Method for biotransformation of isorhamnetin

Abstract

The invention discloses a method for biologically converting isorhamnetin, which comprises the following steps: adding a microorganism for producing glucosidase, glycosyl transferase and / or succinyl transferase, or producing glucosidase, glycosyl transferase and / or succinyl transferase into an isorhamnetin substrate, adding a glycosyl donor for synchronous conversion, and separating and purifying, thereby obtaining the isorhamnetin. And the isorhamnetin-3-O-glucoside, the isorhamnetin-3-O-beta-(6 ''-O-succinyl)-D-glucoside and / or the isorhamnetin-3, 7-O-diglucoside are obtained after the reaction is finished. The method is green, safe and high in conversion efficiency, the product has higher water solubility, the bioavailability and additional value of the flavone compound can be improved, and high-valued utilization of natural plant product raw materials is achieved.

Description

Technical Field

[0001] The invention relates to a method for biologically transforming isorhamnetin, in particular to an application and a transformation method of transforming isorhamnetin into isorhamnetin-3-O-glucoside, isorhamnetin-3-O-β-(6"-O-succinyl)-D-glucoside and / or isorhamnetin-3,7-O-diglucoside by using Bacillus subtilis, and belongs to the technical field of biological transformation. Background Art

[0002] Isorhamnetin (also known as 3,5,7-trihydroxy-2-(4-hydroxy-3-methoxyphenyl)benzopyran-4-one) is a plant flavonoid compound with multiple pharmacological effects. It is mainly extracted and separated from some medicinal plants (such as sea buckthorn, ginkgo, etc.), and also exists in the flowers, leaves and fruits of many plants.

[0003] Isorhamnetin, as a natural flavonoid compound, has multiple biological activities and has received extensive attention in the medical field in recent years. Isorhamnetin has multiple biological activities and has shown potential application value in the fields of acute lung injury, anti-oxidation and anti-aging, anti-tumor and Parkinson's disease.

[0004] The preparation of flavonoids by biotransformation has long attracted wide attention from scholars. Existing studies have also disclosed a series of methods for preparing flavonoid derivatives by biotransformation, including the conversion of flavonoids and flavonoids such as quercetin, genistein, luteolin, and oroxylin by microorganisms producing glycosyltransferase and succinyl synthase to generate different glucosylation and succinylation products. These methods have the advantages of simple process, high conversion rate, few by-products, less use of organic reagents, and green environmental protection.

[0005] At present, there are few studies on isorhamnetin-3-O-glucoside, isorhamnetin-3-O-β-(6”-O-succinyl)-D-glucoside and isorhamnetin-3,7-O-diglucoside. The transformation products have high research value, and the bioavailability of the products is improved, which can be better absorbed and utilized by the human body to exert their efficacy. Therefore, it is necessary to structurally modify isorhamnetin and synthesize derivatives to improve bioavailability, safety and effectiveness. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a method for converting isorhamnetin into isorhamnetin-3-O-glucoside, isorhamnetin-3-O-β-(6"-O-succinyl)-D-glucoside and / or isorhamnetin-3,7-O-diglucoside using Bacillus subtilis. The method can improve the solubility of isorhamnetin and its bioavailability, and has the advantages of being green, safe and having high conversion efficiency.

[0007] Technical solution: A method for biotransformation of isorhamnetin of the present invention comprises the following steps:

[0008] Add glucosidase, glycosyltransferase and / or succinyltransferase, or microorganisms producing glucosidase, glycosyltransferase and / or succinyltransferase to the isorhamnetin substrate, add a glycosyl donor for synchronous conversion, separate and purify, and obtain a conversion product.

[0009] Further, the isorhamnetin structural formula is as follows:

[0010]

[0011] Further, the conversion product includes isorhamnetin-3-O-glucose, isorhamnetin-3-O-β-(6″-O-succinyl)-D-glucoside and / or isorhamnetin-3,7-O-diglucoside.

