Fermentation extract C21 steroid compound as well as biological fermentation method and application thereof

Abstract

The invention discloses a radix cynanchi bungei C21 steroid compound as well as a biological fermentation method and application thereof. Fresh radix cynanchi bungei tuberous root slurry is subjected to enzymolysis treatment through a magnetic nano-composite enzyme preparation, obtained saccharified liquid is fermented through agrobacterium tumefaciens F-45, obtained fermentation liquid is freeze-dried, obtained freeze-dried powder is extracted with absolute ethyl alcohol, supernate is collected, finally, the supernate is added into a precipitation agent for treatment, and the radix cynanchi bungei tuberous root extract is obtained. And volatilizing the obtained precipitate in a water bath at 40 DEG C until no liquid is left, wherein the precipitate is the C21 steroid compound. According to the fermentation system constructed by the invention, the absolute amount of the C21 steroid compound is increased by 4.3 times compared with that of a root tuber raw material, and the C21 steroid compound is rich in variety, high in yield, short in production period and easy for large-scale industrial production; the obtained fermentation extract C21 steroid compound has a good protection effect on liver cell injury caused by oxidative stress.

Description

Technical Field

[0001] The present invention belongs to the field of biological fermentation technology and particularly relates to a fermentation extract C 21 Steroidal compounds and their biofermentation methods and applications. Background Art

[0002] C 21 Steroid compounds are the main components of the pharmacological activity of Baishouwu, which have multiple pharmacological effects such as protecting the liver, resisting oxidative damage and resisting liver fibrosis. 21 Steroid compounds can protect the liver by increasing the metabolic function of liver cells and antagonizing lipid peroxidation. 2 Combined modeling induced inflammatory response and found that C 21 Steroidal compounds can not only significantly reduce the levels of AST, ALT, ALP, BUG, ​​and LDH in serum. 21 Steroid compounds have antioxidant effects and can inhibit oxidative damage caused by strenuous exercise. Fang Xixiu and other studies have shown that Radix Polygoni Multiflori can reduce lipid peroxidation by reducing the amount of metabolite malondialdehyde. A large number of studies have shown that Radix Polygoni Multiflori C 21 Steroidal compounds have good preventive and therapeutic effects on liver fibrosis. Steroidal total glycosides of Cynanchum multiflorum can significantly reduce the liver and spleen index, serum GPT, HA, PCIII levels and HyP content in liver tissue in rats with liver fibrosis induced by carbon tetrachloride. 2 In rats with liver and kidney fibrosis caused by 21 Steroidal compounds can not only significantly reduce the levels of AST, ATL, ALP, BUN, and LDH in serum, but also significantly reduce the levels of TBIL and UA in serum.

[0003] In summary, 21 Steroid compounds have good pharmacological effects such as anti-liver fibrosis and anti-tumor. 21 The extraction of steroidal compounds is all done with the root tuber of Cynanchum multiflorum as raw material, and the extraction process is first ethanol reflux, and then purification by macroporous resin adsorption. However, this method is limited by the source of raw materials, the method cost is high, and the extraction time is long. Summary of the invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a fermentation extract C 21 Steroidal compounds and their biofermentation methods and applications.

[0005] The present invention is achieved by a fermentation extract C 21 A biological fermentation method for steroidal compounds, the method comprising the following steps:

[0006] (1) Fresh tubers of Radix Cynanchum multiflorum and water were mixed at a mass volume ratio of 5-7 g:1 mL and then pulped. A magnetic nanocomposite enzyme preparation was added at a mass ratio of pulp to enzyme preparation of 2.5-3:1. The mixture was subjected to enzymatic hydrolysis at 37°C for 2 h, and the enzyme preparation was recovered by magnetic force to obtain a saccharified liquid.

