CN116535292A - Isopentenyl bibenzyl derivative, preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of medicines, discloses an isopentenyl bibenzyl derivative, a preparation method and application thereof, and in particular discloses an isopentenyl bibenzyl derivative, a biosynthesis preparation method based on engineering escherichia coli cell factory and application thereof as a neuroprotective medicament, wherein the isopentenyl bibenzyl derivative comprises novel bibenzyl synthase DoBBS8 and F2 derived from dendrobium candidum, nucleic acid molecules respectively encoding the proteins, engineering microorganisms containing the corresponding nucleic acid molecules, and bioactive isopentenyl bibenzyl derivatives synthesized by the engineering microorganisms, a production method thereof, the neuroprotective medicament and an antioxidant.

Description

Isopentenyl bibenzyl derivative, preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and in particular relates to application of a genetic engineering means to create a microbial cell factory and biologically synthesize bibenzyl and isopentenyl derivatives thereof, and the isopentenyl bibenzyl derivatives are applied to neuroprotective medicines and used as novel antioxidants.

Background

The dibenzyl compounds are important plant polyphenol compounds and have pharmacological activities of resisting tumor, resisting diabetes, neuroprotection, resisting oxidation, resisting inflammation, resisting platelet coagulation, relieving spasm and the like. However, the plant resources containing the compounds are limited, the content of the target compounds is low, and meanwhile, the problems of poor selectivity, environmental pollution and the like are involved in chemical synthesis. Therefore, the development of a method for efficiently synthesizing the dibenzyl compounds has important practical significance and application value.

In recent years, with the rapid development of synthetic biology, mass production of many important natural drugs or their precursors, intermediates (such as precursor compounds of artemisinin, paclitaxel, etc.) has been achieved by constructing microbial cell factories. Based on synthetic biology concepts, creating artificial cell factories to produce active natural products has become a new model for drug resource acquisition. Therefore, by using a synthetic biological means to construct a microbial cell factory, a natural or unnatural dibenzyl compound with various structure types and drug development potential is synthesized by taking a cheap and easily available compound as a raw material, and the method is one of alternative methods.

The synthesis of the dibenzyl skeleton structure is catalyzed by dibenzyl synthase (Bibenzyl synthase, BBS), which is the key for synthesizing the dibenzyl compounds. Currently, such enzymes are only identified in Xinjiang firefly (Gehlert R, kindl H. Induced formation of dihydrophenanthrenes and bibenzyl synthase upon destruction of orchid mycorriza. Phytochemistry,1991,30 (2): 457-460.), bletillae (Reinecke T, kindl H. Characination of bibenzyl synthase catalysing the biosynthesis of phytoalexins of organs, phytochemistry,1993,35 (1): 63-66.) and Phalaenopsis varieties (Preisigmiller R, gnau P, kindl H. The indable 9,10-dihydrophenanthrene pathway: characterization and expression of bibenzyl synthase and S-adenosyl hydrolase. Arches of Biochemistry and Biophysics,1995,317 (1): 201-207.), but there are no reports of their application to enzymatic synthesis of dibenzyl compounds.

The basic framework compound of the bibenzyl can be catalyzed and synthesized into the isopentenyl derivative by the isopentenyl transferase, and no report on the aspect exists until now.

In view of the above, the studies related to benzyl and its isopentenyl derivatives currently have the following limitations and disadvantages:

1. the dibenzyl compound has few natural resources, difficult chemical synthesis and limited sources;

2. the related studies of bibenzyl synthase and isopentenyl transferase responsible for bibenzyl skeleton biosynthesis are limited, and the biosynthesis of active bibenzyl derivatives by using a multienzyme combination has not been reported.

Therefore, by excavating novel dibenzyl synthase in plants and combining isopentenyl transferase with substrate broad property, an artificial approach for constructing structurally diverse isopentenyl dibenzyl derivatives in microorganisms is adopted, the defects can be overcome by preparing structurally diverse and bioactive dibenzyl compounds in engineering microorganism cell factories, a novel method is provided for obtaining the compounds, and a novel compound entity is provided for discovering novel drugs.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide isopentenyl dibenzyl derivatives with novel pharmacological activity; it is a further object of the present invention to provide a process for its preparation; a third object of the invention is its use in the preparation of neuroprotective medicaments and in the preparation of novel antioxidants.

In order to achieve the purpose of the invention, the following technical scheme is adopted:

the first aspect of the technical scheme of the invention is to provide isopentenyl dibenzyl derivatives with novel pharmacological activity and pharmaceutically acceptable salts thereof, wherein the structures of the isopentenyl dibenzyl derivatives are shown as a general formula 1. Wherein the substituents R ₁ 、R ₂ Each independently selected from hydrogen, hydroxy.

The second aspect of the technical scheme of the invention provides a preparation method of the isopentenyl dibenzyl derivatives with novel pharmacological activity, which is characterized by comprising the following steps:

culturing the engineering bacteria containing the bibenzyl skeleton synthesis module of the bibenzyl synthase gene DoBBS8 and the engineering bacteria containing the isopentenyl transferase gene FD2 in proper culture mediums, inducing expression, centrifuging to collect thalli, washing and re-suspending thalli to proper concentration with proper culture mediums, culturing the two strains in combination, and adding substrate malonate and phenylpropionic acid compounds for whole cell reaction to synthesize the isopentenyl bibenzyl derivative. Separating and purifying the centrifuged reaction liquid by macroporous resin column chromatography and reversed-phase semi-preparative HPLC in sequence to obtain a product, and adopting UV, MS, ¹ H NMR、 ¹³ Analysis and identification of product structure by using C NMR, HSQC, HMBC and other spectroscopy technologies. The engineering bacteria can be recycled.

The third aspect of the technical scheme of the invention is to provide the application of the isopentenyl dibenzyl derivatives in preparing neuroprotective medicines.

The neuroprotection of the isopentenyl dibenzyl derivative (5) was evaluated using an in vitro glutamate-induced neuronal injury model, and an in vivo ICR mouse hypoxia and acute hypoxia tolerance experimental model, an acetic acid torsion experimental model, and an SD rat permanent ischemia model.

1. In a glutamic acid-induced neuron damage model, the compound 5 shows a neuron protection effect, and can remarkably improve the survival rate by 15.32%, which is equivalent to that of a positive control medicine resveratrol.

2. In the mouse hypoxia tolerance experimental model, the compound 5 increases the survival time of the ICR mouse hypoxia tolerance experiment in a dose-dependent manner at 10mg/kg and 50mg/kg, and is superior to that of the positive control drug atenolol.

3. In a mouse acute hypoxia tolerance experimental model, the compound 5 obviously increases the survival time of ICR mice in the acute hypoxia tolerance experiment at dosages of 10mg/kg and 50 mg/kg; the respiration frequency of ICR mice in the acute hypoxia tolerance experiment is obviously increased at the dosage of 10mg/kg, and the method is superior to that of nimodipine which is a positive control medicine.

4. In the ICR mouse acetic acid torsion experimental model, the compound 5 obviously reduces the torsion times in the ICR mouse acetic acid torsion experiment at the dosage of 10 mg/kg.

5. In the SD rat permanent ischemia model (pMCAO), compound 5 significantly reduced the brain infarct volume 24h post pMCAO surgery and significantly reduced the neurobehavioral score 24h post pMCAO surgery in SD rats at a dose of 30 mg/kg.

According to a fourth aspect of the technical scheme, the invention provides application of the isopentenyl dibenzyl derivatives in preparation of novel antioxidants.

