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Application of beta-glucosidase to converting total flavonoids of herba epimedii to prepare baohuoside I

A technology of glucosidase and icariin, which is applied in the direction of glycosylase, biochemical equipment and methods, enzymes, etc., can solve the problems of increased cost, single biotransformation method, and the efficiency of icariin needs to be improved. Achieve high conversion efficiency, excellent temperature stability, and high-efficiency expression

Active Publication Date: 2017-09-15
NANJING FORESTRY UNIV
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Problems solved by technology

[0004] Taking the above problems into consideration, the target product, baojoside I, can be obtained by selectively removing glycosyl groups that are different from baojoside I. According to the published literature, the current biotransformation method for preparing baojoside I is relatively single, almost All use multi-component icariin as the substrate, and have not achieved efficient utilization of the total flavonoids of Epimedium. For example, CN103160553 prepares baojoside I by hydrolyzing icariin with glucanase; CN103305572 uses yeast anaerobic fermentation Transformation of only icariin in Epimedium to baochoside I
Therefore, in the preparation process, firstly obtain the epimedium extract, then isolate and obtain icariin, and then transform through enzymatic or microbial methods, which not only greatly increases the cost, but also does not fully and efficiently utilize Chaohuodine A, Chaohuoding Ding B, facing Huo Ding C
On the other hand, the problem of obtaining a large amount of icariin resources is very prominent, and it is impossible to guarantee the large-scale preparation of baochoside I
Moreover, the efficiency of existing enzymatic or microbial methods to transform icariin needs to be improved

Method used

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  • Application of beta-glucosidase to converting total flavonoids of herba epimedii to prepare baohuoside I
  • Application of beta-glucosidase to converting total flavonoids of herba epimedii to prepare baohuoside I
  • Application of beta-glucosidase to converting total flavonoids of herba epimedii to prepare baohuoside I

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Effect test

Embodiment 1

[0022] Embodiment 1: the acquisition of β-glucosidase gene of the present invention and the construction of recombinant plasmid pET-DthBGL3

[0023] 1.1 Culture of Dictyoglomus thermophilum DSM 3960

[0024]Dictyoglomus thermophilum DSM 3960 was purchased from the DSMZ Culture Collection Center (www.dsmz.de) and the number was 13995. The medium formula was: potassium dihydrogen phosphate 1.5g / L, disodium hydrogen phosphate dodecahydrate 4.2g / L, chlorine Ammonium chloride 0.5g / L, magnesium chloride hexahydrate 0.38g / L, calcium chloride dihydrate 0.06g / L, ferric ammonium sulfate hexahydrate 0.04g / L, cobalt chloride hexahydrate 2.9mg / L, sodium molybdate dihydrate 2.4mg / L, sodium selenate pentahydrate 1.7mg / L, manganese chloride tetrahydrate 2mg / L, zinc sulfate 2.8mg / L, soluble starch 5g / L, peptone 2g / L, yeast extract 2g / L, carbonic acid Sodium 1g / L, cysteine ​​hydrochloride 1g / L, resazurin sodium 1g / L, deoxidize under nitrogen environment, adjust pH to 7.2. Inoculate with a syr...

Embodiment 2

[0039] Embodiment 2: the preparation of β-glucosidase Dth3 of the present invention

[0040] The recombinant plasmid pET-DthBGL3 was transformed into Escherichia coli JM109 (DE3) host bacteria (purchased from Novagen), on LB plates containing kanamycin (50 μg / mL) (LB medium: tryptone 10 g / L, yeast extract 5g / L, NaCl 5g / L, agar 15g / L) after culturing overnight at 37°C, pick the transformants into 200mL LB medium (50μg / mL kanamycin) at 37°C, shake at 200rpm until the OD600 is 0.6 , add a final concentration of 0.005-0.01mM isopropyl β-D-thiogalactopyranoside (IPTG) inducer, culture at 30°C for 6h, and use a high-speed refrigerated centrifuge to cool the culture medium at 4°C at 13,000 Centrifuge at rpm for 15 min to collect the cells.

[0041] Since the recombinant plasmid pET-DthBGL3 contains a His-tag tag, it was purified by His·Bind Purification Kit (purchased from Novagen) to obtain a purified recombinant enzyme. Specific operation process:

[0042] A. Processing of sampl...

Embodiment 3

[0058] Embodiment 3: the qualitative determination of β-glucosidase of the present invention

[0059] 1. Determination method of enzyme activity

[0060] Reaction system 100 μL, add 85 μL 100 mmol / L citric acid-disodium hydrogen phosphate buffer (pH 5.0) to 5 μL 20 mmol / L p-nitrophenyl-β-L-glucoside (pNPG), first incubate at 90 ° C for 2 min, then add 10 μL of enzyme solution diluted to an appropriate multiple was reacted for 10 minutes, and after color development, 600 μL of 1 mol / L sodium carbonate solution was added to terminate the reaction. Absorbance was measured at 405 nm. Enzyme activity unit (U) is defined as: under the assay conditions, the amount of enzyme required to produce 1 μmol p-nitrophenol per minute is 1 enzyme activity unit.

[0061] 2. Determination of the optimum reaction temperature

[0062] In the range of 60-100°C, the enzyme activity was measured every 5°C. The buffer is 100mmol / L citric acid-disodium hydrogen phosphate buffer solution, pH 5.0, th...

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Abstract

The invention discloses application of beta-glucosidase to converting total flavonoids of herba epimedii to prepare baohuoside I. According to a method, the specific beta-glucosidase is utilized for converting the total flavonoids of herba epimedii in an oriented mode, so that multiple ingredients rich in the total flavonoids of herba epimedii are all converted into the baohuoside I. The beta-glucosidase obtained through screening can be used for effective enzymolysis of icariin, epimedin A and sagittatoside A rich in the total flavonoids of herba epimedii in preparing the baohuoside I; and meanwhile, epimedin B and sagittatoside B can also be specifically used for preparing the baohuoside I. The beta-glucosidase is low in application cost, and only one type of enzyme is needed; and the converting efficiency of the total flavonoids is high, and the molar conversion ratio is greater than 95%. Meanwhile, the pharmacological activity of the prepared baohuoside I in the aspect of an inhibiting effect on proliferation of tumors such as breast cancer, liver cancer, colon cancer, lung cancer and the like is remarkably higher than that of other main icariin in the total flavonoids of herba epimedii.

Description

technical field [0001] The invention belongs to the field of biomedicine and health care products, and specifically relates to the application of β-glucosidase in transforming total flavonoids of Epimedium to prepare baojoside I and the preparation of baojoside I in the preparation of drugs for treating breast cancer, lung cancer, colon cancer and liver cancer in the application. Background technique [0002] Flavonoids have a wide range of pharmacological activities. In vitro, in vivo and clinical trials have shown that some flavonoids are of great significance for the prevention and treatment of cancer. Their antioxidant properties can reduce the nutritional damage of cell DNA, thereby reducing DNA mutations, and can also inhibit the activation of various carcinogens in vivo, and improve the body's removal of carcinogens. For cancer cells, flavonoids can also inhibit their proliferation. The transduction of cancer cells can block the cycle of cancer cells and induce apopt...

Claims

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Application Information

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IPC IPC(8): C12P19/60C12P19/14A61K31/7048A61P35/00
CPCA61K31/7048C12N9/2445C12P19/14C12P19/60C12Y302/01021Y02A50/30
Inventor 赵林果裴建军解静聪赵顺懿房仙颖陈安娜
Owner NANJING FORESTRY UNIV
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