An isopentenyl bibenzyl derivative, its preparation method and use

Abstract

The present invention belongs to the field of pharmaceutical technology, and discloses an isopentenyl bibenzyl derivative, a preparation method and uses thereof. Specifically, it discloses an isopentenyl bibenzyl derivative, a biosynthetic preparation method based on an engineered Escherichia coli "cell factory" and its use as a neuroprotective drug, including a new class of bibenzyl synthase DoBBS8 derived from Dendrobium officinale and isopentenyltransferase FD2 of Periconia atropurpurea, nucleic acid molecules encoding the above proteins respectively, an engineered microorganism containing the corresponding nucleic acid molecules, and also includes a bioactive isopentenyl bibenzyl derivative synthesized by the engineered microorganism, its production method, and uses as a neuroprotective drug and an antioxidant.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the use of genetic engineering methods to create a microbial cell factory and biosynthesize bibenzyl and its isopentenyl derivatives, the use of isopentenyl bibenzyl derivatives in neuroprotective drugs, and the use of isopentenyl bibenzyl derivatives as novel antioxidants. Background Art

[0002] Bibenzyl compounds are an important class of plant polyphenols with pharmacological activities including antitumor, antidiabetic, neuroprotective, antioxidant, anti-inflammatory, antiplatelet aggregation, and antispasmodic activities. However, due to limited plant resources containing these compounds and low target compound content, chemical synthesis also presents challenges such as poor selectivity and environmental pollution. Therefore, developing an efficient method for the synthesis of bibenzyl compounds has important practical significance and application value.

[0003] In recent years, with the rapid development of synthetic biology, the mass production of many important natural drugs or their precursors and intermediates (such as precursor compounds of artemisinin and paclitaxel) has been achieved through the construction of microbial cell factories. Based on the concept of synthetic biology, the creation of artificial cell factories to produce active natural products has become a new model for drug resource acquisition. Therefore, applying synthetic biology methods to construct microbial cell factories, using inexpensive and readily available compounds as raw materials, is one of the possible approaches to synthesize a diverse range of natural and non-natural bibenzyl compounds with drug development potential.

[0004] The synthesis of the bibenzyl skeleton structure is catalyzed by bibenzyl synthase (BBS), which is the key to the synthesis of bibenzyl compounds. At present, this type of enzyme is only found in Xinjiang Huo Shao Lan (Gehlert R, Kindl H. Induced formation of dihydrophenanthrenes and bibenzyl synthase upon destruction of orchidmycorrhiza. Phytochemistry, 1991, 30 (2): 457-460.) and Bai Ji (Reinecke T, Kindl H. Characterization of bibenzyl synthase catalysing the biosynthesis ofphytoalexins of orchids. Phytochemistry, 1993, 35(1):63-66.) and Phalaenopsis varieties (Preisigmuller R, Gnau P, Kindl H. The inducible 9,10-dihydrophenanthrenepathway: characterization and expression of bibenzyl synthase and S-adenosylhomocysteine ​​hydrolase. Archives of Biochemistry and Biophysics, 1995, 317(1): 201-207.), but there is no report on its application in the enzymatic synthesis of bibenzyl compounds.

[0005] Bibenzyl basic skeleton compounds can be synthesized into their isopentenyl derivatives by isopentenyl transferase catalysis, but there is no report on this aspect so far.

[0006] In summary, the current research on benzyl and its isopentenyl derivatives has the following limitations and deficiencies:

[0007] 1. Bibenzyl compounds have few natural resources and are difficult to chemically synthesize, so their sources are limited;

[0008] 2. There is limited research on bibenzyl synthase, which is responsible for the biosynthesis of the bibenzyl skeleton, and prenyl transferase, which is prenylated. There are no reports on the biosynthesis of active bibenzyl derivatives using a combination of multiple enzymes.

[0009] Therefore, by exploring novel bibenzyl synthases in plants and combining them with isopentenyl transferases with broad substrate activity, an artificial pathway for producing structurally diverse isopentenyl bibenzyl derivatives in microorganisms can be constructed. The above-mentioned shortcomings can be overcome by preparing structurally diverse and biologically active bibenzyl compounds through engineered microbial cell factories, providing a new method for obtaining such compounds and new compound entities for new drug discovery. Summary of the Invention

[0010] In order to overcome the deficiencies in the prior art, the present invention aims to provide an isopentenyl bibenzyl derivative with novel pharmacological activity; another object of the present invention is to provide a method for preparing the same; and a third object of the present invention is to use the same in the preparation of neuroprotective drugs and in the preparation of novel antioxidants.

[0011] To achieve the purpose of the present invention, the following technical solutions are adopted:

[0012] The first aspect of the technical solution of the present invention is to provide a class of isopentenylbibenzyl derivatives with novel pharmacological activity and pharmaceutically acceptable salts thereof, which have a structure as shown in Formula 1, wherein the substituents R1 and R2 are each independently selected from hydrogen and hydroxyl.

[0013]

[0014] The second aspect of the technical solution of the present invention is to provide a method for preparing the aforementioned novel pharmacologically active isopentenylbibenzyl derivatives, which are characterized by:

[0015] The bibenzyl skeleton synthesis module engineered bacteria containing the bibenzyl synthase gene DoBBS8 and the isopentenyl transferase gene FD2 were cultured in appropriate culture media, and after induction of expression, the cells were collected by centrifugation, washed and resuspended in appropriate culture media to a suitable concentration, and the two strains were combined and co-cultured. The substrates malonate and phenylpropionic acid compounds were added for whole-cell reaction to synthesize isopentenyl bibenzyl derivatives. The reaction solution after centrifugation was separated and purified by macroporous resin column chromatography and reversed-phase semi-preparative HPLC to obtain the product, and UV, MS, 1 H NMR, 13 The product structure is analyzed and identified using spectral techniques such as CNMR, HSQC and HMBC. The above-mentioned engineered bacteria can be recycled and reused.

[0016] The third aspect of the technical solution of the present invention is to provide the use of the above-mentioned isopentenylbibenzyl derivatives in the preparation of neuroprotective drugs.

[0017] The neuroprotective effects of isopentenyl bibenzyl derivatives (5) were evaluated using an in vitro glutamate-induced neuronal injury model, as well as in vivo ICR mouse hypoxia tolerance and acute hypoxia tolerance experimental models, acetic acid writhing experimental models, and SD rat permanent focal cerebral ischemia model.

[0018] 1. In the glutamate-induced neuronal injury model, compound 5 exhibited neuroprotective effects, significantly increasing the survival rate by 15.32%, which was comparable to the positive control drug resveratrol.

[0019] 2. In the mouse hypoxia tolerance experimental model, compound 5 at doses of 10 mg / kg and 50 mg / kg increased the survival time of ICR mice in the hypoxia tolerance experiment in a dose-dependent manner, and was superior to the positive control drug atenolol.

[0020] 3. In the mouse acute hypoxia tolerance experimental model, compound 5 significantly increased the survival time of ICR mice in the acute hypoxia tolerance experiment at doses of 10 mg / kg and 50 mg / kg; and significantly increased the number of respirations in the acute hypoxia tolerance experiment of ICR mice at a dose of 10 mg / kg, and was superior to the positive control drug nimodipine.

[0021] 4. In the acetic acid writhing test model of ICR mice, compound 5 at a dose of 10 mg / kg significantly reduced the number of writhings in the acetic acid writhing test of ICR mice.

[0022] 5. In the SD rat permanent focal cerebral ischemia model (pMCAO), compound 5 at a dose of 30 mg / kg significantly reduced the cerebral infarction volume of SD rats 24 hours after pMCAO surgery, and significantly reduced the neurobehavioral score of SD rats 24 hours after pMCAO surgery.

