CN117925589A - Oxidation squalene cyclase gene NiOSC2 and its product heterocycle triterpene - Google Patents
Oxidation squalene cyclase gene NiOSC2 and its product heterocycle triterpene Download PDFInfo
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- CN117925589A CN117925589A CN202410031801.8A CN202410031801A CN117925589A CN 117925589 A CN117925589 A CN 117925589A CN 202410031801 A CN202410031801 A CN 202410031801A CN 117925589 A CN117925589 A CN 117925589A
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- 238000001514 detection method Methods 0.000 description 1
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- 239000003480 eluent Substances 0.000 description 1
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- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
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- 239000002207 metabolite Substances 0.000 description 1
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- JURZHOVRCOWZFN-UHFFFAOYSA-N notoginsenoside R1 Natural products CC(=CCCC(C)(OC1OC(CO)C(O)C(O)C1O)C2CCC3(C)C2C(O)CC4C5(C)CCC(O)C(C)(C)C5C(CC34C)OC6OC(COC7OCC(O)C(O)C7O)C(O)C(O)C6O)C JURZHOVRCOWZFN-UHFFFAOYSA-N 0.000 description 1
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- 229940035893 uracil Drugs 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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Abstract
The invention discloses an application of an oxidation squalene cyclase gene NiOSC and a coding product thereof, belonging to the technical fields of synthetic biology and natural medicines. The invention starts from cucurbitaceae plant clavicle cucurbit (Neoalsomitra integrifoliola), clones and functionally identifies a functional hybrid triterpene synthase NiOSC coding gene, the nucleotide sequence of which is shown as Seq ID No.2, and can cyclize 2,3, 22, 23-double epoxy squalene to generate heterocyclic triterpene (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24R) -20, 24-epoxy dammarenediol), and the heterocyclic triterpene can be used for preparing medicines for resisting arrhythmia, myocardial ischemia injury, bacteria, inflammation, cancer, nerve protection and the like by carrying out gene cloning and connecting with an expression vector pYES2 to construct a recombinant plasmid of pYES2-NiOSC2, and then converting the recombinant plasmid into engineering saccharomycetes, thereby realizing heterogeneous efficient synthesis of the heterocyclic triterpene by the saccharomyces cerevisiae.
Description
Technical Field
The invention belongs to the technical fields of synthetic biology and natural medicines. In particular to an oxidation squalene cyclase NiOSC and a coded product (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24R) -20, 24-epoxy dammarenediol thereof, and application of the coded product in preparing anti-inflammatory and antitumor drugs.
Background
The octocrocetin type ginsenoside (Ocotillol type ginsenosides) is a tetracyclic triterpenoid type saponin with tetrahydrofuran ring at the side chain, and can be divided into (20S, 24S) according to different configurations of C-20 and C-24; (20S, 24R); (24R, 24S); four types (20R, 24R). Modern pharmacological studies show that the octopus type ginsenoside and aglycone thereof have various pharmacological effects, mainly including antiarrhythmic, antibacterial, anti-inflammatory, anticancer, nerve protecting and the like. In recent years, in vivo and in vitro metabolism studies of secondary ginsenoside (ginsenoside Rg3, rh2, rg2 and Rh 1) and aglycones (PPD and PPT) of C-20 non-linked glycosyl indicate that the octopus type metabolites of C-20 and C-24 epoxy can be truly effective components for in vivo functions. The octopus type ginsenoside is mainly present in Panax ginseng (Panax vietnamensis), panax quinquefolium (P.quinquefoil), panax ginseng (P.japonica. Major), panax japonicus (P.japonica), and Gynostemma pentaphyllum (Gynostemma pentaphyllum) belonging to the genus Cucurbitaceae. Since naturally occurring octopus type ginsenosides are less in variety and mainly have (20S, 24S) configuration and are low in content, researchers convert the ginsenosides into (20R, 24S), (20S, 24R) and (20R, 24R) configuration octopus type compounds through semi-synthetic means. The dammarane type ginsenoside with anticancer activity, which is unique to heterologous synthesized ginseng (Panax l.), is one of the hot spots in research on synthetic biology. Therefore, the method utilizes means of synthetic biology and metabolic engineering to excavate key genes in the biological synthesis path of the okadayl alcohol type saponin, and realizes the efficient heterologous biosynthesis of the okadayl alcohol type saponin, in particular to the key precursor skeleton epoxy dammarenediol.
