CN109504727B

CN109504727B - Method for preparing reduced glucoraphanin by adopting intestinal bacterium metabolism and detection method

Info

Publication number: CN109504727B
Application number: CN201811470518.6A
Authority: CN
Inventors: 朱立俏; 盛华刚; 张茜; 周洪雷
Original assignee: Shandong University of Traditional Chinese Medicine
Current assignee: Shandong University of Traditional Chinese Medicine
Priority date: 2018-12-04
Filing date: 2018-12-04
Publication date: 2020-08-28
Anticipated expiration: 2038-12-04
Also published as: CN109504727A

Abstract

The invention provides a method for preparing reduced type glucoraphanin by intestinal bacterium metabolism and a detection method, wherein an anaerobic culture solution and an intestinal bacterium solution are prepared, the anaerobic culture solution and the intestinal bacterium solution are mixed and incubated to obtain an intestinal bacterium culture solution, a glucoraphanin extract is added into the intestinal bacterium culture solution to obtain a crude product solution containing metabolites under the action of intestinal flora, and the high-purity reduced type glucoraphanin is obtained by separating and purifying metabolites by using sephadex. The reduced glucoraphanin obtained by the invention has high purity and good purification effect.

Description

Method for preparing reduced glucoraphanin by adopting intestinal bacterium metabolism and detection method

Technical Field

The invention relates to a method for preparing reduced glucoraphanin by adopting intestinal bacterium metabolism and a detection method, belonging to the field of traditional Chinese medicines.

Background

The cruciferous vegetables contain abundant reduced glucoraphanin and myrosinase, and researches show that the reduced glucoraphanin can be hydrolyzed by the myrosinase and then is converted into reduced glucoraphanin through non-enzymatic intramolecular rearrangement reaction. Reduced sulforaphane (4-methylthio-3-butenyl isothiocyanate), an isothiocyanate compound having induced phase II detoxification enzyme activity, not only enhances the elimination ability of human body to carcinogen, but also inhibits and kills cancer cells. The reduced sulforaphane is a phytochemical component with the strongest anti-cancer activity found in cruciferous vegetables, and has strong inhibition effects on breast cancer, lung cancer, prostatic cancer and various cancers.

Reduced sulforaphane (glucoraphanin), i.e. 4-methylthio-3-butenyl thioglucoside (4-Methylsulfanyl-3-butylglucoraphaninate). Since the R group is CH₃SCH＝CH(CH₂)₂The reduced sulforaphane belongs to one of aliphatic glucosinolates, and the enzymolysis product of the reduced sulforaphane is reduced sulforaphane (Rhaphasatin) with obvious anticancer activity. The isothiocyanate group in the molecular structure of the reduced sulforaphane is very active and is very easy to react with a compound containing sulfydryl or hydroxyl, so that the anti-cancer activity of the reduced sulforaphane is lost.

The conventional method for preparing the reduced glucoraphanin is to extract the reduced glucoraphanin from cruciferous plants by an alcohol extraction and water precipitation method and then remove impurities to obtain purified reduced glucoraphanin, the preparation method is low in conversion efficiency, many impurities are obtained after reaction, multiple times of filtration are needed, and the content and the purity of the reduced glucoraphanin obtained after filtration are low. This method not only increases the cost, but also increases the difficulty of subsequent purification.

Disclosure of Invention

The invention provides a method for preparing high-purity reduced glucoraphanin aiming at the defects of low conversion efficiency, complex purification process and low purity of the reduced glucoraphanin, and the method has the advantages of simple and convenient purification process, high purity, high conversion efficiency, low energy consumption and no pollution.

The invention is realized by adopting the following technical scheme: the method for preparing reduced glucoraphanin by adopting intestinal bacterium metabolism comprises the following steps:

(1) preparation of sample liquid

Adding distilled water into the glucoraphanin extract to be ultrasonically dissolved until the mass concentration is 1.5-3 mg/mL^-1Then adding the mixture into an enterobacteria culture solution, placing the mixture into an anaerobic incubator for incubation for 7-9 h, taking out the mixture, and adding methanol to stop reaction to obtain a sample solution;

(2) preparation of reducing glucoraphanin

Centrifuging the sample solution in the step (1), concentrating the supernatant under reduced pressure to be nearly dry, transferring the supernatant with a proper amount of methanol, passing through a 0.45-micron microporous filter membrane to obtain a crude product solution containing the metabolite, passing the crude product solution through a SephadexLH-20 sephadex gel column, eluting with methanol, collecting the eluate containing the metabolite by stages, detecting the eluate containing the metabolite by HPLC, combining the eluates containing the metabolite, concentrating the eluate containing the metabolite to be nearly dry under reduced pressure below 50 ℃, transferring with a proper amount of distilled water, and freeze-drying to obtain a pure metabolite, namely a pure reduced glucoraphanin product.

