Panax japonicus fermentation product as well as preparation method and application thereof

Abstract

The invention relates to the technical field of traditional Chinese medicine microbial fermentation pharmacy, in particular to a panax japonicus fermentation product and a preparation method and application thereof. The preparation method of the panax japonicus fermentation product comprises the following steps: carrying out mixed fermentation on crushed panax japonicus and Aspergillus niger CICC 40048, and obtaining fermentation liquor after fermentation, namely the panax japonicus fermentation product. The fermented panax japonicus fermentation product has obvious effects in the aspects of relieving vascular wall calcification deposition, renal fibrosis area and kidney calcium salt crystallization and reducing biochemical indexes of creatinine and urea nitrogen, and can be used for preparing medicines for treating and relieving vascular calcification diseases caused by chronic kidney diseases.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine microbial fermentation pharmaceutical production, and in particular to a Panax notoginseng fermentation product and a preparation method thereof, and an application thereof in alleviating vascular calcification caused by chronic kidney disease. Background Art

[0002] The prevalence of chronic kidney disease (CKD) is increasing year by year. Vascular calcification (VC) is a common disease associated with CKD. VC is a pathological process that supports major cardiovascular diseases and has attracted public attention due to its high morbidity and mortality. In addition to kidney damage, vascular stiffness caused by related VC can also lead to increased cardiovascular mortality. However, the development of drugs to relieve CKD is currently hindered, and both traditional Chinese and Western medicine are aimed at alleviating this process.

[0003] Panax japonicus was first recorded in Baicaojing (Mirror of Herbs) and is named after its rhizome, which is like a bamboo node. In the herbal works of successive dynasties, Zhao Xuemin of the Qing Dynasty recorded in Bencao Gangmu Shiyi that it is "shaped like ginseng", "sweet and quite similar to ginseng", "some with reed-like patterns on the skin", "white like a silkworm, and each has a dent like a mortar." Therefore, it is named Panax japonicus CAMey. It is a commonly used Chinese herbal medicine in the Tujia and Miao ethnic groups. It has outstanding effects in tonifying deficiency, strengthening, dispersing blood stasis, stopping bleeding, and relieving pain. It is mostly used for diseases such as weak body, cough and sputum, rheumatic pain, etc. Modern pharmacological studies have shown that Panax japonicus has anti-tumor, liver protection, and antioxidant effects, among which triterpenoid saponins largely determine its pharmacological activity. Studies have reported that Panax japonicus saponin compounds can alleviate tissue damage through anti-inflammatory and antioxidant processes in animal models of renal fibrosis, alcoholic liver, and myocardial infarction.

[0004] In clinical practice, there are limited means to prevent and treat CKD vascular calcification, mainly through prevention and other means, and the effect is not ideal; therefore, it is necessary to explore new diagnostic and treatment ideas and technical solutions to broaden the prevention and treatment of CKD vascular calcification, a major health problem. Fermentation of Chinese medicine technology belongs to the traditional Chinese medicine processing method. The related technology of using microorganisms to process medicinal materials provides development opportunities for the use of traditional Chinese medicine as a prescription. Fermentation technology has the advantages of low cost, simple operation, and strong selectivity. In addition, there are many types of microorganisms in nature, and the number of strains available for fermentation of Chinese medicine has also increased. The main effects of microbial transformation on Chinese medicine include improving the absorption and utilization of effective ingredients in Chinese medicine, producing new compounds, improving the medicinal properties of Chinese medicine, and reducing toxicity and increasing efficacy. Therefore, it is a very necessary study to apply microbial fermentation technology to the research of Chinese medicinal materials to broaden its medicinal market, which provides a new way to enhance the value of Chinese medicinal materials, while enhancing its medicinal value and enhancing its application potential in clinical medicine and health care. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a Panax notoginseng fermentation product and a preparation method and application thereof.

[0006] The object of the present invention is achieved through the following technical scheme: a bamboo ginseng fermentation product, the preparation method of the bamboo ginseng fermentation product comprises the following steps: mixing the crushed bamboo ginseng with Asperillus niger CICC40048 for anaerobic fermentation, and the fermentation liquid obtained after fermentation is the bamboo ginseng fermentation product. Wherein, the Asperillus niger CICC 40048 is purchased from the China Industrial Microbiological Culture Collection Management Center.

[0007] Furthermore, the particle size of the crushed Panax notoginseng is in the range of 50 to 60 meshes.

[0008] Furthermore, the method for extracting the fermentation broth is: ethanol heating reflux extraction.

[0009] Preferably, the method for extracting the fermentation broth is: using ethanol to ultrasonicate the fermentation product twice, heating and refluxing to obtain a fermentation broth extract, recovering the ethanol solvent, and drying under reduced pressure to obtain a Panax japonicus fermentation product.

[0010] Furthermore, the fermentation temperature ranges from 23 to 33° C., and the fermentation is carried out in a dark environment.

[0011] Furthermore, the Panax notoginseng is a dried Panax notoginseng tuber.

[0012] Preferably, the fermentation method is solid-state fermentation, specifically: provide a nitrogen source and a carbon source 2.5 times the mass of the japonica ginseng, mix them with the crushed japonica ginseng, sterilize them at high temperature, cool them to below 35°C, and then add Asperillus niger CICC 40048 bacterial liquid to ferment for 5 to 50 days.

[0013] Preferably, the fermentation method is liquid fermentation, specifically: the amount of bamboo ginseng added in liquid fermentation is 0.4 times that of potato culture medium, the potato culture medium is mixed with the crushed bamboo ginseng and sterilized at high temperature, then cooled to below 35°C, and then mixed with Asperillus niger CICC 40048 bacterial liquid and fermented for 3 to 10 days.