The cucumber (Neoalsomitra integrifoliola (cognin.) Hutch) is plant of genus Cucurbita (Neoalsomitra) of family Cucurbitaceae, and is grown in rainforest or secondary forest or shrub with an altitude of 550-840 m. The genus plant is about 22 species, distributed in india to brinesian and australia, and there are the n.integrifolia (cogni.) Hutch species and the n.clavigera (wall.) hutch.2 species, the former species distributed in the south of the sea and taiwan, guangxi, yunnan, and the latter species produced in the southeast part of the tibetan (ink drop). The clavulanate saponins are rich in dammarane saponins, and are a new resource plant which contains high ginsenoside content in cucurbitaceae plants, wherein the content of the triterpenoid A of the octopus loltype saponins reaches more than 3%, so that the key gene research of the synthesis of the octopus loltype saponins in the clavulanate is very necessary, particularly the key gene involved in the synthesis of the rare (20R) and (24R) configuration epoxy dammarenediol is found, and the rare compounds are synthesized in a heterogeneous manner.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides an oxidation squalene cyclase gene NiOSC and a coding product thereof, wherein the gene is a key enzyme gene participating in the synthesis of triterpenoid sapogenin of the clavus fruit, can be used as a biosynthesis regulatory gene of (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol and can be applied to the preparation of (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol. Thus, a novel method for biosynthesis of (20R, 24S) -20, 24-epoxydammarenediol, (20S, 24S) -20, 24-epoxydammarenediol and (20S, 24R) -20, 24-epoxydammarenediol is provided.
In order to achieve the above object of the present invention, the present invention adopts the following technical scheme:
An oxidosqualene cyclase NiOSC2 which is:
(1) A protein comprising the amino acid sequence shown in Seq ID No. 1;
(2) The amino acid sequence shown in the Seq ID No.1 is a derivative protein with the same function by substituting and/or deleting and/or adding one or more amino acid residues.
The coding gene of the oxidation squalene cyclase NiOSC is as follows:
(a) A nucleotide sequence shown as Seq ID No. 2;
(b) The nucleotide sequence shown in Seq ID No.2 is a nucleotide sequence which is substituted and/or deleted and/or added with one or several nucleotides and expresses the same functional protein.
A recombinant vector containing the gene encoding the oxidosqualene cyclase NiOSC.
Recombinant bacteria containing the gene encoding the oxidosqualene cyclase NiOSC.
The oxidation squalene cyclase NiOSC or the encoding gene thereof is applied to the preparation of recombinant vectors, expression cassettes, transgenic cell lines and recombinant bacteria containing (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol.
The application of the oxidation squalene cyclase NiOSC or the coding gene thereof in preparing fermentation liquor containing the compound (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol is characterized in that the application adopts: constructing an expression vector containing the coding gene, transforming the recombinant vector into saccharomyces cerevisiae cells, and fermenting and culturing the obtained genetically engineered saccharomycetes to obtain fermentation liquor containing (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol.
The application of the oxidation squalene cyclase NiOSC or the coding gene thereof in the synthesis or preparation of heterocyclic triterpene (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol.
The oxidation squalene cyclase NiOSC or the encoding gene thereof is applied to the preparation of antiarrhythmic, antibacterial, anti-inflammatory, anticancer and nerve-protecting drugs.
A process for the preparation of the compound (20 r,24 s) -20, 24-epoxydammarenediol, (20 s,24 r) -20, 24-epoxydammarenediol, comprising the steps of:
Constructing an expression vector containing a coding gene for coding the squalene cyclase NiOSC2, converting the recombinant vector into saccharomyces cerevisiae, fermenting and culturing the obtained genetically engineered saccharomyces cerevisiae to obtain a fermentation broth containing (20R, 24S) -20, 24-epoxydammarenediol, (20S, 24R) -20, 24-epoxydammarenediol, extracting by petroleum ether or ethyl acetate or dichloromethane or chloroform to obtain an extract containing (20R, 24S) -20, 24-epoxydammarenediol, (20S, 24R) -20, 24-epoxydammarenediol, separating and purifying by a silica gel column chromatography method and a high performance liquid chromatography method to finally obtain the compound (20R, 24S) -20, 24-epoxydammarenediol, (20S, 24-epoxydammarenediol) shown in the following structural formula,
The compound (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol and the application of (20S, 24R) -20, 24-epoxy dammarenediol in preparing medicines for resisting arrhythmia, myocardial ischemia injury, bacteria, inflammation, cancer and nerves are disclosed.
