Cynanchum bungei C21 steroidal glycoside fermentation purified product as well as preparation method and application thereof

Abstract

The invention discloses a cynanchum bungei C21 steroidal glycoside fermentation purified product as well as a preparation method and application thereof. The method comprises the following steps: fermenting and culturing an endophytic strain of the radix cynanchi bungei under the conditions that the temperature is 32-37 DEG C, the speed is 120rpm and the pH value is 8; wherein the radix cynanchi bungei endophytic strain is selected from any one of colletotrichum gloeosporioides Z-44 (the collection number of the strain is CCTCC (China Center for Type Culture Collection) NO: M20211164), bacillus cereus X-32 (the collection number of the strain is CCTCC NO: M20211165) and agrobacterium tumefaciens F-45 (the collection number of the strain is CCTCC NO: M20211166); and carrying out extraction and precipitation treatment on the fermentation liquor to obtain a cynanchum bungei C21 steroidal glycoside fermentation purified product. The medicine prepared from the cynanchum bungei C21 steroidal glycoside fermentation purified product can effectively improve liver injury, is mainly reflected in the aspects of remarkably relieving liver function injury indexes, remarkably resisting oxidation, relieving liver fibrosis degree and the like, is wide in pharmacological action and obvious in acting effect, and has a relatively good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of medical technology and particularly relates to a 21 Steroidal glycoside fermentation purification product and its preparation method and application. Background Art

[0002] C 21 Steroidal glycosides 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 Steroidal glycosides 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 glycosides can not only significantly reduce the levels of AST, ALT, ALP, BUG, ​​and LDH in serum. 21 Steroidal glycosides 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 glycosides 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 glycosides 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 Steroidal glycosides have good application prospects in improving liver damage, but currently 21 Steroidal glycosides are mainly extracted from plants, which cannot meet the needs of large-scale industrial production due to the constraints of planting area, pests and diseases, climate and other environmental conditions. In addition, according to incomplete statistics, there are currently 450 million patients with various liver injuries and 140 million patients with liver fibrosis in my country. The raw materials for the treatment of liver injury and liver fibrosis are in short supply on the market. Summary of the invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a 21 Steroidal glycoside fermentation purification product and its preparation method and application.

[0005] The present invention is achieved by: a Radix Cynanchum multiflorum C 21A method for preparing a steroidal glycoside fermentation purified product, the method comprising the steps of:

[0006] (1) fermenting and culturing an endophytic strain of Radix Cynoglossum at 32° C. to 37° C., 120 rpm, and pH 8; wherein the endophytic strain of Radix Cynoglossum is selected from any one of Colletotrichum gloeosporioides Z-44 (strain collection number: CCTCC NO: M20211164), Bacillus cereus X-32 (strain collection number: CCTCC NO: M20211165), and Agrobacterium tumefaciens F-45 (strain collection number: CCTCC NO: M20211166);

[0007] (2) The fermentation liquid is subjected to extraction and precipitation to obtain Radix Cynanchum multiflorum C 21 Steroidal glycosides fermentation purified product; wherein the fermentation purified product of Colletotrichum gloeosporioides Z-44 includes cyperoside C 3 N, the fermentation purified product of Bacillus cereus X-32 includes cyperoside C 1 N, the fermentation purified product of Agrobacterium tumefaciens F-45 includes kadenin.

[0008] Preferably, in step (1), the fermentation temperature of Colletotrichum gloeosporioides Z-44 is 35°C, the fermentation temperature of Bacillus cereus X-32 is 37°C, and the fermentation temperature of Agrobacterium tumefaciens F-45 is 32°C.

[0009] Preferably, in step (2), in the precipitation treatment, the volume ratio of the extraction supernatant to the precipitating agent is 1:3-4.

[0010] Preferably, in step (2), the precipitating agent is composed of ether and acetone in a volume ratio of 1:1 to 2.

[0011] The present invention further discloses the Radix Cynanchum multiflorum C obtained by the above preparation method. 21 Steroidal glycosides fermentation purification.

[0012] The present invention further discloses the above-mentioned Radix Cynanchum multiflorum C 21 Application of steroidal glycoside fermentation purification products in the preparation of drugs for improving liver damage.

[0013] Preferably, the liver damage includes alcohol damage to hepatocytes, oxidative damage and liver fibrosis.