It is preferable that: the preparation process of the intestinal fungus culture solution comprises the steps of preparing an anaerobic culture solution for later use; adding normal saline into the feces of the healthy human or animals, mixing the feces and the feces, fully and uniformly mixing the feces and the feces to obtain a suspension, putting the suspension into a centrifuge, centrifuging the suspension for 8-13 min, and taking the supernatant after centrifugation to obtain intestinal bacteria liquid for later use; and finally, adding the intestinal bacteria liquid into a sterile anaerobic culture solution, fully mixing, and culturing in an anaerobic culture box for 24 hours to obtain an intestinal bacteria culture solution.

It is preferable that: the glucoraphanin extract in the step (1) is fried radish seed extract, radish seed thioglycoside effective parts or glucoraphanin pure products.

It is preferable that: the preparation process of the fried radish seed extracting solution comprises the steps of crushing fried radish seed decoction pieces into coarse powder, adding 20 times of the coarse powder, extracting for 15-30 min for 3 times, combining extracting solutions, and concentrating to 0.5 g/mL^-1(ii) a The preparation process of the sulforaphane effective part of the radish seed comprises the steps of crushing fried radish seed decoction pieces into coarse powder, adding 20 times of the coarse powder, extracting for 15-30 min for 3 times, combining extracting solutions, and concentrating to 0.5 g/mL^-1Adding the concentrated radish seed extract into AB-8 macroporous adsorbent resin at a sample loading amount of 0.5-0.75 BV and a sample loading flow rate of 0.5-1 BV.h^-1Eluting with water at an elution flow rate of 1-2 BV.h^-1Collecting water eluent with the elution volume of 3-5 BV, concentrating, and drying under reduced pressure to obtain the product; the preparation process of the pure glucoraphanin comprises the steps of crushing fried radish seed decoction pieces into coarse powder, adding 20 times of the coarse powder, extracting for 15-30 min for 3 times, combining extracting solutions, and concentrating to 0.5 g/mL^-1Obtaining a fried radish seed extracting solution, and concentrating the fried radish seed extracting solution under reduced pressure until the mass concentration is 0.1-0.2 g/mL^-1The D301 chlorine type anion exchange resin is passed through, the sample loading amount is 7BV, and the adsorption flow rate of the sample loading liquid is 1-3 BV.h^-1Eluting with 3BV water, eluting with 3-5 BV NaCl solution with the concentration of 5% -10%,the elution flow rate is 1-2 BV.h^-1(ii) a Concentrating the collected eluate under reduced pressure, filtering, removing most of salt, adding water into the concentrated solution, performing nanofiltration to further remove salt, concentrating under reduced pressure, and freeze drying to obtain pure glucoraphanin.

The method for detecting the molecular weight and the structure of the pure metabolite comprises the following steps:

s1: taking a proper amount of the pure metabolite prepared in the step (2), adding distilled water for dissolving, passing through a 0.22 mu m microporous filter membrane, and determining the molecular weight of the pure metabolite by using a high performance liquid chromatography-time-of-flight mass spectrometer;

s2: taking a proper amount of the pure metabolite prepared in the step (2), adding DMSO (dimethyl sulfoxide) for dissolving, and obtaining a hydrogen spectrum of the compound by adopting a nuclear magnetic resonance spectrometer¹H-NMR and carbon Spectroscopy¹³C-NMR data; and (3) integrating mass spectrum and nuclear magnetic resonance detection results to confirm that the product is reduced glucoraphanin, namely 4-methylthio-3-butenyl thioglucose.

It is preferable that: the chromatographic conditions of the detection method for the molecular weight and the structure of the pure metabolite are as follows: the chromatographic column is an octadecylsilane chemically bonded silica gel column; the mobile phase A is 0.1% acetonitrile formate, and the mobile phase B is 0.1% formic acid water; the gradient elution time was: 0-5 min, 2% A, 5-8 min, 2% A-5% A; 8-10 min, 5-12.5% A; 10-12 min, 12.5% A-14% A; 12-25 min, 14% -25% A; 25-32 min, 25% A-100% A; 32-40 min, 100% A; the column temperature is 25-30 ℃; the flow rate was 0.3 mL/min^-1(ii) a 3 mu L of sample volume; the wavelength is 225 nm;

the time-of-flight mass spectrometer is an Agilent6230 time-of-flight mass spectrometer provided with a standard electrospray ion source, and the ion source is an electrospray ion source; in the negative ion detection mode, the scanning range is m/z 50-1000; the flow rate of the dry gas is 10 L.min^-1(ii) a The atomizer pressure is 30 Pa; the dryer temperature was 350 ℃ and the capillary voltage was 4 KV.

It is preferable that: the chromatographic column is a Halo-C18 column with the column length of 2.1mm multiplied by 100mm and the filler particle size of 2.7 mu m.