[0023] The fourth aspect of the technical solution of the present invention is to provide the use of the above-mentioned isopentenylbibenzyl derivatives in the preparation of new antioxidants.

[0024] 1. In the DPPH free radical scavenging experiment, the EC of the positive control drug vitamin C 50 The value was 11.53 μM, and the EC of compound 5 50 The value is 6.12μM, which is better than positive drugs and shows strong free radical scavenging ability.

[0025] 2. Detection of superoxide anion radicals in pyrogallol autooxidation method In the experiment, compound 5 has a certain effect of scavenging superoxide anion free radicals.

[0026] 3. In the Feton reaction to determine the hydroxyl radical (·OH) experiment, compound 5 showed a strong hydroxyl radical scavenging ability, and its effect was better than that of the positive drug vitamin C.

[0027] 4. In the experiment of determining the oxygen free radical scavenging ability using the 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 the positive drug vitamin C was 8.55±3.33, indicating that the antioxidant ability of compound 5 is stronger than that of the positive drug vitamin C.

[0028] The present invention has the following technical advantages:

[0029] 1. This invention provides a modular engineered bacterial strain for the efficient synthesis of bibenzyl backbone compounds, which is economical, efficient, and environmentally friendly. Compared with existing technologies, this system avoids the drawbacks of chemical synthesis, such as functional group protection and deprotection, and environmental pollution, while enabling the rapid production of large quantities of target compounds. Furthermore, because the bibenzyl synthase involved has a broad substrate spectrum, it can be developed as a platform cell for the synthesis of a range of bibenzyl derivatives.

[0030] 2. The present invention provides a modular engineered bacterium for efficient isopentenylation. The isopentenylation reaction is completed by the engineered bacterium itself synthesizing an isopentenyl donor combined with a highly catalytic isopentenyl transferase, thereby achieving self-sufficiency of the isopentenyl donor in the bacterium, being economical and practical, and effectively reducing the cost of synthesizing isopentenylation products.

[0031] 3. The modular engineered bacteria involved in the present invention effectively avoid the shortcomings of heavy cell load and weak vitality caused by multiple genes in the same host cell through combined co-cultivation, fully utilize the catalytic ability of each functional bacteria, and provide a new method for the efficient synthesis of structurally diverse compounds (not limited to bibenzyl).

[0032] 4. The isopentenylbibenzyl derivative synthesis system of the present invention has recycling capabilities and can realize multiple recycling synthesis of target products, which is simple, efficient, economical and sustainable.

[0033] 5. The isopentenyl bibenzyl derivative 5 of the present invention can counteract glutamate-induced neuronal damage and improve cell survival rate, significantly prolong the survival time of mice under acute hypoxic conditions and hypoxic conditions, improve the neurobehavior and cerebral infarction volume of rats with permanent focal cerebral ischemia, and has an analgesic effect.

[0034] 6. The isopentenylbibenzyl derivative 5 of the present invention has a strong free radical scavenging ability and can scavenge superoxide anions, hydroxyl radicals, and oxygen free radicals. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 .Summary of the structural formulas of bibenzyl derivatives

[0036] Figure 2 Bibenzyl skeleton synthesis engineered bacteria whole cell synthesis of bibenzyl skeleton compounds.

[0037] (A), (B), (C), Whole-cell synthesis of the bibenzyl skeleton-synthesizing bacteria when malonic acid and S1 (4-hydroxyphenylpropionic acid), malonic acid and S2 (3,4-dihydroxyphenylpropionic acid), or malonic acid and S3 (3-hydroxyphenylpropionic acid) were added. (1), HPLC spectrum of the control S1 / S2 / S3; (2), HPLC spectrum of the whole-cell synthesis of the bibenzyl skeleton-synthesizing bacteria when no substrate was added; (3), HPLC spectrum of the whole-cell synthesis of the bibenzyl skeleton-synthesizing bacteria when malonic acid and S1 / S2 / S3 were added.

[0038] Figure 3 Co-culture of engineered bacteria for bibenzyl skeleton synthesis and prenylation to synthesize isopentenylbibenzyl derivatives.

[0039] (A), (B), (C), Whole-cell synthesis of bibenzyl skeleton-synthesizing bacteria co-cultured with isopentenyl-forming bacteria when malonic acid and S1 (4-hydroxyphenylpropionic acid), malonic acid and S2 (3,4-dihydroxyphenylpropionic acid), or malonic acid and S3 (3-hydroxyphenylpropionic acid) were added. (1), HPLC spectrum of whole-cell synthesis of bibenzyl skeleton-synthesizing bacteria co-cultured with malonic acid and S1 / S2 / S3 were added. (2) HPLC spectrum of co-culture of bibenzyl skeleton-synthesizing bacteria co-cultured with isopentenyl-forming bacteria when malonic acid and S1 / S2 / S3 were added.

[0040] Figure 4 Compound 1 1 H NMR spectrum (DMSO-d6, 400 MHz)

[0041] Figure 5 Compound 1 13 C NMR spectrum (DMSO-d6, 100 MHz)

[0042] Figure 6 Compound 2 1 H NMR spectrum (DMSO-d6, 400 MHz)

[0043] Figure 7 Compound 2 13 C NMR spectrum (DMSO-d6, 100 MHz)

[0044] Figure 8 Compound 3 1 H NMR spectrum (DMSO-d6, 400 MHz)

[0045] Figure 9 Compound 3 13 C NMR spectrum (DMSO-d6, 100 MHz)

[0046] Figure 10Compound 4 1 H NMR spectrum (DMSO-d6, 400 MHz)

[0047] Figure 11 Compound 4 13 C NMR spectrum (DMSO-d6, 100 MHz)

[0048] Figure 12 Compound 5 1 H NMR spectrum (DMSO-d6, 400 MHz)

[0049] Figure 13 Compound 5 13 C NMR spectrum (DMSO-d6, 100 MHz)

[0050] Figure 14 Compound 6 1 H NMR spectrum (DMSO-d6, 400 MHz)

[0051] Figure 15 Compound 6 13 C NMR spectrum (DMSO-d6, 100 MHz)

[0052] Figure 16 Recycling biosynthesis of compound 5 DETAILED DESCRIPTION

[0053] For a further understanding of the present invention, the following examples are only used to further illustrate the present invention, but are not intended to limit the present invention in any way.

[0054] Example 1: Cloning of the DoBBS8 gene

[0055] 1. Total RNA Extraction and First-Strand cDNA Synthesis

[0056] Select appropriate amount of fresh leaves of Dendrobium officinale as material, quickly transfer to a mortar pre-cooled with liquid nitrogen, and grind thoroughly into powder; take 100 mg of powder sample and transfer to a centrifuge tube, add 500 μL Buffer RCL / 2-mercaptoethanol (EZNA TMPlant RNA Kit), vortex vigorously; incubate at 55°C for 3 minutes; centrifuge at 14,000g for 2 minutes, transfer the supernatant to a filter column, and centrifuge at 14,000g for 2 minutes. Add an equal volume of Buffer RCB to the filtered liquid and invert 5-10 times. Transfer the liquid to an RNA adsorption column and centrifuge at 10,000g for 1 minute; discard the filtrate, add 500μL RNA wash Buffer II to the adsorption column, centrifuge at 10,000g for 1 minute, discard the filtrate, repeat twice, and centrifuge the empty column at 10,000g for 1 minute; after the adsorption column is dried at room temperature, elute the total RNA with 50μL DEPC water. The integrity of the total RNA was detected by 1.0% non-denaturing agarose gel electrophoresis, and the OD was measured using a UV spectrophotometer. 260 / OD 280 ratio and RNA concentration. TM cDNA was synthesized using the RACE cDNA amplification kit (Clontech, USA).