[0014] The present invention overcomes the deficiencies of the prior art and provides a 21The present invention discloses a steroidal glycoside fermentation purified product and a preparation method and application thereof. The present invention ferments and cultures an endophytic strain of Radix Cynoglossum at 32°C to 37°C, 120rpm, and a pH of 8; wherein the endophytic strain of Radix Cynoglossum is selected from Colletotrichum gloeosporioides Z-44 (Colletotrichum gloeosporioides Z-44, strain collection number is CCTCC NO: M20211164, preserved in China Center for Type Culture Collection on September 13, 2021, with a preservation address of: Wuhan University Collection Center, Luojia, Wuchang District, Wuhan City, zip code: 430072), Bacillus cereus X-32 (Bacillus cereus X-32, strain collection number is CCTCC NO: M20211165, deposited in China Center for Type Culture Collection on September 13, 2021, the deposit address is: Wuhan University Collection Center, Luojia, Wuchang District, Wuhan, zip code: 430072), Agrobacterium tumefaciens F-45 (Agrobacterium tumefaciens F-45, strain deposit number is CCTCC NO: M20211166, deposited in China Center for Type Culture Collection on September 13, 2021, the deposit address is: Wuhan University Collection Center, Luojia, Wuchang District, Wuhan, zip code: 430072); the fermentation broth is subjected to extraction and precipitation treatment to obtain Radix Cynomorii multiflori C 21 Steroidal glycosides fermentation purified product; wherein the fermentation purified product of Colletotrichum gloeosporioides Z-44 includes cyperoside C 3 N, the fermentation purified product of Bacillus cereus X-32 includes cyperoside C 1 N, the fermentation purified product of Agrobacterium tumefaciens F-45 includes kadenin.

[0015] The present invention further verifies that the above-mentioned Radix Cynanchum multiflorum C 21 The present invention constructs an ethanol-induced liver cell injury model, adds different concentrations of Radix Cynoglossifolia C 21 After a period of time, the ALT and GOT enzyme activities of liver cells were measured by fermentation and purification of steroidal glycosides. The results showed that Radix Polygoni Multiflori C 21 The steroidal glycoside fermentation purification can significantly alleviate the liver function damage index. In addition, the present invention constructs a liver cell model damaged by formaldehyde oxidation, adds different concentrations of Radix Cynanchum multiflorum C 21 After a period of time, the LDH and GOT enzyme activities of liver cells were measured by fermentation and purification of steroidal glycosides. The results showed that Radix Polygoni Multiflori C 21 The steroidal glycosides fermentation purified product has significant antioxidant effect. In addition, the present invention uses liver fibrosis cell model Lx-2 cells as materials, adds different concentrations of Radix Cynanchum multiflorum C 21After a period of time, the purified steroidal glycosides were subjected to Masson staining and the hydroxyproline content was determined. The results showed that 21 The fermentation purified products of steroidal glycosides can effectively alleviate the degree of liver fibrosis in model cells.

[0016] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects:

[0017] (1) Radix Cynanchum multiflorum C of the present invention 21 The preparation of steroidal glycoside fermentation purified products is simple, high in yield and purity;

[0018] (2) Radix Cynanchum multiflorum C of the present invention 21 Drugs prepared from steroidal glycoside fermentation purification products can effectively improve liver damage, mainly manifested in aspects such as significantly alleviating liver function damage indicators, significant antioxidant effects, and alleviating the degree of liver fibrosis. They have a wide range of pharmacological actions and obvious effects, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a purified product of the fermentation broth of Bacillus cereus X-32 (Bai Shou Wu C 21 HPLC results of steroidal glycoside fermentation purified product 1);

[0020] Figure 2 It is a purified product of the fermentation broth of Colletotrichum gloeosporioides Z-44 (Bai Shou Wu C 21 HPLC results of steroidal glycoside fermentation purified product 2);

[0021] Figure 3 It is a purified product of Agrobacterium tumefaciens F-45 fermentation broth (Bai Shou Wu C 21 HPLC results of steroidal glycosides fermentation purified product 3)

[0022] Figure 4 It is a purified product of the fermentation broth of Bacillus cereus X-32 (Bai Shou Wu C 21 LC-MS test results of steroidal glycoside fermentation purification product 1);

[0023] Figure 5 It is a purified product of the fermentation broth of Colletotrichum gloeosporioides Z-44 (Bai Shou Wu C 21 LC-MS test results of steroidal glycoside fermentation purification product 2);