[0012] Furthermore, the structural formula of the isorhamnetin-3-O-glucoside is as follows:

[0013]

[0014] Furthermore, the structural formula of the isorhamnetin-3-O-β-(6"-O-succinyl)-D-glucoside is as follows:

[0015]

[0016] Furthermore, the structural formula of the isorhamnetin-3,7-O-diglucoside is as follows:

[0017]

[0018] Furthermore, the isorhamnetin substrate is isorhamnetin, or a plant extract containing isorhamnetin and / or its derivatives.

[0019] Furthermore, the microorganism producing glucosidase, glycosyltransferase and / or succinyltransferase is Bacillus subtilis and / or Aspergillus aculeatus ZJ.

[0020] Furthermore, Bacillus subtilis was deposited in the General Microbiology Center of the China Microorganism Culture Collection (CGMCC) on November 24, 2023, with the deposit number CGMCC No. 29125. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the classification name is: Bacillus subtilis.

[0021] Furthermore, Aspergillus aculeatus ZJ was deposited in China Center for Type Culture Collection (CCTCC) on July 25, 2011, with the deposit number: CCTCC NO.M2011264, and the deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the classification name is: Aspergillus aculeatus ZJ.

[0022] Furthermore, the glycosyltransferase is cyclodextrin glycosyltransferase (CGTase).

[0023] Furthermore, the glycosyl donor is dextrin, cyclodextrin, sucrose or lactose.

[0024] Furthermore, the concentration of the isorhamnetin substrate is 0.05-200 g / L.

[0025] Furthermore, the microorganism producing glucosidase, glycosyltransferase and / or succinyltransferase is added in the form of microbial strain, microbial liquid, microbial powder, or microbial enzyme powder.

[0026] Furthermore, the volume or weight of the added microbial culture liquid is 0.1%-10% of the volume or weight of the isorhamnetin substrate.

[0028] Furthermore, the amount of microbial powder added is 0.05%-8% of the mass of the isorhamnetin substrate.

[0029] Furthermore, the amount of the added microbial enzyme powder is 0.01%-6% of the amount of the isorhamnetin substrate.

[0030] Furthermore, the amount of the glycosyl donor added is 1-6 times the amount of the isorhamnetin substrate equivalent.

[0031] Furthermore, the synchronous conversion is carried out at 15-45° C. and 180-220 r / min for 2-48 hours.

[0032] Furthermore, when the isorhamnetin substrate is added, a co-solvent may be added and then filtered.

[0033] Furthermore, the co-solvent may be methanol, ethanol or DMSO.

[0034] Furthermore, separation and purification is carried out by silica gel column chromatography, macroporous resin, polyamide resin, preparative liquid chromatography or high performance countercurrent chromatography.

[0035] Furthermore, the macroporous resin is one or more of D101, AB-8, DA201, LSA-20, LSD-280, XDA-8D, HPD-500, LSA-10, and XAD-1.

[0036] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0037] (1) The isorhamnetin of the present invention is rich in sources and has a high content in plants, and is an ideal material for preparing rare isorhamnetin-3-O-glucoside, isorhamnetin-3-O-β-(6"-O-succinyl)-D-glucoside and / or isorhamnetin-3,7-O-diglucoside.

[0038] (2) The microbial transformation method of the present invention has strong specificity and high transformation efficiency, which can reach 99%.

[0039] (3) The conversion process of the method of the present invention is simple and green, and has good environmental compatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the result of HPLC analysis of isorhamnetin standard;

[0041] Figure 2 This is the result of HPLC analysis of isorhamnetin-3-O-glucoside standard;

[0042] Figure 3 This is the analysis result of the new product of isorhamnetin after 24 hours of biotransformation;

[0043] Figure 4 This is the analysis result of the new product of isorhamnetin after 48 hours of biotransformation;

[0044] Figure 5 This is the LC / MS analysis result of the new product (isorhamnetin-3,7-O-diglucoside) after biotransformation of isorhamnetin;