[0014] Furthermore, the formula of the potato culture medium per 1000 ml is: 200 g of the filtrate filtered after boiling potatoes, 20 g of analytically pure glucose, 1.5 g of analytically pure magnesium sulfate, 3.0 g of analytically pure potassium dihydrogen phosphate and V B1 40mg.

[0015] The present invention also provides an application of the above-mentioned Panax notoginseng fermentation product in preparing a drug for treating and alleviating vascular calcification caused by chronic kidney disease.

[0016] Furthermore, the dosage form of the drug is selected from one or more of decoction, powder, pill, granule, and paste.

[0017] Preferably, the Panax notoginseng medicinal material is cultivated in a natural planting base in Baoshan City, Yunnan Province.

[0018] The principle of the present invention is: vascular calcification (VC) is caused by calcium and phosphorus metabolism disorders due to kidney damage, and calcium and phosphorus are ectopically deposited on the blood vessel wall. The abnormal metabolism of calcium and phosphorus can stimulate the transdifferentiation of vascular smooth muscle cells to osteoblasts and cause calcification of the vascular media, resulting in increased hardness and reduced compliance of the blood vessel wall. The clinical outcome of CKD vascular calcification can be manifested as peripheral small blood vessel calcification causing ischemic necrosis of surrounding tissues and skin ulcers. In recent years, under the guidance of the theory of "kidney storing essence and responsible for bone and marrow production", chronic kidney disease vascular calcification has been treated according to the syndrome differentiation prescription of "tonifying the kidney and filling essence, tonifying qi and producing essence". The 2020 edition of the "Chinese Pharmacopoeia" records that bamboo joint ginseng has the effects of tonifying deficiency and strengthening, relieving cough and expectoration, dispersing blood stasis and stopping bleeding, and relieving swelling and pain. Bamboo-jointed ginseng grows in high mountain thickets or beside rocks in shady and damp valleys. It has the dual effects of both the tonic effect of the northern medicine ginseng and the blood-activating and swelling-reducing effect of the southern medicine Panax notoginseng. In the areas where bamboo-jointed ginseng is distributed, it is called the "King of Herbs" by local herbalists. In the Tujia dialect, it is called "Baisanqi" and "Bamboo-jointed God". It is a classic "Qi" medicine in Tujia ethnic medicine, and has unique therapeutic effects on Qi stagnation and blood stasis type diseases.

[0019] It is currently determined that vascular calcification is an irreversible process that can only be controlled through proactive preventive regulation. Panax notoginseng and its active ingredients have a significant inhibitory effect on both immune inflammation and general inflammation, have a bidirectional regulatory effect on immune function, and can restore immune system abnormalities. Panax notoginseng fermentation products are prepared by fermenting Panax notoginseng with Asperillus niger CICC 40048. The types and contents of active ingredients in Panax notoginseng fermentation products change, making them more suitable for alleviating CKD caused by vascular calcification.

[0020] The beneficial effects of the present invention are:

[0021] 1. Panax notoginseng contains a large amount of saponin compounds. Fermentation with Asperillus niger (CICC40048) will change the chemical composition content of Panax notoginseng, so that the fermentation product of Panax notoginseng has better relief and treatment effects on vascular calcification of abdominal aorta in mice, better relief and treatment effects on calcium salt deposition in mice, and significant effects on reducing creatinine and urea nitrogen in CKD mice. It has better protective effect on the kidneys and can increase the relevant components that can reduce creatinine and urea nitrogen.

[0022] 2. During the powdering process, the bitter smell of bamboo joint ginseng diffuses in the air. The bitter smell in the raw medicinal materials will be alleviated by fermentation with Aspergillus niger. The extract finally prepared not only has better efficacy, but also is more convenient for drug storage and drinking. After drying into powder, it is convenient to reproduce and prepare decoctions, powders, pills, granules, pastes, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The content change diagram of ginsenoside V and ginsenoside IVa in the ginsenoside microbial fermentation products of Examples 1-3 and Comparative Examples 2-6; the ginsenoside V in the figure is a reduced content, and the ginsenoside IVa in the figure is an increased content;

[0024] Figure 2 HE staining sections of kidneys of mice in each group;

[0025] Figure 3 The MASOON-stained sections of kidneys of mice in each group;

[0026] Figure 4 VON-Kossa staining of blood vessels of mice in each group;

[0027] Figure 5 Alizarin red staining of blood vessels of mice in each group;

[0028] Figure 6 is the levels of creatinine and urea nitrogen in serum of mice in each group. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings, but the protection scope of the present invention is not limited to the following.

[0030] Example 1

[0031] Raw materials: 0.24 kg of Panax notoginseng powder, 0.6 kg of oats, Asperillus niger (CICC 40048) bacterial solution (total colony count: 1.0-2.0×10 6 cfu / ml)

[0032] Preparation method: Take 0.24kg of bamboo ginseng superfine powder and set aside for later use. Take oats, wash them, transfer them to an insulation bucket, add 80℃ hot water, soak the grain surface for more than 15cm, keep warm for 10-12 hours, and after all the oats bloom, drain the hot water, mix it with the bamboo ginseng powder, steam it for 60min, take it out and cool it to room temperature, add 450ml of activated Aspergillus niger (CICC 40048), stir evenly, and keep warm for saccharification. After sufficient saccharification, transfer it to a clean sterile container and ferment it in a closed container for 20 days. After sufficient fermentation, distill and take out the fermentation liquid, dry the remaining residue at 60℃, grind it into coarse powder, and obtain a total of 0.282kg of fermentation product. Take the fermentation product, add 5 times the amount of 70% (v / v) alcohol, heat and reflux to extract for 2 hours, filter, take the filtrate, extract the residue again in the same way, and combine the filtrate; the filtrate and the filtrate obtained by distillation are jointly reduced pressure to recover ethanol, further concentrated to obtain an extract, the extract is dried under reduced pressure at 60°C, and ground into fine powder to obtain 8.23g of crude fermentation extract.