1. In DPPH free radical scavenging experiments, vitamin C as positive control agent is EC ₅₀ EC of compound 5 with a value of 11.53 μm ₅₀ The value is 6.12 mu M, the effect is better than that of a positive medicine, and the strong free radical scavenging capability is shown.

2. Detection of superoxide anion radical in pyrogallol autoxidationIn the experiment of (2), the compound 5 has a certain effect of scavenging superoxide anion free radicals.

3. In the hydroxyl radical (.OH) assay by the Feton reaction, compound 5 exhibited a stronger hydroxyl radical scavenging capacity and was superior to the positive drug vitamin C.

4. In the experiment of determining oxygen radical scavenging ability by ORAC method, the area under the fluorescence decay curve (net AUC) of compound 5 at a dose of 10 μm was 48.84+ -2.84, while the net AUC value of vitamin C as a positive drug was 8.55+ -3.33, suggesting that the antioxidant ability of compound 5 is stronger than that of vitamin C as a positive drug.

The invention has the following technical advantages:

1. the invention provides a modularized engineering bacterium for efficiently synthesizing a dibenzyl skeleton compound, which is economical, efficient and environment-friendly. Compared with the prior art, the method not only avoids the defects of protecting, deprotecting, polluting the environment and the like of functional groups existing in chemical synthesis, but also can obtain a large amount of target compounds in a short time, and meanwhile, the related bibenzyl synthase has a wider substrate spectrum and can be developed into a series of chassis cells synthesized by bibenzyl derivatives.

2. The invention provides a high-efficiency isopentenyl modularized engineering bacterium, which completes the isopentenyl reaction by combining an isopentenyl donor synthesized by the engineering bacterium with high catalytic capability and isopentenyl transferase, realizes the self-sufficiency of the isopentenyl donor in the bacterium, is economical and practical, and effectively reduces the cost of synthesizing an isopentenyl product.

3. The modularized engineering bacteria effectively avoid the defects of cell load, weak activity and the like caused by the fact that multiple genes are in the same host cell through combined co-culture, fully exert the catalytic capability of each functional bacteria, and provide a novel method for efficiently synthesizing structurally diverse compounds (not limited to bibenzyl).

4. The isopentenyl dibenzyl derivative synthesis system has recycling capability, can realize repeated recycling synthesis of target products, and is simple, efficient, economical and sustainable.

5. The isopentenyl dibenzyl derivative 5 can improve the cell survival rate against glutamate-induced neuronal injury, can obviously prolong the survival time of mice under acute hypoxia condition and hypoxia condition, improves the neural behavior and cerebral obstruction volume of a permanently ischemic rat, and has an analgesic effect.

6. The isopentenyl dibenzyl derivative 5 has stronger free radical scavenging capability and can scavenge superoxide anions, hydroxyl radicals and oxygen radicals.

Drawings

FIG. 1A summary of the structural formulas of the bibenzyl derivatives

Figure 2. Full cell synthesis of bibenzyl skeleton compound by bibenzyl skeleton synthesis engineering bacteria.

(A), (B) and (C) adding malonic acid and S1 (4-hydroxy-phenylpropionic acid), malonic acid and S2 (3, 4-dihydroxy-phenylpropionic acid), and carrying out whole cell synthesis of the engineering bacteria for synthesizing the dibenzyl skeleton during malonic acid and S3 (3-hydroxy-phenylpropionic acid), (1) and comparing HPLC spectrograms of the reference substances S1/S2/S3; (2) Synthesizing an HPLC spectrogram of whole cell synthesis of engineering bacteria by using a dibenzyl skeleton without adding a substrate; (3) HPLC spectrogram of whole cell synthesis of engineering bacteria synthesized by dibenzyl skeleton when malonic acid and S1/S2/S3 are added.

FIG. 3 shows that the engineering bacteria for synthesizing the dibenzyl skeleton and the engineering bacteria for prenyl are co-cultured to synthesize the isopentenyl dibenzyl derivative.

(A), (B), (C), malonic acid and S1 (4-hydroxy-benzene propionic acid), malonic acid and S2 (3, 4-dihydroxy-benzene propionic acid), and total cell synthesis of co-culture of the engineering bacteria for synthesizing the dibenzyl skeleton and the engineering bacteria for prenylation when malonic acid and S1/S2/S3 are added, (1) HPLC spectrograms of total cell synthesis of the engineering bacteria for synthesizing the dibenzyl skeleton when malonic acid and S1/S2/S3 are added, and (2) HPLC spectrograms of co-culture of the engineering bacteria for synthesizing the dibenzyl skeleton and the engineering bacteria for prenylation when malonic acid and S1/S2/S3 are added.

FIG. 4. Compound 1 ¹ H NMR spectrum (DMSO-d) ₆ ,400MHz)

FIG. 5 Compound 1 ¹³ C NMR spectrum (DMSO-d) ₆ ,100MHz)

FIG. 6 Compound 2 ¹ H NMR spectrum (DMSO-d) ₆ ,400MHz)

FIG. 7 Compound 2 ¹³ C NMR spectrum (DMSO-d) ₆ ,100MHz)

FIG. 8 Compound 3 ¹ H NMR spectrum (DMSO-d) ₆ ,400MHz)

FIG. 9. Compound 3 ¹³ C NMR spectrum (DMSO-d) ₆ ,100MHz)

FIG. 10. Compound 4 ¹ H NMR spectrum (DMSO-d) ₆ ,400MHz)

FIG. 11 Compound 4 ¹³ C NMR spectrum (DMSO-d) ₆ ,100MHz)

FIG. 12 Compound 5 ¹ H NMR spectrum (DMSO-d) ₆ ,400MHz)

FIG. 13 Compound 5 ¹³ C NMR spectrum (DMSO-d) ₆ ,100MHz)

FIG. 14. Compound 6 ¹ H NMR spectrum (DMSO-d) ₆ ,400MHz)

FIG. 15 Compound 6 ¹³ C NMR spectrum (DMSO-d) ₆ ,100MHz)

FIG. 16 recycle biosynthesis of Compound 5

Detailed Description

For a further understanding of the present invention, the following examples are provided for further illustration of the invention and are not meant to be limiting in any way.

Example 1: cloning of the DoBBS8 Gene

1. Extraction of Total RNA and Synthesis of first strand cDNA

Selecting a proper amount of fresh leaves of dendrobium candidum as a material, rapidly transferring the fresh leaves into a mortar precooled by liquid nitrogen, and fully grinding the fresh leaves into powder; 100mg of the powdered sample was transferred to a centrifuge tube, and 500. Mu.L Buffer RCL/2-mercaptoethanol (E.Z.N.A ^TM Plant RNA Kit), vigorous vortexing; incubating at 55 ℃ for 3min; centrifuge at 14,000g for 2min, transfer the supernatant to a filter column, centrifuge at 14,000g for 2min. The filtered liquid was added to an equal volume Buffer RCB and inverted 5-10 times. Transferring the liquid to an RNA adsorption column, and centrifuging at 10,000g for 1min; the filtrate was discarded and 500. Mu. L RNA wash Buffer II was added toCentrifuging the adsorption column at 10,000g for 1min, discarding the filtrate, repeating the steps twice, and centrifuging the empty column at 10,000g for 1min; after the column was dried at room temperature, the total RNA was eluted with 50. Mu.L DEPC water. 1.0% non-denaturing agarose gel electrophoresis to detect total RNA integrity and UV spectrophotometry to determine OD ₂₆₀ /OD ₂₈₀ Ratio and RNA concentration. Using smart ^TM cDNA was synthesized using RACE cDNA amplification kit (Clontech, USA).