The method for detecting the purity of the pure metabolite comprises the following steps:

taking a proper amount of the pure metabolite prepared in the step (2), adding the initial mobile phase for dissolving, filtering the mixture through a 0.22 mu m microporous filter membrane, and calculating the purity of the mixture by adopting an area normalization method in a high performance liquid chromatograph.

It is preferable that: the chromatographic conditions are as follows: the chromatographic column adopts an octadecylsilane chemically bonded silica gel column, the mobile phase A is acetonitrile, and the mobile phase B is 0.1% phosphoric acid; the gradient elution time was: 0-5 min, 2% A, 5-8 min, 2% A-5% A; 8-10 min, 5-12.5% A; 10-12 min, 12.5% A-14% A; 12-25 min, 14% -25% A; 25-40 min, 25% A-50% A; 40-50 min, 50-70% A; 50-60 min, 70% A-100% A; the column temperature is 25-30 ℃; the flow rate is 1 mL/min^-1(ii) a The wavelength was 225 nm.

It is preferable that: the specification of the chromatographic column is an InertSuataiNAQ-C18 chromatographic column with the column length of 4.6mm multiplied by 250mm and the packing particle size of 5 mu m.

The invention has the beneficial effects that: under the action of intestinal bacteria liquid, most of glucoraphanin is converted into reduced glucoraphanin, the conversion efficiency is high, fewer impurities are generated, the subsequent purification difficulty is reduced, and the degradation rate of glucoraphanin is high; the medicine is inevitably contacted with intestinal flora in organisms, and certain medicine components can be absorbed only after metabolic conversion under the action of the intestinal flora. The pure reduced glucogel is adopted for separation to prepare the reduced glucogel, the purity of the obtained reduced glucogel is higher than 90%, and the pure product is pure white in color and strong in hygroscopicity. And carrying out structural identification on the prepared pure reduced glucoraphanin by adopting mass spectrum and nuclear magnetic resonance, and confirming that the product finally obtained is the reduced glucoraphanin by confirming that the product conforms to the molecular structure characteristics of the reduced glucoraphanin. The method can provide reference for extraction, purification and structure identification of reduced glucoraphanin in radish raw materials and other cruciferous vegetables, and also lay a foundation for development of health care products and functional foods containing reduced glucoraphanin.

Drawings

FIG. 1 is an HPLC chart of crude metabolite

FIG. 2 is an HPLC chart after metabolite purification

FIG. 3 is a chromatogram of three groups of comparative experiments

FIG. 4 shows the peak mass-to-charge ratio m/z of the control group at t-1.464: 434

FIG. 5 shows that the peak mass-to-charge ratio m/z of the administration group at t 1.464 is 434

FIG. 6 shows the peak mass-to-charge ratio m/z 418 at t 10.563 for the administration group

FIG. 7 shows the metabolic reaction equation of glucoraphanin

FIG. 8 is the NMR spectrum of pure metabolite

FIG. 9 is the NMR spectrum of pure metabolite

FIG. 10 shows the molecular formula of reduced glucoraphanin

FIG. 11 is a graph showing the degradation of glucoraphanin and the generation of metabolites

Detailed Description

The first embodiment is as follows: a method for preparing reduced glucoraphanin by intestinal bacteria metabolism comprises the following steps:

(1) preparation of anaerobic culture solution

Solution A: 0.2925g of dipotassium hydrogen phosphate was weighed out accurately, and distilled water was added thereto to 37.5ml to dissolve it.

And B, liquid B: 0.1763g of dipotassium hydrogen phosphate, 0.4425g of sodium chloride, 0.4500g of ammonium sulfate, 0.0450g of calcium chloride and MgSO are accurately weighed₄·H₂O0.0938 g, and distilled water 37.5ml were added and dissolved.

And C, liquid C: 4g of sodium carbonate is accurately weighed, and distilled water is added to 50ml for dissolution.

Then adding 0.5g of L-cysteine, 2ml of 25% L-ascorbic acid by volume fraction, 1g of beef extract, 1g of tryptone and 1g of nutrient agar, adding distilled water to 1L, adjusting the pH value to 7.0-7.5 by hydrochloric acid to obtain an anaerobic culture solution, and placing the anaerobic culture solution into a refrigerator at 4 ℃ for later use.

(2) Preparation of rat intestinal bacteria liquid

Taking 2 healthy SD male rats, fasting for 12h without water prohibition, weighing 0.4g of fresh excrement, placing the fresh excrement into a 2mL EP tube, adding 4 times of physiological saline, mixing, fully and uniformly mixing in a tissue homogenizer to obtain a suspension, placing the suspension into a centrifuge, and performing 5000 r.min^-1Centrifuging for 10min, collecting supernatant as rat intestinal canal bacterial liquid, and placing in a refrigerator at 4 deg.C for use.