[0033] Example 2

[0034] Raw materials: 0.08 kg of Panax notoginseng powder, 0.2 kg of potato, Asperillus niger (CICC 40048) bacterial solution (total colony count: 1.0-2.0×10 6 cfu / ml)

[0035] Preparation method: Take the powder of Panax notoginseng, add anhydrous ethanol to reflux and extract for 60 minutes, the solid-liquid ratio is (1:15), filter, retain the filtrate, and reflux and extract the residue with water for 30 minutes again, the solid-liquid ratio is 1:10. The two filtrates are mixed and concentrated, and then evaporated to an extract for use. The extract is added to 1000ml potato culture medium (the culture medium contains 200g fresh potatoes (the filtrate is taken after boiling purified water for 30 minutes), 20g analytical pure glucose, 1.5g analytical pure magnesium sulfate, 3.0g analytical pure potassium dihydrogen phosphate, 40mg VB1), and sterilized at 121℃ for 30min. When the temperature drops below 35℃, take 200ml and add 30ml Asperillus niger (Asperillus niger CICC 40048) bacterial solution and mix well. Ferment aseptically at 26℃, 220r / min on a shaker for 7 days. The fermentation liquid and ethanol were extracted at a ratio of (1:2), and further concentrated to obtain an extract, which was dried under reduced pressure at 60°C to obtain 2.567 g of crude fermentation extract. After passing the test, the extract was sealed and packaged, and stored in a warehouse.

[0036] Example 3

[0037] Raw materials: 0.24 kg of bamboo ginseng powder, 0.6 kg of sorghum and round glutinous rice, Asperillus niger (CICC 40048) bacterial solution (total colony count: 1.0-2.0×10 6 cfu / ml)

[0038] Preparation method: Take 0.24kg of bamboo ginseng superfine powder and set aside for later use. Take sorghum and round glutinous rice, wash them, mix the sorghum and round glutinous rice and transfer them into a heat preservation barrel, add 80℃ hot water to soak the grain surface for more than 15cm, keep warm for 10-12 hours, after all the sorghum is shelled, drain the hot water, mix it with the bamboo ginseng powder, steam it for 60min, take it out and cool it to room temperature, add 150ml of activated Aspergillus niger CICC 40048 bacterial liquid, stir evenly, and keep warm for saccharification. After sufficient saccharification, transfer it to a clean sterile container and ferment it in a closed container for 20 days. After sufficient fermentation, distill and take out the fermentation liquid, dry the remaining residue at 60℃, grind it into coarse powder, and obtain a total of 0.254kg of fermentation residue. Take the fermentation product, add 5 times the amount of 70% (v / v) alcohol, heat and reflux to extract for 2 hours, filter, take the filtrate, extract the residue again in the same way, and combine the filtrate; the filtrate and the filtrate obtained by distillation are jointly reduced pressure to recover ethanol, further concentrated to obtain an extract, the extract is dried under reduced pressure at 60°C, and ground into fine powder to obtain 4.857g of crude fermentation extract.

[0039] Comparative Example 1

[0040] Ingredients: Bamboo Ginseng Powder

[0041] Preparation method: Take 0.2 kg of Panax notoginseng powder, add anhydrous ethanol to reflux and extract for 60 minutes, the solid-liquid ratio is (1:15), retain the filtrate, and reflux and extract the residue with water for 30 minutes again, the solid-liquid ratio is 1:10. The two filtrates are mixed and concentrated, and dried under reduced pressure to powder to obtain 25.66 g of Panax notoginseng medicinal material extract.

[0042] Comparative Example 2

[0043] Raw materials: 0.08 kg of Panax notoginseng powder, 0.2 kg of potato, Bifidobacterium breve CICC6185 (total colony count: 5.0-6.0 × 10 7 cfu / ml)

[0044] Preparation method: Take the powder of bamboo joint ginseng, add anhydrous ethanol to reflux and extract for 60 minutes, the solid-liquid ratio is (1:15), filter, retain the filtrate, and reflux and extract the residue with water for 30 minutes again, the solid-liquid ratio is 1:10. The two filtrates are mixed and concentrated, and rotary evaporated to extract, and set aside. Add the extract to 1000ml potato culture medium containing 200g fresh potatoes (purified water boiled for 30 minutes and then the filtrate was taken), 20g analytical pure glucose, 1.5g analytical pure magnesium sulfate, 3.0g analytical pure potassium dihydrogen phosphate, V B1 40mg), sterilized at 121℃ for 30min. When the temperature drops below 35℃, add 30ml of Bifidobacterium breve (CICC 6185) bacterial solution to 200ml and mix well. Ferment aseptically at 26℃, 220r / min on a shaker for 7 days. Extract the fermentation liquid with ethanol (1:2), further concentrate to obtain an extract, and dry the extract under reduced pressure at 60℃ to obtain 2.234g of fermentation crude extract. After passing the test, seal and package it and store it in a warehouse.