The Open Reading Frame (ORF) of the coding gene of the oxidosqualene cyclase gene NiOSC provided by the invention is 2298bp (Seq ID No. 2), 765 amino acids (Seq ID No. 1) are coded, and when the gene is placed in NCBI for BLASTN analysis and comparison, the result shows that the homology with SgBAS1 of the cucurbitaceae plant fructus momordicae (Siraitia grosvenorii) is 76%.
The coding gene of the oxidation squalene cyclase NiOSC provided by the invention is a gene cloned from the cucumis metuliferus (Neoalsomitra integrifoliola), and can cyclize 2,3;22, 23-bis-epoxy squalene forming compound (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol, and (20S, 24R) -20, 24-epoxy dammarenediol. NiOSC2 is cloned from plants for the first time, and the discovery of the oxidosqualene cyclase NiOSC and its coding gene enriches the diversity of enzymes.
The invention clones and functionally identifies the oxidation squalene cyclase NiOSC from cucurbitaceae plant clavicle, and uses saccharomyces cerevisiae to generate (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol and the application of (20S, 24R) -20, 24-epoxy dammarenediol.
The invention discloses an oxidation squalene cyclase NiOSC and an application of an encoding product (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol thereof, belonging to the technical fields of synthetic biology and natural medicines. According to the invention, starting from cucurbitaceae plant cucurbita pepo (Neoalsomitra integrifoliola), a functional hybrid triterpene synthase NiOSC coding gene is cloned and functionally identified, and the gene can cyclize 2,3; the 22, 23-double epoxy squalene generates heterocyclic triterpene (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24R) -20, 24-epoxy dammarenediol, the nucleotide sequence of which is shown as Seq ID No.2, and the heterocyclic triterpene (20R, 24S) -20, 24-epoxy dammarenediol is connected with an expression vector pYES2 after gene cloning, so that the heterocyclic triterpene (20R, 24S) -20, 24-epoxy dammarenediol is constructed into a recombinant plasmid pYES2-NiOSC, and then the recombinant plasmid is transformed into Saccharomyces cerevisiae to be constructed into engineering saccharomycetes, thereby realizing the heterogenous efficient synthesis of the heterocyclic triterpene (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24-epoxy dammarenediol) 20S, 24-epoxy dammarenediol. The NiOSC product (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol and the application of (20S, 24R) -20, 24-epoxy dammarenediol in preparing medicines for resisting arrhythmia, myocardial ischemia injury, bacteria, inflammation and cancers and protecting nerves are provided.
Drawings
FIG. 1 is a three-dimensional structure prediction diagram of the oxidosqualene cyclase NiOSC in example 1.
FIG. 2 shows the analysis of the expression level of the oxidosqualene cyclase NiOSC2 in roots, stems, leaves and flowers in example 2.
FIG. 3 is a total ion figure of GC-MS analysis of an engineered yeast extract expressing the oxidosqualene cyclase NiOSC2 in example 2.
FIG. 4 is a mass spectrum of the catalytic product (20R, 24S) -20, 24-epoxydammarenediol, (20S, 24S) -20, 24-epoxydammarenediol, and (20S, 24R) -20, 24-epoxydammarenediol of the squalene cyclase NiOSC catalytic product of example 2.
FIG. 5 is a chromatogram of the (20R, 24S) -20, 24-epoxydammarenediol, (20S, 24S) -20, 24-epoxydammarenediol, and (20S, 24R) -20, 24-epoxydammarenediol standard in example 2.
FIG. 6 is a standard quality spectrum of (20R, 24S) -20, 24-epoxydammarenediol, (20S, 24S) -20, 24-epoxydammarenediol, and (20S, 24R) -20, 24-epoxydammarenediol in example 2.