[0057] 2. RT-PCR amplification of target gene fragments

[0058] Based on the transcriptome data from Dendrobium officinale, specific primers for the candidate gene DoBBS8 were designed. PCR amplification was performed using KOD DNA Polymerase to obtain the full-length DoBBS8 gene. The PCR product (target gene fragment) was recovered from gels, ligated into the pEASY-blunt vector, and transformed into Trans1-T1 competent cells. Blue-white spot screening and colony PCR screening were performed, and positive clones were shaken and sent for sequencing to confirm the sequence of the candidate gene.

[0059] 2.1.PCR reaction system:

[0060]

[0061]

[0062] 2.2.PCR Cycles:

[0063] 1) 94℃ for 2 min; 2) 98℃ for 10 sec; 3) 58℃ for 30 sec; 4) 68℃ for 1 min; 5) 2-4 cycles 35 times; 6) 68℃ for 10 min; 7) hold at 10℃.

[0064] Example 2: Construction of pCDFDuet-1-DoBBS8 recombinant expression vector

[0065] pCDFDuet-1 has two multiple cloning sites. The EcoRV restriction site located in the second multiple cloning site is selected for single enzyme digestion to obtain the linear vector of pCDFDuet-1. At the same time, according to the gene sequence on both sides of the expression vector pCDFDuet-1-EcoRV site, primers containing vector homology arms are designed. The homology arms are introduced on both sides of the target gene DoBBS8 by PCR technology (the specific method is the same as in Example 1), and the PCR product is recovered by gel. The target gene is connected to the linearized pCDFDuet-1 vector by homologous recombination, and Trans1-T1 competent cells are transformed. Colony PCR screening is performed, and positive clones are sent for sequencing to confirm the sequence information of the candidate gene. The transformant with the correct sequence is extracted to obtain the recombinant plasmid pCDFDuet-1-DoBBS8.

[0066] Example 3: Construction of pETDuet-1-MatB-MatC recombinant expression vector

[0067] Similarly, pETDuet-1 has two multiple cloning sites. First, the EcoRV restriction site located in the second multiple cloning site is selected for single enzyme digestion to obtain the linear vector of pETDuet-1; at the same time, the target genes MatB (AAP03025) and MatC (KF765784.1) are amplified from the template plasmid respectively. According to the gene sequence on both sides of the expression vector pCDFDuet-1-EcoRV site, primers containing vector homology arms are designed. By PCR technology (the specific method is the same as in Example 1), homology arms are introduced on both sides of the target gene MatC, and the PCR products are recovered by gel. Homologous recombination is used to achieve the connection between the target gene and the linearized pETDuet-1 vector, Trans1-T1 competent cells are transformed, colony PCR screening is performed, and positive clones are sent for sequencing to confirm the sequence information of the candidate gene. The transformants with the correct sequence are extracted to obtain the recombinant plasmid pETDuet-1-MatC. Based on the recombinant plasmid pETDuet-1-MatC, the Not1 restriction site of the first multiple cloning site was selected for single enzyme digestion to obtain the linearized vector of pETDuet-1-MatC; according to the gene sequence on both sides of the expression vector pETDuet-1-NotI site, primers containing vector homology arms were designed, and homology arms were introduced on both sides of the target gene MatB by PCR technology (the specific method is the same as in Example 1), and the PCR product was recovered by gel. The target gene was connected to the linearized pETDuet-1 vector by homologous recombination, and Trans1-T1 competent cells were transformed. Colony PCR screening was performed, and positive clones were sent for sequencing to confirm the sequence information of the candidate gene. The transformants with correct sequences were extracted to obtain the recombinant plasmid pETDuet-1-MatB-MatC.

[0068] Example 4: Construction of pACYCDuet-1-At4CL1 recombinant expression vector

[0069] Similarly, pACYCDuet-1 has two multiple cloning sites. The EcoRV restriction site located at the second multiple cloning site is selected for single enzyme digestion to obtain the linear vector of pACYCDuet-1. At the same time, the target gene At4CL1 (AY376729.1) is amplified from the template plasmid. According to the gene sequence on both sides of the expression vector pACYCDuet-1-EcoRV site, primers containing vector homology arms are designed. By PCR technology (the specific method is the same as in Example 1), homology arms are introduced on both sides of the target gene At4CL1, and the PCR product is recovered by gel. The target gene is connected to the linearized pACYCDuet-1 vector by homologous recombination, and transformed into Trans1-T1 competent cells. Colony PCR screening is performed, and positive clones are sent for sequencing to confirm the sequence information of the candidate gene. The transformants with the correct sequence are extracted to obtain the recombinant plasmid pACYCDuet-1-At4CL1.

[0070] Example 5: Cloning of the FD2 gene

[0071] 1. Total RNA Extraction and First-Strand cDNA Synthesis

[0072] Select an appropriate amount of black spore mycelium as the material, quickly transfer it to a mortar pre-cooled with liquid nitrogen, grind it thoroughly into powder, take 100 mg of the powder sample and transfer it to a centrifuge tube, add 500 μL Buffer RB / 2-mercaptoethanol (EZNA TM Fungal RNA Kit), vortex vigorously; centrifuge at 14,000g for 5 minutes, transfer the supernatant to a homogenizer column, and centrifuge at 14,000g for 2 minutes. Add 0.5 volumes of anhydrous ethanol to the filtered liquid, mix well, and transfer the mixture to an RNA adsorption column. Centrifuge at 10,000g for 1 minute, and discard the filtrate. Add 400μL RNA wash Buffer I to the adsorption column, centrifuge at 10,000g for 1 minute, and discard the filtrate and collection tube. Place the adsorption column in a new collection tube, add 500μL RNA wash Buffer II to the adsorption column, centrifuge at 10,000g for 1 minute, and discard the filtrate. Repeat this process twice, then centrifuge the empty column at 10,000g for 1 minute. After the adsorption column is dried at room temperature, elute the total RNA with 50μL DEPC water. The integrity of the total RNA was checked by 1.0% non-denaturing agarose gel electrophoresis, and the OD was measured using a UV spectrophotometer. 260 / OD 280 ratio and RNA concentration. TMcDNA was synthesized using the RACE cDNA amplification kit (Clontech, USA).

[0073] 2. RT-PCR amplification of target gene fragments

[0074] Based on the transcriptome information of Aspergillus niger, specific primers for gene FD2 were designed. PCR amplification was performed using KOD DNA Polymerase (the specific method was the same as in Example 1) to obtain the full-length FD2 gene. The PCR product (target gene fragment) was recovered from the gel, ligated into the pEASY-blunt vector, and transformed into Trans1-T1 competent cells. Blue-white spot screening and colony PCR screening were performed, and positive clones were shaken and sent for sequencing to confirm the sequence information of gene FD2.

[0075] Example 6: Construction of pCDFDuet-1-FD2 recombinant expression vector

[0076] The EcoRV restriction site of the second multiple cloning site of pCDFDuet-1 was selected for single enzyme digestion to obtain the linear vector of pCDFDuet-1 (same as Example 3); at the same time, according to the gene sequence on both sides of the linear vector, primers containing vector homology arms were designed, and homology arms were introduced on both sides of the target gene FD2 by PCR technology (the specific method is the same as Example 1), and the PCR product was recovered by gel. The target gene was connected to the linearized pCDFDuet-1 vector by homologous recombination, and transformed into Trans1-T1 competent cells for colony PCR screening. The positive clones were sent for sequencing to confirm the sequence information of the candidate gene. The transformants with correct sequences were extracted to obtain the recombinant plasmid pCDFDuet-1-FD2.