RT-PCR amplification of Gene fragments of interest

And designing a specific primer of a candidate gene DoBBS8 according to the information of the dendrobium candidum transcriptome, and performing PCR amplification by using KOD DNA Polymerase to obtain the full length of the DoBBS8 gene. And (3) recovering PCR products (target gene fragments) by glue, connecting the PCR products to a pEASY-blue vector, transforming Trans1-T1 competent cells, carrying out blue white spot screening and colony PCR screening, and sequencing positive clone shaking bacteria to confirm sequence information of candidate genes.

PCR reaction System:

pcr cycle:

1) 94 ℃ for 2min; 2) 10sec at 98 ℃; 3) 58 ℃ for 30sec; 4) 68 ℃ for 1min; 5) Cycling for 35 times 2-4 times; 6) 68 ℃ for 10min; 7) Maintained at 10 ℃.

Example 2: construction of pCDFDuet-1-DoBBS8 recombinant expression vector

The pCDFDuet-1 has 2 multiple cloning sites, and EcoRV restriction sites positioned at the second multiple cloning site are selected for single restriction to obtain a linear vector of the pCDFDuet-1; meanwhile, primers containing homologous arms of the vector are designed according to the gene sequences at two sides of the pCDFDuet-1-EcoRV locus of the expression vector, and homologous arms are respectively introduced at two sides of the target gene DoBBS8 by a PCR technology (the specific method is the same as that of example 1), and the PCR product is recovered by glue. The connection of the target gene and the linearized pCDFDuet-1 vector is realized by utilizing homologous recombination, trans1-T1 competent cells are transformed, colony PCR screening is carried out, positive cloning is carried out, sequencing is carried out, sequence information of candidate genes is confirmed, and a transformant with correct sequence is obtained by extracting plasmids to obtain recombinant plasmid pCDFDuet-1-DoBBS8.

Example 3: construction of pETDuet-1-MatB-MatC recombinant expression vector

Similarly, pETDuet-1 has 2 multiple cloning sites, firstly selecting EcoRV restriction sites positioned at the second multiple cloning site for single restriction to obtain a linear vector of pETDuet-1; meanwhile, target genes MatB (AAP 03025) and MatC (KF 765784.1) are obtained by amplifying the template plasmids respectively, primers containing homologous arms of the vector are designed according to gene sequences at two sides of the pCDFDuet-1-EcoRV locus of the expression vector, and the homologous arms are introduced at two sides of the target gene MatC respectively through a PCR technology (the specific method is the same as that of example 1), and the PCR products are recovered through glue. The connection of the target gene and the linearized pETDuet-1 vector is realized by utilizing homologous recombination, trans1-T1 competent cells are transformed, colony PCR screening is carried out, positive cloning is carried out, sequencing is carried out, sequence information of candidate genes is confirmed, and a transformant with correct sequence is obtained by extracting plasmids to obtain recombinant plasmid pETDuet-1-MatC. On the basis of recombinant plasmid pETDuet-1-MatC, selecting a Not1 restriction enzyme cutting site of a first multiple cloning site for single restriction enzyme cutting to obtain a linearization vector pETDuet-1-MatC; according to the gene sequence at two sides of the expression vector pETDuet-1-NotI locus, designing a primer containing a vector homology arm, respectively introducing homology arms at two sides of the target gene MatB by a PCR technology (the specific method is the same as that of the example 1), and recovering a PCR product by glue. The connection of the target gene and the linearized pETDuet-1 vector is realized by utilizing homologous recombination, trans-1-T1 competent cells are transformed, colony PCR screening is carried out, positive cloning is carried out, sequencing is carried out, sequence information of candidate genes is confirmed, and a transformant with correct sequence is obtained by extracting plasmids to obtain recombinant plasmids pETDuet-1-MatB-MatC.

Example 4: construction of pACYCDuet-1-At4CL1 recombinant expression vector

Similarly, pACYCDuet-1 has 2 multiple cloning sites, and EcoRV restriction sites positioned at the second multiple cloning sites are selected for single restriction to obtain a linear vector of pACYCDuet-1; meanwhile, amplifying from a template plasmid to obtain a target gene At4CL1 (AY 376729.1), designing a primer containing a vector homology arm according to the gene sequences At two sides of the pACYCDuet-1-EcoRV locus of an expression vector, respectively introducing homology arms At two sides of the target gene At4CL1 by a PCR technology (the specific method is the same as that of the example 1), and recycling a PCR product by glue. The connection of the target gene and the linearized pACYCDuet-1 vector is realized by utilizing homologous recombination, the target gene is transformed into a Trans1-T1 competent cell, colony PCR screening is carried out, positive cloning is carried out for sequencing, sequence information of candidate genes is confirmed, and a transformant with the correct sequence is subjected to plasmid extraction to obtain a recombinant plasmid pACYCDuet-1-At4CL1.

Example 5: cloning of FD2 Gene

1. Extraction of Total RNA and Synthesis of first strand cDNA

Selecting appropriate amount of Mortierella gracilis filaments as material, rapidly transferring into a mortar precooled by liquid nitrogen, grinding to powder, transferring 100mg of powder sample into a centrifuge tube, adding 500 μl Buffer RB/2-mercaptoethanol (E.Z.N.A) ^TM Fungal RNA Kit), vigorous vortexing; centrifuging at 14,000g for 5min, transferring the supernatant to a homogenizing column, and centrifuging at 14,000g for 2min. Adding the filtered liquid into 0.5 times volume of absolute ethyl alcohol, uniformly mixing, transferring the mixed liquid to an RNA adsorption column, centrifuging for 1min at 10,000g, and discarding the filtrate; adding 400 mu L RNA wash Buffer I to an adsorption column, centrifuging at 10,000g for 1min, and discarding filtrate and collecting tube; sleeving the adsorption column on a new collecting pipe, adding 500 mu L RNA wash Buffer II to the adsorption column, centrifuging for 1min at 10,000g, discarding filtrate, repeating for two times, and centrifuging for 1min at 10,000 g; after the column was dried at room temperature, the total RNA was eluted with 50. Mu.L DEPC water. 1.0% non-denaturing agarose gel electrophoresis to detect total RNA integrity and UV spectrophotometry to determine OD ₂₆₀ /OD ₂₈₀ Ratio and RNA concentration. Using smart ^TM cDNA was synthesized using RACE cDNA amplification kit (Clontech, USA).

RT-PCR amplification of Gene fragments of interest

Specific primers for gene FD2 were designed based on the information of the transcript of Mortierella gracilis, and PCR amplification was performed using KOD DNA Polymerase (the specific method was the same as in example 1) to obtain the full length of FD2 gene. And (3) recovering PCR products (target gene fragments) by glue, connecting the PCR products to a pEASY-blue vector, transforming Trans1-T1 competent cells, carrying out blue white spot screening and colony PCR screening, and sequencing positive clone shaking bacteria to confirm sequence information of the gene FD2.

Example 6: construction of pCDFDuet-1-FD2 recombinant expression vector

A linear vector of pCDFDuet-1 was obtained by single cleavage at EcoRV cleavage site of the second multiple cloning site of pCDFDuet-1 (same as example 3); meanwhile, primers containing homologous arms of the vector are designed according to gene sequences at two sides of the linear vector, homologous arms are respectively introduced at two sides of the target gene FD2 through a PCR technology (the specific method is the same as that of the embodiment 1), and PCR products are recovered through gel. The connection of the target gene and the linearized pCDFDuet-1 vector is realized by utilizing homologous recombination, the target gene is transformed into Trans1-T1 competent cells, colony PCR screening is carried out, positive cloning is carried out, sequencing is carried out, sequence information of candidate genes is confirmed, and a transformant with correct sequence is obtained by extracting plasmids to obtain recombinant plasmids pCDFDuet-1-FD2.

