A method for preparing 4-vinylphenol-1,2-diol by biotransformation of forsythoside A

Abstract

The application belongs to the technical field of natural organic chemistry, and relates to a method for preparing 4-vinylbenzene-1,2-diol by biotransformation of forsythoside A. The method comprises the following steps: inoculating a strain into PDA culture medium to activate the strain; inoculating the activated strain into PDB culture medium to culture the strain, then adding forsythoside A to the culture medium to culture again; centrifuging the culture medium, taking supernatant, adding water-saturated butanol extraction liquid, extracting several times, combining the extraction liquids, and rotary evaporating the combined extraction liquids to dryness; after redissolving the extraction liquids with a solvent, filtering the extraction liquids, separating the product by using a preparation liquid phase separation method, rotary evaporating the separated product solution to dryness, redissolving the separated product solution with a small amount of water, and then transferring the redissolved solution to a high specific surface container, precooling, and drying in a freeze dryer; and 4-vinylbenzene-1,2-diol is obtained. The application is the first to use the biotransformation technology to transform forsythoside A to prepare 4-vinylbenzene-1,2-diol. The application has the characteristics of low preparation cost, simple operation, low solvent residue, environmental friendliness and the like.

Description

Technical Field

[0001] This invention belongs to the field of natural organic chemistry technology and relates to a method for preparing 4-vinylbenzene-1,2-diol by biotransformation of forsythoside A. Technical Background

[0002] 4-Vinylbenzene-1,2-diol was first discovered in roasted coffee bean extract. This compound is one of the components that contribute to the aroma of baked goods. 4-Vinylbenzene-1,2-diol exhibits strong reactivity, possessing antioxidant properties, promoting wound healing in diabetic patients, and also acting as an antioxidant and preservative.

[0003] Forsythosides are widely distributed active and characteristic substances in the plant kingdom, and are also a major secondary metabolite in plants. The characteristic structure of these compounds is a hydroxyphenylethanol group linked to β-glucose via a glycosidic bond. Due to the polyphenolic hydroxyl structure of phenylethanol glycosides, these compounds possess antioxidant, anti-inflammatory, antibacterial, anticancer, antiviral, anti-allergic, and neuroprotective activities. Among them, forsythoside A is considered one of the main components of Forsythia due to its high content and potential biological activity.

[0004] Currently, researchers mainly use organic synthesis and organic solvent extraction methods to prepare 4-vinylbenzene-1,2-diol. The organic solvents used in these processes primarily include methanol, ethanol, and ethyl acetate, and the large quantities used result in environmentally unfriendly emissions and organic solvent pollution. Therefore, there is an urgent need to find a mild and environmentally friendly method for preparing 4-vinylbenzene-1,2-diol. Summary of the Invention

[0005] The technical problem to be solved by this invention is to address the shortcomings of the existing methods and provide a method for preparing 4-vinylbenzene-1,2-diol by biotransformation of forsythoside A. This method is convenient, rapid, green, economical, and environmentally friendly, providing a new approach for the industrial production of 4-vinylbenzene-1,2-diol.

[0006] This invention provides a method for preparing 4-vinylbenzene-1,2-diol using forsythoside A through biotransformation, comprising the following steps:

[0007] (1) Activation of bacterial strains: The bacterial strains are inoculated into PDA medium for activation;

[0008] (2) Substrate transformation: The activated strain was inoculated into PDB medium for culture, and then forsythoside A was added at a material-to-liquid ratio of 1:2000-1:20000 g / mL, and cultured again.

[0009] (3) Crude extraction of the conversion product: Centrifuge the culture medium, take the supernatant and add water-saturated n-butanol extract, the ratio of conversion liquid to extract is 1:1-1:2, extract for 0.5-1h, repeat 3 times, combine the extracts and evaporate to dryness at 40-60℃.

[0010] (4) Purification of the conversion product: After reconstitution with solvent, filter, separate the product using preparative liquid phase separation method, evaporate the separated solution to dryness, reconstitute with a small amount of water, transfer to a high specific surface area container, pre-cool and then dry in a freeze dryer;

[0011] 4-vinylbenzene-1,2-diol was obtained.

[0012] In the above-mentioned method for preparing 4-vinylbenzene-1,2-diol by biotransformation of forsythoside A, in step (1), the bacterial strain is Fusarium moniliforme CGMCC 3.4759.

[0013] In the above-mentioned method for preparing 4-vinylbenzene-1,2-diol by biotransformation of forsythoside A, in step (1), the activation is carried out in an incubator at 26-29℃ with a shaking speed of 60-110 rpm for 48-72 h.

[0014] In the above-mentioned method for preparing 4-vinylbenzene-1,2-diol by biotransformation of forsythoside A, in step (2), the culture is carried out at 26-29℃ for 24-72h with a shaking speed of 60-110rpm; the second culture is carried out at 24-31℃ for 72-192h with a shaking speed of 60-110rpm.

[0015] In the above-mentioned method for preparing 4-vinylbenzene-1,2-diol by biotransformation of forsythoside A, in step (4), the solvent is redissolved and the solvent is prepared by mixing methanol and aqueous solution in a volume ratio of (20-60):(40-80).