[0024] Figure 6 It is a purified product of Agrobacterium tumefaciens F-45 fermentation broth (Bai Shou Wu C 21 LC-MS test results of steroidal glycoside fermentation purified product 3);

[0025] Figure 7 It is Bai Shou Wu C 21Statistical results of the effects of steroidal glycoside fermentation purified products 1 to 3 on the GOT and ALT enzyme activities of ethanol-damaged hepatocytes; Figure 7 A is ALT enzyme activity, Figure 7 B is GOT enzyme activity;

[0026] Figure 8 It is the Radix Cynanchum multiflorum C of the present invention 21 Effects of steroidal glycoside fermentation purified products 1 to 3 on the activity of GOT and LDH in liver damaged by formaldehyde oxidation; Figure 8 A is ALT enzyme activity, Figure 8 B is GOT enzyme activity;

[0027] Fig. 9 It is the Radix Cynanchum multiflorum C of the present invention 21 Effect of steroidal glycoside fermentation purified product 3 on hydroxyproline content in LX-2 cells;

[0028] Fig.10 In the present invention, Radix Cynanchum multiflorum C 21 Masson staining results of collagen production by LX-2 cells under the action of steroidal glycoside fermentation purified product 3; Fig.10 A is CK group, Fig.10 B is the low concentration group (20 mg / L). Fig.10 C is the high concentration group (80 mg / L). DETAILED DESCRIPTION

[0029] 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.

[0030] Example 1

[0031] (1) Bacillus cereus X-32 (strain accession number: CCTCCNO: M20211165) was fermented at 37°C, 120 rpm, and pH 7 for 120 h; the inoculation amount was 1%, and the fermentation medium formula was: 2.5 g of fructose, 12.5 g of tryptone, 10 g of sodium chloride, and 1 L of distilled water.

[0032] (2) The fermentation broth was pre-frozen (-20°C, 12 h) and then freeze-dried (-60°C, 48 h). Anhydrous ethanol was added to the freeze-dried product at a ratio of 3-4:1 (volume-to-mass ratio mL / g). After sufficient mixing, the product was extracted at room temperature (3-4 h). The extract was collected by centrifugation (10,000 rpm, 10 min). A precipitant (ether: acetone volume ratio of 1:1) was added at a volume ratio of (1:4) and the precipitate was collected to obtain Radix Cynomorii Multiflori C. 21 Steroidal glycosides fermentation purification 1.

[0033] Example 2

[0034] (1) Colletotrichum gloeosporioides Z-44 (CCTCC NO: M20211164) was fermented at 37°C, 120 rpm, and pH 8 for 48 h; the inoculation amount was 1%, and the fermentation medium formula was: sucrose 10 g, potato extract powder 12 g, peptone 2 g, potassium dihydrogen phosphate 2 g, magnesium sulfate 2 g, and distilled water 1 L.

[0035] (2) The fermentation broth was pre-frozen (-20°C, 12 h) and then freeze-dried (-60°C, 48 h). Anhydrous ethanol was added to the freeze-dried product at a ratio of 3-4:1 (volume-to-mass ratio mL / g). After sufficient mixing, the product was extracted at room temperature (3-4 h). The extract was collected by centrifugation (10,000 rpm, 10 min). A precipitant (ether: acetone volume ratio of 1:1) was added at a volume ratio of (1:4) and the precipitate was collected to obtain Radix Cynomorii Multiflori C. 21 Steroidal glycoside fermentation purification 2.

[0036] Example 3

[0037] (1) Agrobacterium tumefaciens F-45 (strain accession number: CCTCCNO: M20211166) was fermented at 32°C, 120 rpm, and pH 8 for 216 h; the inoculation amount was 1%, and the fermentation medium formula was: 30 g sucrose, 1 g beef extract, 0.5 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate, 0.5 g sodium chloride, 0.01 g ferrous sulfate, and 1 L distilled water.

[0038] (2) The fermentation broth was pre-frozen (-20°C, 12 h) and then freeze-dried (-60°C, 48 h). Anhydrous ethanol was added to the freeze-dried product at a ratio of 3-4:1 (volume-to-mass ratio mL / g). After sufficient mixing, the product was extracted at room temperature (3-4 h). The extract was collected by centrifugation (10,000 rpm, 10 min). A precipitant (ether: acetone volume ratio of 1:1) was added at a volume ratio of (1:4) and the precipitate was collected to obtain Radix Cynomorii Multiflori C. 21 Steroidal glycoside fermentation purification 3.

