CN116004747A - Method for extracting 2,3-butanediol monoglucoside from Raffaelea lauricola - Google Patents

Abstract

The invention relates to a method for extracting 2,3-butanediol monoglucoside. The method uses Raffaelea lauricola (symbiotic fungus) as raw material to ferment, extract with ethyl acetate, concentrate, and pass through a reversed-phase silica gel column. and HPLC purification to extract the secondary metabolite of the strain: 2,3-butanediol monoglucoside. The method of the invention can effectively extract the pure product of the compound, has the advantages of easy large-scale production and popularization and application, and provides a reference for the application and development of 2,3-butanediol monoglucoside in functional medicines in the future.

Description

The method for extracting 2,3-butanediol monoglucoside from Raffaelea lauricola

technical field

The invention belongs to the technical field of isolating active substances from bacterial strains, in particular to a method for extracting 2,3-butanediol monoglucoside from Raffaelea lauricola .

Background technique

Alkyl glucoside is a kind of multifunctional detergent and cosmetic raw material, because of its excellent ecological and toxicological properties such as non-toxic, non-irritating, good biodegradability, sterilization, improving enzyme activity and reducing the irritation of other surfactants nature has received widespread attention. 2,3-Butanediol monoglucoside is a polyalcohol preglucoside, which has strong hydrophilicity and water holding capacity, and at the same time has no toxic and side effects, and has good applications in the chemical field and cosmetics.

Raffaelea lauricola is a highly pathogenic companion fungus carried by the ambrosia beetle ( Ambrosia beetle ) and is a pathogenic fungus that causes tree blight. Currently, there are few reports on the secondary metabolites of this genus of microorganisms. Therefore, the study extracts its metabolic The compound 2,3-butanediol monoglucoside is of great significance.

Contents of the invention

The purpose of the present invention is to provide a method for extracting 2,3-butanediol monoglucoside from Raffaelea lauricola , so as to promote its further research and development in the field of medicine.

In order to achieve the above object, the technical scheme that the present invention takes is as follows:

A method for extracting 2,3-butanediol monoglucoside from Raffaelea lauricola comprises the following steps:

1) After activating Raffaelea lauricola , take the bacterial block and inoculate it into the PDB medium, and cultivate it continuously for 5-10 days under the conditions of 140-180r/min and 20-28°C, which is the primary culture, and inoculate at 15wt%~20wt% The amount is transferred to the improved rice culture medium, and the static culture is continued for 28-40 days under the condition of 20-28° C., that is, secondary culture to obtain mycelia. 2) Dry the mycelia obtained in step 1) at room temperature, grind and pulverize, soak in 1-2 times the volume of ethyl acetate for 12-24 hours, ultrasonicate for 40-60 minutes, collect the extract, and add bacteria to the residue Ethyl acetate with 1-2 times the volume of silk, ultrasonic for 40-60 minutes, collect the extract, repeat the above steps to obtain a total of three extracts, combine and concentrate to obtain the crude extract extract.

3) Dissolve the extract in chloroform or dichloromethane, then mix the sample with reversed-phase silica gel C ₁₈ , dry-pack the column, elute with methanol:water gradient from volume ratio 1:9→9:1, and collect methanol: The water volume ratio is [1] 9:1 part of the eluate, concentrated by rotary evaporation to obtain component A, which is prepared and separated by HPLC, and washed isocratically with mobile phase volume percentage of 4% chromatographic grade methanol: 96% pure water Eluting, flow rate 2 mL/min, retention time t _R =9.6min, collecting 9.0 min-10.0 min eluate, suspension steaming to obtain white powder 2,3-butanediol monoglucoside, its structural formula is as follows:

.

Further, the improved rice medium in the step 1): 100 g of rice, 10 g of wood chips, 120 mL of pure water, and autoclave at 121°C for 20 min.

Further, the size of the inoculum block in the step 1) is 0.5×0.5 cm, and the inoculum size is 4 blocks.

Further, the ultrasonic power in step 2) is 400 W, and the frequency is 35 KHz.