[0045] Comparative Example 3

[0046] Raw materials: 0.08 kg of Panax notoginseng powder, 0.2 kg of potato, Aspergillus oryzae CICC 40857 (total colony count: 3.0-4.0 × 10 6 cfu / ml)

[0047] Preparation method: Take the powder of bamboo joint ginseng, add anhydrous ethanol to reflux and extract for 60 minutes, the solid-liquid ratio is (1:15), filter, retain the filtrate, and reflux and extract the residue with water for 30 minutes again, the solid-liquid ratio is 1:10. The two filtrates are mixed and concentrated, and rotary evaporated to extract, and set aside. Add the extract to 1000ml potato culture medium containing 200g fresh potatoes (purified water boiled for 30 minutes and then the filtrate was taken), 20g analytical pure glucose, 1.5g analytical pure magnesium sulfate, 3.0g analytical pure potassium dihydrogen phosphate, V B1 40mg), sterilized at 121℃ for 30min. When the temperature drops below 35℃, take 200ml and add Aspergillus oryzae CICC40857, mix well. Ferment aseptically at 26℃, 220r / min on a shaker for 7 days. Take the fermentation liquid: ethanol (1:2) for extraction, further concentrate to obtain an extract, and dry the extract under reduced pressure at 60℃ to obtain 2.356g of fermentation crude extract. After passing the test, seal and package, and store in warehouse.

[0048] Comparative Example 4

[0049] Raw materials: 0.08 kg of bamboo ginseng powder, 0.2 kg of potato, Acetobacter pasteurianus CICC 20002 (total colony count: 6.0-8.0 × 10 7 cfu / ml)

[0050] Preparation method: Take the powder of bamboo joint ginseng, add anhydrous ethanol to reflux and extract for 60 minutes, the solid-liquid ratio is (1:15), filter, retain the filtrate, and reflux and extract the residue with water for 30 minutes again, the solid-liquid ratio is 1:10. The two filtrates are mixed and concentrated, and rotary evaporated to extract, and set aside. Add the extract to 1000ml potato culture medium containing 200g fresh potatoes (purified water boiled for 30 minutes and then the filtrate was taken), 20g analytical pure glucose, 1.5g analytical pure magnesium sulfate, 3.0g analytical pure potassium dihydrogen phosphate, V B1 40mg), sterilized at 121℃ for 30min. When the temperature drops below 35℃, take 200ml and add Acetobacter pasteurianusCICC 20002 and mix well. Ferment aseptically at 26℃, 220r / min on a shaker for 7 days. Take the fermentation liquid: ethanol (1:2) for extraction, further concentrate to obtain an extract, and dry the extract under reduced pressure at 60℃ to obtain 2.339g of fermentation crude extract. After passing the test, seal and package it and store it in a warehouse.

[0051] Comparative Example 5

[0052] Ingredients: 0.08kg of Panax notoginseng powder, 0.2kg of potato, 0.6g of Mucor sp.CICC 3116

[0053] Preparation method: Take the powder of bamboo joint ginseng, add anhydrous ethanol to reflux and extract for 60 minutes, the solid-liquid ratio is (1:15), filter, retain the filtrate, and reflux and extract the residue with water for 30 minutes again, the solid-liquid ratio is 1:10. The two filtrates are mixed and concentrated, and rotary evaporated to extract, and set aside. Add the extract to 1000ml potato culture medium containing 200g fresh potatoes (purified water boiled for 30 minutes and then the filtrate was taken), 20g analytical pure glucose, 1.5g analytical pure magnesium sulfate, 3.0g analytical pure potassium dihydrogen phosphate, V B1 40mg), sterilized at 121℃ for 30min. When the temperature drops below 35℃, take 200ml and add 1.4g of Mucor sp.CICC 3116 powder, mix well. Ferment aseptically at 26℃, 220r / min on a shaker for 7 days. Extract the fermentation liquid with ethanol (1:2), further concentrate to obtain an extract, dry the extract under reduced pressure at 60℃, and obtain 2.234g of fermentation crude extract. After passing the test, seal and package, and store in warehouse.

[0054] Comparative Example 6

[0055] Raw materials: 0.08 kg of Panax notoginseng powder, 0.2 kg of potato, Saccharomyces cerevisiae CICC 1307 (total colony count: 2.0-5.0 × 10 7 cfu / ml)

[0056] Preparation method: Take the powder of bamboo joint ginseng, add anhydrous ethanol to reflux and extract for 60 minutes, the solid-liquid ratio is (1:15), filter, retain the filtrate, and reflux and extract the residue with water for 30 minutes again, the solid-liquid ratio is 1:10. The two filtrates are mixed and concentrated, and rotary evaporated to extract, and set aside. Add the extract to 1000ml potato culture medium containing 200g fresh potatoes (purified water boiled for 30 minutes and then the filtrate was taken), 20g analytical pure glucose, 1.5g analytical pure magnesium sulfate, 3.0g analytical pure potassium dihydrogen phosphate, V B1 40mg), sterilized at 121℃ for 30min. When the temperature drops below 35℃, take 200ml and add 30ml of Saccharomyces cerevisiae CICC 1307 bacterial solution, mix well. Ferment aseptically at 26℃, 220r / min on a shaker for 7 days. Take the fermentation liquid: ethanol (1:2) for extraction, further concentrate to obtain an extract, and dry the extract under reduced pressure at 60℃ to obtain 2.041g of fermentation crude extract. After passing the test, seal and package, and store in warehouse.

[0057] Experimental Example 1 HPLC analysis of changes in chemical components of microbial fermentation

[0058] Preparation of control solution:

[0059] Take 1.0 g of the extract of Panax notoginseng in Comparative Example 1, accurately weigh it, put it in a stoppered conical flask, accurately add 25 ml of 60% methanol, weigh it, ultrasonically treat it at 25°C (power 500 W, frequency 28 kHz) for 40 minutes, cool it, weigh it again, make up the lost weight with 60% methanol, shake it well, filter it with a 0.22 μm microporous filter membrane, and take the filtrate to obtain it.