FIG. 7 is a schematic diagram showing the construction of recombinant expression plasmid pYES2-NiOSC2 in example 2.
Detailed description of the preferred embodiments
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless otherwise indicated, the examples were conducted under conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular cloning: a laboratory manual, 2001), or under conditions recommended by the manufacturer's instructions.
Example 1
Cloning of the Oxidation squalene cyclase Gene in Calophyllum Instrongylus.
1. According to the sequenced data of the gloriope clavatus transcriptome, the candidate oxidation squalene cyclase gene cDNA sequence in the gloriope clavatus saponin synthesis pathway is obtained through operations such as splicing, annotation, screening and the like.
2. Designing a primer of the candidate oxidation squalene cyclase gene, wherein the primer sequence is as follows:
Forward primer (NiOSC-F):
gggaatattaagcttggtaccATGTGGAGACTAACAATGGGAGAGG，Seq ID NO.3；
reverse primer (NiOSC-R):
ccctctagatgcatgctcgagTTAACGAGGCATTGAAACCAAATTACGG，Seq ID NO.4；
primers were synthesized by Kunming division, inc. of Biotech, beijing.
3. The individual tissues (root, stem, leaf and flower) of the plant of the Hamamelis mollis were taken, total RNA was extracted using TRIzol kit (Invitrogen, carlsbad, calif., USA), cDNA was obtained by reverse transcription using PRIMESCRIPT TM RT kit (Takara, china), and the gene sequence of NiOSC was amplified using the cDNA as a template.
4. The amplified product agarose gel electrophoresis shows a specific band at about 2.3kb, the target band is subjected to gel cutting recovery, the gel recovery product is connected to a vector pYES2, and E.coli DH5 alpha is transformed, positive clones are selected for sequencing (Kunming division of Beijing qing biological science and technology Co., ltd.), and NiOSC gene clones with correct sequences are selected and saved for the construction of subsequent expression vectors.
Sequencing to obtain the triterpenoid saponin anabolism pathway oxidation squalene cyclase gene NiOSC with the length of 2298bp and nucleotide sequence of Seq ID No.1; codes for 765 amino acids, the amino acid sequence is as set forth in Seq ID No.2.
By performing multiple sequence alignment and phylogenetic tree analysis of NiOSC2 with the identified OSCs, niOSC is known to have the highly conserved domains QW and DCTAE of the OSCs gene family.
Example 2
NiOSC2 eukaryotic expression and functional analysis of the gene.
NiOSC2 function preliminary analysis.
RNA of roots, stems, leaves and flowers of Hamamelis mollis was extracted, respectively, reverse transcribed into cDNA by referring to PRIMESCRIPT TM RT kit (Takara, china) and amplified by real-time fluorescent quantitative PCR on Applied Biosystems QuantStudio TM platform (Life Technologies) using 2X ChamQ Universal SYBR QPCR MASTER Mix (Vazyme).
Forward primer (NiOSC 2-qRT-F): TGTGGTAGAGCTCGCAAATG, seq ID No.5;
reverse primer (NiOSC 2-qRT-R): CAACCGACAGTAGCAAAGCA, seq ID No.6.
From the results of the real-time fluorescent quantitative PCR analysis, niOSC.sup.2 was found to be most expressed in the leaves, and NiOSC.sup.2 was presumed to be involved in the biosynthesis of triterpene compounds in the leaves of Hamamelis mollis, as shown in the results of the real-time fluorescent quantitative PCR analysis (FIG. 2).
2. Construction of a Yeast expression vector.
ERG 7-deficient Saccharomyces cerevisiae mutant GIL77, which lacks lanosterol synthase gene, can endogenously accumulate 2,3;22, 23-bisoxy squalene, 2,3;22, 23-bisoxasqualene can be used as a substrate for carrying out NiOSC functional assays.
By analyzing the coding sequence and the cleavage site of the gene NiOSC, primers with KpnI and XhoI cleavage sites were designed as follows, and NiOSC full-length ORF amplification was performed.