Example 7: bibenzyl skeleton synthetic engineering bacterium BL21DE3:

construction of pCDFDuet-1-DoBBS8-pETDuet-1-MatB-MatC-pACYCDuet-1-At4CL1

The recombinant plasmids pCDFDuet-1-DoBBS8, pETDuet-1-MatB-MatC and pACYCDuet-1-At4CL1 in examples 3-5 are transferred into competent cells of escherichia coli BL21DE3 together for PCR screening, and positive transformants are engineering bacteria containing the three recombinant plasmids At the same time and are named as S01.

Example 8: construction of prenylated engineering bacterium BL21DE3 pCDFDuet-1-FD2-pXL13-pXL17

Three recombinant plasmids pCDFDuet-1-FD2 and pXL13, pXL17 (Wang J, li S, xiong Z, et al Pathway mining-based integration of critical enzyme parts for DE novo biosynthesis of steviolglycosides sweetener in Escherichia coll.cell Research,2016,26 (2): 258-261) were simultaneously transformed into competent cells of E.coli BL21DE3, and the positive transformants were selected to obtain engineering bacteria carrying 3 recombinant plasmids simultaneously, designated as S02.

Example 9: synthesis of bibenzyl skeleton compound

1) Inoculating engineering bacteria S01 with correct sequence verified by sequencing into LB culture medium containing ampicillin (pETDuet-1 carrier carrying resistance), chloramphenicol (pACYCDuet-1 carrier carrying resistance) and streptomycin (pCDFDuet-1 carrier carrying resistance) at 37deg.C under shaking culture at 200rpm for 12 hr to obtain seed solution;

2) Inoculating the activated seed solution into fresh LB culture medium (500 mL triangular flask with 200mL liquid volume per flask) containing the same concentration of antibiotics until the bacterial solution concentration is OD ₆₀₀ ＝0.1；

3) Culturing at 37deg.C and 200rpm to OD ₆₀₀ =0.6, IPTG was added to each flask of culture at a final concentration of 0.15 mM; inducing the target protein to express at 16 ℃ and 200 rpm;

4) After 16h of induction culture, the cells were collected, centrifuged at 5,000rpm for 5min, and the supernatant was discarded, followed by ddH ₂ O, M9 Medium (containing 1 XM 9 salts, 2.0mM MgSO) ₄ 、0.1mM CaCl ₂ 20g/L glucose) to remove residual LB medium and ddH, respectively ₂ O；

5) Suspending the bacterial cells in M9 culture medium, and adjusting the bacterial liquid concentration to OD ₆₀₀ =5.0, selecting 250mL triangular flask, and adding substrate malonic acid and phenylpropionic acid compounds to final concentration of 4.5mM and 1.5mM respectively, wherein the liquid amount of each flask is not more than 100mL (if 500mL triangular flask is selected, the liquid amount of each flask is not more than 200 mL); shaking culture at 30 ℃ and 200rpm for 24 hours;

6) Centrifuging at 5,000rpm for 10min, collecting supernatant, subjecting to macroporous resin column chromatography, eluting with water to remove high-polarity impurities, eluting with 80% ethanol water solution to obtain target fraction, concentrating under reduced pressure, evaporating to dryness, redissolving with methanol, centrifuging at high speed, and preparing bibenzyl product by semi-preparative HPLC (chromatographic column Shiseido capcell pak C) ₁₈ column (250 mm x 10mm i.d., shiseido co., ltd., tokyo, japan); the mobile phase is chromatographic acetonitrile (B)/pure water (A), and gradient elution is carried out: 30% -45% of B for 15min;45-100% B,5min;100% b,5min, flow rate: 3mL/min; column temperature: 30 ℃; lambda (lambda) _max ＝280nm。)。

When 4-hydroxy phenylpropionic acid is used as a phenylpropionic acid substrate, the yield of the obtained dibenzyl compound is 190mg/L, and the structure is identified as the dihydro resveratrol (1) by MS, NMR (figures 2, 4 and 5) and the like, the spectrum data are as follows:

dihydro resveratrol (1): c (C) ₁₄ H ₁₄ O ₃ :ESI-MS m/z 231.05[M+H] ⁺ ； ¹ H NMR(DMSO-d ₆ ,400MHz):δ _H 9.13(s,4′-OH),9.04(s,3,5-OH),6.98(d,J＝8.4Hz,H-2',6′),6.64(d,J＝8.4Hz,H-3′,5′),6.05(d,J＝2.1Hz,H-2,6),6.01(t,J＝2.1Hz,H-4),2.63(m,H-α,α′)； ¹³ C NMR(DMSO-d ₆ ,100MHz):δ _C 158.2(C-3,5),155.3(C-4′),143.6(C-1),131.7(C-1′),129.1(C-2′,6′),115.0(C-3′,5′),106.4(C-2,6),100.1(C-4),37.6(C-α),36.1(C-α′).；

When 3, 4-dihydroxybenzene propionic acid is used as a phenylpropionic acid substrate, the obtained dibenzyl compound has the structure of 3,3',4', 5-tetrahydroxydibenzyl (2) identified by MS, NMR (shown in figures 2, 6 and 7) and the like, and the spectrum data are as follows:

3,3',4', 5-tetrahydroxybibenzyl (2): c (C) ₁₄ H ₁₄ O ₄ ,ESI-MS m/z 290.91[M+HCOOH-H] ^- ； ¹ H NMR(DMSO-d ₆ ,400MHz):δ _H 9.01(s,3,5-OH),8.66(s,3′-OH),8.59(s,4′-OH),6.60(d,J＝8.0Hz,H-5′),6.58(d,J＝1.8Hz,H-2′),6.44(dd,J＝1.8Hz,8.0Hz,H-6′),6.05(d,J＝1.9Hz,H-2,6),6.01(t,J＝1.9Hz,H-4),2.59(m,H-α,α′)； ¹³ C NMR(DMSO-d ₆ ,100MHz):δ _C 158.2(C-3,5),144.9(C-3′),143.7(C-4′),143.2(C-1),132.5(C-1′),118.8(C-6′),115.7(C-5′),115.4(C-2′),106.4(C-2,6),100.1(C-4),37.6(C-α),36.3(C-α′).

When 3-hydroxy phenylpropionic acid is used as a phenylpropionic acid substrate, the obtained dibenzyl compound has the following identification structure of 3,3', 5-trihydroxy dibenzyl (3) by MS and NMR (shown in figures 2, 8 and 9), and the spectrum data are as follows:

3,3', 5-trihydroxybenzyl (3): c (C) ₁₄ H ₁₄ O ₃ ,ESI-MS m/z 231.06[M+H] ⁺ ； ¹ H NMR(DMSO-d ₆ ,400MHz):δ _H 9.28(s,3'-OH),9.08(s,3,5-OH),7.04(t,J＝7.7Hz,H-5′),6.63(m,H-2′,4′),6.56(dd,J＝1.7Hz,8.0Hz,H-6′),6.07(d,J＝2.0Hz,H-2,6),6.02(t,J＝2.0Hz,H-4),2.66(m,H-α,α′)； ¹³ C NMR(DMSO-d ₆ ,100MHz):δ _C 158.2(C-3,5),157.3(C-3′),143.5(C-1),143.1(C-1′),129.1(C-5′),118.9(C-6′),115.2(C-2′),112.7(C-4′),106.3(C-2,6),100.2(C-4),37.1(C-α),36.8(C-α′).

