical Field The invention belongs to the technical field of deodorization additives, and particularly relates to an aloin composition for inhibiting growth of vibrio desulphurisation and generation of hydrogen sulfide, and a screening method and application thereof. Background Hydrogen sulfide (H 2 S) is colorless, extremely toxic, inflammable and has strong odor of smelly eggs, and widely exists in environments such as oil fields, sewage treatment plants, livestock and poultry farms, chemical production, intestinal microecology and the like. The source mainly comprises Sulfate-reducing bacteria (Sulfate-Reducing Bacteria, SRB) which are produced by a catabolic Sulfate reduction process under anaerobic conditions. Vibrio desulphurisation (Desulfovibrio) is a typical and widely distributed genus of SRB, and H 2 S produced in the metabolic process not only corrodes metal pipelines and pollutes the environment, but also poses serious threats to animal and human health, such as causing intestinal inflammation, metabolic disorders and even nervous system toxicity. Currently, the control means for hydrogen sulfide generation mainly include physical adsorption, chemical oxidation, biological inhibition, and the like. Although the common chemical inhibitors such as molybdate, nitrate, aldehydes and the like have certain effects, the common chemical inhibitors have the problems of high cost, environmental residue, low biological safety, easy generation of drug resistance and the like. Therefore, the development of efficient, safe, and environmentally friendly inhibitors of natural origin is an important direction of current research. In recent years, natural plant extracts have good application potential in the fields of bacteriostasis and metabolic regulation due to the characteristics of wide sources, good biocompatibility, environmental friendliness and the like. Natural components such as aloin, flavone, sea buckthorn extract, sappan wood extract, erigeron breviscapus extract and the like have been reported to have various biological activities such as antibiosis, antioxidation, anti-inflammatory and the like, but a synergistic action mechanism of the natural components in the aspects of inhibiting the growth of sulfate reducing bacteria, particularly vibrio desulphurisicus and the generation of H 2 S has not been systematically studied. In addition, the traditional inhibitor screening method is mostly dependent on experimental trial and error, and is low in efficiency and high in cost. With the development of computational biology and structural biology, virtual screening methods based on target protein structures have become an important means for rapidly discovering efficient inhibitors. The dissimilated sulfite reductase (Dsr) is a key enzyme in SRB for catalyzing the reduction of sulfite into hydrogen sulfide, and is an ideal drug target. However, current screening studies for natural inhibitors of Dsr remain relatively limited, lacking systematic screening strategies and effective composition design. Therefore, a virtual screening method based on a Dsr target structure is developed, and a natural active ingredient is optimized from the virtual screening method to form a high-efficiency composite inhibitor, so that the virtual screening method has important scientific significance and application value for solving the problem of H 2 S pollution caused by vibrio desulphurisation. Disclosure of Invention In view of the above problems, an object of the present invention is to provide an aloin composition that inhibits growth of Vibrio desulphurized and hydrogen sulfide production, and a screening method and application thereof. The technical content of the invention is as follows: The invention provides an aloin composition for inhibiting the growth of vibrio desulphurisation and the generation of hydrogen sulfide, which comprises aloin, flavone, sea buckthorn extract, sappan wood extract and erigeron breviscapus extract; 200-250 parts of aloin, 100-120 parts of flavone, 30-40 parts of sea buckthorn extract, 20-30 parts of sappan wood extract and 10-20 parts of erigeron breviscapus extract. The invention provides a screening method of an aloin composition for inhibiting the growth of vibrio desulphurisation and the generation of hydrogen sulfide, which comprises the following steps: 1) Homologous protein phylogenetic analysis and sequence alignment to confirm protein target-target protein catabolite sulfite reductase (Dsr) 1.1 Obtaining the sequence and crystal structure of the enzyme of interest (Dsr) from Uniprot and PDB databases, downloading Desulfovibrio vulgaris the amino acid sequence and protein crystal structure of the produced catabolic sulfite reductase from Uniprot and PDB databases, respectively; 1.2 Phylogenetic analysis and sequence multiplex a