[0039] Example 4

[0040] The Radix Cynanchum multiflorum C prepared in the above embodiment 21Steroidal glycoside fermentation purified products 1 to 3 were subjected to HPLC detection and LC-MS identification. Chromatographic parameters: Alltima C18 chromatographic column (250 mm × 4.6 mm, 5 μm); acetonitrile (A)-water (B) gradient elution (0 to 10 min, 35% A.); flow rate 1 mL / min; column temperature 35°C; detection wavelength 263 nm; injection volume 10 μL. Mass spectrometry parameters: ESI positive ion mode, MSE Continum acquisition mode; spray voltage 5 kV; desolvation gas is nitrogen, flow rate 1000 L / h, desolvation temperature 550°C, ion source temperature 100°C, scanning range m / z 50 to 1500, collision gas is argon.

[0041] Test results such as Figures 1 to 3 As shown in the figure, the HPLC results of the purified products of X-32, Z-44 and F-45 fermentation broths are shown. The HPLC results show an obvious main peak, and the peak is relatively concentrated. It can be judged that the purification method is good. The yields of the purified products are 64.6%, 68.55% and 71.45%, respectively, and the purities are 72.04%, 75.58% and 82.78%, respectively.

[0042]

[0043]

[0044] In addition, Cynanchum multiflorum C 21 LC-MS analysis of steroidal glycoside fermentation purified products 1 to 3 showed that they were all C 21 Steroid compound, the fermentation product purified from Bacillus cereus X-32 is cyperoside C 1 N( Figure 4 ), the fermentation product of Colletotrichum gloeosporioides Z-44 is diatomaceous glycoside C 3 N( Figure 5 ), the fermentation product purified by Agrobacterium tumefaciens F-45 is aglycone ( Figure 6 ).

[0045] Application Examples

[0046] 1. Cynanchum multiflorum C 21 Improvement of alcohol-damaged hepatocytes by purified steroidal glycoside fermentation products 1-3

[0047] (1) Establishment of alcohol injury model

[0048] 10 μL of ethanol solution of different concentrations (40, 80, 120 mmol / L) was added to AML-12 cells growing in the exponential growth phase in a 96-well culture plate, and a blank group (normal cells) and a model group (10 μL complete culture medium) were set up, with 6 replicates in each group. The MTT reaction of hepatocytes was analyzed after 6 hours of action.

[0049] (2) Determination of liver protection indexes

[0050] In the mouse AML-12 cells growing in the exponential growth phase in a 48-well culture plate, 50 μL of the optimal concentration of ethanol solution (modeling concentration) was added, and simultaneously, Radix Polygoni Multiflori C was added. 21 Steroidal glycoside fermentation purified products 1-3 (20, 80, 160 mg / L) were set up as blank group (normal cells) and model group (50 μL damaging agent), with 6 replicates in each group. After 6 hours of action, the cell MTT reaction was detected, and the ALT and GOT enzyme activities of the hepatocytes were determined after the cells were collected and ultrasonically disrupted.

[0051] 2. Cynanchum multiflorum C 21 Improvement of steroidal glycoside fermentation purified products 1-3 on oxidatively damaged hepatocytes

[0052] (1) Establishment of formaldehyde oxidative damage model

[0053] 10 μL of formaldehyde solution of different concentrations (1.7, 3.3, 5.0 μmol / L) was added to AML-12 cells cultured in 96-well culture plates for 12 hours, and a blank group (normal cells) and a model group (10 μL double distilled water) were set up. After 6 hours of action, the survival rate of hepatocytes was analyzed by MTT method.

[0054] (2) Determination of antioxidant index

[0055] After culturing the cells in a 48-well culture plate for 12 hours, the supernatant was removed and the culture medium was replaced. 50 μL of formaldehyde at the optimal concentration (modeling concentration) was added to the well plate, and Radix Polygoni Multiflori C was added. 21 Steroidal glycoside fermentation purified products 1-3 (20, 80, 160 mg / L) were set up as blank group (normal cells), model group (50 μL damaging agent) and positive model group (50 μL Vc solution), with 6 replicates in each group. After 6 hours of action, the cell MTT reaction was detected and the MDA content was determined. At the same time, the cells were collected and ultrasonically disrupted, and the LDH and GOT enzyme activities of the hepatocytes were determined.