After sequencing and verifying the amplification product, connecting a target gene NiOSC to a yeast expression vector pYES2 by a homologous recombination method, screening a transformed colony by using an LB solid plate containing ampicillin (100 mu g/mL), selecting a monoclonal for verification, sequencing and verifying correctness to obtain a pYES2-NiOSC2 vector, inoculating the verified correct monoclonal into 5mL of LB liquid culture with the same resistance, fermenting and culturing, and extracting a pYES2-NiOSC2 plasmid.
3. Yeast transformation.
The pYES2-NiOSC2 plasmid was transferred into Saccharomyces cerevisiae strain GIL77 by lithium acetate transformation, and an empty pYES2 transformation control group was set. Positive clones GIL77-pYES2-NiOSC2 and GIL77-pYES2 were selected by colony PCR.
4. Induction of expression and incubation.
Positive yeast monoclonal GIL77-pYES2-NiOSC and GIL77-pYES2 were picked separately and inoculated in 50ml of uracil-free synthetic complete medium [ SC-U; comprises ergosterol (20. Mu.g/ml), tween 80 (5 mg/ml) and hemin (13. Mu.g/ml), and is then cultured at 30℃for 2 days with shaking at 200 rpm.
Yeast cells were harvested, resuspended in 50mL of SC-U medium containing 2% galactose and induced by shaking at 200rpm for 2 days at 30 ℃.
After induction for 2 days, the cells were harvested and resuspended in the same volume of 0.1M potassium phosphate buffer (pH 7.0; 2% glucose and hemin (13. Mu.g/ml) were added and incubated at 30℃for 12 hours with shaking at 200 rpm.
5. And (5) extracting and identifying a catalytic product.
After 12 hours of incubation, the cells were refluxed with the same volume of saponification reagent (20% KOH/50% EtOH) for 10 minutes at 92℃and then extracted twice with the same volume of petroleum ether, the extracts were combined, concentrated to dryness under reduced pressure and the residue was derivatised with 200. Mu.l of cyano trimethylsilane at 65℃for 30 minutes.
The derived products are subjected to GC-MS analysis, and a catalytic group containing NiOSC gene recombination expression vector pYES2-NiOSC is compared with a control group containing empty pYES2, namely new substances are generated, and the specific products are identified as (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol.
The experiment proves that NiOSC gene participates in the biosynthesis of triterpenoid saponins of the clavus, niOSC gene can be used for carrying out the regulation of the biosynthesis of (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol in the clavus, and the heterologous synthesis of (20R, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol.
Example 3
Preparation of the compound (20R, 24S) -20, 24-epoxydammarenediol, (20S, 24S) -20, 24-epoxydammarenediol, and (20S, 24R) -20, 24-epoxydammarenediol.
Extracting RNA of the Hamamelis mollis, reversely transcribing the RNA into cDNA by referring to PRIMESCRIPT TM RT kit (Takara, china), amplifying NiOSC2 gene sequence by taking the cDNA as a template, carrying out agarose gel electrophoresis on an amplified product, generating a specific band at about 2.3kb, carrying out gel cutting recovery on a target band, connecting a gel recovery product to a vector pYES2, converting the gel recovery product into escherichia coli DH5 alpha, picking up positive clones for sequencing (Kunming division of biological technology Co., ltd. In Beijing) and selecting NiOSC2 gene with correct sequencing, connecting the target gene NiOSC2 to a yeast expression vector pYES2 by adopting a homologous recombination method, and obtaining a pYES2-NiOSC2 vector, and carrying out sequencing verification on the correctness. Extracting pYES2-NiOSC2 plasmid, introducing the pYES2-NiOSC plasmid into Saccharomyces cerevisiae GIL77 strain by adopting a lithium acetate method, selecting positive clones GIL77-pYES2-NiOSC and GIL77-pYES2 by adopting a colony PCR method, and inoculating the positive yeast monoclonal GIL77-pYES2-NiOSC2 into 50ml of uracil-free synthetic complete medium [ SC-U; comprises ergosterol (20. Mu.g/ml), tween 80 (5 mg/ml) and hemin (13. Mu.g/ml), and is incubated at 30℃for 2 days. 50ml of the bacterial liquid is inoculated to 10L of synthetic complete medium [ SC-U ] without uracil; comprising ergosterol (20. Mu.g/ml), tween 80 (5 mg/ml) and hemin (13. Mu.g/ml), followed