(3) Preparation of Enterobacter culture solution

2mL of the prepared rat intestinal bacteria liquid for later use in the refrigerator is placed in a 50mL triangular flask sterilized by high-pressure steam, 18mL of sterile anaerobic culture solution is added, and the mixture is fully mixed to obtain 20mL of the intestinal bacteria culture solution. Placing the intestinal bacteria culture solution in an anaerobic culture box, adding an anaerobic gas-producing bag, quickly covering an anaerobic culture box cover, placing the anaerobic culture box in a 37 ℃ gas bath constant temperature oscillation box, culturing for 24h, and taking out.

(4) Preparation of sample liquid

4.1 preparation of test solution sample

a. Crushing the fried radish seed decoction pieces into coarse powder, adding 20 times of the coarse powder, extracting for 15-30 min for 3 times, combining extracting solutions, and concentrating to 0.5 g/mL^-1。

b. Precisely weighing 20mg of fried radish seed extract, adding distilled water into a 10mL volumetric flask, and ultrasonically dissolving to a constant volume. The mass concentration is 2 mg/mL^-1The glucoraphanin extract solution is ready for use.

4.2 Enterobacter culture experiment

And (4) taking out the enterobacteria culture solution in the step (3) from the culture bottle. 5mL of the glucoraphanin extract solution was added to 10mL of the enterobacter culture solution to obtain a sample solution. Placing the triangular flask containing the sample solution in an anaerobic incubator, adding an anaerobic gas bag, and quickly covering an anaerobic culture cover to ensure an anaerobic environment. Placing the mixture in a 37 ℃ gas bath constant temperature oscillation box, and taking out after 8 hours. The instruments used in the experiment were sterilized with high pressure steam at 121 ℃ for 20 min.

(5) Preparation of metabolites

The sample solution obtained in 4.2 was placed in a rotary evaporator at a temperature of 50 ℃, rotated to dryness, transferred with a small amount of methanol, and passed through a 0.45 μm microporous membrane to obtain a crude solution containing the metabolite, the liquid phase detection of which is shown in FIG. 1. And (3) putting the crude product solution on SephadexLH-20, eluting with methanol, collecting in sections, carrying out HPLC detection, collecting eluent containing metabolites, putting the eluent into a rotary evaporator, carrying out rotary evaporation at the temperature of 50 ℃ until the eluent is dried, transferring with a small amount of water, and carrying out freeze drying to obtain a pure metabolite, namely the reduced glucoraphanin, wherein liquid phase detection is shown in figure 2.

(6) Determination of molecular weight and Structure of pure metabolite

S1: and (3) adding distilled water into a proper amount of the pure metabolite prepared in the step (5) for dissolving, passing through a 0.22-micron microporous filter membrane, and determining the molecular weight of the reduced glucoraphanin by using a high performance liquid chromatography-time of flight mass spectrometer, wherein as can be seen in fig. 6, the molecular ion peak mass-to-charge ratio m/z of the pure metabolite is 418. A16 reduction was found by comparison with the glucoraphanin mass-to-charge ratio m/z 434, which presumably was a reduction of one O atom, by the glucoraphanin molecular formula [ glu-S-C ]₆H₉NS₂O₅]The molecular formula of the metabolite can be presumed to be [ glu-S-C ]₆H₉NS₂O₄]The reaction equation is shown in figure 7, the chromatographic column is a Halo-C18 column (2.1mm × 100mm, 2.7 mu m), the mobile phase A is 0.1% of formic acid acetonitrile, the mobile phase B is 0.1% of formic acid water, the gradient elution time is 0-5 min, 2% A, 5-8 min, 2% A-5% A, 8-10 min, 5% A-12.5% A, 10-12 min, 12.5% A-14% A, 12-25 min, 14% A-25% A, 25-32 min, 25% A-100% A, 32-40 min and 100% A, the column temperature is 27 ℃, and the flow rate is 0.3 mL-min^-1(ii) a 3 mu L of sample volume; the wavelength is 225 nm;

the time-of-flight mass spectrometer is an Agilent6230 time-of-flight mass spectrometer equipped with a standard electrospray ion source, and the ion source is an electrospray ion source; in the negative ion detection mode, the scanning range is m/z 50-1000; the flow rate of the dry gas is 10 L.min^-1(ii) a The atomizer pressure is 30 Pa; the dryer temperature was 350 ℃ and the capillary voltage was 4 KV.