Example 10: synthesis of isopentenyl bibenzyl derivative

1) Inoculating engineering bacteria S01 with correct sequencing verification into LB culture medium containing ampicillin (pETDuet-1 vector carrying resistance), chloramphenicol (pACYCDuet-1 vector carrying resistance) and streptomycin (pCDFDuet-1 vector carrying resistance); inoculating engineering bacteria S02 with correct sequence verified by sequencing into LB culture medium simultaneously containing kanamycin (pXL 13 recombinant plasmid carrying resistance), ampicillin (pXL 17 recombinant plasmid carrying resistance) and streptomycin (pCDFDuet-1 vector carrying resistance), and carrying out shake culture at 37 ℃ for 12h at 200rpm to obtain seed liquid;

2) Inoculating the activated seed solution into fresh LB culture medium (500 mL triangular flask with 200mL liquid volume per flask) containing the same concentration of antibiotics until the bacterial solution concentration is OD ₆₀₀ ＝0.1；

3) Culturing at 37deg.C and 200rpm to OD ₆₀₀ =0.6, IPTG was added to each flask of culture at a final concentration of 0.15 mM; inducing the target protein to express at 16 ℃ and 200 rpm;

4) After 16h of induction culture, the cells were collected, centrifuged at 5,000rpm for 5min, and the supernatant was discarded, and the cells were subjected to ddH ₂ O, M9 Medium (containing 1 XM 9 salts, 2.0mM MgSO) ₄ 、0.1mM CaCl ₂ 20g/L glucose) was washed 1 time each to remove the residual LB medium and ddH, respectively ₂ O；

5) Suspending the bacterial cells in M9 culture medium, and adjusting the bacterial liquid concentration to OD ₆₀₀ ＝5.0；

6) Mixing the S01 and the S02 bacterial liquid in equal volume, selecting a 250mL triangular flask, adding substrate malonic acid and phenylpropionic acid compounds to the final concentration of 2.25mM and 0.75mM respectively, wherein the liquid filling amount of each flask is not more than 100 mL; shaking culture at 30 ℃ and 200rpm for 24 hours; the bacterial liquid working-up procedure was as in example 9, and the corresponding product was prepared by semi-preparative HPLC (chromatographic column Shiseido capcell pak C column (250 mm. Times.10 mm I.D., shiseido Co., ltd., tokyo, japan), mobile phase chromatography acetonitrile (B)/pure water (A), gradient elution: 15% -40% B,20min, 40-100% B,5min, 100% B,5min, flow rate: 3mL/min; column temperature: 30 ℃; lambda (lambda) _max ＝280nm)。

When 4-hydroxy phenylpropionic acid is used as a phenylpropionic acid substrate, the obtained dibenzyl compound has been identified as 2-isopentenyl-3, 4', 5-trihydroxybenzyl (4) by MS and NMR (FIGS. 3, 10, 11) and the like. The spectral data are as follows:

2-isopentenyl-3, 4', 5-trihydroxybenzyl (4): c (C) ₁₉ H ₂₂ O ₃ ,ESI-MS m/z 298.99[M+H] ⁺ ； ¹ H NMR:(DMSO-d ₆ ,400MHz):δ _H 9.15(s,3-OH),8.98(s,5-OH),8.83(s,4′-OH),6.98(d,J＝8.4Hz,H-2′,6′),6.66(d,J＝8.4Hz,H-3′,5′),6.14(d,J＝2.3Hz,H-2,6),6.07(d,J＝2.3Hz,H-4),4.99(t,J＝6.6Hz,H-8),3.13(d,J＝6.6Hz,H-7),2.60(s,H-α,α′),1.66(s,H-10),1.60(s,H-11)； ¹³ C NMR(DMSO-d ₆ ,100MHz):δ _C 156.7(C-3),156.5(C-5),156.3(C-4′),141.4(C-1),132.0(C-1′),129.0(C-2′,6′),128.8(C-9),124.7(C-8),116.3(C-2),115.0(C-3′,5′),106.9(C-6),100.3(C-4),37.6(C-α),36.1(C-α′),25.5(C-11),23.9(C-7),17.7(C-10).

When 3, 4-dihydroxybenzene propionic acid is used as a phenylpropionic acid substrate, the obtained dibenzyl compound is identified as 2-isopentenyl-3, 3',4', 5-tetrahydroxydibenzyl (5) by MS and NMR (shown in figures 3, 12 and 13) and the like. The spectral data are as follows:

2-isopentenyl-3, 3',4', 5-tetrahydroxybibenzyl (5): c (C) ₁₉ H ₂₂ O ₄ ,ESI-MS m/z 313.17[M-H] ^- ； ¹ H NMR(DMSO-d ₆ ,400MHz):δ _H 6.62(d,J＝8.0Hz,H-5′),6.58(d,J＝2.0Hz,H-2′),6.44(dd,J＝2.0Hz,8.0Hz,H-6′),6.14(d,J＝2.4Hz,H-4),6.06(d,J＝2.4Hz,H-6),5.00(t,J＝5.4Hz,H-8),3.14(d,J＝6.4Hz,H-7),2.56(m,H-α,α′),1.67(s,H-10),1.61(s,H-11)； ¹³ C NMR(DMSO-d ₆ ,100MHz):δ _C 156.2(C-3),156.2(C-5),145.5(C-3′),143.7(C-4′),142.0(C-1),133.3(C-1′),129.3(C-9),125.2(C-8),119.1(C-6′),116.7(C-5′),116.1(C-2′),115.9(C-2),107.4(C-6),100.7(C-4),37.0(C-α),36.7(C-α′),26.0(C-11),24.4(C-7),18.3(C-10).

When 3-hydroxy phenylpropionic acid is used as substrate of phenylpropionic acid, the obtained dibenzyl compound has been identified as 2-isopentenyl-3, 3', 5-trihydroxybenzyl (6) by MS and NMR (FIG. 3, 14, 15) etc. The spectral data are as follows:

2-isopentenyl-3, 3', 5-trihydroxybenzyl (6): c (C) ₁₉ H ₂₂ O ₄ ,HRESIMS:m/z 297.1492[M-H] ^- (calcd for C ₁₉ H ₂₁ O ₃ 297.1491)；ESI-MS m/z 299.02[M+H] ⁺ ； ¹ H NMR(DMSO-d ₆ ,400MHz):δ _H 7.06(t,J＝7.7Hz,H-5′),6.64-6.60(m,H-6′,2′),6.57(ddd,J＝0.8Hz,2.4Hz,7.7Hz,H-4′),6.14(d,J＝2.4Hz,H-4),6.08(d,J＝2.4Hz,H-6),5.00(t,J＝6.7Hz,H-8),3.14(d,J＝6.7Hz,H-7),2.63(s,H-α,α′),1.67(s,H-10),1.61(s,H-11)； ¹³ C NMR(DMSO-d ₆ ,100MHz):δ _C 157.4(C-3′),155.8(C-3),155.6(C-5),143.5(C-1′),141.4(C-1),129.3(C-9),128.9(C-5′),124.8(C-8),118.7(C-6′),115.0(C-2'),116.3(C-2),112.8(C-4′),106.9(C-6),100.4(C-4),37.0(C-α′),34.7(C-α),25.5(C-11),23.8(C-7),17.8(C-10).