[0045] Figure 6 This is the LC / MS analysis result of the new product (isorhamnetin-3-O-glucoside) after biotransformation of isorhamnetin;

[0046] Figure 7 This is the LC / MS analysis result of the new product (isorhamnetin-3-O-β-(6”-O-succinyl)-D-glucoside) after biotransformation of isorhamnetin;

[0047] Figure 8 This is the carbon NMR spectrum of isorhamnetin-3-O-glucoside;

[0048] Fig. 9 This is the hydrogen spectrum NMR diagram of isorhamnetin-3-O-glucoside. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0050] Example 1

[0051] Preparation of Bacillus subtilis bacterial solution, bacterial powder and enzyme powder:

[0052] After the liquid fermentation of Bacillus subtilis, the fermentation liquid is centrifuged to obtain bacterial mud, which is then dried by conventional methods to obtain bacterial powder (see Li Dapeng, Gao Yurong. Study on the preparation and storage conditions of the composite freeze-dried fermentation agent of probiotic Lactobacillus plantarum G1-28 [J]. Food Industry Science and Technology, 2021, 42(10): 100-104). Preparation of Bacillus subtilis enzyme powder: After the obtained bacterial mud is added to phosphate buffer, the enzyme is released by conventional cell wall breaking treatment, and the enzyme solution is subjected to conventional vacuum freeze drying to obtain enzyme powder.

[0053] Example 2

[0054] Inoculate 400 mL of broth medium with Bacillus subtilis at 5% (v / v) and culture at 200 rpm and 35°C for 24 h until the concentration of Bacillus subtilis reaches 10 8 -10 9CFU / mL, 400mg isorhamnetin and 1g dextrin were added to the transformation bottle, and the transformation was carried out under the same culture conditions for 24h. The transformed solution was centrifuged at 8000rpm for 20min, and the supernatant was concentrated by rotary evaporation at a temperature of 50°C until the liquid volume was 5-20mL. The concentrated sample was separated and purified by D101 macroporous resin, and 10% ethanol aqueous solution was used as the eluent at a flow rate of 3BV / h, eluting 10BV, collecting the eluent, recovering the solvent under reduced pressure, and refrigerating for crystallization. The substance was detected to be isorhamnetin-3,7-O-diglucoside. A 60% ethanol aqueous solution was used as the eluent, the flow rate was 3BV / h, 15BV was eluted, the eluate was collected, the solvent was recovered under reduced pressure and then loaded onto an HPD-850 resin column; a 20% ethanol aqueous solution was used as the eluent, the flow rate was 3BV / h, 10BV was eluted, the eluate was collected, the solvent was recovered under reduced pressure, and the solution was refrigerated for crystallization. The substance was detected to be isorhamnetin-3-O-glucoside; a 30% ethanol aqueous solution was used as the eluent, the flow rate was 3BV / h, 10BV was eluted, the eluate was collected, the solvent was recovered under reduced pressure, and the solution was refrigerated for crystallization. The substance was detected to be isorhamnetin-3-O-β-(6"-O-succinyl)-D-glucoside.

[0055] HPLC analysis of isorhamnetin and isorhamnetin-3-O-glucoside standards showed the following results: Figure 1-2 As shown. Figure 1-2 It can be seen that the retention time of isorhamnetin is 27.373 min, and the retention time of isorhamnetin-3-O-glucoside is 20.726 min.

[0056] The samples after 24 hours of conversion were analyzed by HPLC. Figure 3 As shown. Figure 3 It can be seen that after 24 hours of conversion, the conversion of isorhamnetin was basically completed, and the peak times were 13.299min, 20.952min and 23.498min. The peak time of 20.952min was close to the peak time of the standard isorhamnetin-3-O-glucoside (see Figure 2 ), the purified product with a peak time of 20.952 min was analyzed by LC-MS, and the results are shown in Figure 6 .Depend on Figure 6 It can be seen that the molecular ion peak [MH]- of the substance with an elution time of 20.952 min is 477.1. The NMR spectrum data of this product after separation and purification were analyzed, and the results are as follows:

[0057] ESI-MS, m / z 477.1, [MH]-.