[0060] Preparation of fermentation product solution:

[0061] (1) Take 1.0 g of the crude fermentation extract in Example 1 and put it into a 25 ml volumetric flask, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 60% methanol, weigh it, and treat it with ultrasound at 25° C. (power 500 W, frequency 28 kHz) for 40 minutes. Let it cool, weigh it again, make up the lost weight with 60% methanol, shake it well, filter it with a 0.22 μm microporous filter membrane, and take the filtrate to obtain the test solution 1 of the solid fermentation product of Panax japonicus.

[0062] (2) Take 1.0 g of the crude fermentation extract in Example 2 and put it into a 25 ml volumetric flask, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 60% methanol, weigh it, and treat it with ultrasound at 25°C (power 500 W, frequency 28 kHz) for 40 minutes. Let it cool and weigh it again. Make up the lost weight with 60% methanol, shake it well, filter it with a 0.22 μm microporous filter membrane, and take the filtrate to obtain the test solution 2 of the liquid fermentation product of Panax japonicus.

[0063] (3) Take 1.0 g of the crude fermentation extract in Example 3 and put it into a 25 ml volumetric flask, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 60% methanol, weigh it, and treat it with ultrasound at 25°C (power 500 W, frequency 28 kHz) for 40 minutes. Let it cool, weigh it again, make up the lost weight with 60% methanol, shake it well, filter it with a 0.22 μm microporous filter membrane, and take the filtrate to obtain the test solution 3 of the solid-state fermentation product of Panax japonicus.

[0064] (4) Take 1.0 g of the crude fermentation extract in Comparative Example 2 and put it into a 25 ml volumetric flask, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 60% methanol, weigh it, and treat it with ultrasound at 25°C (power 500 W, frequency 28 kHz) for 40 minutes. Let it cool and weigh it again. Make up the lost weight with 60% methanol, shake it well, filter it with a 0.22 μm microporous filter membrane, and take the filtrate to obtain the test solution 4 of the liquid fermentation product of Panax japonicus.

[0065] (5) Take 1.0 g of the crude fermentation extract in Comparative Example 3 and put it into a 25 ml volumetric flask, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 60% methanol, weigh it, and treat it with ultrasound at 25°C (power 500 W, frequency 28 kHz) for 40 minutes. Let it cool, weigh it again, make up the lost weight with 60% methanol, shake it well, filter it with a 0.22 μm microporous filter membrane, and take the filtrate to obtain the test solution 5 of the liquid fermentation product of Panax japonicus.

[0066] (6) Take 1.0 g of the crude fermentation extract in Comparative Example 4 and put it into a 25 ml volumetric flask, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 60% methanol, weigh it, and treat it with ultrasound at 25°C (power 500 W, frequency 28 kHz) for 40 minutes. Let it cool and weigh it again. Make up the lost weight with 60% methanol, shake it well, filter it with a 0.22 μm microporous filter membrane, and take the filtrate to obtain the test solution 6 of the liquid fermentation product of Panax japonicus.

[0067] (7) Take 1.0 g of the crude fermentation extract in Comparative Example 5 and put it into a 25 ml volumetric flask, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 60% methanol, weigh it, and treat it with ultrasound at 25°C (power 500 W, frequency 28 kHz) for 40 minutes. Let it cool and weigh it again. Make up the lost weight with 60% methanol, shake it well, filter it with a 0.22 μm microporous filter membrane, and take the filtrate to obtain the test solution 7 of the liquid fermentation product of Panax japonicus.

[0068] (8) Take 1.0 g of the crude fermentation extract in Comparative Example 6 and put it into a 25 ml volumetric flask, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 60% methanol, weigh it, and treat it with ultrasound at 25°C (power 500 W, frequency 28 kHz) for 40 minutes. Let it cool and weigh it again. Make up the lost weight with 60% methanol, shake it well, filter it with a 0.22 μm microporous filter membrane, and take the filtrate to obtain the test solution 8 of the liquid fermentation product of Panax japonicus.

[0069] Preparation of reference solution:

[0070] Take appropriate amount of Panax notoginseng saponin V and Panax notoginseng saponin V reference substance, weigh accurately, add methanol to dissolve, and prepare a reference substance solution with a concentration of 0.2 mg / ml.

[0071] Testing conditions:

[0072] Chromatographic column: Nano-Micro UniSil C18 (250mm×4.6mm, 10μm); mobile phase: 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B), gradient elution according to phase B from 25% to 55%; volume flow rate: 1.0mL / min; wavelength: 203nm; column temperature: 30℃; injection volume: 10μL. The reference solution, comparative example 1, and test solutions 1 to 8 were analyzed in turn.

[0073] Results Analysis

[0074] The HPLC analysis of the microbial fermentation products of Examples 1-3 and Comparative Examples 2-6 was performed, and the ginsenoside V and ginsenoside IVa in each sample before and after fermentation were calibrated according to the chromatographic retention time, and then the content was analyzed. The results are shown in Figure 1 The main active substances of Panax japonicus include saponins, sugars, amino acids, volatile oils, etc. Saponins are the main active ingredients, and Panax japonicus saponin Ⅳa has high biological activity. Figure 1It can be seen that the contents of ginsenoside V and ginsenoside IVa in the ginsenoside fermentation products prepared by most microbial fermentation did not change significantly, while the ginsenoside V and ginsenoside IVa contents of the ginsenoside fermentation products prepared by fermentation with Asperillus niger CICC 40048 changed significantly (the content of ginsenoside V decreased, and the content of ginsenoside IVa increased). It can be seen that Asperillus niger CICC 40048 has the most obvious effect on the changes in the types and contents of the main active ingredients of ginsenosides during the fermentation process, while other microorganisms are difficult to affect the types and contents of the active ingredients of ginsenosides during the fermentation process.