Example 11: recycle of dibenzyl isopentenyl derivative synthesis system

Separating the supernatant from the bacterial liquid after co-culture for one time (see example 10) by centrifugation (5,000 g,6 min) under the sterile environment, separating and enriching the target product from the supernatant, re-suspending the bacterial liquid to be uniform by using a 1 XM 9 culture medium, fixing the volume to be the same as the volume of the first time, adding the same amount of malonic acid and 3, 4-dihydroxybenzene propionic acid S2, uniformly mixing, culturing for 24 hours at 30 ℃ and 200rpm, and repeating the above operation for 5 times in a circulating way. After each cycle, 400. Mu.L of the sample was sampled, 800. Mu.L of cold methanol was added, the mixture was vortexed and centrifuged at 15,000g for 30min, the supernatant was subjected to HPLC-UV analysis, the sample loading was 60. Mu.L, and the liquid phase conditions were: column Shiseido capcellpak C MG III column (250 mm×4.6mm, i.d.,5 μm, shiseido co., ltd., japan); mobile phase a (0.1% aqueous formic acid)/B (acetonitrile), gradient elution: 15-40% B for 15min;40-100% B,10min;100% B,15min; flow rate: 1.0mL/min; column temperature: 30 ℃; lambda (lambda) _max =280 nm. Each cycle is set to 3 parallels.

As a result, as shown in FIG. 16, the yield of the objective product 5 in the first cycle was 33mg/L, and the yields of the objective product 5 in each subsequent cycle were 64%, 33%, 21%, 17% of the first cycle in order, indicating that the modular biosynthetic system for the synthesis of dibenzyl isopentenyl derivatives had the ability to recycle the objective product, and efficient and sustainable production of the objective dibenzyl compound could be achieved by recycling the synthetic system.

Example 12: protection of glutamate-induced neuronal injury by Compound 5

The neuroblastoma SK-N-SH cell strain is plated by adopting an MTT method, and the density is 7.5x10 ⁴ After the cells are completely attached, compound 5 and resveratrol (final concentration 10 μm) which is a positive drug are added, and the compound to be tested is incubated with the cells for 4 hours. Changing the liquid, sucking and discarding the culture medium of the 96-well plate, adding the complete culture medium into the control group, and 100 mu L of the culture medium is added into each well; the model group, the positive medicine group and the test group are added with GLU injury agent with the concentration of 29mM, and 90 mu L of GLU injury agent is added in each hole; the positive group was then supplemented with 10. Mu.L of resveratrol at a concentration of 100. Mu.M (final concentration of 10. Mu.M), and the test group was further supplemented with 10. Mu.L of Compound 5 at a concentration of 100. Mu.M. Glutamate damaging cells for 4h; and adding MTT to continue the reaction for 4 hours, measuring the absorbance value of the cells at 570nm, measuring each reaction for 3 times, taking the average value of the three times, and calculating the relative blank survival rate of the cells of each hole and the improvement survival rate of the cells of each hole relative to the model.

The results are shown in Table 1, and compound 5 can resist neuronal damage induced by glutamate to a certain extent, and improve cell survival by 15.32%, indicating that it has potential neuronal protection effect.

Table 1 effect of compound 5 on cell viability of glutamate-induced neuronal damage (n=3)

Example 13: compound 5 effect on mice hypoxia tolerance under hypoxic conditions

Male ICR mice were randomized and the positive drug group was given atenolol (50 mg/kg), and experiments were performed after 30min of gastric administration, and the control group and the different concentration experimental groups (10 mg/kg and 50 mg/kg) were performed after 15min of intraperitoneal injection. After the administration, the mice were put into wide-mouth bottles (one for each bottle) pre-filled with 10g of soda lime, the caps were uniformly coated with vaseline, and the bottles were sealed with a sealing film to prevent air leakage, and immediately timed, and the whole survival time of the mice from death due to hypoxia was recorded by using a timer with respiratory stop as an index. Statistical analysis of the data was performed on Graph Prism 8.0.1, all results being expressed as means±sem. Statistical analysis was performed using one-way ANOVA test, comparing differences between groups, P <0.05 considered significant differences.

Table 2 effect of compound 5 on survival of mice under hypoxic conditions

* P <0.05vs control group, P <0.01vs control group, n=10.

The results are shown in Table 2, and compound 5 increases the survival time of ICR mice in hypoxia tolerance experiments in a dose-dependent manner at 10mg/kg and 50mg/kg, and is superior to the positive drug group, and the fact that compound 5 can increase the survival time of animals in a closed space indicates that the animals have a certain hypoxia tolerance effect.

Example 14: compound 5 effect on mice hypoxia tolerance under acute hypoxia conditions

After random grouping of male ICR mice, nimodipine (120 mg/kg) was administered to the positive drug group, experiments were performed after 1 hour of oral administration, and experiments were performed after intraperitoneal injection for 30 minutes in the control group and the different concentration experimental groups (10 mg/kg and 50 mg/kg). After administration, the specific experimental time was waited for, and the mouse head was cut off by surgical scissors. The survival time of the mice was recorded using a timer, and the number of times the mice were opened. The data processing procedure was as in example 13.

Table 3 effect of compound 5 on survival time of mice under acute hypoxic conditions

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* P <0.05vs control group, P <0.01vs control group, n=10.

Table 4 effect of compound 5 on the number of breaths in mice under acute hypoxic conditions

* P <0.05vs control, n=10.

The results are shown in tables 3 and 4, and compound 5 significantly increased survival time in ICR mice acute hypoxia tolerance experiments at doses of 10mg/kg and 50mg/kg, and significantly increased respiratory rate in ICR mice acute hypoxia tolerance experiments at doses of 10 mg/kg. The results suggest that the compound 5 can increase the survival time and the respiration frequency of animals after head breakage, and shows that the compound has a certain anti-hypoxia effect.

Example 15: analgesic effect of Compound 5 on mice

After random grouping of male ICR mice, sodium carboxymethyl cellulose solution and solvent control of compound 5 at the corresponding concentrations were administered 1h before the test torsion reaction, respectively, and 0.6% acetic acid was administered 1h later, and the number of torsion times was recorded. Statistical analysis of the data was performed on Graph Prism 8.0.1, all results being expressed as means+ -SEM. Statistical analysis was performed using t-test, comparing differences between groups, P <0.05 considered significant differences.

TABLE 5 influence of Compound 5 on the number of torsions in the acetic acid torsions of mice

* P <0.05vs control, n=15.

The results are shown in Table 5, and the compound 5 obviously reduces the number of writhing times in the ICR mouse acetic acid writhing experiment at the dosage of 10mg/kg, which suggests that the compound 5 has a certain analgesic effect in the ICR mouse acetic acid writhing model.

Example 16: effect of Compound 5 on permanent ischemic rats

After completion of the preparation of the permanent ischemia model (pMCAO) in male SD rats, the rats were incubated (34-35 ℃) for 2 hours. Compound 5 was administered intraperitoneally 5min after cerebral ischemia in different dose groups and control groups. The room temperature is kept at 24-25 ℃ in the whole rest process.

Animals were scored behaviorally 24h after ischemia, as detailed in methods of pharmacological experiments (Xu Shuyun, third edition p 1066-1067.). The tail of the hand-lifting tail leaves the ground by about 1 ruler, and the conditions of two forelimbs are observed; placing the rat on the horizontal ground, pushing the two shoulders of the rat, and observing whether the resistance of the two sides is different; the rats were placed on the ground and observed for walking.