[0058] 1H-NMR (300MHz, DMSO) δ: 6.81 (1H, d, J = 3.5 Hz, H-5'), 6.80 (1H, d, J = 3.7 Hz, H-2'), 6.79 (1H, d, J = 4.5 Hz, H-6'), 6.06 (1H, s, H-8), 5.95 (1H, s, H-6), 5.71(1H,d,J=9.0Hz,H-2), 5.70(1H,s,H-4”), 5.68(1H,d,J=10.5Hz,H-3), 3.90(1H,t,J=9.5Hz,H-1”), 3.70(2H,d,J=7.5Hz,H-2”,3”).

[0059] 13C-NMR (300MHz, DMSO) δ: 196.9 (C-4), 164.9 (C-7), 163.8 (C-5), 163.6 (C-9 ), 147.7(C-3'), 147.6(C-4'), 128.6(C-1'), 119.7(C-6'), 115.8(C-5'), 109 .8(C-2'), 105.9(C-1”), 104.7(C-5”), 100.6(C-10), 95.1(C-6), 94.6(C-8) , 93.7(C-3), 85.5(C-2), 73.8(C-2”), 69.8(C-4”), 69.7(C-3”), 56.1(C-7’).

[0060] Its carbon and hydrogen NMR spectra are shown in Figure 8 and Fig. 9 shown.

[0061] According to the NMR results, the substance is judged to be isorhamnetin-3-O-glucoside with a molecular weight of 478, which is consistent with the molecular ion peak [MH]- of the substance in LC-MS at 477.1. The structural formula of the product is as follows:

[0062]

[0063] The product of 13.299 min was purified and analyzed by LC-MS. Figure 5 .Depend on Figure 5 It can be seen that the molecular ion peak [MH]- of the substance with an elution time of 13.299 min is 639.2. The nuclear magnetic resonance spectrum data of the purified product was analyzed, and the results are as follows:

[0064] ESI-MS, m / z 639.2, [MH]-.

[0065] 1H-NMR (300MHz, DMSO) δ: 6.81(1H,s,H-5'), 6.80(1H,s,H-2'), 6.79(1H,s,H-6'), 6.19(1H,d ,J=1.5Hz,H-8), 6.18(1H,d,J=1.8Hz,H-6), 5.80(1H,s,H-1”), 5.71(1H,s,H-2), 5.68(1H,s, H-3), 5.40(1H,d,J=7.5Hz,H-1”), 4.20(1H,s,H-2”), 3.90(1H,t,J=14Hz,H-2”), 3.70(2H,d, J=10.5Hz,H-3”,9”), 3.60(4H,t,J=7.5Hz,H-4”,5”,10”,11”), 3.57-3.51(2H,m,H-6”,12”).

[0066] 13C-NMR (300MHz, DMSO) δ: 196.9 (C-4), 166.5 (C-7), 163.0 (C-5), 162.8 (C-9), 147.7 (C-3' ), 147.6(C-4'), 128.6(C-1'), 119.7(C-6'), 115.8(C-5'), 109.8(C-2'), 109.6(C-1”), 10 9.2(C-7”), 99.6(C-10), 94.9(C-6), 94.1(C-8), 93.7(C-3), 85.5(C-2), 81.5(C-5”,11”), 76.8(C-3”,9”), 73.8(C-2”), 73.4(C-8”), 71.5(C-4”,10”), 62.2(C-6”,12”), 56.1(C-7’).

[0067] According to the NMR results, the substance is isorhamnetin-3,7-O-diglucoside with a molecular weight of 640, which is consistent with the molecular ion peak [MH]- of 639.2 in LC-MS. The structural formula of the product is as follows:

[0068]

[0069] The product at 23.498 min was purified and analyzed by LC-MS. Figure 7 .Depend on Figure 7 It can be seen that the molecular ion peak [MH]- of the substance with an elution time of 23.498 min is 577.1. The nuclear magnetic resonance spectrum data of the purified product was analyzed, and the results are as follows:

[0070] ESI-MS, m / z 577.1, [MH]-.