S2: taking a proper amount of the pure metabolite prepared in the step (5), adding DMSO to dissolve, and obtaining a hydrogen spectrum of the compound (a)¹HNMR) (see FIG. 8) and carbon spectrum (C: (C)¹³C-NMR) data (see FIG. 9).¹H-NMR(400MHz，D₂O-d6)H：4.95(d，1H，H-1′)，3.30～3.55(m，H-2′，3′，4′，5′)，3.64(m，1H，H₁-6a)，3.80(m，1H，H₁-6b)，2.70(t，1H，H₁-8)，2.42(q，1H，H₁-9)，5.47(m，1H，H₁-10)，6.10(d，1H，H₁-11)，2.17(s，3H，H₃-12)。¹³CNMR(400MHz，DMSO-d6)C：82.47(C1)，73.29(C2)，78.54(C3)，70.28(C4)，81.69(C5)，61.32(C6)，155.82(C7)，32.28(C8)，30.88(C9)，125.34(C10), 125.11(C11), 14.52 (C12). Obtaining NMR spectra of metabolites using an AV400 NMR spectrometer (Bruker Corp.) (¹H-NMR) and nuclear magnetic resonance carbon Spectroscopy (C¹³C-NMR) data, and determining that the molecular structure data conforms to the molecular structure characteristics of the reduced glucoraphanin through the analysis of hydrogen spectrum data and carbon spectrum data. The mass spectrum and nuclear magnetic resonance detection results are integrated to confirm that the extracted and prepared product is reduced glucoraphanin, namely 4-methylthio-3-butenyl thioglucoside (4-Methylsulfanyl-3-butenylglucosinolate), the specific chemical structure of the product is shown in figure 10, and the nuclear magnetic resonance hydrogen spectrum and the nuclear magnetic resonance carbon spectrum are used for confirming the metabolite structure.

Example two: a method for preparing reduced glucoraphanin by intestinal bacteria metabolism comprises the following steps:

(1) preparation of Enterobacter culture solution

Please refer to the experimental procedure for preparing the culture solution of Enterobacter in example I;

(2) preparation of sample liquid

2.1 preparation of test solution sample

a. Crushing the fried radish seed decoction pieces into coarse powder, adding 20 times of the coarse powder, extracting for 15-30 min for 3 times, combining extracting solutions, and concentrating to 0.5 g/mL^-1Adding the concentrated radish seed extract into AB-8 macroporous adsorbent resin at a sample loading amount of 0.5-0.75 BV and a sample loading flow rate of 0.5-1 BV.h^-1Eluting with water at an elution flow rate of 1-2 BV.h^-1And (3) collecting water eluent with the elution volume of 3-5 BV, concentrating, and drying under reduced pressure to obtain the active ingredients of the sulforaphane seed.

b. Precisely weighing 20mg of the sulforaphane effective part of the radish seed, adding distilled water into a 10mL volumetric flask, ultrasonically dissolving, and fixing the volume. The mass concentration is 2 mg/mL^-1The glucoraphanin extract solution is ready for use.

2.2 Enterobacter culture experiment

The culture solution of the Enterobacter according to the step (3) in example one was taken out from the culture flask. 5mL of the glucoraphanin extract solution was added to 10mL of the enterobacter culture solution to obtain a sample solution. Placing the triangular flask containing the sample solution in an anaerobic incubator, adding an anaerobic gas bag, and quickly covering an anaerobic culture cover to ensure an anaerobic environment. Placing the mixture in a 37 ℃ gas bath constant temperature oscillation box, and taking out after 8 hours. The instruments used in the experiment were sterilized with high pressure steam at 121 ℃ for 20 min.

(3) Preparation of metabolites

Placing the sample solution obtained in step (2) in a rotary evaporator, setting the temperature at 50 deg.C, rotating to dry, transferring with a small amount of methanol, and passing through 0.45 μm microporous membrane to obtain crude product solution containing metabolite, and detecting liquid phase as shown in FIG. 1. And (3) loading the crude product solution on SephadexLH-20, eluting with methanol, collecting by sections, carrying out HPLC detection, combining eluates containing metabolites, placing in a rotary evaporator, carrying out rotary evaporation at the temperature of 50 ℃ until the eluates are dried, transferring with a small amount of water, and carrying out freeze drying to obtain a pure metabolite product, wherein liquid phase detection is shown in figure 2.

Example three: a method for preparing reduced glucoraphanin by intestinal bacteria metabolism comprises the following steps:

(1) preparation of Enterobacter culture solution

(2) preparation of samples

2.1 preparation of test solution sample

a. Crushing the fried radish seed decoction pieces into coarse powder, adding 20 times of the coarse powder, extracting for 15-30 min for 3 times, combining extracting solutions, and concentrating to 0.5 g/mL^-1Obtaining a fried radish seed extracting solution, and concentrating the fried radish seed extracting solution under reduced pressure until the mass concentration is 0.1-0.2 g/mL^-1The D301 chlorine type anion exchange resin is passed through, the sample loading amount is 7BV, and the adsorption flow rate of the sample loading liquid is 1-3 BV.h^-1Eluting with 3BV water and 3-5 BV NaCl solution with the concentration of 5% -10%, and the elution flow rate is 1-2 BV.h^-1(ii) a The collected eluate was concentrated under reduced pressure, filtered and most of the salts were removed. Adding water into the concentrated solution, performing nanofiltration to further remove salt, concentrating under reduced pressure, and freeze drying to obtain pure glucoraphanin.

b. Precisely weighing 20mg of pure glucoraphanin, adding distilled water into a 10mL volumetric flask, ultrasonically dissolving, and fixing the volume. The mass concentration is 2 mg/mL^-1The glucoraphanin extract solution is ready for use.