[0075] Experimental Example 2 Pharmacological activity of ginsenosides and ginsenoside V against renal fibrosis in CKD mice

[0076] The experiment used SPF-grade C57 mice, which were purchased from Chengdu Dashuo Experimental Animal Co., Ltd., license number: SCXK (Chuan) 2020-030. The temperature of the animal room was controlled at 23±2°C, and the humidity was controlled at 50±10%. The bedding was changed weekly, and the mice were numbered with ear tags. During the experiment, the mice were free to move, eat and drink freely. After 7 days of adaptive feeding, the mice were modeled.

[0077] Control group (Comparative Example 1 Panax notoginseng group): Extract Panax notoginseng extract from Comparative Example 1 with methanol, and dry to obtain Panax notoginseng saponins. According to the dosage in the Chinese Pharmacopoeia, the maximum daily intake of Panax notoginseng by mice before extraction was estimated, and the mass of the same mass of medicinal material extract was calculated. 0.5% sodium carboxymethyl cellulose was used to prepare a solution.

[0078] Experimental group (Example 1 Panax notoginseng microbial fermentation product group): Extract the Panax notoginseng fermentation liquid of Example 1 and dry it to obtain Panax notoginseng saponin fermentation product. Refer to the dosage in the Chinese Pharmacopoeia to calculate the maximum daily intake of Panax notoginseng by mice before fermentation, and calculate the mass of the same mass of medicinal material fermentation product. Prepare a solution with 0.5% sodium carboxymethyl cellulose.

[0079] Monomer group (ginsenoside extract monomer): Ginsenoside V standard (also known as ginsenoside Ro) was purchased from Xi'an Chenguang Biological Reference Material Co., Ltd. The standard batch number is HS19115B2, and the purity is ≥98%. According to the dosage in the Chinese Pharmacopoeia, the maximum daily intake of ginsenoside V by mice before fermentation was calculated, and the mass of ginsenoside V in the same mass of medicinal materials was calculated. The solution was prepared with 0.5% sodium carboxymethyl cellulose.

[0080] The blank control group was fed with AIN-76A purified rodent diet, and the model group, control group, experimental group and monomer group (comparative example 1 bamboo ginseng group, example 1 bamboo ginseng microbial fermentation product group and bamboo ginseng saponin V group, respectively) were first fed with high adenine (0.75%) high phosphorus (0.923%) high calcium (1.062%) AIN-76A purified rodent diet (Modified AIN-76A Purified Rodent Diet With0.75% Adenine, 0.923% P, 1.062% Ca) for 2 days, and then maintained with high phosphorus high adenine AIN-76A purified rodent diet (Modified AIN-76A Purified Rodent Diet With 1.8% Phosphorusand 0.2% Adenine) until the end of the experiment. During the period, the control group, experimental group and monomer group were added with oral administration for 2.0 months after 1.5 months of maintenance. All mice were fed for 3.5 months, fasted for 12 hours, anesthetized with sodium pentobarbital, and dissected. The kidneys were removed and fixed in 10% formaldehyde fixative for pathological section detection.

[0081] HE staining: Embedding: The tissue samples were fixed with 10% paraformaldehyde, rinsed with running water, and then the tissues were trimmed and placed in a plastic pathological embedding basket for gradient alcohol dehydration, xylene transparency, wax immersion and embedding. Sectioning: The tissue was cut into 5 μm thick slices using a Leica RM2235 slicer, spread in warm water, and fixed on a glass slide. Staining: The sections were dewaxed to water, stained with hematoxylin-eosin, dehydrated with gradient alcohol, transparentized with xylene, and sealed with neutral resin glue. Changes in kidney structure can be observed under a microscope.

[0082] MASSON staining: Embedding: The tissue samples fixed with 4% paraformaldehyde are rinsed with running water, and then the tissue blocks are trimmed and placed in a pathological embedding plastic basket for gradient alcohol dehydration, xylene transparency, wax immersion and embedding. Sectioning: The tissue is cut into 5μm thick slices using a Leica RM2235 slicer, spread in warm water, and fixed on a slide. Staining: The sections are dewaxed to water, stained with hematoxylin-ponceau acid fuchsin solution-aniline blue, dehydrated with gradient alcohol, transparent with xylene, and sealed with neutral resin glue. The renal fibrosis deposition can be observed under a microscope, and the collagen fiber deposition in the tissue is dyed blue, which is convenient for observing the degree of tissue fibrosis.

[0083] Results Analysis

[0084] Mouse kidney pathological section results are shown in Figure 2 and Figure 3. The results of HE staining of the kidneys show that: no mineralized foci were found in the samples of the NC group (normal control group); the number of mineralized foci in the M group (model group) was the largest and the area was larger; the number of mineralized foci in the C group (comparative example 1) was slightly more; the E group (example 1) had mineralized foci with a smaller area; the number of mineralized foci in the V group (monomer ginsenoside V) was relatively large and the area was moderate. The severity of mineralization of the samples in each group is from mild to severe: NC group < E group < C group < V group < M group. No interstitial fibrosis or capsule depression was found in the samples of the NC group; the samples of the other groups all had varying degrees of interstitial fibrosis and capsule depression. The severity of fibrosis in the samples of each group is from mild to severe: NC group < E group < C group < V group < M group.