[0071] 1H-NMR (300MHz, DMSO) δ: 6.81(1H,s,H-5'), 6.80(1H,s,H-2'), 6.79(1H,s,H-6'), 6.06(1 H,s,H-8), 5.95(1H,s,H-6), 5.71(1H,d,J=3.5Hz,H-2), 5.68(1H,d,J=3.0Hz,H-3), 5.40(1 H,d,J=1.5Hz,H-1”), 4.32-4.07(2H,m,H-6”), 4.19(1H,s,H-5”), 3.77(3H,s,H-7’), 3.70 (1H,s,H-3”), 3.60(1H,s,H-4”), 2.71(2H,d,J=7.0Hz,H-3”), 2.61(2H,d,J=3.5Hz,H-2”).

[0072] 13C-NMR (300MHz, DMSO) δ: 196.9 (C-4), 174.7 (C-4″′), 173.1 (C-1″′), 164.9 (C-7), 163.8 (C-5 ), 163.6(C-9), 147.7(C-3'), 147.6(C-4'), 128.6(C-1'), 119.7(C-6'), 115.8(C-5'), 109.8(C -2'), 109.3(C-1”), 100.6(C-10), 95.1(C-6), 94.6(C-8), 93.7(C-3), 85.5(C-2), 78.2(C-5”) , 76.5(C-3”), 73.8(C-2”), 71.7(C-4”), 63.1(C-6”), 56.1(C-7′), 29.2(C-2″′), 29.1(C-3″′).

[0073] According to the NMR results, the substance is isorhamnetin-3-O-β-(6"-O-succinyl)-D-glucoside, with a molecular weight of 578, which is consistent with the molecular ion peak [MH]- of the substance in LC-MS at 577.1. The structural formula of the product is as follows:

[0074]

[0075] Therefore, isorhamnetin is transformed by Bacillus subtilis to generate isorhamnetin-3-O-glucoside, isorhamnetin-3-O-β-(6"-O-succinyl)-D-glucoside and isorhamnetin-3,7-O-diglucoside. The quantitative results show that the conversion rate of isorhamnetin is 99.2%, the yield of isorhamnetin-3-O-glucoside is 74.7%, the yield of isorhamnetin-3-O-β-(6"-O-succinyl)-D-glucoside is 11.6%, and the yield of isorhamnetin-3,7-O-diglucoside is 3.4%.

[0076] Example 3

[0077] The experimental process was the same as that in Example 2. 5 g of dried seabuckthorn leaves were ground and sieved through a 40-mesh sieve. The seabuckthorn leaves were ultrasonically extracted with a 60% ethanol aqueous solution at 40° C. for 1 h at a solid-liquid ratio of 1:10 to obtain a seabuckthorn leaf extract.

[0078] Bacillus subtilis cells were inoculated into the broth at a rate of 10% (v / v) and cultured for 24 h under the same conditions as in Example 2 until the bacterial count reached 10 8 -10 9 cfu / mL or more. Add all 1g of dextrin and seabuckthorn leaf extract to the cultured bacterial culture medium and transform under the same culture conditions for 24h. Carry out rotary evaporation concentration under the same conditions as Example 2, and separate and purify with macroporous resin D101. According to LC-MS and NMR analysis, the conversion rate of isorhamnetin is 98.1%, the yield of isorhamnetin-3-O-glucoside is 73.9%, the yield of isorhamnetin-3-O-β-(6"-O-succinyl)-D-glucoside is 11.4%, and the yield of isorhamnetin-3,7-O-diglucoside is 3.3%.