[0007] (2) diluting the saccharified liquid with Gao's medium No. 1 and inoculating Agrobacterium tumefaciens F-45 at an inoculation amount of 1 to 3%, and culturing for 120 hours at 32° C., pH 8, and shaking at 120 rpm to obtain a fermentation liquid;

[0008] (3) freeze-drying the fermentation broth, adding the resulting freeze-dried powder into anhydrous ethanol, homogenizing, and standing at room temperature for 1 to 2 hours, and collecting the supernatant alcohol extract;

[0009] (4) Add the alcohol extract to the precipitant, mix well and let stand at room temperature for 2 to 3 hours, filter the precipitate and collect the precipitate, place the precipitate in a 40°C water bath and evaporate until no liquid remains. The precipitate is C 21 Steroid compounds.

[0010] Preferably, in step (2), the volume ratio of the saccharification solution to Gao's medium No. 1 is 1:5-7.

[0011] Preferably, in step (3), the freeze-drying is specifically: pre-freezing at -20°C for 24 hours, and then freeze-drying in a vacuum environment at -60°C for 48 hours.

[0012] Preferably, in step (3), the mass volume ratio of the lyophilized powder to anhydrous ethanol is 1 g: 3-4 mL.

[0013] Preferably, in step (4), the volume ratio of the alcohol extract to the precipitating agent is 1:3-4.

[0014] Preferably, in step (4), the precipitating agent is composed of ether and acetone in a volume ratio of 1:1 to 2.

[0015] The present invention further discloses the fermentation extract C prepared by the above method. 21 Steroid compounds.

[0016] The present invention further discloses the fermentation extract C 21 Application of steroidal compounds in the preparation of drugs for repairing liver cell damage.

[0017] Preferably, the hepatocyte damage is hepatocyte damage caused by oxidative stress.

[0018] The present invention overcomes the deficiencies of the prior art and provides a 21Steroid compound and its biofermentation method and application. In the present invention, the fresh tuberous root pulp of Radix Cynanchus multiflori is enzymatically treated by a magnetic nanocomposite enzyme preparation, the obtained saccharified liquid is fermented by Agrobacterium tumefaciens F-45, the obtained fermentation liquid is freeze-dried, the obtained freeze-dried powder is extracted with anhydrous ethanol, the supernatant is collected, and finally the supernatant is treated with a precipitant, and the obtained precipitate is placed in a 40°C water bath to evaporate until no liquid remains, and the precipitate is C 21 Steroid compounds. In the present invention, Agrobacterium tumefiaciens F-45 was deposited in the China Center for Type Culture Collection on September 13, 2021, with a deposit number of CCTCCNO: M 20211166, and a deposit address of: Wuhan University Collection Center, Luojia, Wuchang District, Wuhan City, with a postal code of 430072.

[0019] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention fully considers the interaction between endophytes and plants to produce C 21 The principle of steroidal compounds, the development of a new fermentation system to produce C 21 Steroids;

[0021] (2) Based on the magnetic nanocarrier constructed in the early stage, the present invention creates a composite enzyme (amylase + saccharifying enzyme) cross-linked enzyme preparation, which efficiently degrades the root pulp of Radix Cynanchifolii with a high material recovery rate;

[0022] (3) The fermentation medium formula of the present invention is to add the saccharified liquid of Radix Cynanchum multiflorum to the basic medium, which effectively promotes the growth of endophytic bacteria F-45 and increases the C 21 Metabolic levels of steroids;

[0023] (3) The present invention constructs a fermentation system, C 21 The absolute amount of steroidal compounds is 4.3 times higher than that of the root raw material;

[0024] (4) Fermentation product C in the present invention 21 Steroid compounds are rich in variety, high in yield, short in production cycle, and easy to produce on a large scale industrially;

[0025] (5) Fermentation extract C of the present invention 21 The steroid compounds can reduce the MDA content, LDH enzyme activity and GOT enzyme activity in formaldehyde-induced mouse liver injury cells AML-12, and all indicators are better than those of the root source C 21 The steroid treatment group showed that the fermentation extract C 21 Steroidal compounds have a good protective effect on hepatocyte damage caused by oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the colony morphology of the endophytic actinomycete F-45 of Cynanchum multiflorum in the present invention;

