Black ginseng extract for relieving acute kidney injury caused by chemotherapy as well as preparation method and application of black ginseng extract

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a preparation method of a scrophularia ningpoensis extract for relieving chemotherapy-induced acute kidney injury and application of the scrophularia ningpoensis extract in preparation of a product for relieving chemotherapy-induced acute kidney injury. The preparation method comprises the following steps: putting fresh ginseng into a high-temperature and high-pressure sterilization pot, steaming at 110-120 DEG C for 1-3 hours, taking out, cooling to room temperature, and repeating the steaming cooling process for 2-5 times; and crushing the treated ginseng materials, adding a mixed strain of saccharomyces cerevisiae and lactobacillus plantarum in a mass ratio of 1: 1, fermenting for 24-72 hours, filtering fermentation liquor, and drying filter residues to obtain the black ginseng extract. The cisplatin-induced acute kidney injury is prevented and / or treated by improving oxidative stress and mitochondrial functional activity, reducing the content of lipid peroxide, inhibiting expression of inflammatory factors TNF alpha, IL1beta and COX2 and remarkably reducing the cell apoptosis rate and the level of inflammatory factors related to endoplasmic reticulum stress.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a method for preparing a black ginseng extract through a high-temperature steaming and probiotic mixed fermentation process, and an application of the black ginseng extract in preparing a drug for preventing and / or treating acute kidney injury caused by chemotherapy drugs. Background Art

[0002] Although chemotherapy drugs such as cisplatin are widely used in cancer treatment, approximately 30% of patients experience dose-limiting nephrotoxicity (Oun R, et al. Cancer Treat Rev. 2018;70:240244). Its mechanisms mainly include: 1. Accumulation of platinum in renal tubules: Cisplatin accumulates in renal tubular epithelial cells through organic anion transporters (OATs), directly damaging mitochondrial function (Ishimoto Y, et al. J Am Soc Nephrol. 2021;32(7):16211632); 2. Oxidative stress: Mitochondrial damage leads to a burst of reactive oxygen species (ROS), increased malondialdehyde (MDA) levels, and decreased superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) activities (Manohar S, et al. Free Radic Biol Med. 2022;178:226240); 3. Inflammatory cascade reaction: Activation of proinflammatory cytokines such as TNFα and IL1β, and upregulation of cyclooxygenase 2 (COX2) expression (SharpCN, et al. J Clin Invest. 2020;130(3):12631277); 4. Endoplasmic reticulum stress: GRP78 / BiP, PERK, and IRE1α pathways are continuously activated, inducing CHOP-mediated cell apoptosis (Inagi R, et al. Kidney Int. 2014;86(5):918926).

[0003] Currently, there is a lack of effective renal protective agents in clinical practice. Although rare ginsenosides (such as Rh2) have renal protective potential, their natural content is less than 0.01% (Christensen LP. Adv Food Nutr Res. 2008;55:199), and traditional extraction methods have the following drawbacks:

[0004] Boiling water extraction method: saponin yield <2%, high impurity content (polysaccharide >40%); ethanol reflux method: total saponin yield 35%, but Rh2 content is extremely low; microbial conversion method: Lactobacillus plantarum fermentation can only produce Rg3 and cannot efficiently convert Rh2 (KimBG, et al. Process Biochem. 2010;45(8):13191324). Summary of the Invention

[0005] 1. Technical Issues

[0006] The invention solves the problems in the prior art of low preparation efficiency of rare saponin Rh2 and insufficient kidney protective activity of traditional extracts.