[0077] Example 7: Bibenzyl skeleton synthesis engineering bacteria BL21DE3:

[0078] Construction of pCDFDuet-1-DoBBS8-pETDuet-1-MatB-MatC-pACYCDuet-1-At4CL1

[0079] The recombinant plasmids pCDFDuet-1-DoBBS8, pETDuet-1-MatB-MatC, and pACYCDuet-1-At4CL1 in Example 3-5 were co-transformed into Escherichia coli BL21DE3 competent cells and screened by drop PCR. The positive transformants, i.e., the engineered bacteria containing the above three recombinant plasmids, were named S01.

[0080] Example 8: Construction of prenylated engineered bacteria BL21DE3:pCDFDuet-1-FD2-pXL13-pXL17

[0081] The three recombinant plasmids pCDFDuet-1-FD2, pXL13, and pXL17 (Wang J, Li S, Xiong Z, et al. Pathwaymining-based integration of critical enzyme parts for de novo biosynthesis of steviolglycosides sweetener in Escherichia coli. Cell Research, 2016, 26(2): 258-261.) were simultaneously transformed into Escherichia coli BL21DE3 competent cells. By screening positive transformants, an engineered bacterium carrying all three recombinant plasmids was obtained and named S02.

[0082] Example 9: Synthesis of Bibenzyl Skeleton Compound

[0083] 1) Inoculate the engineered bacteria S01 with the correct sequence verified by sequencing into LB medium containing ampicillin (pETDuet-1 vector carries resistance), chloramphenicol (pACYCDuet-1 vector carries resistance), and streptomycin (pCDFDuet-1 vector carries resistance), and culture at 37°C, 200 rpm, and shake for 12 hours to obtain seed solution;

[0084] 2) Inoculate the activated seed liquid into fresh LB medium containing the same concentration of antibiotics (use 500mL triangular flasks, each bottle contains 200mL of liquid) until the bacterial liquid concentration reaches OD 600 =0.1;

[0085] 3) Cultivate at 37°C, 200 rpm until OD 600 =0.6, add IPTG to each flask at a final concentration of 0.15 mM; induce the expression of the target protein at 16°C and 200 rpm;

[0086] 4) After 16 h of induction culture, cells were collected and centrifuged at 5,000 rpm for 5 min. The supernatant was discarded and the cells were washed sequentially with ddH2O and M9 medium (containing 1× M9 salts, 2.0 mM MgSO4, 0.1 mM CaCl2, and 20 g / L glucose) to remove residual LB medium and ddH2O.

[0087] 5) Use M9 medium to suspend the bacteria and adjust the concentration of the bacterial solution to OD 600 =5.0, use a 250 mL Erlenmeyer flask, with the volume per bottle not exceeding 100 mL (if a 500 mL Erlenmeyer flask is used, the volume per bottle should not exceed 200 mL), add the substrate malonate and phenylpropionic acid compounds to final concentrations of 4.5 mM and 1.5 mM, respectively; culture at 30°C, 200 rpm, and shake for 24 h;

[0088] 6) Centrifuge at 5,000 rpm for 10 min, collect the supernatant, and pass it through a macroporous resin column chromatography. After eluting with water to remove highly polar impurities in the supernatant, the target fraction was eluted with 80% ethanol aqueous solution. After vacuum concentration and evaporation to dryness, it was reconstituted with methanol and centrifuged at high speed to prepare the bibenzyl product using semi-preparative HPLC (chromatographic column: Shiseido capcell pak C 18 column (250 mm × 10 mm ID, Shiseido Co., Ltd., Tokyo, Japan); mobile phase: acetonitrile (B) / pure water (A), gradient elution: 30%-45% B, 15 min; 45-100% B, 5 min; 100% B, 5 min, flow rate: 3 mL / min; column temperature: 30°C; λ max =280nm. ).

[0089] When 4-hydroxyphenylpropionic acid was used as the phenylpropionic acid substrate, the yield of the obtained bibenzyl compound was 190 mg / L. Figure 2 、 4 , 5) and other identifications as dihydro-resveratrol (1), and the spectral data are as follows:

[0090] Dihydro-resveratrol (1): C 14 H 14 O3:ESI-MS m / z 231.05[M+H] + ; 1 H NMR (DMSO-d6, 400 MHz): 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-d6, 100 MHz): 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′).;

[0091] When 3,4-dihydroxyphenylpropionic acid was used as the phenylpropionic acid substrate, the obtained bibenzyl compound was characterized by MS and NMR (Attached Figure 2 、 6, 7) and other identifications showed that the structure was 3,3′,4′,5-tetrahydroxybibenzyl (2). The spectral data were as follows:

[0092] 3,3′,4′,5-Tetrahydroxybibenzyl(2):C 14 H 14 O4,ESI-MS m / z 290.91[M+HCOOH-H] - ; 1 H NMR (DMSO-d6, 400 MHz): 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-d6, 100 MHz): 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′).

[0093] When 3-hydroxyphenylpropionic acid was used as the phenylpropionic acid substrate, the obtained bibenzyl compound was characterized by MS and NMR (see Figure 2 、 8 , 9) and other identifications showed that the structure was 3,3′,5-trihydroxybibenzyl (3), and the spectral data were as follows:

[0094] 3,3′,5-Trihydroxybibenzyl(3):C 14 H 14 O3,ESI-MS m / z 231.06[M+H] + ; 1 H NMR (DMSO-d6, 400 MHz): 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-d6, 100 MHz): 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′).

[0095] Example 10: Synthesis of isopentenylbibenzyl derivatives

[0096] 1) The engineered bacteria S01 verified to be correct by sequencing was inoculated into LB medium containing ampicillin (resistance carried by the pETDuet-1 vector), chloramphenicol (resistance carried by the pACYCDuet-1 vector), and streptomycin (resistance carried by the pCDFDuet-1 vector); the engineered bacteria S02 verified to be correct by sequencing was inoculated into LB medium containing kanamycin (resistance carried by the pXL13 recombinant plasmid), ampicillin (resistance carried by the pXL17 recombinant plasmid), and streptomycin (resistance carried by the pCDFDuet-1 vector), and cultured at 37°C and 200 rpm for 12 h to obtain seed solution;

[0097] 2) Inoculate the activated seed liquid into fresh LB medium containing the same concentration of antibiotics (use 500mL triangular flasks, each bottle contains 200mL of liquid) until the bacterial liquid concentration reaches OD 600 =0.1;

[0098] 3) Cultivate at 37°C, 200 rpm until OD 600 =0.6, add IPTG to each flask at a final concentration of 0.15 mM; induce the expression of the target protein at 16°C and 200 rpm;

[0099] 4) After 16 h of induction culture, cells were collected and centrifuged at 5,000 rpm for 5 min. The supernatant was discarded and the cells were washed once with ddH2O and once with M9 medium (containing 1× M9 salts, 2.0 mM MgSO4, 0.1 mM CaCl2, and 20 g / L glucose) to remove residual LB medium and ddH2O.