Immediately after 24h ischemia, the rat was cut off its head to take out the brain, the olfactory tract, cerebellum and low brain stem were removed, and the head was cut into 6 pieces (first to fifth pieces 2 mm/piece, sixth piece 4 mm), and rapidly placed in 5mL containing 1.5mL4% TTC and 0.1mL 1M K ₂ HPO ₄ Is dyed (at 37 ℃ and protected from light) for 20-30min, and is turned over every 5 min. After TTC staining, normal tissues were deeply stained red and infarcted tissues were white. And (5) arranging each group of brain slices in order, photographing and storing. The infarct area of each slice was calculated by using the image analysis system software Photoshop to process and count, and finally the infarct volume was converted by superposition.

To eliminate the effect of cerebral edema, the percent infarct volume was calculated as follows:

cerebral infarction volume (%) = (non-operative hemispheric uninjured volume)/non-operative hemispheric volume×100%

Statistical analysis of the data was performed on Graph Prism 8.0.1, all results being expressed as means+ -SEM. Statistical analysis was performed using t-test, comparing differences between groups, P <0.05 considered significant differences.

TABLE 6 Effect of Compound 5 on post-pMCAO cerebral infarction volumes in SD rats

* P <0.01vs control group, n=8-9.

TABLE 7 Effect of Compound 5 on post-pMCAO behavior impairment in SD rats

* P <0.05vs control, n=8 to 9.

The results are shown in tables 6 and 7, compound 5 significantly reduced brain infarct volume 24h post-pMCAO surgery and significantly reduced neurobehavioral score 24h post-surgery SD rats at a dose of 30mg/kg in the SD rat permanent ischemia model. The results suggest that the compound 5 has a certain therapeutic effect on SD rat permanent cerebral apoplexy.

EXAMPLE 17 antioxidant effect of Compound 5

DPPH radical scavenging action

Solutions of Compound 5 at different concentrations (5. Mu.M, 10. Mu.M, 20. Mu.M, 40. Mu.M) were combined with 1X 10 ^-4 100 mu L of each mol/L DPPH solution is sequentially added into a 96-well plate to be used as a test solution, and an antioxidant vitamin C is used as a positive control drug. Taking test sample solutions with various concentrations without adding DPPH (100 mu L of absolute ethyl alcohol is used for replacing the DPPH) as a control group so as to eliminate the interference of the color of the test sample on the test result; a negative control group (100. Mu.L of absolute ethyl alcohol was used instead of the test sample) was simultaneously set, and three duplicate wells were set in parallel for each group. The 96-well plate was put into an microplate reader and oscillated for 1min, and after light shielding at room temperature for 30min, its absorbance value was measured at 517nm wavelength and the radical clearance was calculated. Statistical analysis of the data was performed using GraphPad 8.0.1 software and the results are expressed as mean ± standard deviation.

TABLE 8 clearance of Compound 5 to DPPH

The results are shown in Table 8, followingWith increasing drug concentration, DPPH clearance of both compound 5 and positive drug was enhanced, wherein compound 5 was significantly more cleared than vitamin C at the first three concentrations (5. Mu.M, 10. Mu.M, 20. Mu.M) of the experiment. Furthermore, at different concentrations in this experiment, the EC of vitamin C ₅₀ EC of compound 5 with a value of 11.53 μm ₅₀ The value was 6.12. Mu.M.

17.2 self-oxidation of pyrogallol

180. Mu.L of 0.05M Tris-HCl buffer solution (pH 8.2) is added into a 96-well plate, 40. Mu.L of test solution with different concentrations and 16. Mu.L of 9mM pyrogallol solution are respectively added, the mixture is fully and uniformly mixed, after 5min, 8. Mu.L of 3M HCl is added to terminate the reaction, and an antioxidant vitamin C is selected as a positive control. The model set replaced the sample solution with 40 μl of water; the blank group replaced the pyrogallol and sample solution with 56 μl of water; in addition, 40. Mu.L of the sample solution was added to 180. Mu.L of a 0.05M Tris-HCl buffer solution (pH 8.2), and 16. Mu.L of water was added as a reference control group after thoroughly mixing. Three duplicate wells were placed in parallel for each group, absorbance was measured at 299nm and radical clearance was calculated. Statistical analysis of the data was performed using GraphPad 8.0.1 software and the results are expressed as mean ± standard deviation.

TABLE 9 different compounds are described forClearance of (2)

The results are shown in Table 9, with increasing drug concentration, compound 5 and positive drug are clearedIs enhanced. EC of vitamin C at four concentrations (0.1 mM,0.2mM,0.4mM,0.8 mM) in the test ₅₀ The value was 0.33mM, while EC of Compound 5 ₅₀ Values were not calculated within the range of the test concentrations.

Feton reaction

0.75mM of O-diAdding 30 μl of azophenanthrene solution into 96-well plate, adding 60 μl PBS solution (pH 7.4) and 30 μl sample solution, mixing, adding 30 μl 0.75mM ferrous sulfate solution, mixing, and adding 1%H ₂ O ₂ 30 mu L of solution, placing the uniformly mixed solution in a water bath at 37 ℃ for 60min, and taking the solution as a test group of a test sample and vitamin C as a positive control; 1%H is replaced by 30 mu L of distilled water ₂ O ₂ Repeating the above operation as a control group; the above procedure was repeated with 30. Mu.L of distilled water instead of 30. Mu.L of the sample solution as a model set; adding only PBS solution and sample solution, and replacing other reagents with distilled water to supplement, repeating the above operation to obtain reference control group; the above procedure was repeated except that the PBS solution was added and the other reagents were replaced with distilled water as a blank. Three complex wells were placed in parallel in each group, and absorbance values were measured at 536nm wavelength and radical clearance was calculated. Statistical analysis of the data was performed using GraphPad 8.0.1 software and the results are expressed as mean ± standard deviation.

TABLE 10 clearance of different compounds to OH

As shown in Table 10, the compound 5 had a gradually increased OH-scavenging ability with increasing drug concentration, and the OH-scavenging ability at different concentrations in the test was significantly higher than that of the positive control vitamin C. In addition, at four concentrations in the assay (0.1 mM,0.2mM,0.4mM,0.8 mM), the EC of Compound 5 ₅₀ The value was 0.46mM, and the EC of vitamin C was not calculated in the range of the test concentration ₅₀ Values.

17.4. Oxygen radical scavenging capacity method (ORAC method)

20 mu L of test sample solutions with different concentrations are respectively added into a fluorescent 96-well plate, then 20 mu L of 75mM potassium phosphate buffer solution and 140 mu L of 18.3mM AAPH are sequentially added, after being fully mixed, 20 mu L of 630nM sodium fluorescein is added for starting reaction, continuous measurement is carried out at the excitation wavelength of 485nM and the emission wavelength of 538nM at 37 ℃, and the fluorescence intensity of each well is measured every 2min until the fluorescence intensity is attenuated to a base line. With vitamin C as positive control, three duplicate wells were placed in parallel for each group. According to Net AUC (i.e. the integral area under the fluorescence decay curve minus the area under the blank curve without antioxidant) of the Trolox at different concentrations, the ORAC value of each sample is calculated, and the ORAC value of the sample to be measured is expressed in mu mol TE/g. Statistical analysis of the data was performed using GraphPad 8.0.1 software and the results are expressed as mean ± standard deviation.

Table 11 Net AUC values for different compounds

The results are shown in Table 11, where the area under the fluorescence decay curve of Compound 5 is significantly greater than vitamin C at the same concentration. In addition, compound 5 had an ORAC value of (9514.0.+ -. 1160.0) μmol/g and vitamin C had an ORAC value of (2607.0.+ -. 594.9) μmol/g, suggesting that compound 5 has a greater antioxidant capacity than vitamin C.