[0007] 2. Technical Solution

[0008] 1. Preparation method of black ginseng extract

[0009] Step 1: Steaming at high temperature

[0010] Ingredients: Fresh ginseng (Panax ginseng CA Meyer), taproot diameter ≥ 1.5 cm

[0011] Parameters: 110-120℃ high pressure steam treatment, single time 13 hours, cooling to 25-30℃

[0012] Cycle times: 2-5 times (preferably 120°C x 2 hours / time x 3 times)

[0013] Step 2: Mixed strains for fermentation

[0014] Bacterial strain combination: Saccharomyces cerevisiae CICC 1009 and Lactobacillus plantarum CICC 22213

[0015] Inoculation ratio: 1:1 (w / w), total inoculation volume 25%

[0016] Fermentation conditions: 25-37°C (preferably 30°C), 24-72 hours (preferably 48 hours), solid-liquid ratio 1:31:5

[0017] Mechanism of action:

[0018] Yeast secretes β-glucosidase to hydrolyze the C3 glucose moiety of Rg3

[0019] Lactic acid bacteria produce arabinosidase to hydrolyze C20 arabinosyl groups

[0020] Synergistic effect: Dual bacterial symbiosis promotes Rh2 conversion (refer to Kim SH, et al. Molecules. 2022;27(15):4940)

[0021] Step 3: Post-processing

[0022] Filter and separate the bacteria, and vacuum dry the residue at 60℃

[0023] Crushed through 80 mesh sieve to obtain brown powder extract

[0024] 2. Characteristics of Black Ginseng Extract

[0025] Physical and chemical properties: moisture ≤8% (GB 5009.32016), ash ≤5% (GB 5009.42016)

[0026] Active ingredient: Rh2 content ≥5.0% (HPLC method, detection conditions as in Example 2)

[0027] Fingerprint: Rh2 characteristic peak appears at retention time 41.5±0.5 min in HPLC chromatogram

[0028] 3. Drug Application

[0029] Indications: Acute kidney injury caused by platinum chemotherapy drugs such as cisplatin and carboplatin

[0030] Dosage form and dosage:

[0031] Oral preparations (capsules, tablets, granules)

[0032] The extract accounts for 30-90% of the preparation (preferably 50%)

[0033] Clinical daily dose 20-100 mg / kg

[0034] 3. Beneficial Effects

[0035] 1. Process innovation:

[0036] Steaming and fermentation synergistically increased Rh2 yield to 8 times that of the traditional method (>5% vs <0.6%)

[0037] Dual-bacteria symbiotic fermentation shortens the conversion cycle to 48 hours (compared to 7 days for single-bacteria fermentation)

[0038] 2. Multi-target renal protection:

[0039] In vitro model: 50 μg / mL extract increased the survival rate of cisplatin-injured HK2 cells from 52.3% to 85.6%

[0040] Animal experiments: A dose of 50 mg / kg reduced serum creatinine in cisplatin model mice by 42.2% (p<0.001)

[0041] 3. Security:

[0042] Acute toxicity test (ICR mice): LD 50 >2000 mg / kg

[0043] No interference with chemotherapy anti-tumor activity (difference in in vitro tumor inhibition rate <5%) Example

[0044] Example 1: Preparation of Black Ginseng Extract

[0045] 1. Steaming: Take 500 g of fresh ginseng from Changbai Mountain and autoclave it at 120°C for 2 hours. Then cool it to room temperature and repeat this process three times.

[0046] 2. Fermentation: Crush the ginseng and inoculate with a mixed strain (Saccharomyces cerevisiae CICC 1009: Lactobacillus plantarum CICC22213 = 1:1) and ferment at 30°C for 48 hours.

[0047] 3. Drying: The filter residue was vacuum dried at 60℃ for 12 hours, crushed and passed through an 80-mesh sieve to obtain 128 g of extract (yield 25.6%).

[0048] Example 2: Rh2 content determination (HPLC method)

[0049] Chromatographic conditions:

[0050] Column: Agilent ZORBAX SBC18 (250 × 4.6 mm, 5 μm)

[0051] Mobile phase: acetonitrile (A) 0.1% phosphoric acid water (B), gradient elution (0-30 min, 20%→45% A)

[0052] Flow rate: 1.0 mL / min, column temperature: 30°C, detection wavelength: 203 nm

[0053] Results: Rh2 content 8.2%, total saponin content 71.3% (quantitative by external standard method)

[0054] Example 3: In vitro renal protective activity

[0055] 1. Model establishment: HK2 cells were incubated with 20 μM cisplatin for 24 h;

[0056] 2. Group processing:

[0057] Control group: no treatment

[0058] Injury group: cisplatin 20 μM

[0059] Treatment group: cisplatin + extract (10 / 20 / 50 μg / mL)

[0060] 3. Test results:

[0061] Cell viability (MTT): 85.6±3.2% in the 50 μg / mL group (vs 52.3±4.1% in the injury group, p<0.01)

[0062] Apoptosis rate (Annexin V / PI): 50 μg / mL group decreased to 15.2±1.8% (vs 64.9±5.3% in injury group, p<0.001)

[0063] Western blot: GRP78 protein expression decreased by 52.7% (p<0.01)

[0064] Example 4: In vivo efficacy verification

[0065] 1. Model: C57BL / 6 mice received a single intraperitoneal injection of cisplatin (20 mg / kg);

[0066] 2. Administration: Oral administration of the extract (50 mg / kg / d) began 3 days before cisplatin injection and continued for 12 days;

[0067] 3. Results:

[0068] Serum creatinine: 0.48±0.05 mg / dL in the treatment group (vs 0.83±0.07 mg / dL in the model group, p<0.001)

[0069] Renal tissue pathology score: treatment group 1.2±0.3 (vs model group 3.8±0.4, p<0.001)

[0070] Kidney GRP78 mRNA: expression was downregulated by 58.3% (p<0.01)

[0071] Example 5: Capsule Preparation

[0072] prescription:

[0073] Black ginseng extract 50 g

[0074] Microcrystalline cellulose 40 g

[0075] Sodium carboxymethyl starch 8 g

[0076] Magnesium stearate 2 g

[0077] Process: Mix well and fill into size 0 capsules, each capsule contains 50 mg of extract.

Claims

1. A method for preparing a black ginseng extract for alleviating chemotherapy-induced acute kidney injury, characterized in that: The following steps are involved: (1) Steaming: Steam the fresh ginseng at 110°C to 120°C for 13 hours, cool it to room temperature, and repeat the steaming and cooling process 2-5 times; (2) Fermentation treatment: crush the ginseng material treated in step (1), add a mixed strain of Saccharomyces cerevisiae and Lactobacillus plantarum in a mass ratio of 1:1, and ferment at 25-37°C for 24-72 hours; (3) Post-processing: Filter the fermentation liquid and dry the residue to obtain the black ginseng extract.

2. The preparation method according to claim 1, wherein: In step (1), the steaming temperature is 120° C., the single steaming time is 2 hours, and the steaming and cooling are repeated 3 times.

3. The preparation method according to claim 1, wherein: The fermentation time in step (2) is 48 hours and the fermentation temperature is 30°C.

4. A black ginseng extract for alleviating chemotherapy-induced acute kidney injury, characterized in that: Prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the black ginseng extract for alleviating chemotherapy-induced acute kidney injury according to claim 4 in the preparation of a medicament for preventing and / or treating chemotherapy-induced acute kidney injury.

6. The use according to claim 5, characterized in that: The chemotherapy-induced acute kidney injury was induced by cisplatin or carboplatin.

7. The use according to claim 5, characterized in that: The drug works by at least one of the following mechanisms: (a) Improves oxidative stress and mitochondrial functional activity in renal tissue; (b) reduce the content of lipid peroxide MDA; (c) inhibiting the expression of inflammatory factors TNFα, IL1β, and COX2; (d) Reduced the apoptosis rate of renal tubular cells and the levels of endoplasmic reticulum stress-related factors.

8. The use according to claim 5, characterized in that: The medicine is an oral preparation, contains a pharmaceutically acceptable carrier, and the mass percentage of a black ginseng extract for alleviating chemotherapy-induced acute kidney injury in the preparation is 30-90%.

9. A pharmaceutical composition for alleviating chemotherapy-induced acute kidney injury, characterized in that: The invention comprises the black ginseng extract for alleviating chemotherapy-induced acute kidney injury as claimed in claim 4 and pharmaceutical excipients.

10. The pharmaceutical composition according to claim 9, characterized in that: The composition is in the form of capsules, tablets or granules.