[0100] 5) Use M9 medium to suspend the bacteria and adjust the concentration of the bacterial solution to OD 600 =5.0;

[0101] 6) Equal volumes of the bacterial cultures of S01 and S02 were mixed and placed in 250 mL Erlenmeyer flasks, with no more than 100 mL of solution per flask. The substrates malonic acid and phenylpropionic acid compounds were added to final concentrations of 2.25 mM and 0.75 mM, respectively. The culture was shaken at 30° C. and 200 rpm for 24 h. The bacterial culture post-treatment steps were the same as in Example 9, and the corresponding products were prepared by semi-preparative HPLC (Shiseido capcellpak C18 column (250 mm×10 mm ID, Shiseido Co., Ltd., Tokyo, Japan); the mobile phase was acetonitrile (B) / purified water (A), gradient elution: 15%-40% B, 20 min; 40-100% B, 5 min; 100% B, 5 min, flow rate: 3 mL / min; column temperature: 30° C.; λ max =280nm).

[0102] When 4-hydroxyphenylpropionic acid was used as the phenylpropionic acid substrate, the obtained bibenzyl compound was characterized by MS and NMR (see Figure 3 、 10 , 11) and other identifications of the structure as 2-isopentenyl-3,4′,5-trihydroxybibenzyl (4). The spectral data are as follows:

[0103] 2-Isopentenyl-3,4',5-trihydroxybibenzyl (4): C 19 H 22 O3,ESI-MS m / z 298.99[M+H] + ; 1 HNMR: (DMSO-d6, 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-d6, 100 MHz): δ C156.7(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).

[0104] When 3,4-dihydroxyphenylpropionic acid was used as the phenylpropionic acid substrate, the obtained bibenzyl compound was characterized by MS and NMR (Attached Figure 3 、 12 , 13) and other methods to identify the structure as 2-isopentenyl-3,3′,4′,5-tetrahydroxybibenzyl (5). The spectral data are as follows:

[0105] 2-Isopentenyl-3,3′,4′,5-tetrahydroxybibenzyl (5): C 19 H 22 O4,ESI-MS m / z 313.17[MH] - ; 1 HNMR (DMSO-d6, 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-d6, 100 MHz): 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).

[0106] When 3-hydroxyphenylpropionic acid was used as the phenylpropionic acid substrate, the obtained bibenzyl compound was characterized by MS and NMR (see Figure 3 、 14 , 15) and other methods to identify the structure as 2-isopentenyl-3,3',5-trihydroxybibenzyl (6). The spectral data are as follows:

[0107] 2-Isopentenyl-3,3′,5-trihydroxybibenzyl (6): C 19 H 22 O4,HRESIMS:m / z 297.1492[MH] - (calcd for C 19 H 21 O3 297.1491); ESI-MS m / z 299.02[M+H] + ; 1 H NMR (DMSO-d6, 400 MHz): 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-d6, 100 MHz): 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).

[0108] Example 11: Recycling of the Bibenzyl Isopentyl Derivative Synthesis System

[0109] Under a sterile environment, the bacterial liquid after one co-culture (see Example 10) was centrifuged (5,000 g, 6 min) to separate the supernatant and the bacterial cells. The supernatant was used to separate and enrich the target product, and the bacterial cells were resuspended in 1×M9 medium to a uniform state and then adjusted to the same volume as the first co-culture. The same amount of malonic acid and 3,4-dihydroxyphenylpropionic acid S2 were added, mixed well, and cultured at 30°C and 200 rpm for 24 h. The above operation was then repeated up to 5 times. After each cycle, 400 μL of the sample was taken, 800 μL of cold methanol was added, the mixture was vortexed and centrifuged at 15,000 g for 30 min, and the supernatant was analyzed by HPLC-UV with an injection volume of 60 μL. The liquid chromatography conditions were as follows: chromatographic column Shiseido capcellpak C18 MG III column (250 mm × 4.6 mm, ID, 5 μm, Shiseido Co., Ltd., Japan); mobile phase A (0.1% formic acid in water) / B (acetonitrile), gradient elution: 15-40% B, 15 min; 40-100% B, 10 min; 100% B, 15 min; flow rate: 1.0 mL / min; column temperature: 30°C; λ max =280nm. Each cycle was repeated three times.

[0110] The results are as attached Figure 16 As shown, the yield of the target product 5 in the first cycle is 33 mg / L, and the yield of the target product 5 in each subsequent cycle is 64%, 33%, 21%, and 17% of the first cycle, respectively. This indicates that the modular biosynthetic system for the synthesis of bibenzyl isopentenyl derivatives has the ability to recycle the production of the target product. By recycling the synthesis system, efficient and sustainable production of the target bibenzyl compound can be achieved.

[0111] Example 12: Protective effect of compound 5 on glutamate-induced neuronal damage

[0112] Using the MTT method, neuroblastoma SK-N-SH cell line was plated at a density of 7.5×10 4After the cells were completely adhered, compound 5 and the positive drug resveratrol (final concentration of 10 μM) were added, and the compounds to be tested were incubated with the cells for 4 hours. The medium was changed and the culture medium in the 96-well plate was discarded. Complete culture medium was added to the control group (100 μL per well); 29 mM GLU damaging agent was added to the model group, positive drug group, and test group (90 μL per well); 10 μL of 100 μM resveratrol (final concentration of 10 μM) was added to the positive drug group, and 10 μL of 100 μM compound 5 was added to the test group. The cells were damaged by glutamate for 4 hours; MTT was added and the reaction continued for 4 hours. The cell absorbance was measured at 570 nm. Each reaction was measured three times, and the average of the three measurements was taken to calculate the relative blank survival rate of each well and the enhanced survival rate of each well relative to the model.

[0113] The results are shown in Table 1. Compound 5 can, to a certain extent, counteract the neuronal damage induced by glutamate and increase the cell survival rate by 15.32%, indicating that it has potential neuroprotective effects.

[0114] Table 1 Effect of compound 5 on cell survival rate of glutamate-induced neuronal damage (n=3)

[0115]

[0116] Example 13: Effect of Compound 5 on the Ability of Mice to Tolerate Hypoxia under Hypoxic Conditions

[0117] Male ICR mice were randomly divided into groups. The positive control group received atenolol (50 mg / kg) by gavage 30 minutes after the experiment. The control group and the experimental groups with different concentrations (10 mg / kg and 50 mg / kg) received intraperitoneal injection 15 minutes after the experiment. After dosing, mice were placed in a wide-mouth bottle (one per bottle) pre-filled with 10 g of soda lime. The bottle cap was evenly coated with petroleum jelly and sealed with sealing film to ensure airtightness. The mice were immediately measured, using respiratory arrest as the indicator. The timer recorded the total survival time of the mice until they died due to hypoxia. Data were analyzed using Graph Prism 8.0.1. All results are expressed as mean ± SEM. Statistical analysis was performed using a one-way ANOVA test to compare differences between groups. P < 0.05 was considered significant.

[0118] Table 2 Effect of compound 5 on the survival time of mice under hypoxic conditions

[0119]

[0120] *P<0.05 vs control group, **P<0.01 vs control group, n=10.

[0121] The results are shown in Table 2. Compound 5 at doses of 10 mg / kg and 50 mg / kg increased the survival time of ICR mice in the hypoxia tolerance experiment in a dose-dependent manner, and was superior to the positive drug group, suggesting that compound 5 can increase the survival time of animals in a confined space, indicating that it has a certain anti-hypoxia effect.

[0122] Example 14: Effect of Compound 5 on the Ability of Mice to Tolerate Hypoxia under Acute Hypoxic Conditions

[0123] Male ICR mice were randomly divided into groups. The positive drug group was given nimodipine (120 mg / kg) orally, and the experiment was conducted 1 hour after oral administration. The control group and the experimental groups with different concentrations (10 mg / kg and 50 mg / kg) were intraperitoneally injected 30 minutes after the experiment. After waiting for the specific experimental time described above, the mouse heads were cut off with surgical scissors. A timer was used to record the mouse survival time and the number of times the mouse opened its mouth. The data processing process was the same as in Example 13.