by shaking culture at 200rpm for 2 days at 30℃to obtain yeast cells, which were resuspended in 10L of SC-U medium containing 2% galactose and the protein expression was induced at 200rpm for 2 days at 30 ℃. After 2 days of induction, yeast cells were harvested and resuspended in the same volume of 0.1M potassium phosphate buffer (pH 7.0; after 12 hours of incubation, the cells were refluxed with the same volume of saponification reagent (20% KOH/50% EtOH) at 92℃for 10 minutes, then extracted 3 times with the same volume of petroleum ether, the extracts were combined and concentrated under reduced pressure to dryness to give an extract containing (20R, 24S) -20, 24-epoxydamageenediol, (20S, 24R) -20, 24-epoxydamageenediol) which was then stirred with 1.5 times the amount of silica gel, column chromatographed on 15 times the amount of silica gel, and dried over petroleum ether: ethyl acetate (10:1, v/v-5:1, v/v), collecting fractions, eluting with 6 column volumes of eluent for each gradient, detecting by thin layer chromatography, collecting fractions containing mixture (20R, 24S) -20, 24-epoxydammarenediol, (20S, 24R) -20, 24-epoxydammarenediol, concentrating under reduced pressure to dryness, separating by preparative high performance liquid chromatography, using Agilent Zorbax SB-C18 chromatographic column (9.4X250 mm,5 μm), acetonitrile (A) -water (B) as mobile phase, 0-10min,83% A-85% A,10-18min,85% A-85% A,18-21min,85% A-100% A, running for 25min, the subsequent operation is carried out for 5min, the flow rate is 8ml/min, the detection wavelength is 194nm, the (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol and the (20S, 24R) -20, 24-epoxy dammarenediol are prepared by preparing liquid phase, and the structures of the (20R, 24S) -20, 24-epoxy dammarenediol and the structural data are shown in the table 1.
TABLE 1 1 H NMR and 13 C NMR data for three compounds of the invention
a Measured at 151 MHz; b Measured at 600 MHz; "m" refers to overlapping or multiple repetition with other signals.
Pharmaceutical formulation examples 1-8:
In the following formulation examples, conventional reagents are selected and formulation preparation is performed according to the conventional methods, and this application example only represents the compound of the present invention, (20 r,24 s) -20, 24-epoxydammarenediol, (20 s,24 s) -20, 24-epoxydammarenediol, and (20 s,24 r) -20, 24-epoxydammarenediol can be prepared into different formulations, and specific reagents and operations are not specifically limited:
1. dissolving one or a mixture of the compounds (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol with absolute ethyl alcohol, adding water for injection according to a conventional method, finely filtering, filling and sterilizing to prepare injection, wherein the concentration of the injection is 0.5-5mg/mL.
2. Dissolving one or a mixture of the compounds (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol in water for sterile injection by dimethyl sulfoxide, stirring to dissolve, filtering by a sterile suction filter funnel, performing sterile fine filtration, split charging into ampoule, performing low-temperature freeze drying, and performing sterile fusion sealing to obtain powder injection.
3. Adding excipient into one or mixture of (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol according to the mass ratio of the excipient to the excipient of 9:1, and preparing into powder.
4. Adding excipient into one or mixture of the compounds (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol according to the mass ratio of the excipient to the excipient of 5:1, granulating and tabletting.
5. The compound (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol, or one or a mixture of the compounds (20S, 24S) -20, 24-epoxy dammarenediol) is prepared into oral liquid according to a conventional oral liquid preparation method.
6. Adding excipient into one or mixture of (20R, 24S) -20, 24-epoxy dammarenediol and (20S, 24S) -20, 24-epoxy dammarenediol, and making into capsule with the ratio of excipient to excipient being 5:1.
7. Adding excipient into one or mixture of (20R, 24S) -20, 24-epoxy dammarenediol and (20S, 24S) -20, 24-epoxy dammarenediol according to the mass ratio of the excipient to the excipient of 5:1, and making into granule.