2.2 Enterobacter culture experiment

(3) Preparation of metabolites

Placing the sample solution obtained in step (2) in a rotary evaporator, setting the temperature at 50 deg.C, rotating to dry, transferring with a small amount of methanol, and passing through 0.45 μm microporous membrane to obtain crude product solution containing metabolite, and detecting liquid phase as shown in FIG. 1. Loading the crude product solution on SephadexLH-20 sephadex column, eluting with methanol, collecting by stages, performing HPLC detection, mixing eluates containing metabolite, placing in rotary evaporator, rotary evaporating at 50 deg.C to dry, transferring with small amount of water, and freeze drying to obtain pure metabolite, wherein the liquid phase detection is shown in FIG. 2.

Example four: a detection method of reduced glucoraphanin comprises the following steps:

(1) preparation of samples

Referring to the procedure for preparing a pure reduced glucoraphanin in example one, the enterobacter culture solution obtained in step (3) in example one was taken out from the culture flask and evenly divided into 2 portions of 10mL each. The experiment was divided into administration group, blank group, and control group. In the step (5) of example, 5mL of the glucoraphanin extract was added to 10mL of the culture medium of enterobacteria to obtain an administration group, 10mL of the culture medium of enterobacteria to obtain a blank group, and 5mL of the glucoraphanin extract was added to 10mL of the anaerobic culture medium to obtain a control group. The triangular flask is placed in an anaerobic incubator, an anaerobic air bag is added, an anaerobic culture cover is quickly covered, and an anaerobic environment is ensured. Placing the mixture in a 37 ℃ gas bath constant temperature oscillation box, and taking out after 8 hours. The instruments used in the experiments were all sterilized by high pressure steam at 121 ℃ for 20 minutes.

(2) Sample processing method

The administration group (medicine + enterobacteria culture solution), the control group (medicine + anaerobic culture solution) and the blank in the step (1)Transferring the group (Enterobacter culture solution) into 50mL centrifuge tubes, adding 5mL methanol to terminate the reaction, vortex, mixing, and shaking for 3min, 8000r min^-1Centrifugation was carried out for 10min, and the supernatant was passed through a 0.22 μm microfiltration membrane and into an Agilent6230 time-of-flight mass spectrometer (Agilent, USA, equipped with a standard electrospray ion source).

(3) Analysis of

3.1Agilent6230 model time-of-flight mass spectrometer atlas analysis

The TIC under the negative ion mode is adopted for analysis, and the Qualitativeanalysis mass spectrometry software is applied to the analysis of three chromatograms of an empty group, a control group and an administration group, so that the peaks in the groups are well separated and are favorable for observation, and the figure 3 shows that the peaks in the groups are well separated. By comparing the administered group and the control group, it was found that both groups showed peaks at a retention time t of 1.464min and a mass-to-charge ratio m/z of 434, confirming that both the administered group and the control group contained glucoraphanin, as shown in fig. 4 and 5. However, it can be seen from FIG. 3 that the peak area (1353244) of glucoraphanin in the administered group is significantly smaller than that in the control group (3098980), i.e., glucoraphanin content is significantly reduced and glucoraphanin is metabolized. In addition, as can be seen from the administration group in fig. 3, a new molecular ion peak with a peak area of 6344342 appeared at a retention time t of 10.563min, whereas the blank group and the control group did not have the peak at the same retention time, and thus it was presumed that the peak was a new metabolite with a molecular ion peak mass-to-charge ratio m/z of 418 (see fig. 6). A16 reduction was found by comparison with the glucoraphanin mass-to-charge ratio m/z 434, which presumably was a reduction of one O atom, by the glucoraphanin molecular formula [ glu-S-C ]₆H₉NS₂O₅]The molecular formula of the metabolite can be presumed to be [ glu-S-C ]₆H₉NS₂O₄]The reaction equation is shown in FIG. 7.

Example five: a method for detecting the purity of reduced glucoraphanin comprises the following steps:

(1) preparation of sample liquid

Please refer to the preparation process of the reduced glucoraphanin in the first embodiment.