[0079] Example 4

[0080] The experimental process was the same as that in Example 3. 5 g of dried seabuckthorn leaves were ground and sieved through a 40-mesh sieve. The seabuckthorn leaves were ultrasonically extracted with a 60% ethanol aqueous solution at 40° C. for 1 h at a solid-liquid ratio of 1:10 to obtain a seabuckthorn leaf extract.

[0081] 1% (v / v) Bacillus subtilis and 3% (v / v) Aspergillus aculeatus ZJ were added to 400 mL of broth medium, and cultured for 24 h under the same conditions as in Example 3 until the concentration of Bacillus subtilis reached 10 8 -10 9cfu / mL or more. Add all the seabuckthorn leaf extract to the culture medium, add 1g of dextrin at the same time, and transform for 24 hours under the same culture conditions as in Example 3. Centrifuge the converted solution at 8000rpm for 20min as in Example 3, take the supernatant and concentrate it by rotary evaporation at a temperature of 45°C until the liquid volume is 5-20mL, and use a macroporous resin of model DA201 to separate and purify the concentrated sample. Analysis showed that the conversion rate of isorhamnetin was 96.5%, the yield of isorhamnetin-3-O-glucoside was 70.7%, the yield of isorhamnetin-3-O-β-(6"-O-succinyl)-D-glucoside was 10.9%, and the yield of isorhamnetin-3,7-O-diglucoside was 3.1%.

[0082] Example 5

[0083] The experimental process was the same as in Example 2. 1% (w / w) Bacillus subtilis cells and 3% (w / w) Aspergillus aculeatus ZJ cells were inoculated into 400 mL of broth medium, and cultured for 24 h under the same conditions as in Example 2 until the bacterial solution concentration reached 10 8 -10 9 cfu / mL or more. 400 mg of isorhamnetin and 1 g of cyclodextrin were introduced into the transformation bottle. The transformation conditions were basically the same as those in Example 2, and the transformation was carried out at a temperature of 25°C for 24 hours. The transformed solution was centrifuged at 8000 rpm for 20 minutes, and the supernatant was concentrated by rotary evaporation at a temperature of 50°C. The liquid volume was evaporated to 5-20 mL, and the concentrated sample was separated and purified using a macroporous resin of model DA201. Analysis showed that the conversion rate of isorhamnetin was 85.3%, the yield of isorhamnetin-3-O-glucoside was 63.7%, the yield of isorhamnetin-3-O-β-(6"-O-succinyl)-D-glucoside was 9.8%, and the yield of isorhamnetin-3,7-O-diglucoside was 2.8%.

[0084] Example 6

[0085] The experimental process was the same as in Example 2. 5% (v / v) Bacillus subtilis cells were inoculated into 400 mL of broth medium and cultured for 24 h until the bacterial solution concentration reached 10 8 -10 9cfu / mL or more. 800 mg of isorhamnetin and 3 g of dextrin were introduced into the transformation bottle. The transformation conditions were the same as those in Example 2 and the transformation was carried out for 24 hours. The transformed solution was centrifuged at 8000 rpm for 20 min, and the supernatant was concentrated by rotary evaporation at a temperature of 50°C. The liquid volume was evaporated to 5-20 mL, and the concentrated sample was separated and purified using a macroporous resin of model DA201. Analysis showed that the conversion rate of isorhamnetin was 97.8%, the yield of isorhamnetin-3-O-glucoside was 72.6%, the yield of isorhamnetin-3-O-β-(6"-O-succinyl)-D-glucoside was 11.2%, and the yield of isorhamnetin-3,7-O-diglucoside was 3.2%.