[0124] Table 3 Effect of compound 5 on the survival time of mice under acute hypoxia

[0125]

[0126]

[0127] *P<0.05 vs control group, **P<0.01 vs control group, n=10.

[0128] Table 4 Effects of compound 5 on respiratory rate of mice under acute hypoxia

[0129]

[0130] *P<0.05vs control group, n=10.

[0131] As shown in Tables 3 and 4, Compound 5 significantly increased survival time in an acute hypoxia tolerance test in ICR mice at doses of 10 mg / kg and 50 mg / kg, and significantly increased respiratory rate in an acute hypoxia tolerance test in ICR mice at a dose of 10 mg / kg. These results suggest that Compound 5 can increase survival time and respiratory rate after decapitation, indicating a certain anti-hypoxic effect.

[0132] Example 15: Analgesic effect of compound 5 on mice

[0133] Male ICR mice were randomly divided into groups and administered with corresponding concentrations of compound 5 in sodium carboxymethylcellulose solution or a solvent control 1 hour before writhing test. One hour later, 0.6% acetic acid was administered, and the number of writhing events was recorded. Data were analyzed using GraphPrism 8.0.1, and all results are presented as means ± SEM. Statistical analysis was performed using the t-test to compare differences between groups. P < 0.05 was considered significant.

[0134] Table 5 Effects of compound 5 on the number of writhing times in the acetic acid writhing model in mice

[0135]

[0136] *P<0.05vs control group, n=15.

[0137] The results are shown in Table 5. Compound 5 at a dose of 10 mg / kg significantly reduced the number of writhings in the acetic acid writhing test in ICR mice, indicating that Compound 5 has a certain analgesic effect in the acetic acid writhing model in ICR mice.

[0138] Example 16: Effects of Compound 5 on Permanent Focal Cerebral Ischemia in Rats

[0139] After the permanent focal cerebral ischemia (pMCAO) model was established in male SD rats, the rats were kept warm (34-35°C) for 2 hours. Compound 5 (at different doses) and the control group were intraperitoneally administered 5 minutes after cerebral ischemia. The room temperature was maintained at 24-25°C throughout the entire process.

[0140] After 24 hours of ischemia, perform behavioral assessments of the animals. For details, see Pharmacological Experimental Methods (Edited by Xu Shuyun, 3rd edition, pp. 1066-1067). Lift the rat's tail approximately 1 foot off the ground and observe the condition of its forelimbs. Place the rat on a level surface and push on both shoulders to observe any difference in resistance. Observe the rat's gait while it remains on the ground.

[0141] After 24 hours of ischemia, rats were immediately decapitated and their brains removed. The olfactory tracts, cerebellum, and lower brainstem were removed and the brains were cut coronally into six slices (2 mm / slice for the first to fifth slices, 4 mm for the sixth slice). The slices were quickly placed in 5 mL of a solution containing 1.5 mL of 4% TTC and 0.1 mL of 1 M K2HPO4 and stained (37°C, protected from light) for 20-30 minutes, with the slices stirred every 5 minutes. After TTC staining, normal tissue stains dark red, while infarcted tissue appears white. Each group of brain slices was neatly arranged, photographed, and stored. Using the image analysis system Photoshop, the infarct area of ​​each slice was calculated and statistically analyzed, and finally the infarct volume was converted by superposition.

[0142] To eliminate the influence of cerebral edema, the percentage of infarct volume was calculated using the following formula:

[0143] Cerebral infarction volume (%) = (volume of the unoperated hemisphere - volume of the uninjured hemisphere on the operated side) / volume of the unoperated hemisphere × 100%

[0144] Data were analyzed using Graph Prism 8.0.1, and all results are expressed as means ± SEM. Student's t-test was used to compare differences among groups, with P < 0.05 considered significant.

[0145] Table 6. Effects of Compound 5 on cerebral infarction volume after pMCAO in SD rats

[0146]

[0147] **P<0.01 vs control group, n=8-9.

[0148] Table 7. Effects of Compound 5 on behavioral deficits after pMCAO in SD rats

[0149]

[0150] *P<0.05 vs control group, n=8-9.

[0151] The results, as shown in Tables 6 and 7, show that in the SD rat model of permanent focal cerebral ischemia, Compound 5 at a dose of 30 mg / kg significantly reduced cerebral infarct volume and neurobehavioral scores 24 hours after pMCAO in SD rats. These results suggest that Compound 5 has a therapeutic effect on permanent cerebral ischemia in SD rats.

[0152] Example 17. Antioxidant effect of compound 5

[0153] 17.1. DPPH Free Radical Scavenging

[0154] Compound 5 solutions with different concentrations (5 μM, 10 μM, 20 μM, 40 μM) were mixed with 1×10 -4 100 μL of each 10 mol / L DPPH· solution was sequentially added to a 96-well plate as the test solution, with the antioxidant vitamin C serving as a positive control. Controls were prepared with the test solution of each concentration without DPPH· (100 μL of anhydrous ethanol was substituted for DPPH·) to eliminate interference from the test sample's inherent color. A negative control group (100 μL of anhydrous ethanol was substituted for the test sample) was also established. Three replicate wells were set up for each group. The 96-well plate was shaken in a microplate reader for 1 minute and incubated in the dark at room temperature for 30 minutes. The absorbance was measured at 517 nm and the free radical scavenging rate was calculated. GraphPad 8.0.1 software was used for statistical analysis, and the results are expressed as mean ± standard deviation.

[0155] Table 8. Scavenging rate of compound 5 for DPPH·

[0156]

[0157] The results are shown in Table 8. As the drug concentration increases, the DPPH scavenging ability of compound 5 and the positive drug increases. Among them, the scavenging ability of compound 5 at the first three concentrations (5μM, 10μM, 20μM) of this experiment is much stronger than that of vitamin C. In addition, at different concentrations of this experiment, the EC 50 The value was 11.53 μM, and the EC of compound 5 50 The value was 6.12 μM.

[0158] 17.2 Pyrogallol Auto-Oxidation Method

[0159] 180 μL of 0.05 M Tris-HCl buffer (pH 8.2) was added to a 96-well plate. Then, 40 μL of test sample solution at various concentrations and 16 μL of 9 mM pyrogallol solution were added and mixed thoroughly. After 5 minutes, the reaction was terminated by adding 8 μL of 3 M HCl. The antioxidant vitamin C was used as a positive control. The model group replaced the sample solution with 40 μL of water; the blank control group replaced the pyrogallol and sample solution with 56 μL of water. Additionally, 40 μL of the sample solution was added to 180 μL of 0.05 M Tris-HCl buffer (pH 8.2), mixed thoroughly, and then 16 μL of water was added to serve as a reference control. Three replicate wells were set up in each group. Absorbance was measured at 299 nm, and free radical scavenging rates were calculated. Data were analyzed using GraphPad 8.0.1 software, and results are expressed as mean ± standard deviation.

[0160] Table 9 Different compounds for Clearance rate

[0161]

[0162] The results are shown in Table 9. As the drug concentration increases, compound 5 and positive drug clearance At the four concentrations in the experiment (0.1mM, 0.2mM, 0.4mM, 0.8mM), the EC 50 The value was 0.33 mM, while the EC 50 Values ​​within the test concentration range were not calculated.