8. The capsule comprises the following components: the compound (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol, one of (20S, 24R) -20, 24-epoxy dammarenediol or a mixture thereof is 20mg, lactose is 180mg, and magnesium stearate is 5mg.
The preparation method comprises the following steps: the compound or mixture was mixed with a cosolvent uniformly, sieved, and the resulting mixture was filled into gelatin capsules each weighing 205mg and having an active ingredient content of 20mg.
The generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. An oxidosqualene cyclase NiOSC2, characterized in that it is:
(1) A protein comprising the amino acid sequence shown in Seq ID No. 1;
(2) The amino acid sequence shown in the Seq ID No.1 is a derivative protein with the same function by substituting and/or deleting and/or adding one or more amino acid residues.
2. The gene encoding the oxidosqualene cyclase NiOSC as described in claim 1, characterized in that it is:
(a) A nucleotide sequence shown as Seq ID No. 2;
(b) The nucleotide sequence shown in Seq ID No.2 is a nucleotide sequence which is substituted and/or deleted and/or added with one or several nucleotides and expresses the same functional protein.
3. A recombinant vector comprising the gene encoding the oxidosqualene cyclase NiOSC as described in claim 2.
4. A recombinant bacterium comprising the gene encoding the oxidosqualene cyclase NiOSC as described in claim 2.
5. Use of an oxidosqualene cyclase NiOSC2 or a gene encoding it according to claim 1 or 2 for the preparation of a recombinant vector, an expression cassette, a transgenic cell line, a recombinant bacterium comprising (20 r,24 s) -20, 24-epoxydammarenediol, (20 s,24 r) -20, 24-epoxydammarenediol.
6. Use of an oxidosqualene cyclase NiOSC or a gene encoding it according to claim 1 or 2 for the preparation of a fermentation broth comprising the compound (20 r,24 s) -20, 24-epoxydammarenediol, (20 s,24 r) -20, 24-epoxydammarenediol, characterized in that the use is as follows: constructing an expression vector containing the coding gene, transforming the recombinant vector into saccharomyces cerevisiae cells, and fermenting and culturing the obtained genetically engineered saccharomycetes to obtain fermentation liquor containing (20R, 24S) -20, 24-epoxy dammarenediol, (20S, 24S) -20, 24-epoxy dammarenediol and (20S, 24R) -20, 24-epoxy dammarenediol.
7. Use of the oxidosqualene cyclase NiOSC2 or its coding gene according to claim 1 or 2 for the synthesis or preparation of heterocyclic triterpene (20 r,24 s) -20, 24-epoxydammarenediol, (20 s,24 r) -20, 24-epoxydammarenediol.
8. Use of an oxidosqualene cyclase NiOSC as described in claim 1 or 2 or a gene encoding it for the preparation of a medicament for antiarrhythmic, antibacterial, antiinflammatory, anticancer, neuroprotection.
9. A process for the preparation of a heterocyclic triterpene (20 r,24 s) -20, 24-epoxydammarenediol, (20 s,24 s) -20, 24-epoxydammarenediol, and (20 s,24 r) -20, 24-epoxydammarenediol, characterized in that the process comprises the steps of:
Constructing an expression vector containing a coding gene for coding the squalene cyclase NiOSC2, converting the recombinant vector into saccharomyces cerevisiae, fermenting and culturing the obtained genetically engineered saccharomyces cerevisiae to obtain a fermentation broth containing (20R, 24S) -20, 24-epoxydamagel, (20S, 24R) -20, 24-epoxydamagel, extracting by petroleum ether or ethyl acetate or dichloromethane or chloroform to obtain an extract containing (20R, 24S) -20, 24-epoxydamagel, (20S, 24R) -20, 24-epoxydamagel, separating and purifying by a silica gel column chromatography method and high performance liquid chromatography to finally obtain the compound (20R, 24S) -20, 24-epoxydamagel, (20S, 24-epoxydamagel) shown in the following structural formula,
10. The use of the compound (20R, 24S) -20, 24-epoxydammarenediol, (20S, 24R) -20, 24-epoxydammarenediol obtained by the preparation method of claim 9 in the preparation of drugs for resisting arrhythmia, myocardial ischemia injury, bacteria, inflammation, cancer and nerves.
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