CN111956677A

CN111956677A - Black ginseng extract for treating renal interstitial fibrosis, black ginseng quick-release pellet and application thereof

Info

Publication number: CN111956677A
Application number: CN202010894333.9A
Authority: CN
Inventors: 郭建鹏; 陈林; 崔福顺; 李官浩; 左阿龙; 蒋世翠
Original assignee: Yanbian University
Current assignee: Yanbian University
Priority date: 2020-08-31
Filing date: 2020-08-31
Publication date: 2020-11-20
Anticipated expiration: 2040-08-31
Also published as: CN111956677B

Abstract

The invention provides a black ginseng extract for treating renal interstitial fibrosis, a black ginseng quick-release pellet and application thereof, and belongs to the technical field of medicines. The black ginseng extract is used for efficiently extracting the ginsenosides Rg3, Rg5 and Rk1 from the black ginseng by an ultrasonic extraction method. The invention also provides a black ginseng quick-release pellet which comprises the following components in percentage by mass: 15-35% of black ginseng extract, 25-47% of microcrystalline cellulose, 23-26% of lactose, 5-8% of cross-linked sodium carboxymethyl starch, 5-8% of low-substituted hydroxypropyl cellulose and the balance of 50-65% of ethanol aqueous solution by volume fraction. The pharmacodynamic action and pharmacological mechanism of the black ginseng extract or the black ginseng quick-release pellet for intervening the renal interstitial fibrosis are investigated through in vivo and in vitro experiments, and the black ginseng extract or the black ginseng quick-release pellet has the potential of preparing the medicament for treating the renal interstitial fibrosis.

Description

Black ginseng extract for treating renal interstitial fibrosis, black ginseng quick-release pellet and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a black ginseng extract for treating renal interstitial fibrosis, a black ginseng quick-release pellet and application thereof.

Background

Chronic Kidney Disease (CKD) is a serious clinical problem, and Renal fibrosis, especially Renal Interstitial Fibrosis (RIF), is now recognized as a common pathway of End-stage Renal disease (ESRD), in which the progression of Renal fibrosis is significantly associated with the occurrence of CKD. RIF is a constantly changing and progressive process characterized by the continuous production of activated myofibroblasts (myofibroblasts) and excess extracellular matrix (ECM) in the renal tubules, which are thought to be a major factor in the pathogenesis of RIF. The origin of activated myofibroblasts remains unclear, and there is evidence that they may originate from Epithelial-mesenchymal transdifferentiation (EMT). The development of EMT goes through the following process: (ii) severe loss of E-cadherin; high expression of alpha-SMA; ③ glomerular basement membrane (TBM) is seriously damaged; has enhanced cell migration and invasion capacity. During EMT, tubular epithelial cells and capillary endothelial cells gradually transform first into mesenchymal cells that phenotypically and functionally display myofibroblast characteristics. Current studies also demonstrate that EMT is regulated by a variety of cytokines. TGF-. beta.1 is widely accepted as an essential fibrotic factor, and TGF-. beta.1 and its downstream Smad3 expression have been shown to be a major regulator in fibrogenesis. TGF- β 1 signaling is transduced intracellularly via Smad and non-Smad pathways, myofibroblast markers are increased, ECM synthesis is increased; TGF- β 1 increases the number of myofibroblasts, promoting fibroblast recruitment from myelopoiesis. High upregulation of TGF- β 1 was observed in injured renal tissue, with severe renal fibrosis prevalent in the downstream Smad cascade. Smad2, 3, 4, 6 and 7 are involved in TGF-beta 1 signal transduction in the Smads protein family, and related research shows that renal fibrosis is positively regulated by Smad2 and Smad3 and negatively regulated by Smad 7. There is evidence that TGF- β 1 can initiate and complete the entire EMT process in patient and animal disease models. Therefore, intervention in the TGF-. beta.1/Smads-induced EMT process is considered as a target for treatment of various anti-fibrosis.