[0027] Figure 2 It is a magnetic representation diagram of the cross-linked enzyme preparation carrier in the present invention;

[0028] Figure 3 is C in the fermentation liquid of Agrobacterium tumefaciens F-45 of the present invention 21 Steroidal glycoside content;

[0029] Figure 4 The C 21 Comparison of the mass of steroidal compounds;

[0030] Figure 5 The effect of different volume ratios of alcohol extract and precipitant on the purification effect of the extract in the present invention;

[0031] Figure 6 The root C in the present invention 21 Full-band scanning results of steroid compound samples;

[0032] Figure 7 The bacterial source C of the present invention 21 Full-band scanning results of steroid compound samples;

[0033] Figure 8 C in the fermentation system of the present invention 21 Full-band scanning results of steroid compound samples;

[0034] Fig. 9 The fermentation extract C of the present invention 21 Effects of steroidal compounds on the survival rate of hepatocytes damaged by formaldehyde oxidation;

[0035] Fig.10 The fermentation extract C of the present invention 21 Effects of steroidal compounds on GOT enzyme activity in formaldehyde-damaged hepatocytes;

[0036] Fig.11 The fermentation extract C of the present invention 21 Effects of steroidal compounds on LDH enzyme activity in formaldehyde-damaged hepatocytes; Fig.12 The fermentation extract C of the present invention 21 Effects of steroidal compounds on MDA content in formaldehyde-damaged hepatocytes. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0038] 1. Raw material pretreatment

[0039] 1. Preparation of magnetic nanocomposite enzyme preparation

[0040] (1) Take 5g of graphite and NaNO 3 2g was mixed and 120mL of concentrated H 2 SO 4 , react for 2h, add 20g KMnO 4 , react for a while, continue to react in a 40℃ warm water bath for a while, add 230mL of deionized water, stir in a 98℃ water bath for 5min, add H 2 O 2 The mixture was washed with 5% hydrochloric acid and deionized water for several times until no bubbles were generated, and the washing solution was neutral, and centrifuged at 10,000 r / min for 5 min. After freeze-drying, graphene oxide GO with a high degree of oxidation was obtained;

[0041] (2) Solution A: 0.5 g GO was added to 200 mL ultrapure water and ultrasonicated for 3 h; Solution B: 3 g FeCl 3 6H 2 O and 2.1g FeSO 4 7H 2 O, add 100mL ultrapure water, stir vigorously in a water bath at 80℃, add ammonia-ammonium nitrate buffer. Add solution A to solution B, ultrasonicate at 80℃ for 45min, and stir magnetically for 6h. Wash with anhydrous ethanol and ultrapure water three times respectively, and precipitate and dry in a vacuum oven to obtain GO / Fe 3 O 4 .

[0042] (3) 100 mg GO / Fe 3 O 4 Dispersed in 100 mL of distilled water for 2 h, 100 mg of EDC and 80 mg of NHS were added, stirred for 2 h, and the activated GO / Fe was collected by a magnet. 3 O 4 (See Figure 1 );

[0043] (4) Compound enzyme (amylase and saccharifying enzyme in a mass ratio of 1:1) and activated GO / Fe 3 O 4 The cross-linking was carried out at a mass ratio of 1:99 to prepare a cross-linked enzyme preparation.

[0044] 2. Preparation of saccharification solution

[0045] Fresh tuberous roots of Cynanchum multiflorum and distilled water were mixed at a mass volume ratio of 5-7 g:1 mL, and the mixture was slurried. A magnetic nanocomposite enzyme preparation was added at a mass ratio of slurry to enzyme preparation of 2.5-3:1. The mixture was enzymatically hydrolyzed at 37°C for 2 h, and the enzyme preparation was recovered by magnetic force to obtain a saccharified liquid.