[0085] The results of MASSON staining showed that the kidneys of mice in groups M, E, and C all showed obvious fibrosis, and groups C and E showed improvement compared with group M. From the data in Table 1, it can be seen that the severity of fibrosis in each group of samples is NC group < E group < C group < V group < M group, which shows that the microbial fermentation product of Panax japonicus in Example 1 has the most significant effect on anti-mouse kidney fibrosis, as shown in Table 1.

[0086] Table 1 Fibrosis area of ​​mice in each group

[0087]

[0088] Experimental Example 3 Pharmacological activity of Panax japonicus, Panax japonicus biofermentation products, and Panax japonicus saponin V against vascular calcification in CKD mice

[0089] The experiment used SPF-grade C57 mice, which were purchased from Chengdu Dashuo Experimental Animal Co., Ltd., license number: SCXK (Chuan) 2020-030. The temperature of the animal room was controlled at 23±2°C, and the humidity was controlled at 50±10%. The bedding was changed every week. The mice were free to move, eat and drink freely during the experiment. After the mice were adaptively fed for 7 days, the model was established.

[0090] The blank control group was fed with AIN-76A purified rodent diet, and the model group, control group, experimental group and monomer group (divided into comparative example 1 bamboo ginseng group, example 1 bamboo ginseng microbial fermentation product group and bamboo ginseng saponin V group) were first fed with high adenine (0.75%) high phosphorus (0.923%) high calcium (1.062%) AIN-76A purified rodent diet (Modified AIN-76A Purified Rodent Diet With0.75% Adenine, 0.923% P, 1.062% Ca) for 2 days, and then maintained with high phosphorus high adenine AIN-76A purified rodent diet (Modified AIN-76A Purified Rodent Diet With 1.8% Phosphorus and 0.2% Adenine) until the end of the experiment. During the period, the control group, experimental group and monomer group were added with oral administration for 2.0 months after 1.5 months of maintenance. All mice were fed for 3.5 months, fasted for 12 hours, anesthetized with sodium pentobarbital, and dissected. The abdominal aorta was removed and fixed in 10% formaldehyde fixative for pathological section detection.

[0091] Alizarin red staining: Embedding: The tissue samples fixed with 10% paraformaldehyde are rinsed with running water, and then the tissue blocks are trimmed and placed in a pathological embedding plastic basket for gradient alcohol dehydration, xylene transparency, wax immersion and embedding. Sectioning: The tissue is cut into 5μm thick slices using a Leica RM2235 slicer, spread in warm water, and fixed on a slide. Staining: The sections are dewaxed to water, and stained according to the instructions of the "Alizarin Red S Staining Solution". After gradient alcohol dehydration, the xylene transparency is used, and the sections are sealed with neutral resin glue. The calcium salt deposition in the blood vessels can be observed under an inverted microscope. The calcium salt will chelate with alizarin red to form an orange-red precipitate.

[0092] Von Kossa staining: Embedding: The tissue samples fixed with 10% paraformaldehyde are rinsed with running water, and then the tissue blocks are trimmed and placed in a pathological embedding plastic basket for gradient alcohol dehydration, xylene transparency, wax immersion and embedding. Sectioning: The tissue is cut into 5μm thick slices using a Leica RM2235 slicer, spread in warm water, and fixed on a slide. Staining: The sections are dewaxed to water, stained according to the instructions of the "Calcium Salt Staining Kit", counterstained with hematoxylin-eosin, dehydrated with gradient alcohol, transparent with xylene, and sealed with neutral resin glue. Observe the appearance of the sections with the naked eye and whether the cover glass is in the appropriate position. Then perform quality inspection under a microscope to observe the integrity and coloring of the tissue sections. Calcium salts will be replaced by silver ions, which are reduced to black under the action of light, generating reddish-brown or dark black calcium salts.

[0093] Results Analysis

[0094] The results of VON-Kossa staining and Alizarin red staining are shown in Figure 4and Figure 5 . Through Alizarin red staining and VON-Kossa staining, it can be seen that the mice in group NC (normal control group), group E (Example 1) and group V (Jiangsu saponin V) did not have calcium salt deposition, while the mice in group M (model group) and group C (Comparative Example 1) had calcium salt deposition, among which the mice in group M had the most calcium salt deposition, and only two mice in group C had calcium salt deposition. It can be seen that the fermentation product of the Jiangsu microorganism in Example 1 can better alleviate the calcium salt deposition of CKD, restore vascular elasticity, and the calcium salt ions do not form deposits in the blood vessels.

[0095] Experimental Example 4 Effects of Panax japonicus, Panax japonicus biological fermentation products, and Panax japonicus saponin V on serum creatinine and urea nitrogen

[0096] The experiment used SPF-grade C57 mice, which were purchased from Chengdu Dashuo Experimental Animal Co., Ltd., license number: SCXK (Chuan) 2020-030. The temperature of the animal room was controlled at 23±2°C, and the humidity was controlled at 50±10%. The bedding was changed every week. The mice were free to move, eat and drink freely during the experiment. After the mice were adaptively fed for 7 days, the model was established.

[0097] The blank control group was fed with AIN-76A purified rodent diet, and the model group, control group, experimental group and monomer group (divided into comparative example 1 bamboo ginseng group, embodiment 1 bamboo ginseng microbial fermentation product group and bamboo ginseng saponin V group) were first fed with high adenine, high phosphorus and high calcium AIN-76A purified rodent diet (Modified AIN-76A Purified Rodent Diet With 0.75% Adenine, 0.923% P, 1.062% Ca) for 2 days, and then maintained with high phosphorus and high adenine AIN-76A purified rodent diet (Modified AIN-76APurified Rodent Diet With 1.8% Phosphorus and 0.2% Adenine) until the end of the experiment. During this period, the control group, experimental group and monomer group were added for gavage administration for 2.0 months after 1.5 months of maintenance. After all mice were fed for 3.5 months, they were fasted for 12 hours, and blood was collected from the eyeballs after anesthesia with sodium pentobarbital for biochemical index detection.