The Ginseng radix is dried root of Panax ginseng C.A.Mey belonging to Araliaceae family, and is effective in spleen, lung and heart channels; has effects of invigorating primordial qi, restoring pulse, relieving depletion, invigorating spleen, benefiting lung, promoting fluid production, and tranquilizing. Black Ginseng (BG) is a processed product of fresh ginseng prepared by repeatedly steaming and drying. Black ginseng is tough and has a black color throughout. The black ginseng obtained by steaming and sun-drying ginseng for nine times has remarkably enhanced anti-inflammatory, antioxidant and blood sugar lowering effects. Under the processing conditions of the black ginseng, the original common saponin components in the ginseng are converted, wherein the contents of the ginsenosides Rg3, Rg5, Rk1 and dammarane aglycone PPT and PPD are higher, and the ginsenosides Rg3, Rg5 and Rk1 are the main characteristic saponins which are newly appeared in the black ginseng, have multiple and obvious pharmacological effects, and have the effects of improving hemorheology, resisting apoptosis and platelet aggregation, improving microcirculation, resisting blood coagulation and the like. The extraction method of black ginseng adopts a preparation method of a test solution under the item of a ginseng content determination method in Chinese pharmacopoeia in the local standard black ginseng (DB 22/T2758-. The reflux extraction method commonly used for traditional Chinese medicines strengthens disturbance between a solution and a matrix by utilizing the boiling state of the solution, reduces diffusion resistance of a fluid membrane on the surface of the matrix, but is not beneficial to further dissolution of components because the solution lacks concentration gradient in a certain stage of extraction, and when the method is used for extracting black ginseng, the total content of rare ginsenosides Rg3, Rg5 and Rk1 is low.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for efficiently extracting Black Ginseng Extract (BGE) for treating renal interstitial fibrosis, wherein the main components of the extract include ginsenosides Rg3, Rg5 and Rk1, which have high extraction rates.

The invention also aims to provide the quick-release black ginseng pellet with quick release of medicinal components and high yield.

The invention also aims to provide the application of the black ginseng extract or the black ginseng quick-release pellet in preparing the medicine for treating the renal interstitial fibrosis diseases.

The invention provides a preparation method of a black ginseng extract, which comprises the following steps:

mixing black ginseng powder with 50-70% ethanol water solution by volume fraction, carrying out ultrasonic extraction for 20-50 min under the condition of 40-80 KHz for 3-4 times, combining extracting solutions, filtering to obtain a filtrate, concentrating the filtrate, and freeze-drying to obtain a black ginseng extract;

the volume ratio of the black ginseng powder to the ethanol aqueous solution is 1 g: (10-20) mL.

Preferably, the volume fraction of the ethanol aqueous solution is 55-65%; the volume ratio of the black ginseng powder to the ethanol aqueous solution is 1 g: (15-20) mL; the frequency of the ultrasonic wave is 60-80 KHz; the time of single ultrasonic extraction is 35-48 min.

Preferably, the volume fraction of the ethanol aqueous solution is 65%, and the ratio of the mass of the black ginseng powder to the volume of the ethanol aqueous solution is 1 g: 20mL, 3 times of extraction, 48min of single ultrasonic extraction time, and 80KHz of ultrasonic frequency.

Preferably, the black ginseng is prepared from ginseng which is more than 4 years old; the black ginseng comprises the black ginseng with fibrous root and the black ginseng without fibrous root.

The invention provides a black ginseng extract prepared by the preparation method, which comprises ginsenoside Rg3, Rg5 and Rk 1; the content of the Rg3 is 1.14-7.62 mg ∙ g^-1(ii) a The content of the Rg5 is 1.22-8.38 mg ∙ g^-1(ii) a The Rk1 content is 0.52-8.26 mg ∙ g^-1(ii) a The total content of the ginsenoside is 2.92-24.26 mg ∙ g^-1。

The invention provides a black ginseng quick-release pellet for treating renal interstitial fibrosis, which comprises the following components in percentage by mass: 15-35% of black ginseng extract, 25-47% of microcrystalline cellulose, 23-26% of lactose, 5-8% of cross-linked sodium carboxymethyl starch, 5-8% of low-substituted hydroxypropyl cellulose and the balance of 50-70% of ethanol aqueous solution by volume fraction.

Preferably, the composition comprises the following components in percentage by weight: 25% of black ginseng extract, 36% of microcrystalline cellulose, 24% of lactose, 7% of cross-linked sodium carboxymethyl starch, 7% of low-substituted hydroxypropyl cellulose and the balance of 50% ethanol water solution.

Preferably, the black ginseng quick-release pellet is a brownish yellow pellet, the appearance is round and uniform, the particle size is 0.09-0.13 mm, the disintegration time is 30s, and the cumulative dissolution rate is more than 90% after 10 min.

The invention provides an application of the black ginseng extract or the black ginseng quick-release pellet in preparing a medicament for treating renal interstitial fibrosis.