[0086] Example 7

[0087] The experimental process was the same as in Example 2. 5% (v / v) Bacillus subtilis cells were inoculated into 400 mL of broth medium and cultured for 24 h until the bacterial solution concentration reached 10 8 -10 9 cfu / mL or more, 400 mg of isorhamnetin and 2 g of lactose were introduced into the transformation bottle, the transformation conditions were the same as those in Example 2, the transformation was carried out for 24 h, the transformed solution was centrifuged at 8000 rpm for 20 min, the supernatant was concentrated by rotary evaporation, the rotary evaporation temperature was 50 ° C, and the liquid volume was 5-20 mL. The concentrated sample was separated and purified by a macroporous resin of model DA201. After analysis, the isorhamnetin conversion rate was 95.9%, the isorhamnetin-3-O-glucoside yield was 70.5%, the isorhamnetin-3-O-β-(6"-O-succinyl)-D-glucoside yield was 10.8%, and the isorhamnetin-3,7-O-diglucoside yield was 3.1%.

[0088] Example 8

[0089] The experimental process was the same as in Example 2. 5% (v / v) of Bacillus subtilis cells were inoculated into 400 mL of broth medium and cultured at 200 rpm and 35°C for 24 h until the bacterial solution concentration reached 10 8 -10 9 cfu / mL or more. 400 mg of isorhamnetin and 1 g of dextrin were added to the transformation bottle, and the transformation was carried out under the same conditions as in Example 2 for 48 h. The transformed solution was centrifuged at 8000 rpm for 20 min, and the supernatant was concentrated by rotary evaporation at a temperature of 50°C until the liquid volume was 5-20 mL. The concentrated sample was separated and purified using a macroporous resin of model DA201. HPLC analysis was performed, and the results were as follows Figure 4As shown. Analysis showed that the conversion rate of isorhamnetin was 99.9%, the yield of isorhamnetin-3-O-glucoside was 47.3%, the yield of isorhamnetin-3-O-β-(6"-O-succinyl)-D-glucoside was 5.6%, and the yield of isorhamnetin-3,7-O-diglucoside was 30.2%.

[0090] Example 9

[0091] The experimental process was the same as in Example 2. 5% (v / v) of Bacillus subtilis cells were inoculated into 400 mL of broth medium and cultured at 200 rpm and 35°C for 24 h until the bacterial solution concentration reached 10 8 -10 9 cfu / mL or more. 400 mg of isorhamnetin and 1 g of dextrin were introduced into the transformation bottle, and the transformation was carried out under the same conditions as in Example 2 for 2 h. The transformed solution was centrifuged at 8000 rpm for 20 min, and the supernatant was concentrated by rotary evaporation at a temperature of 50°C until the liquid volume was 5-20 mL. The concentrated sample was separated and purified using a macroporous resin of model DA201. HPLC analysis was performed, and the conversion rate of isorhamnetin was 15.7%, the yield of isorhamnetin-3-O-glucoside was 10.6%, and the yield of isorhamnetin-3-O-β-(6"-O-succinyl)-D-glucoside was 1.6%.

[0092] Example 10

[0093] The experimental process was the same as in Example 2. 10 mg of Bacillus subtilis powder was added to 400 mL of broth medium and cultured for 24 h as in Example 2 until the bacterial solution concentration reached 10 8 -10 9 cfu / mL or more, add 400mg isorhamnetin and 1g dextrin to the cultured bacterial solution, transform at 200rpm and 35℃ for 24h, centrifuge the transformed solution at 8000rpm for 20min, take the supernatant and perform rotary evaporation concentration at 50℃ until the liquid volume is 5-20mL. Separation, purification and identification are the same as in Example 2. Analysis shows that the conversion rate of isorhamnetin is 98.9%, the yield of isorhamnetin-3-O-glucoside is 74.2%, the yield of isorhamnetin-3-O-β-(6"-O-succinyl)-D-glucoside is 11.5%, and the yield of isorhamnetin-3,7-O-diglucoside is 3.3%.