[0098] Creatinine: Creatine is generated under the catalysis of creatinine amide hydrolase, and creatine is hydrolyzed into sarcosine and urea under the catalysis of creatine amino hydrolase. Sarcosine is then catalyzed by sarcosine oxidase to generate glycine, formaldehyde and hydrogen peroxide. Hydrogen peroxide reacts with 2,4-(6-triiodo-3-hydroxybenzoic acid) and 4-aminoantipyrine under the catalysis of peroxidase to generate a purple-red compound. It is measured by colorimetry at a wavelength of 546nm.

[0099] Urea nitrogen: It is hydrolyzed to produce ammonia ions and carbon dioxide under the action of urease. The ammonia ions react with phenol color developer in alkaline medium to produce a blue substance. The amount of this substance produced is proportional to the urea content and is measured colorimetrically at a wavelength of 640nm.

[0100] Results Analysis

[0101] The test results and contents of creatinine and urea nitrogen in mouse serum are shown below Figure 6 . It can be seen that the blood creatinine and blood urea nitrogen of the mice in the model group were significantly higher than those in the control group, indicating that the chronic renal failure mouse model was successfully established. Example 1 The microbial fermentation products of Panax notoginseng have a better effect on reducing creatinine and urea nitrogen in CKD mice. Under the intervention of the microbial fermentation products of Panax notoginseng, it can be seen that the kidney function indicators improved most significantly, indicating that the microbial fermentation products of Panax notoginseng have a better protective effect on the kidneys.

[0102] Example 1, Comparative Example 1 and Panax notoginseng saponin V as the drug intervention group all had a good effect on reducing creatinine and urea nitrogen in CKD mice, which shows that Panax notoginseng medicinal materials are effective in improving renal function indicators in the treatment of CKD, indicating that Panax notoginseng has a good effect on protecting the kidneys. Among them, the Panax notoginseng group fermented with Asperillus niger (Asperillus niger CICC 40048) has a more prominent effect on reducing renal function levels and relieving vascular calcification. If the method of fermenting Panax notoginseng with Asperillus niger enzyme is added to the application of drugs for treating and recovering chronic kidney disease patients and relieving vascular calcification, its prospects are relatively considerable, while improving its medicinal value and enhancing its application potential in clinical medicine and health care.

[0103] In summary, the present invention verifies in a specific embodiment that the Aspergillus niger microorganism has the most obvious effect on the active ingredients of Panax notoginseng during the fermentation process, and verifies that under the same dosage conditions, the product of Panax notoginseng fermented by Asperillus niger (Asperillus niger CICC 40048) has better biological activity than Panax notoginseng or Panax notoginseng saponin V, has better relief and therapeutic effects on vascular calcification of the abdominal aorta of mice, has better relief and therapeutic effects on calcium salt deposition in mice, has a significant effect on reducing creatinine and urea nitrogen in CKD mice, has a better protective effect on the kidneys, and can increase the relevant components that can reduce creatinine and urea nitrogen.

[0104] The above is only a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art shall not deviate from the spirit and scope of the present invention, and shall be within the scope of protection of the claims attached to the present invention.

Claims

1. A fermented product of Panax notoginseng, characterized in that: The preparation method of the bamboo ginseng fermentation product comprises the following steps: mixing the crushed bamboo ginseng with Asperillus niger CICC 40048 for anaerobic fermentation, and the fermentation liquid obtained after fermentation is the bamboo ginseng fermentation product.

2. A Panax notoginseng fermentation product according to claim 1, characterized in that: The particle size of the crushed bamboo ginseng is in the range of 50 to 60 meshes.

3. A Japonica ginseng fermentation product according to claim 1, characterized in that: The method for extracting the fermentation liquid is: ethanol heating reflux extraction.

4. A Panax notoginseng fermentation product according to claim 1, characterized in that: The fermentation temperature ranges from 23 to 33° C., and the fermentation is performed in a light-proof manner.

5. A Panax notoginseng fermentation product according to claim 1, characterized in that: The fermentation method is solid-state fermentation, specifically: provide 2.5 times the mass of the bamboo ginseng nitrogen source and carbon source, mix with the crushed bamboo ginseng, sterilize at high temperature, cool to below 35°C, and then add Aspergillus niger CICC 40048 bacterial liquid to ferment for 5 to 50 days.

6. A Panax notoginseng fermentation product according to claim 1, characterized in that: The fermentation method is liquid fermentation, specifically: providing a bamboo ginseng addition amount of 0.4 times that of a potato culture medium, mixing the prepared potato culture medium with the crushed bamboo ginseng and sterilizing it at high temperature, then cooling it to below 35°C, and then adding Aspergillus niger CICC 40048 bacterial liquid to ferment for 3 to 10 days.

7. A Panax japonicus fermentation product according to claim 6, characterized in that: The formula of the potato culture medium per 1000 ml is: 200 g of the filtrate filtered after boiling potatoes, 20 g of analytical pure glucose, 1.5 g of analytical pure magnesium sulfate, 3.0 g of analytical pure potassium dihydrogen phosphate and V B1 40mg.

8. Use of a Panax notoginseng fermentation product according to any one of claims 1 to 7 for preparing a drug for treating and alleviating vascular calcification caused by chronic kidney disease.

9. The use according to claim 8, characterized in that: The dosage form of the medicine is selected from one or more of decoction, powder, pill, granule and paste.

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