(2) Detection of metabolites

2.1 determination of purity and analysis thereof

Examples of the inventionTransferring the sample solution obtained by culturing in step one (5) to 50ml centrifuge tubes, adding 5ml methanol to terminate reaction, vortex mixing, shaking for 3min, 8000r min^-1Centrifuging for 10min, collecting supernatant, filtering with 0.22 μm microporous membrane, collecting appropriate amount of reducing sulforaphane prepared in step (2), dissolving with initial mobile phase, filtering with 0.22 μm microporous membrane, and performing high performance liquid chromatograph with area normalization method to calculate its purity.

Detecting and analyzing metabolite purity by using a high performance liquid chromatograph under the conditions of using an InertSuataiQ-C18 (4.6mm × 250mm, 5 mu m) chromatographic column and gradient elution (0-5 min, 2% A, 5-8 min, 2% A-5% A, 8-10 min, 5% A-12.5% A, 10-12 min, 12.5% A-14% A, 12-25 min, 14% A-25% A, 25-40 min, 25% A-50% A, 40-50 min, 50% A-70% A, 50-60 min, 70% A-100% A) by using acetonitrile (A) -0.1% phosphoric acid (B), wherein the column temperature is 27 ℃, and the flow rate is 1 mL/min^-1Wavelength: 225 nm.

Detecting and analyzing the metabolite purity by a high performance liquid chromatograph, wherein the purity of the prepared reduced glucoraphanin is more than 90%.

Example six: glucoraphanin intestinal bacteria degradation experiment

First, experiment content

(1) Sample preparation and sample purification

Please refer to the sample preparation and purification steps in example one.

(2) Detection of

Samples with different incubation times are respectively prepared by the test method of the step 4.2 in the example, and are put into a chromatograph for analysis and detection, and the peak area change of characteristic chromatographic peaks of different intestinal flora incubation times is shown in fig. 11 and table 1.

Second, recording experimental data

TABLE 1 Peak area Change of characteristic chromatographic Peak for incubation time of different intestinal flora

Third, conclusion and analysis

From fig. 11, under the action of the intestinal flora of the rat, the peak area of the peak 1 plus the peak 2 (glucoraphanin) both decrease with the increase of the co-incubation time, and the peak area decreases slowly in the first 8 h; when the peak area of the No. 1 peak is changed to 69.97% of 0h and the peak area of the No. 2 peak is 68.47% of 0h by 8 h; by 10h, the peak areas of the No. 1 and No. 2 peaks are obviously reduced, the No. 1 peak is 32.39% of 0h, and the No. 2 peak is 28.81% of 0 h. The peak area of the No. 3 chromatographic peak gradually increases along with the reduction of the No. 1 and No. 2 peaks; and when the time is 24 hours, the peak area of the No. 3 peak reaches the maximum value, and the UV spectrums of the three peaks are basically the same. Therefore, it is assumed that

peaks

1 and 2 are degraded by the action of rat intestinal flora, and the metabolite peak is peak 3.

As can be seen from Table 1, the degradation rates of glucoraphanin and the methanol converted products thereof are respectively 0%, 1.5%, 2.5%, 4.0% and 4.5% at 0h, 0.25h, 0.5h, 1h and 2h, and the degradation rate is slow; the degradation rate of the glucoraphanin is 5.2%, 8.1%, 18.3%, 30.0% and 68.8% in sequence at 3h, 4h, 6h, 8h and 10h, the glucoraphanin is greatly degraded under the action of the intestinal bacteria in the period of time, and the degradation rate is fastest within 8-10 h; the degradation curve tended to be flat after 10h, but was still degrading.

The generation rates of the glucoraphanin metabolites are 0, 0.90%, 1.13%, 1.23%, 5.10%, 8.79%, 18.76% and 26.49% in sequence at 0h, 0.25h, 0.5h, 1h, 2h, 3h, 4h and 6h, and the generation rates are slowly increased; the generation rate of the glucoraphanin metabolite is 50.00 percent and 111.50 percent at 8h and 10h, and the glucoraphanin metabolite is produced in a large amount in the period; the metabolite production rate of glucoraphanin rose slowly after 10 hours, but it was still rising.

Although particular embodiments of the present description have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these particular embodiments without departing from the principles and spirit of the description, the scope of which is defined in the appended claims and their equivalents.

Claims

1. The method for preparing reduced glucoraphanin by adopting intestinal bacterium metabolism is characterized by comprising the following steps:

(1) preparation of sample liquid

the preparation process of the intestinal fungus culture solution comprises the steps of preparing an anaerobic culture solution for later use; adding normal saline into the feces of the healthy human or animals, mixing the feces and the feces, fully and uniformly mixing the feces and the feces to obtain a suspension, putting the suspension into a centrifuge, centrifuging the suspension for 8-13 min, and taking the supernatant after centrifugation to obtain intestinal bacteria liquid for later use; finally, adding the intestinal bacteria liquid into a sterile anaerobic culture solution, fully mixing, and culturing in an anaerobic culture box for 24 hours to obtain an intestinal bacteria culture solution;

(2) preparation of reducing glucoraphanin

2. The method for producing reduced glucoraphanin according to claim 1, wherein: the glucoraphanin extract in the step (1) is fried radish seed extract, radish seed thioglycoside effective parts or glucoraphanin pure products.