[0046] 2. Construction of Agrobacterium tumefaciens fermentation system

[0047] 1. Bacteria activation

[0048] Agrobacterium tumefaciens F-45 was inoculated into Gao's No. 1 medium and activated for 48 to 72 hours, with an inoculation amount of 1%, 37°C, and 150 rpm.

[0049] Gao's medium No. 1: soluble starch 20.0 g, potassium nitrate 1.0 g, dipotassium hydrogen phosphate 0.5 g, sodium chloride 0.5 g, magnesium sulfate 0.5 g, ferrous sulfate 0.01 g, water 1,000 mL, pH 7.2-7.4.

[0050] 2. Fermentation medium formula

[0051] The saccharified liquid obtained from the pretreatment was homogenized with Gao's medium No. 1 at a volume ratio of 1:5-7 to prepare a fermentation medium, and the pH was adjusted to 8.

[0052] 3. Fermentation culture

[0053] The activated Agrobacterium tumefaciens F-45 was transferred to the fermentation medium with an inoculation amount of 1-3%, 32°C, 120rpm, and pH 8. The C content of the fermentation system was measured every 24h. 21 Total steroidal glycosides content.

[0054] 4. C in the fermentation system 21 Dynamic detection of steroid compound content

[0055] Take an appropriate amount of fermentation liquid and centrifuge at 10,000rpm for 10 minutes. Take 1mL of supernatant in a test tube and react with the optimized vanillin colorimetric method. After the reaction is completed, transfer to an ice water bath immediately, stop the reaction and add 10mL of glacial acetic acid and mix well. Measure the data at a wavelength of 450nm and record it. Figure 2 Dynamic Detection C 21 Steroid compounds. Figure 3 It can be seen that C 21 The mass of steroidal compounds is 4.3 times that of the raw material of Cynanchum multiflorum root tuber.

[0056] 3. C in fermentation broth 21 Extraction technology of steroidal compounds

[0057] 1. C in the sample21 Steroid compound concentration process

[0058] F-45 was fermented for 120 hours, the fermentation liquid was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was freeze-dried. The freeze-drying conditions were: pre-freezing at -20°C for 24 hours, and vacuum freezing (-60°C) drying for 48 hours.

[0059] After the above treatment, a freeze-dried sample was obtained.

[0060] 2. C in freeze-dried samples 21 Separation and extraction technology of steroidal compounds

[0061] (1)C 21 Extraction of steroidal compounds

[0062] Anhydrous ethanol was added to the freeze-dried sample at a mass volume ratio (g / mL) of 1:3-4, and the mixture was homogenized with a homogenizer. The mixture was allowed to stand for 1-2 hours, and centrifuged at 10,000 rpm for 5 minutes to collect the supernatant alcohol extract.

[0063] (2)C 21 Precipitation of steroidal compounds

[0064] Add precipitant (ether and acetone in a volume ratio of 1:1) to the above alcohol extract in a volume ratio of 1:1 to 7, shake well, let stand for 2 to 3 hours, centrifuge at 1,000 rpm for 5 minutes, evaporate in a 40°C water bath until no liquid remains, and the precipitate is the fermentation extract C 21 Steroid compounds.

[0065] like Figure 4 As shown in the figure, with the increase of the addition ratio of the precipitating agent, the precipitation rate first increases and then decreases. Among them, when the volume ratio of the two is 1:4, the precipitation rate is the highest.

[0066] 4. C in fermentation broth 21 Verification of the effect of steroid compound extraction process

[0067] The above established method was used to extract C 21 Steroid compounds were detected and analyzed to verify the effectiveness of the method.

[0068] 1. Sample pretreatment

[0069] The fermentation extract C 21 The steroid compound was diluted to 2 mL with 60% ethanol aqueous solution, shaken well, filtered through a microporous filter membrane (0.22 μm), diluted 10 times and set aside.

[0070] 2. Ultraviolet full-band scanning

[0071] Take 2 μL of the above treated sample and perform UV full wavelength scanning. The results are as follows: Figures 5 to 7As shown in the figure, this method can fully utilize the interaction and metabolism between the root material of Radix Cynanchum multiflorum and the endophytic bacteria F-45 to accumulate C 21 Characteristics of steroidal compounds produced by fermentation products.