[0056] 3. Cynanchum multiflorum C 21 Improvement of liver fibrosis by purified steroidal glycoside fermentation products 1-3

[0057] (1) Cultivation of Lx-2 cells, a liver fibrosis cell model

[0058] After Lx-2 cells were subcultured to 96-well culture plates and cultured for 48 h, the cell density was high and they showed obvious characteristics of liver fibrosis.

[0059] (2) Determination of anti-liver fibrosis indexes

[0060] 100 μL of different concentrations of Radix Polygoni Multiflori C were added to Lx-2 cells cultured in 96-well culture plates for 48 h.21 Steroidal glycoside fermentation purified product 3 (20, 80 mg / L), set up a blank group (normal cells), and set up 6 replicates for each group. After 12 hours of action, Masson staining was performed and the hydroxyproline content was determined at the same time.

[0061] 4. Results and explanation

[0062] The present invention has three kinds of C 21 In the alcoholic liver injury and oxidative damage protection test, the ALT activity and GOT activity of the low-concentration treatment group (20 mg / L) of steroidal glycosides fermentation purified products 1, 2, and 3 were lower than those of the model group. Among them, compared with the alcoholic liver injury model group, the ALT activity of the low-concentration treatment group (20 mg / L) of fermentation purified product 2 decreased by 21.54%, and the GOT activity decreased by 34.95% (see Figure 7 ). Compared with the oxidative damage model group, the Radix Cynanchum multiflorum C in the present invention 21 After treatment with steroidal glycosides fermentation purified product 2 (80 mg / L), the cell survival rate increased by 3 times and the LDH activity decreased by 59.28%; 21 The LDH activity was reduced by 35.36% under the treatment of steroidal glycoside fermentation purified product 3 (160 mg / L) (see Figure 8 In the anti-liver fibrosis protection test, low concentration (20 mg / L) of Radix Polygoni Multiflori C 21 When Lx-2 cells were treated with steroidal glycoside fermentation purified product 3 for 8 h, the hydroxyproline content was significantly lower than that of the control group, and collagen staining showed that the color of the low-concentration treatment group was weaker (see Figures 9-10 ), indicating that 21 Steroidal glycoside fermentation purified product 3 has significant anti-hepatic fibrosis effect.

[0063] 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 kind of Cynanchum multiflorum C 21 A method for preparing a steroidal glycoside fermentation purified product, characterized in that: The method comprises the steps of: (1) fermenting and culturing an endophytic strain of Radix Cynoglossum at 32° C. to 37° C., 120 rpm, and pH 8; wherein the endophytic strain of Radix Cynoglossum is selected from any one of Colletotrichum gloeosporioides Z-44 (strain collection number: CCTCC NO: M20211164), Bacillus cereus X-32 (strain collection number: CCTCC NO: M20211165), and Agrobacterium tumefiaciens F-45 (strain collection number: CCTCC NO: M20211166); (2) The fermentation liquid is subjected to extraction and precipitation to obtain Radix Cynanchum multiflorum C 21 Steroidal glycoside fermentation purified product; wherein, the fermentation purified product of Colletotrichum gloeosporioides Z-44 includes cyperoside C3N, the fermentation purified product of Bacillus cereus X-32 includes cyperoside C1N, and the fermentation purified product of Agrobacterium tumefaciens F-45 includes kaidegenin.

2. The preparation method according to claim 1, characterized in that In step (1), the fermentation temperature of Colletotrichum gloeosporioides Z-44 is 35°C, the fermentation temperature of Bacillus cereus X-32 is 37°C, and the fermentation temperature of Agrobacterium tumefaciens F-45 is 32°C.

3. The preparation method according to claim 1, characterized in that: In step (2), in the precipitation treatment, the volume ratio of the extraction supernatant to the precipitating agent is 1:3-4.

4. The preparation method according to claim 3, characterized in that: In step (2), the precipitating agent is composed of ether and acetone in a volume ratio of 1:1 to 2.

5. Radix Cynanchum multiflorum C obtained by the preparation method according to any one of claims 1 to 4 21 Steroidal glycosides fermentation purification.

6. The Radix Cynanchum multiflorum C according to claim 5 21 Application of steroidal glycoside fermentation purification products in the preparation of drugs for improving liver damage.

7. The use according to claim 6, characterized in that The liver damage includes alcohol damage to hepatocytes, oxidative damage and liver fibrosis.

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