Further, the reversed-phase silica gel C ₁₈ used in step 3) has a particle size of 40-60 μm and a pore size of 120 Å.

Further, the chromatographic column used in the HPLC preparation in the step 3) is a YMC C ₁₈ chromatographic column 250 mm×4.6 mm, 5 μm.

Further, the gradient elution ratio in step 3) is methanol:water volume ratio 1:9, 2:8, 3:7, 4:6,6:4, 7:3, 8:2, 9:1 .

The notable advantages of the present invention are: the raw materials of the present invention are convenient to obtain, the separation and purification are simple, and the cost is low; the prepared compound has high purity and good repeatability.

Description of drawings

Figure 1 is the ¹ H NMR spectrum (CD ₃ OD) of 2,3-butanediol monoglucoside;

Figure 2 is the ¹³ C NMR spectrum (CD ₃ OD) of 2,3-butanediol monoglucoside;

Figure 3 is the DEPT135 spectrum (CD ₃ OD) of 2,3-butanediol monoglucoside;

Figure 4 is the COZY spectrum (CD ₃ OD) of 2,3-butanediol monoglucoside;

Figure 5 is the HMBC spectrum (CD ₃ OD) of 2,3-butanediol monoglucoside;

Figure 6 is the HSQC spectrum (CD ₃ OD) of 2,3-butanediol monoglucoside.

Detailed ways

In order to make the above-mentioned features and advantages of the present invention more comprehensible, specific embodiments are given below for detailed description. The methods of the present invention are conventional methods in the art unless otherwise specified.

The strain number of Raffaelea lauricola used in the present invention is Hulcr7161.

Example 1

1) Take Raffaelea lauricola as the material, pick 4 pieces of bacteria with a size of 0.5×0.5 cm under sterile conditions and inoculate them into PDB medium (3.9 g of potato dextrose powder, 100 mL of pure water, put them into a 250 mL Erlenmeyer flask, Sterilized under high pressure at 121°C for 20 min), cultured continuously at 160 r/min and 28°C for 7 days as primary culture, and transferred to improved rice medium (rice 100 g, sawdust 10 g, add 120 mL of pure water, autoclave at 121°C, 20 min), and continue to culture at 28°C for 30 days, which is the secondary culture.

2) Collect and cultivate the mycelium for 30 days, dry it at room temperature, grind and crush it, add 1.5 times the volume of ethyl acetate to soak for 12 hours, ultrasonicate for 60 minutes, filter to obtain the extract, and spin-steam to obtain the extract, and then add the mycelium to the residue 1 times the volume of ethyl acetate, ultrasonicated for 60 minutes, the extract was collected, and the above steps were repeated again to obtain three extracts, which were combined and concentrated to obtain the crude extract extract.

3) Dissolve the extract in chloroform, mix the sample with reversed-phase C ₁₈ silica gel with a particle size of 40-60 μm, conduct a crude fractionation under reduced pressure, and start gradient elution with methanol:water from a volume ratio of 1:9 (methanol:water ratio is 1:9, 2:8, 3:7, 4:6, 6:4, 7:3, 8:2, 9:1), collected methanol: water 9:1 partial rotary evaporation to obtain the crude product.

4) The crude product was dissolved in 6% methanol/water by volume and separated by HPLC. Chromatographic column: YMC C ₁₈ column (250 mm×4.6 mm, 5 μm), mobile phase volume percentage 4% methanol: 96% pure water Isocratic elution, flow rate 2 mL/min, retention time t _R =9.6 min, collection of 9.0 min-10.0 min eluate, rotary evaporation to obtain a white powder product, 2,3-butanediol monoglucoside.

5) The calculated extraction rate is 1.110% (extraction rate%=weight/total amount of mycelium extract×100%).