[0016] In the above method for preparing 4-vinylbenzene-1,2-diol using forsythoside A through biotransformation, in step (4), the chromatographic column used in the liquid phase separation method is C18. 18 The column and the mobile phase are composed of mobile phase A and mobile phase B mixed in a volume ratio of 30-80:60-20, wherein mobile phase A is an aqueous solution and mobile phase B is a methanol solution, and the separation is performed under a preparative liquid chromatography instrument in the preparative liquid chromatography purification method.

[0017] In the above-mentioned method for preparing 4-vinylbenzene-1,2-diol using biotransformation of forsythoside A, the volume ratio of mobile phase A to mobile phase B is 52:48.

[0018] The above-mentioned method for preparing 4-vinylphenyl-1,2-diol using forsythoside A through biotransformation, wherein C18 The column temperature was 30℃, and the preparative column was a ZORBAX RX-C. 18 The preparation column has a size of 5μm and 9.4×250mm.

[0019] The above-mentioned method for preparing 4-vinylbenzene-1,2-diol using biotransformation of forsythoside A, wherein the mobile phase flow rate is 1.5–2.5 mL / min and the injection volume is 60–100 μL.

[0020] The strain Fusarium moniliforme CGMCC 3.4759 was purchased from the China General Microbiological Culture Collection Center.

[0021] The present invention has the following beneficial effects:

[0022] This method combines biotransformation and preparative liquid chromatography, utilizing the fungus *Fusarium solani* CGMCC 3.4759 to efficiently and specifically transform forsythoside A. The entire transformation process is carried out in a water-based medium, avoiding excessive organic solvent loss. The transformation conditions are mild, with low energy consumption. Optimized transformation conditions further improve transformation efficiency. High-carbon-loading ZORBAX RX-C is used. 18 The preparation column can rapidly prepare 4-vinylbenzene-1,2-diol from the crude extract. The method of this invention features precise control at each stage, coordinated system control, simplicity, and environmental friendliness. Attached Figure Description

[0023] Figure 1 The image shows the liquid chromatogram of the crude extract from Example 1.

[0024] Figure 2 The liquid chromatogram is shown after the preparation of 4-vinylbenzene-1,2-diol, the conversion product of Example 1.

[0025] Figure 3 The conversion product of Example 1 1 H-NMR spectrum.

[0026] Figure 4 The conversion product of Example 1 13 C-NMR spectrum.

[0027] Figure 5 The image shows the MS spectrum of the conversion product in Example 1. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0029] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0030] Example 1

[0031] 1. Activation of bacterial strain: Sterilize 80 mL of potato dextrose agar (PDA) medium at 121 °C for 20 min. After sterilization, wait until 70 °C, then pour the agar plates in a clean bench. After cooling, streak Fusarium CGMCC 3.4759 in three zones and incubate at 27 °C for 48 h. Then, select pure bacterial strains and subculture them three times using the same method, with each subculture lasting 24 h.

[0032] 2. Substrate transformation: Under aseptic conditions, the selected fungal fragments were inoculated into PDB seed medium and cultured at 27°C and 100 rpm for 2 days. 2 mL of the bacterial culture was then inoculated into potato dextrose aqueous solution PDB liquid medium and cultured at 27°C and 100 rpm. After vigorous mycelial growth, 100 μL of forsythoside A (50 mg / mL substrate solution, dissolved in pure methanol, filtered and sterilized in a clean bench before being added to the culture medium) was added. The transformation time was 3 days, 27°C, 110 rpm, 70 mL PDB medium.

[0033] 3. Crude extraction of the transformation product: After centrifugation of the biotransformation culture medium (7000 rpm, 15 min), the supernatant was collected. The supernatant was extracted three times with the same volume of water-saturated n-butanol. The filtrates were combined and rotary evaporated at 50-60℃ until dry. The residue was dissolved in about 2 mL of methanol solution and filtered through a 0.22 μL microporous membrane into a liquid chromatography vial for preparative separation. The chromatogram for liquid chromatography analysis is shown in [Figure number missing]. Figure 1 .

