Preparation method of ginsenoside Gyp LXXV and application of ginsenoside Gyp LXXV in treatment of enteritis

Abstract

The invention discloses a preparation method of ginsenoside Gyp LXXV and application of the ginsenoside Gyp LXXV in treatment of enteritis. The preparation method comprises the following steps: preparing Gyp LXXV through bioconversion by using beta-glucosidase and beta-1, 2 glucosidase for hydrolyzing glucose on the inner side of ginsenoside and taking ginsenoside Rb1 as a substrate, a conversion system is a buffer solution with the pH value of 4.0-8.0, the reaction temperature is 25-45 DEG C, the ratio of the glucosidase to the substrate is 0.1 U / mg-0. 5U / mg, the concentration of the substrate is 25-200mg / mL, the reaction time is 1-5 hours, the conversion product is recrystallized and purified, and the Gyp LXXV is obtained. The ginsenoside Gyp LXXV with the purity of 90%-95% is obtained. Meanwhile, by means of DSS-induced mouse colitis model research, the ginsenoside Gyp LXXV is found to be capable of remarkably relieving colitis symptoms including weight loss, colon length, tight junction protein expression and the like through intraperitoneal injection and oral administration.

Claims

1. A large-scale preparation method of ginsenoside Gyp LXXV, comprising the following steps: using β-glucosidase BglPC28 and β-1,2-glucosidase CcBgl1A to hydrolyze the inner glucose of ginsenoside to hydrolyze ginsenoside Rb1 to obtain ginsenoside Gyp LXXV; The BglPC28 is any one of the following A1)-A3): A1) a protein whose amino acid sequence is SEQ ID No. 1; A2) a protein having the function of β-glucosidase by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 1 in the sequence list; A3) a fusion protein obtained by connecting a tag to the N-terminus or / and the C-terminus of A1) or A2); The CcBgl1A is any one of the following B1)-B3): B1) a protein whose amino acid sequence is positions 21 to 410 of SEQ ID No. 2; B2) a protein whose amino acid sequence is SEQ ID No. 2; B3) A protein having the same function as that obtained by replacing and / or deleting and / or adding one or more amino acid residues from positions 21 to 410 of SEQ ID No. 2 in the sequence listing or the amino acid sequence shown in SEQ ID No.

2.

2. The preparation method according to claim 1, characterized in that: The conditions of the enzymolysis are as follows: the enzymolysis is carried out in a buffer solution with a pH of 4.0-8.0; the ratio of β-glucosidase BglPC28 and β-1,2-glucosidase CcBgl1A to substrate is 0.1U / mg-0.5U / mg, the concentration of substrate ginsenoside Rb1 is 10-200mg / mL, the reaction temperature of the enzymolysis is 25-45°C, and the reaction time of the enzymolysis is 1-5 hours.

3. The preparation method according to claim 1 or 2, characterized in that: The conditions of the enzymatic hydrolysis are as follows: the enzymatic hydrolysis is carried out in a buffer solution of pH 6.0; the ratio of β-glucosidase BglPC28 and β-1,2-glucosidase CcBgl1A to substrate is 0.3U / mg, the concentration of substrate ginsenoside Rb1 is 20mg / mL, the reaction temperature of the enzymatic hydrolysis is 30°C, and the reaction time of the enzymatic hydrolysis is 3 hours.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The method further comprises the step of purifying the product ginsenoside Gyp LXXV by recrystallization, wherein the purification step is as follows: Step 1: Add 20% ethanol (based on the volume of the enzymatic hydrolysate as 100%) to the enzymatic hydrolysate containing ginsenoside Gyp LXXV, heat at 60-70°C for 30-60 minutes to denature and precipitate the protein, filter while hot, and dry the filtrate by rotary evaporation to obtain the converted sample; Step 2: Add 60% isopropanol solution to the sample in step 1 to make the sample concentration reach 500 mg / mL, vortex and then perform ultrasonic oscillation and 50°C water bath to mix thoroughly, and let stand for 12 hours; Step 3: Centrifuge the sample in step 2 at 6000 rpm for 5 min, take the supernatant and place it in a dry thermostat to evaporate all the isopropanol, and then dry it to obtain Gyp LXXV with a purity of 90%-95%.

5. Ginsenoside Gyp LXXV prepared by the method according to any one of claims 1 to 4.

6. The use of ginsenoside GypLXXV according to claim 5, wherein the use is at least one of the following: a1) Preparation of products for the prevention and / or treatment of enteritis; a2) Prevention and / or treatment of enteritis.

7. The use according to claim 6, characterized in that: The enteritis is colitis, including ulcerative colitis, infectious colitis, ischemic colitis and pseudomembranous colitis; Alternatively, the prevention and / or treatment of enteritis is embodied in at least one of the following aspects: b1) Anti-inflammatory effect; b2) inhibiting colon atrophy in patients with colitis; b3) inhibiting weight loss in patients with colitis; b4) Reduce the disease activity index (DAI) in patients with colitis; b5) reduce colon tissue damage in patients with colitis; b6) Promote the polarization of M1 macrophages to M2 macrophages; b7) inhibited the production of pro-inflammatory cytokines; b8) Inhibit the reduction of tight junction proteins.

8. A product for preventing and / or treating enteritis, the active ingredient of which comprises the ginsenoside GypLXXV according to claim 5.

9. The product according to claim 8, characterized in that: The product has at least one of the following effects: c1) Anti-inflammatory effect; c2) inhibiting colon atrophy in patients with colitis; c3) inhibiting weight loss in patients with colitis; c4) Reduce the disease activity index (DAI) in patients with colitis; c5) reduce colon tissue damage in patients with colitis; c6) Promote the polarization of M1 macrophages to M2 macrophages; c7) inhibited the production of pro-inflammatory cytokines; c8) inhibit the reduction of tight junction proteins.

10. The use according to claim 6 or 7 or the product according to claim 8 or 9, characterized in that: The pro-inflammatory cytokine is selected from at least one of the following: TNF-α and IL-6; Or, the tight junction protein is selected from at least one of the following: Zo-1, Claudin and Occludin; Alternatively, the product is a drug.

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