Example 2 Purification Effects of Various Types of Chromatographic Columns

1) After concentrating the crude product in Example 1 under reduced pressure, divide the volume into 4 parts (100 mL each, containing 0.50 g of the product), and introduce them into a well-balanced HPLC (the column specifications are all 250 mm×4.6 mm, 5 μm; filler: YMC C ₁₈ , C ₈ , C ₄ ), and then eluted with 4% volume percentage chromatographic grade methanol: 96% pure water mixture, flow rate 2 mL/min, detection wavelength 254nm, and collected Components to form the suction mixture, the calculation results are shown in Table 1:

Table 1 Purification effect of various chromatographic columns

By comparing the results of Examples 1 to 2, it can be found that when using YMC _C18 chromatographic column, when the eluent 4% methanol and 96% pure water mixture, the sample obtained by elution has the highest purity and the highest yield, which is more suitable Used for the separation of this sample.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims (7)

1. A slaveRaffaelea lauricolaThe method for extracting 2, 3-butanediol monoglucoside is characterized by comprising the following steps of:

1) Will beRaffaelea lauricolaAfter activation, inoculating the bacterial blocks into a PDB culture medium, continuously culturing for 5-10 days at the temperature of 160-180 r/min and the temperature of 20-28 ℃, transferring the bacterial blocks into a rice culture medium according to the inoculum size of 15-20 wt%, and continuously performing stationary culture at the temperature of 20-28 ℃ for 28-40 days to obtain secondary culture to obtain mycelium;

2) Drying the mycelium obtained in the step 1) at 35-40 ℃, grinding into powder, soaking for 12-24 hours by using ethyl acetate with the volume being 1-2 times that of the mycelium, carrying out ultrasonic treatment for 40-60 minutes, collecting an extract, adding ethyl acetate with the volume being 1-2 times that of the mycelium into residues, carrying out ultrasonic treatment for 40-60 minutes, collecting the extract, repeating the steps to obtain three extracts, merging and concentrating to obtain a crude extract;

3) Dissolving the crude extract with chloroform or dichloromethane, and then using reverse phase silica gel C ₁₈ Mixing the sample, loading the sample into a column by a dry method, and mixing the sample with methanol: gradient elution is carried out according to the water volume ratio of 1:9-9:1, and methanol is collected: the eluent at the part with the water volume ratio of 9:1 is concentrated by rotary evaporation to obtain a component A;

4) Component a was separated by HPLC prep. as 4% mobile phase volume percent chromatography grade methanol: isocratic elution with 96% pure water, flow rate 2 mL/min, retention time t _R The eluent is collected for 9.0 min-10.0 min and is subjected to suspension evaporation to obtain white powdery 2, 3-butanediol monoglucoside.

2. The slave according to claim 1Raffaelea lauricolaThe method for extracting 2, 3-butanediol monoglucoside is characterized by comprising the following steps: the size of the pellet in step 1) was 0.5X0.5 cm, and the inoculum size was 4 pellets.

3. The slave according to claim 1Raffaelea lauricolaThe method for extracting 2, 3-butanediol monoglucoside is characterized by comprising the following steps: the rice culture medium in the step 1) comprises the following components: 100g of rice, 10 g of wood chips and g, and 120 g mL of pure water are added; sterilizing at 121deg.C for 20 min.

4. The slave according to claim 1Raffaelea lauricolaThe method for extracting 2, 3-butanediol monoglucoside is characterized by comprising the following steps: the power of the ultrasound in step 2) was 400W and the frequency was 35 KHz.

5. The slave according to claim 1Raffaelea lauricolaThe method for extracting 2, 3-butanediol monoglucoside is characterized by comprising the following steps: reversed phase silica gel C used in step 3) ₁₈ Particle size 40-60 μm, pore size 120A.

6. The slave according to claim 1Raffaelea lauricolaThe method for extracting 2, 3-butanediol monoglucoside is characterized by comprising the following steps: the gradient elution proportion in the step 3) is methanol: the water volume ratio was 1:9, 2:8, 3:7, 4:6, 6:4, 7:3, 8:2, 9:1.

7. The slave according to claim 1Raffaelea lauricolaThe method for extracting 2, 3-butanediol monoglucoside is characterized by comprising the following steps: the column used in the HPLC preparation in step 3) was YMC ₁₈ Column 250 mm ×4.6 mm, 5 μm.

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