[0034] 4. Purification of the conversion product: A ZORBAX RX-C18 9.4×250mm 5μm semi-preparative column was used. The mobile phase was 48% methanol-water isocratic elution at a flow rate of 2mL / min, a separation time of 13.5min, a column temperature of 30℃, an injection volume of 80μL, and a detection wavelength of 258nm. After each 5-shot separation, the system was washed with pure methanol to restore the column's separation capacity. The peak at Rt = 10.9-12.2min was collected. The separated solution was rotary evaporated to dryness at 40-60℃, reconstituted with a small amount of water, transferred to a high specific surface area container, pre-cooled at -80℃ for 15-24h, and then dried in a lyophilizer. The purity of the prepared sample reached 95%. The HPLC chromatogram is shown below. Figure 2 The 1H-NMR spectra of the metabolites are shown below. Figure 3The 1H signal δH = 6.88 (d, J = 1.20 Hz, 1H) represents the hydrogen at the 3rd carbon position of the benzene ring, and the signals 6.75–6.66 (m, 2H) represent the hydrogens at the 5th and 6th carbon positions of the benzene ring. δH = 6.55 (dd, J = 17.6, 10.9 Hz, 1H) is coupled with hydrogens at δH = 5.50 (d, J = 17.6 Hz, 1H) and δH = 4.99 (d, J = 10.9 Hz, 1H), respectively. The presence of two independent doublet hydrogen signals in the 1H spectrum confirms that forsythoside A underwent not only hydrolysis but also decarboxylation. The 13C-NMR spectrum of the metabolite is shown below. Figure 4 The 13C signals of the metabolites are δC = 145.0, 144.9, 136.6, 129.85, 118.2, 114.7, 112.0, and 109.2. Compared with the 13C signals of the caffeic acid portion of forsythoside A, δC = 145.0 and 144.9 correspond to the carbons at the hydroxyl substitution positions on the benzene ring, respectively; δC = 136.6 corresponds to the carbons on the olefin connected to the benzene ring; and δC = 129.9 corresponds to the carbons on the olefin. The carbons on the benzene ring have the following 13C signals: δC = 118.2 corresponds to the 6th carbon on the benzene ring, δC = 114.7 corresponds to the 5th carbon, δC = 112.0 corresponds to the 2nd carbon, and δC = 109.2 corresponds to the terminal carbon of the alkene. Forsythoside A, if it undergoes hydrolysis to produce caffeic acid, will contain an acrylic acid group, but no carboxyl carbon signal was found in the 13C spectrum. This confirms that forsythoside A undergoes hydrolysis and decarboxylation after transformation by *Fusarium solani* (CGMCC 3.4759). The MS spectrum of the metabolites is shown below. Figure 5 ESIMS m / z: 137.1 [M+H]+, and data comparison confirmed that the metabolite is 4-vinylbenzene-1,2-diol.

[0035] The above embodiments are preferred embodiments of the present invention, but the actual embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention are equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A method for preparing 4-vinylphenyl-1,2-diol by biotransformation of forsythoside A, characterized in that, Includes the following steps (1) Activation of strain: The strain was inoculated into PDA medium for activation; the strain was Fusarium moniliforme CGMCC3.4759; (2) Substrate transformation: The activated strain was inoculated into PDB medium for culture, and then forsythoside A was added at a material-to-liquid ratio of 1:2000-1:20000 g / mL, and cultured again; (3) Crude extraction of conversion product: Centrifuge the culture medium, take the supernatant and add water-saturated n-butanol extract, the ratio of conversion liquid to extract is 1:1-1:2, extract for 0.5-1 h, repeat 3 times, combine the extracts and evaporate to dryness at 40-60℃; (4) Purification of the conversion product: After reconstitution with solvent, filter, separate the product using preparative liquid phase separation method, evaporate the separated solution to dryness, reconstitute with a small amount of water, transfer to a high specific surface area container, pre-cool and then dry in a freeze dryer to obtain 4-vinylbenzene-1,2-diol.

2. The method for preparing 4-vinylbenzene-1,2-diol by biotransformation of forsythoside A as described in claim 1, characterized in that: In step (1), activation is carried out at 26-29 ℃ and a shaking speed of 60-110 rpm for 48-72 hours.

3. The method for preparing 4-vinylbenzene-1,2-diol by biotransformation of forsythoside A as described in claim 1, characterized in that: In step (2), the culture is carried out at 26-29 ℃ for 24-72 h with a shaking speed of 60-110 rpm; the second culture is carried out at 24-31 ℃ for 72-192 h with a shaking speed of 60-110 rpm.

4. The method for preparing 4-vinylphenyl-1,2-diol by biotransformation of forsythoside A as described in claim 1, characterized in that: In step (4), the solvent is reconstituted by mixing methanol and aqueous solution in a volume ratio of (20~60):(40~80).

5. The method for preparing 4-vinylphenyl-1,2-diol by biotransformation of forsythoside A as described in claim 1, characterized in that: In step (4), the chromatographic column for the liquid phase separation method is C10. 18 The column and the mobile phase are composed of mobile phase A and mobile phase B mixed in a volume ratio of 30-80:60-20, wherein mobile phase A is an aqueous solution and mobile phase B is a methanol solution. The preparative liquid chromatography purification method is performed under a preparative liquid chromatograph.

6. The method for preparing 4-vinylphenyl-1,2-diol by biotransformation of forsythoside A as described in claim 5, characterized in that: The volume ratio of mobile phase A to mobile phase B is 52:

48.

7. The method for preparing 4-vinylbenzene-1,2-diol by biotransformation of forsythoside A as described in claim 5, characterized in that: The C 18 The column temperature was 30℃, and the preparative column was a ZORBAX RX-C. 18 The preparation column has a size of 5μm and a diameter of 9.4×250mm.

8. The method for preparing 4-vinylbenzene-1,2-diol by biotransformation of forsythoside A as described in claim 5, characterized in that: The flow rate of the mobile phase is 1.5~2.5 mL / min, and the injection volume is 60~100 μL.

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