[0072] 5. Fermented Extract C 21 Effect of steroid compounds on protection against oxidative damage

[0073] After mouse AML-12 cells were revived, they were transferred to a 48-well culture plate and cultured for 12 h. The supernatant was removed and the culture medium was replaced. Except for the blank group, 50 μL of formaldehyde solution (5 mmol / L) was added to the other treatment groups. Among them, the F-45 group was added with different concentrations (20, 80, 160 mg / mL) of fermentation extract C 21 Steroidal compounds, and the root group was added with different concentrations (20, 80, 160 mg / mL) of Radix Cynanchum multiflorum root C 21 Steroid compounds, the control group was 50 μL formaldehyde damage group, and the positive group was added with 50 μL Vc solution, with three replicates in each group. After 6 hours of action, the MTT reaction, GOT and LDH enzyme activities, and MDA content of hepatocytes were determined according to the kit method.

[0074] like Figures 8 to 11 As shown, fermentation extract C 21 The steroid compounds can reduce the MDA content, LDH enzyme activity and GOT enzyme activity in formaldehyde-induced mouse liver injury cells AML-12, and all indicators are better than those of the root source C 21 The steroid treatment group showed that the fermentation extract C 21 Steroid compounds have a good protective effect on liver cell damage caused by oxidative stress. 21 Steroid compounds have a good protective effect on the liver and can provide a basis for industrial production.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A fermented extract C 21 A biofermentation method for steroidal compounds, characterized in that: The method comprises the following steps: (1) Fresh tubers of Radix Cynanchum multiflorum and distilled water were mixed at a mass volume ratio of 5-7 g:1 mL and then pulped. A magnetic nanocomposite enzyme preparation was added at a mass ratio of pulp to enzyme preparation of 2.5-3:

1. The mixture was subjected to enzymatic hydrolysis at 37°C for 2 h, and the enzyme preparation was recovered by magnetic force to obtain a saccharified liquid. (2) diluting the saccharified liquid with Gao's medium No. 1 and inoculating Agrobacterium tumefaciens F-45 at an inoculation amount of 1 to 3%, and culturing for 120 hours at 32° C., pH 8, and shaking at 120 rpm to obtain a fermentation liquid; (3) freeze-drying the fermentation broth, adding anhydrous ethanol to the resulting freeze-dried powder, homogenizing, and standing at room temperature for 12 hours, and collecting the supernatant alcohol extract; (4) Add the alcohol extract to the precipitant, mix well and let stand at room temperature for 2 to 3 hours, centrifuge the precipitate to collect the precipitate, and place the precipitate in a 40°C water bath to evaporate until no liquid remains. The precipitate is C 21 Steroid compounds.

2. The method according to claim 1, characterized in that In step (2), the volume ratio of the saccharification solution to Gao's medium No. 1 is 1:5-7.

3. The method according to claim 1, characterized in that In step (3), the freeze drying is specifically: pre-freezing at -20°C for 24 hours, and then freeze drying in a vacuum environment at -60°C for 48 hours.

4. The method according to claim 1, characterized in that In step (3), the mass volume ratio of the lyophilized powder and anhydrous ethanol is 1 g: 3-4 mL.

5. The method according to claim 1, characterized in that In step (4), the volume ratio of the alcohol extract to the precipitating agent is 1:3-4.

6. The method according to claim 1, characterized in that In step (4), the precipitating agent is composed of ether and acetone in a volume ratio of 1:1 to 2.

7. The fermentation extract C prepared by the method according to any one of claims 1 to 6 21 Steroid compounds.

8. The fermentation extract C according to claim 7 21 Application of steroidal compounds in the preparation of drugs for repairing liver cell damage.

9. The use according to claim 8, characterized in that The hepatocyte damage is hepatocyte damage caused by oxidative stress.

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