3. The method for producing reduced glucoraphanin according to claim 2, wherein: the semen Raphani preparata extractive solution is prepared by decocting semen Raphani preparataPulverizing into coarse powder, adding 20 times of the powder, extracting for 15-30 min for 3 times, mixing the extractive solutions, and concentrating to 0.5 g/mL^-1(ii) a The preparation process of the sulforaphane effective part of the radish seed comprises the steps of crushing fried radish seed decoction pieces into coarse powder, adding 20 times of the coarse powder, extracting for 15-30 min for 3 times, combining extracting solutions, and concentrating to 0.5 g/mL^-1Adding the concentrated radish seed extract into AB-8 macroporous adsorbent resin at a sample loading amount of 0.5-0.75 BV and a sample loading flow rate of 0.5-1 BV.h^-1Eluting with water at an elution flow rate of 1-2 BV.h^-1Collecting water eluent with the elution volume of 3-5 BV, concentrating, and drying under reduced pressure to obtain the product; the preparation process of the pure glucoraphanin comprises the steps of crushing fried radish seed decoction pieces into coarse powder, adding 20 times of the coarse powder, extracting for 15-30 min for 3 times, combining extracting solutions, and concentrating to 0.5 g/mL^-1Obtaining a fried radish seed extracting solution, and concentrating the fried radish seed extracting solution under reduced pressure until the mass concentration is 0.1-0.2 g/mL^-1The D301 chlorine type anion exchange resin is passed through, the sample loading amount is 7BV, and the adsorption flow rate of the sample loading liquid is 1-3 BV.h^-1Eluting with 3BV water and 3-5 BV NaCl solution with the concentration of 5% -10%, and the elution flow rate is 1-2 BV.h^-1Concentrating the collected eluent under reduced pressure, filtering, removing most of salt, adding water into the concentrated solution, further removing salt through nanofiltration, concentrating under reduced pressure, and freeze-drying to obtain the pure product of glucoraphanin.

4. The method for preparing reduced glucoraphanin by intestinal bacteria metabolism according to claim 1, wherein the method for detecting the molecular weight and structure of the pure metabolite in step (2) comprises the following steps:

s2: taking a proper amount of the pure metabolite prepared in the step (2), adding DMSO (dimethyl sulfoxide) for dissolving, and obtaining a hydrogen spectrum of the compound by adopting a nuclear magnetic resonance spectrometer¹H-NMR and carbon Spectroscopy¹³C-NMR data; the product is confirmed to be reduced type glucoraphanin, namely 4-methylthio by combining mass spectrum and nuclear magnetic resonance detection results3-butenyl-thioglucose;

the chromatographic conditions comprise that a chromatographic column is a Halo-C18 column with the column length of 2.1mm × 100mm and the filler particle size of 2.7 mu m, a mobile phase A is 0.1% of formic acid acetonitrile, a mobile phase B is 0.1% of formic acid water, the gradient elution time is 0-5 min, 2% A, 5-8 min, 2% A-5% A, 8-10 min, 5% A-12.5% A, 10-12 min, 12.5% A-14% A, 12-25 min, 14% A-25% A, 25-32 min, 25% A-100% A, 32-40 min and 100% A, the column temperature is 25-30 ℃, and the flow rate is 0.3 mL/min^-1(ii) a 3 mu L of sample volume; the wavelength is 225 nm;

5. The method for preparing reduced glucoraphanin by intestinal bacteria metabolism according to claim 1, wherein the method for detecting the purity of the pure metabolite in step (2) comprises the following steps:

taking a proper amount of the pure metabolite prepared in the step (2), adding the initial mobile phase for dissolution, filtering the mixture through a 0.22 mu m microporous filter membrane, feeding the mixture into a high performance liquid chromatograph, and calculating the purity of the mixture by adopting an area normalization method;

the chromatographic conditions comprise that the chromatographic column adopts an InertSuataiNQC 18 chromatographic column with the column length of 4.6mm × 250mm and the filler particle size of 5 mu m, the mobile phase A is acetonitrile, the mobile phase B is 0.1% of phosphoric acid, the gradient elution time is 0-5 min, 2% A, 5-8 min, 2% A-5% A, 8-10 min, 5% A-12.5% A, 10-12 min, 12.5% A-14% A, 12-25 min, 14% A-25% A, 25-40 min, 25% A-50% A, 40-50 min, 50% A-70% A, 50-60 min, 70% A-100% A, the column temperature is 25-30 ℃, and the flow rate is 1 mL/min^-1(ii) a The wavelength was 225 nm.