Sequence listing

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290 295 300

Thr Ala Gly Leu Gln Ala Leu Asp Glu Phe Trp His His Leu Phe Gly

305 310 315 320

Phe Ser Lys Ser Asp Pro Asp Ala Gln His Lys Met Val Met Pro Gly

325 330 335

His Lys Cys Leu Phe Val Phe Glu Met Arg Pro Thr Gln Glu Gly Glu

340 345 350

Gln Asp Ala Thr Pro Asp Ile Glu Val Lys Val His Leu Pro Met Trp

355 360 365

Asp Ile Gly Lys Thr Asp Ala Glu Ile Ser Glu Leu Leu Ala Ser Trp

370 375 380

Phe Gln Ala His Gly His Arg Asp Leu Ala Glu Arg Tyr Gln Ala Asp

385 390 395 400

Leu Asp Ala Ala Phe Pro Lys His Asn Ile Lys Thr Ser Ser Gly Thr

405 410 415

His Thr Phe Leu Ser Leu Thr Tyr Thr His Lys Thr Gly Leu Tyr Met

420 425 430

Thr Met Tyr Tyr Thr Thr Lys Phe Pro Glu Leu Tyr Tyr Leu Pro Asn

435 440 445

<210> 8

<211> 1347

<212> DNA

<213> Mortierella gracilis (Periconia sp. F-31)

<400> 8

atgtctcaca ccgtcgtcaa aacagcctca aacaaggcaa acagccaaga ctgtaccgcc 60

acagctgcgg tcatgaacga aatcgacaaa gaatttcagg caaacagtga agatgatgca 120

ttctggtgga gtgcctcagg acagccactg tgcacactac ttcagcaaaa tcagtacagc 180

cacgaccgac agctctacct cctccgctgg ttccgccaac gagtccttcc gtctctgggg 240

ccccgcccca gcggaaccaa gccgtattat gggtcctggt tgacatacga tgggtctcct 300

ctcgagtaca gtctcaactg gaaggagaag aaacccaatc aaaccatccg cttcaccata 360

gaacctacat cgagcaaggc aggaactgct gcggaccgtc tcaatcaatt gggagcgaaa 420

gagctcctga ctacattgag caaagaaatt ccatccatag acttgaaacg attcaatctt 480

ttcctcgagg atacttacgt acccgacgac gcgatcgagg aggtcatttc taaacatccg 540

gctgggtttc cccagagccg cgtctgggtc gcatttgatc tcgagcgctc tggcgacatc 600

gtggccaagg catattttct cccgcactgg agagaaatct ataccgggac tcctaccaaa 660

acgatcgtct tcgatgccat caagaagtgc aatggaccac tagggtcgta cgacgcttcg 720

attgcggcgc tcgatggcta cttggaaagc ttcccgtccg aagaagcacc aaaaatcgta 780

ctattgtcga acgactgtgt cgccgactcg cctgcagcga gaatgaaggt ttaccttcac 840

acctccgtcg acactctcgc caaagccaag gacatgtttc acctaggagg aaggctttca 900

ggaacagcca ttaccgcggg cttgcaagca ctcgatgaat tttggcacca cctcttcggc 960

ttctccaagt ccgatccaga tgctcaacac aaaatggtca tgcccggaca caaatgcctc 1020

ttcgttttcg aaatgaggcc cacgcaggag ggggaacaag acgcgacgcc cgacatcgaa 1080

gtcaaggtgc acctccccat gtgggacatc ggcaagacgg acgcggaaat cagcgagctg 1140

ctggcatcct ggttccaggc tcacggtcac cgggatctcg cggaacggta tcaggctgac 1200

ctggacgcgg catttccaaa gcataatatc aaaacaagca gcggtactca tacgttcctg 1260

tccctcacgt acacgcacaa gactggtctt tacatgacca tgtactacac gacaaaattc 1320

cctgaacttt actatctccc caactaa 1347

<210> 9

<211> 27

<212> DNA

<213> Forward primer FD2-F (Periconia sp.F-31)

<400> 9

atgtctcaca ccgtcgtcaa aacagcc 27

<210> 10

<211> 26

<212> DNA

<213> reverse primer FD2-R (Periconia sp.F-31)

<400> 10

ctagttgggg agatagtaaa gttcag 26

<210> 11

<211> 32

<212> DNA

<213> forward primer with homology arm FD2-Fe (Periconia sp.F-31)

<400> 11

ggcagatctc aattggatgt ctcacaccgt cg 32

<210> 12

<211> 32

<212> DNA

<213> reverse primer with homology arm FD2-Re (Periconia sp.F-31)

<400> 12

cgtggccggc cgatatctag ttggggagat ag 32

Claims (9)

1. An isopentenyl dibenzyl derivative with a structure shown in a general formula 1 and pharmaceutically acceptable salts thereof,wherein the substituents R ₁ 、R ₂ Each independently selected from hydrogen, hydroxy.

2. The isopentenyl dibenzyl derivative according to claim 1, and pharmaceutically acceptable salts thereof, wherein said compound is selected from the group consisting of 2-isopentenyl-3, 4', 5-trihydroxybenzyl, 2-isopentenyl-3, 3',4', 5-tetrahydroxybibenzyl.

3. A process for the preparation of an isopentenyl-dibenzyl derivative according to claim 1, characterized in that it is obtained by means of an engineered microorganism "cell factory", comprising the following specific steps:

a. respectively carrying out induced expression on a bibenzyl skeleton synthesis engineering microorganism containing a bibenzyl synthase gene DoBBS8 and an isopentenyl engineering microorganism containing an isopentenyl transferase gene FD2 to obtain recombinant bibenzyl synthase DoBBS8 and isopentenyl transferase FD2;

b. respectively centrifugally collecting the bibenzyl skeleton synthesized engineering microorganism and the isopentenyl engineering microorganism, washing and resuspension sequentially by using sterile water and a 1 xM 9 culture medium, regulating cells to proper concentration by using the 1 xM 9 culture medium, mixing, adding a proper amount of compound, and reacting for 24 hours at 30 ℃ and 200 rpm; the compound comprises malonic acid and phenylpropionic acid, malonic acid and 4-hydroxy phenylpropionic acid, malonic acid and 3, 4-dihydroxy phenylpropionic acid, which are all available commercially;

c. centrifuging to collect supernatant, and purifying by macroporous resin column, semi-preparative HPLC, etc. chromatography to obtain target compound.

4. The preparation method of claim 3, wherein the recombinant bibenzyl synthase DoBBS8 is derived from Dendrobium officinale, and has an amino acid sequence shown in SEQ ID No.1 or a sequence which has more than 90% homology with the sequence shown in SEQ ID No.1 and has the same function.

5. The preparation method of claim 3, wherein the nucleic acid sequence of the bibenzyl synthase gene DoBBS8 is shown as SEQ ID No.2 or a sequence which has more than 90% of homology with the sequence shown as SEQ ID No.2 and has the same function.

6. A pharmaceutical composition comprising an effective amount of an isopentenyl-dibenzyl derivative according to any one of claims 1-2 and a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, wherein the pharmaceutical composition is selected from the group consisting of injection, tablet, capsule, pill, granule, oral liquid and suspension.

8. Use of an isopentenyl-dibenzyl derivative according to any one of claims 1-2 and a pharmaceutically acceptable salt thereof and a pharmaceutical composition according to any one of claims 6-7 in the manufacture of a neuroprotective medicament.

9. Use of an isopentenyl dibenzyl derivative according to any one of claims 1-2 and a pharmaceutically acceptable salt thereof and a pharmaceutical composition according to any one of claims 6-7 in the preparation of an antioxidant.