[0163] 17.3. Feton reaction

[0164] 30 μL of 0.75 mM o-phenanthroline solution was added to a 96-well plate, followed by 60 μL of PBS (pH 7.4) and 30 μL of the test sample solution. After thorough mixing, 30 μL of 0.75 mM ferrous sulfate solution was added. After mixing, 30 μL of 1% H₂O₂ solution was added. The mixed solution was placed in a 37°C water bath for 60 min. This was used as the test sample group, and vitamin C was used as a positive control. The above procedure was repeated with 30 μL of distilled water replacing 1% H₂O₂ as the control group. The above procedure was repeated with 30 μL of distilled water replacing 30 μL of the test sample solution as the model group. The above procedure was repeated with only PBS solution and the test sample solution, with all other reagents replaced with distilled water, as the reference control group. The above procedure was repeated with only PBS solution and all other reagents replaced with distilled water, as the blank control group. Three replicate wells were set up for each group. The absorbance of each group was measured at a wavelength of 536 nm, and the free radical scavenging rate was calculated. The data were statistically analyzed using GraphPad 8.0.1 software, and the results were expressed as mean ± standard deviation.

[0165] Table 10 Scavenging rate of ·OH by different compounds

[0166]

[0167] The results are shown in Table 10. As the drug concentration increases, the ability of compound 5 to scavenge ·OH gradually increases, and the ability to scavenge ·OH at different concentrations in this experiment is significantly stronger than that of the positive control drug vitamin C. In addition, at the four concentrations in the experiment (0.1mM, 0.2mM, 0.4mM, 0.8mM), the EC 50 The value was 0.46 mM, while the EC of vitamin C was not calculated within the experimental concentration range. 50 value.

[0168] 17.4. Oxygen Radical Scavenging Capacity Assay (ORAC Assay)

[0169] 20 μL of test solution at different concentrations was added to a 96-well fluorescent plate. 20 μL of 75 mM potassium phosphate buffer and 140 μL of 18.3 mM AAPH were then added. After thorough mixing, 20 μL of 630 nM sodium fluorescein was added to initiate the reaction. Fluorescence intensity was measured continuously at 37°C with an excitation wavelength of 485 nm and an emission wavelength of 538 nm. The fluorescence intensity of each well was measured every 2 minutes until the fluorescence intensity decayed to baseline. Vitamin C was used as a positive control, and three replicate wells were set up in parallel for each group. A standard curve was drawn based on the Net AUC (i.e., the integrated area under the fluorescence decay curve minus the area under the blank curve without antioxidant) of Trolox at different concentrations. The ORAC value of each sample was calculated. The ORAC value of the test sample was expressed in μmol TE / g. Statistical analysis of the data was performed using GraphPad 8.0.1 software, and the results are expressed as mean ± standard deviation.

[0170] Table 11 Net AUC values ​​of different compounds

[0171]