[0094] Embodiment 11

[0095] The experimental process was the same as in Example 2. 25 mg of Bacillus subtilis powder was added to 2 L of broth medium and cultured for 24 h as in Example 2 until the bacterial solution concentration reached 10 8 -109 cfu / mL or more, add 400mg isorhamnetin and 1g dextrin to the cultured bacterial solution, the conversion conditions are the same as in Example 2, and the conversion time is 24h. The converted solution is centrifuged at 8000rpm for 20min, and the supernatant is concentrated by rotary evaporation at a temperature of 50°C until the liquid volume is 5-20mL. Separation, purification and identification are the same as in Example 2. Analysis shows that the conversion rate of isorhamnetin is 97.8%, the yield of isorhamnetin-3-O-glucoside is 73.5%, the yield of isorhamnetin-3-O-β-(6"-O-succinyl)-D-glucoside is 11.3%, and the yield of isorhamnetin-3,7-O-diglucoside is 3.2%.

[0096] Example 12

[0097] The experimental process is the same as that of Example 2. 50 mg of Bacillus subtilis enzyme powder is added to a conversion bottle containing 4 g of isorhamnetin and 8 g of lactose. The conversion is carried out at 200 rpm and 40°C for 12 h. The converted solution is centrifuged at 8000 rpm for 20 min. The supernatant is concentrated by rotary evaporation at a temperature of 50°C until the liquid volume is 5-20 mL. Separation, purification and identification are the same as those of Example 2. Analysis shows that the conversion rate of isorhamnetin is 88.2%, the yield of isorhamnetin-3-O-glucoside is 67.1%, the yield of isorhamnetin-3-O-β-(6"-O-succinyl)-D-glucoside is 10.3%, and the yield of isorhamnetin-3,7-O-diglucoside is 2.9%.

Claims

1. A method for biotransformation of isorhamnetin, characterized in that: The following steps are involved: Add glucosidase, glycosyltransferase and / or succinyltransferase, or microorganisms producing glucosidase, glycosyltransferase and / or succinyltransferase to the isorhamnetin substrate, add a glycosyl donor for synchronous conversion, separate and purify, and obtain a conversion product.

2. The method for biotransformation of isorhamnetin according to claim 1, characterized in that: The conversion products include isorhamnetin-3-O-glucoside, isorhamnetin-3-O-β-(6″-O-succinyl)-D-glucoside and / or isorhamnetin-3,7-O-diglucoside.

3. The method for biotransformation of isorhamnetin according to claim 1, characterized in that: The isorhamnetin substrate is isorhamnetin, or a plant extract containing isorhamnetin and / or its derivatives.

4. The method for biotransformation of isorhamnetin according to claim 1, characterized in that: The microorganism producing glucosidase, glycosyltransferase and / or succinyltransferase is Bacillus subtilis and / or Aspergillus aculeatus ZJ.

5. The method for biotransformation of isorhamnetin according to claim 1, characterized in that: The glycosyltransferase is cyclodextrin glucosyltransferase, and the glycosyl donor is dextrin, cyclodextrin, sucrose or lactose.

6. The method for biotransformation of isorhamnetin according to claim 1, characterized in that: The added form of the microorganism producing glucosidase, glycosyltransferase and / or succinyltransferase is microorganism strain, microorganism liquid, microorganism powder, or microorganism enzyme powder.

7. The method for biotransformation of isorhamnetin according to claim 6, characterized in that: The concentration of the isorhamnetin substrate is 0.05-200 g / L, and the volume or weight of the microbial culture solution added is 0.1%-10% of the volume or weight of the isorhamnetin substrate; or the amount of the added microbial powder is 0.05%-8% of the mass of the isorhamnetin substrate; or the amount of the added microbial enzyme powder is 0.01%-6% of the mass of the isorhamnetin substrate.

8. The method for biotransformation of isorhamnetin according to claim 1, characterized in that: The synchronous conversion is carried out at 15-45°C and 180-220 r / min for 2-72 hours.

9. The method for biotransformation of isorhamnetin according to claim 1, characterized in that: The amount of the added glycosyl donor is 1-6 times the equivalent of the isorhamnetin substrate.

10. The method for biotransformation of isorhamnetin according to claim 1, characterized in that: The separation and purification is carried out by silica gel column chromatography, macroporous resin, polyamide resin, preparative liquid chromatography or high performance countercurrent chromatography.

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