[0172] The results are shown in Table 11. At the same concentration, the area under the fluorescence decay curve of compound 5 was significantly greater than that of vitamin C. In addition, the ORAC value of compound 5 was (9514.0±1160.0) μmol / g, and the ORAC value of vitamin C was (2607.0±594.9) μmol / g, indicating that compound 5 has a stronger antioxidant capacity than vitamin C. Sequence Listing <110> Institute of Materia Medica, Chinese Academy of Medical Sciences <120> An isopentenyl bibenzyl derivative, preparation method and use thereof <160> 12 <170> SIPOSequenceListing 1.0 <210> 1 <211> 390 <212> PRT <213> Dendrobium officinale <400> 1 Met Pro Ser Leu Glu Ser Ile Lys Lys Ala Pro Arg Ala Asp Gly Phe 1 5 10 15 Ala Ser Ile Leu Ala Ile Gly Arg Ala Asn Pro Glu Asn Phe Ile Glu 20 25 30 Gln Ser Ala Tyr Pro Asp Leu Phe Phe Arg Ile Thr Asn Ser Glu His 35 40 45 Leu Val Asp Leu Lys Asn Lys Phe Lys Arg Ile Cys Asp Lys Thr Ala 50 55 60 Ile Arg Lys Arg His Phe Val Trp Thr Glu Glu Phe Ile Thr Ala Asn 65 70 75 80 Pro Cys Phe Ser Thr Phe Met Asp Lys Ser Leu Asn Ile Arg Gln Glu 85 90 95 Val Ala Ile Arg Glu Ile Pro Lys Leu Gly Ala Glu Ala Ala Thr Lys 100 105 110 Ala Ile Gln Glu Trp Gly Gln Pro Lys Ser Arg Ile Thr His Leu Ile 115 120 125 Phe Cys Thr Thr Ser Gly Met Asp Leu Pro Gly Ala Asp Tyr Gln Leu 130 135 140 Thr Gln Ile Leu Gly Leu Asn Pro Asn Val Glu Arg Val Met Leu Tyr 145 150 155 160 Gln Gln Gly Cys Phe Ala Gly Gly Thr Thr Ile Arg Leu Ala Lys Cys 165 170 175 Leu Ala Glu Ser Arg Lys Gly Ala Arg Val Leu Val Val Cys Ala Glu 180 185 190 Thr Thr Thr Val Leu Phe Arg Gly Pro Ser Glu Glu His Gln Asp Asp 195 200 205 Leu Val Thr Gln Ala Leu Phe Ala Asp Gly Ala Ser Ala Leu Ile Val 210 215 220 Gly Ala Asp Pro Asp Glu Ala Ala Asp Glu His Ala Ser Phe Val Ile 225 230 235 240 Val Ser Thr Ser Gln Val Leu Leu Pro Asp Ser Ala Gly Ala Ile Gly 245 250 255 Gly His Val Ser Glu Gly Gly Leu Leu Ala Thr Leu His Arg Asp Val 260 265 270 Pro Gln Ile Val Ser Lys Asn Val Gly Lys Cys Leu Glu Glu Ala Phe 275 280 285 Thr Pro Leu Gly Ile Ser Asp Trp Asn Ser Ile Phe Trp Val Pro His 290 295 300 Pro Gly Gly Arg Ala Ile Leu Asp Gln Val Glu Glu Ser Val Gly Leu 305 310 315 320 Lys Pro Glu Lys Leu Phe Ile Ser Arg His Val Leu Ala Glu Tyr Gly 325 330 335 Asn Met Ser Ser Val Cys Val His Phe Ala Leu Asp Glu Met Arg Lys 340 345 350 Arg Ser Ala Lys Glu Gly Lys Ala Thr Thr Gly Glu Gly Leu Glu Trp 355 360 365 Gly Val Leu Phe Gly Phe Gly Pro Gly Val Thr Val Glu Thr Val Ile 370 375 380 Leu Arg Ser Val Pro Ile 385 390 <210> 2 <211> 1173 <212> DNA <213> Dendrobium officinale <400> 2 atgccgagcc ttgaatccat caagaaggca ccaagagccg acggcttcgc ctccatcttg 60 gccatcggga gggcgaatcc tgaaaacttc attgaacaga gcgcttaccc agacttgttt 120 ttccgtatca ccaacagcga gcacttggtc gacctcaaaa ataaattcaa acgcatatgt 180 gataaaacag caattagaaa gcgccatttt gtttggaccg aggagtttat cactgcaaac 240 ccttgcttca gcactttcat ggataaatct ttgaatataa ggcaagaggt cgcaataagg 300 gagataccga agctcggcgc ggaggcagca accaaggcaa ttcaggagtg ggggcagcct 360 aagtcccgca tcactcacct catattctgc accacgagcg gcatggactt acctggcgct 420 gactatcagc taactcaaat tcttggcctt aacccaaatg tcgagcgtgt catgctctat 480 cagcagggtt gtttcgctgg cggaaccacg atccgtctcg ctaagtgcct tgccgagagc 540 cgcaagggcg cacgcgttct tgtggtttgt gcggagacca ccactgtgct atttcgtgga 600 ccgtctgagg agcaccagga tgaccttgtt actcaagctt tatttgctga tggtgcatct 660 gcgcttatag tgggtgccga tccagatgag gcggctgacg agcacgccag cttcgtcata 720 gtctctacat ctcaagtctt attgccggac tctgcaggtg ctattggagg ccatgtaagt 780 gagggaggtc tcttagccac gcttcataga gatgtcccgc aaattgtttc caaaaatgtt 840 gggaagtgtt tggaagaggc attcacccca cttggtattt cggattggaa ctcaatcttc 900 tgggttccgc atccaggagg tcgagccatt cttgatcagg tggaggagag tgtggggcta 960 aagccagaga agctgtttat ttcaaggcat gtgcttgcag agtacggtaa tatgtcaagt 1020 gtctgtgtgc actttgctct tgatgaaatg cgcaaaaggt ctgcgaaaga aggcaaggct 1080 acaacaggcg aaggccttga gtggggtgtg ctttttggct tcgggccggg cgttaccgtt 1140 gaaaccgtca tcctccgcag tgttccaatt tga 1173 <210> 3 <211> twenty one <212> DNA <213> Forward primer-DoBBS8-F (Dendrobium officinale) <400> 3 atgccgagcc ttgaatccat c 21 <210> 4 <211> twenty one <212> DNA <213> Reverse primer-DoBBS8-R (Dendrobium officinale) <400> 4 ttaaattgga acactgcgga g 21 <210> 5 <211> 33 <212> DNA <213> Forward primer with homology arm-DoBBS8-Fe (Dendrobium officinale) <400> 5 gcagatctca attggatgcc gagccttgaa tcc 33 <210> 6 <211> 30 <212> DNA <213> Reverse primer with homology arm - DoBBS8-Re (Dendrobium officinale) <400> 6 atcgcgtggc cggccttaaa ttggaacact 30 <210> 7 <211> 448 <212> PRT <213> Periconia sp. F-31 <400> 7 Met Ser His Thr Val Val Lys Thr Ala Ser Asn Lys Ala Asn Ser Gln 1 5 10 15 Asp Cys Thr Ala Thr Ala Ala Val Met Asn Glu Ile Asp Lys Glu Phe 20 25 30 Gln Ala Asn Ser Glu Asp Asp Ala Phe Trp Trp Ser Ala Ser Gly Gln 35 40 45 Pro Leu Cys Thr Leu Leu Gln Gln Asn Gln Tyr Ser His Asp Arg Gln 50 55 60 Leu Tyr Leu Leu Arg Trp Phe Arg Gln Arg Val Leu Pro Ser Leu Gly 65 70 75 80 Pro Arg Pro Ser Gly Thr Lys Pro Tyr Tyr Gly Ser Trp Leu Thr Tyr 85 90 95 Asp Gly Ser Pro Leu Glu Tyr Ser Leu Asn Trp Lys Glu Lys Lys Pro 100 105 110 Asn Gln Thr Ile Arg Phe Thr Ile Glu Pro Thr Ser Ser Lys Ala Gly 115 120 125 Thr Ala Ala Asp Arg Leu Asn Gln Leu Gly Ala Lys Glu Leu Leu Thr 130 135 140 Thr Leu Ser Lys Glu Ile Pro Ser Ile Asp Leu Lys Arg Phe Asn Leu 145 150 155 160 Phe Leu Glu Asp Thr Tyr Val Pro Asp Asp Ala Ile Glu Glu Val Ile 165 170 175 Ser Lys His Pro Ala Gly Phe Pro Gln Ser Arg Val Trp Val Ala Phe 180 185 190 Asp Leu Glu Arg Ser Gly Asp Ile Val Ala Lys Ala Tyr Phe Leu Pro 195 200 205 His Trp Arg Glu Ile Tyr Thr Gly Thr Pro Thr Lys Thr Ile Val Phe 210 215 220 Asp Ala Ile Lys Lys Cys Asn Gly Pro Leu Gly Ser Tyr Asp Ala Ser 225 230 235 240 Ile Ala Ala Leu Asp Gly Tyr Leu Glu Ser Phe Pro Ser Glu Glu Ala 245 250 255 Pro Lys Ile Val Leu Leu Ser Asn Asp Cys Val Ala Asp Ser Pro Ala 260 265 270 Ala Arg Met Lys Val Tyr Leu His Thr Ser Val Asp Thr Leu Ala Lys 275 280 285 Ala Lys Asp Met Phe His Leu Gly Gly Arg Leu Ser Gly Thr Ala Ile 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 With Gly Lys Thr Asp With Glu Serving Glu Leu Leu Serving 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> Periconia sp. F-31 <400> 8 atgtctcaca ccgtcgtca aacagcctca aaaaggcaa 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 FD2-Fe with homology arms (Periconia sp. F-31) <400> 11 ggcagatctc aattggatgt ctcacaccgt cg 32 <210> 12 <211> 32 <212> DNA <213> Reverse primer FD2-Re with homology arm (Periconia sp. F-31) <400> 12 cgtggccggc cgatatctag ttggggagat ag 32

Claims

1. A method for preparing an isopentenyl bibenzyl derivative having the structure shown in General Formula 1, wherein the substituents R1 and R2 are each independently selected from hydrogen and hydroxyl, and characterized in that It is prepared by engineering microbial "cell factories", and the specific steps are as follows: a. Induce the expression of the benzyl skeleton synthesis engineering microorganism containing the dibenzyl synthase gene DoBBS8 and the prenylation engineering microorganism containing the prenyltransferase gene FD2 respectively to obtain the recombinant dibenzyl synthase DoBBS8 and prenyltransferase FD2; b. Centrifuge and collect the above-mentioned benzyl skeleton synthesis engineering microorganism and prenylation engineering microorganism respectively, wash and resuspend them successively with sterile water and 1×M9 medium, adjust the cells to an appropriate concentration with 1×M9 medium and mix them, add an appropriate amount of compounds, and react at 30 °C and 200 rpm for 24 h; the compounds include malonic acid and phenylpropionic acid, malonic acid and 4-hydroxy phenylpropionic acid, malonic acid and 3-hydroxy phenylpropionic acid, malonic acid and 3,4-dihydroxy phenylpropionic acid; c. Centrifuge and collect the supernatant, and purify the target compound by macroporous resin column and semi-preparative HPLC chromatography methods.

2. The preparation method according to claim 1, characterized in that, The recombinant dibenzyl synthase DoBBS8 is derived from Dendrobium officinale, and its amino acid sequence is as shown in SEQ ID No.1, or a sequence having more than 90% homology with the sequence shown in SEQ ID No.1 and having the same function.

3. The preparation method according to claim 1, characterized in that, The dibenzyl synthase gene DoBBS8 has a nucleic acid sequence as shown in SEQ ID No.2, or a sequence having more than 90% homology with the sequence shown in SEQ ID No.2 and having the same function.

4. 2-Isopentenyl-3,3',4',5-tetrahydroxybibenzyl with the following structural formula: wherein the substituents R1 and R2 are each a hydroxyl group, and the use of the same and its pharmaceutically acceptable salts in the preparation of neuroprotective drugs.

5. 2-Isopentenyl-3,3',4',5-tetrahydroxy bibenzyl with the following structural formula: wherein the substituents R1 and R2 are each a hydroxyl group, and the use of the same and its pharmaceutically acceptable salts in the preparation of antioxidants.

Images