The invention provides a preparation method of a black ginseng extract, which fully considers the influence of ethanol concentration, ethanol dosage (material-liquid ratio), ultrasonic frequency and single ultrasonic time in a solvent on the contents of ginsenoside Rg3, Rg5 and Rk1 in black ginseng, takes the total contents of rare ginsenoside Rg3, Rg5 and Rk1 as the basis, and takes the design premise that the factors have no interaction and the interference on the design result is less as the design premise, so as to obtain the extraction method with higher extraction rates of the ginsenoside Rg3, Rg5 and Rk 1. The method has the advantages of high extraction efficiency by adopting an ultrasonic extraction method, stable process, simple operation, feasible method and reliable result compared with the traditional reflux alcohol extraction method, and the result can provide a basis for revising the sample preparation method in the black ginseng local standard. The established method for measuring the contents of the rare ginsenosides Rg3, Rg5 and Rk1 in the Yanbian black ginseng is accurate, simple, reliable and good in repeatability, can be used for measuring the content of the marked components in the black ginseng medicinal material, and the result can provide reference for quality control, reasonable medication and further research and development of the black ginseng medicinal material.

Furthermore, the invention specifically limits the growth period of the raw material black ginseng and the existence of fibrous roots of the black ginseng. The total content of the rare ginsenoside Rg3, Rg5 and Rk1 is calculated by an external standard method, and the determination result shows that the fibrous root of the black ginseng is obviously more than the main root and the black ginseng growing for 5 years is more than 4 years is obtained by comparing the content of the three rare saponins.

The invention provides a quick-release pellet of black ginseng, which comprises the following components in percentage by mass: 15-35% of black ginseng extract, 25-47% of microcrystalline cellulose, 23-26% of lactose, 5-8% of cross-linked sodium carboxymethyl starch, 5-8% of low-substituted hydroxypropyl cellulose and the balance of 50-65% of ethanol aqueous solution by volume fraction. The invention takes the yield and the disintegration time as the investigation indexes, and comprehensively scores and evaluates the drug release performance of the quick-release black ginseng pellet. The quick-release black ginseng pellet is a brownish yellow pellet, is round and uniform in appearance, has a particle size of 0.09-0.13 mm, is disintegrated for 30s, and has an accumulated dissolution rate of more than 90% after 10 min. HPLC method for determining the content of 3 rare saponins in the quick-release black ginseng pellet, the content of ginsenoside Rg3, Rg5 and Rk1 should not be less than 0.295mg ∙ g^-1、0.294mg∙g^-1、0.142mg∙g^-1。

The invention provides an application of the black ginseng extract or the black ginseng quick-release pellet in preparing a medicament for treating renal interstitial fibrosis. Blank group (Control), model group (ADR) and positive Control group as Control, and black ginseng extract with high dose, medium dose and low dose as experimental group, and performing gavage for 9 weeks. The results of the physiological and biochemical detection indexes of the urinary protein excretion, the urea nitrogen (BUN) and the Serum Creatinine (SCR) show that the 24-hour urinary protein excretion of the rats in the ADR group is obviously increased (P is less than 0.05) compared with the urinary protein excretion in the Control group; after 9W of experiment, the 24-hour urine protein discharge of rats in the administration group is obviously reduced (P is less than 0.05) compared with that in the ADR group, and the BGE high-dose group is most obvious; the serum BUN content of the rats in the ADR group is obviously increased compared with that in the Control group (P <0.05), and the BUN level of each administration group can be obviously reduced compared with that in the ADR group (P < 0.05); the level of Scr in the ADR group is obviously increased (P <0.05) compared with that in the Control group, and the amount of Scr in each administration group can be obviously reduced (P <0.05) compared with that in the ADR group. The pathological detection result of the kidney tissue shows that the renal tubular morphology of the ADR group is lost, and meanwhile, obvious renal tubular expansion, epithelial cell apoptosis necrosis and cell loss can be seen, and abnormal change is found in renal interstitium; after different dosages of BGE are given, various pathological changes are improved to different degrees, and the degree of interstitial fibrosis is reduced; the BGE high dose group has more remarkable improvement effect than HKSX and LOS groups. The kidney of the rat in the ADR group obviously has a large amount of collagen fibers which are enriched around the renal tubules, the renal interstitium is widened, the collagen fibers are increased, a large amount of protein tubes are formed, and the renal interstitium is distributed in a diffuse manner; the collagen deposition and fibrosis area of each administration group is obviously lower than that of the ADR group, and the BGE high-dosage group only has a small amount of filamentous fibers to deposit, so that the improvement effect is most remarkable. The results of the simultaneous immunohistochemistry showed that the BGE-administered group prevented the elevation of α -SMA and COL-i protein levels in a dose-dependent manner compared to the ADR group, and that the BGE high dose group had the most significant shift in protein compared to the other administered groups (P < 0.05). The semi-quantitative analysis result shows that the average positive area expression rate of the alpha-SMA and the COL-I in the kidney tissue of the rat in the ADR group is obviously higher than that of the Control group, while the average positive area expression rate of the alpha-SMA and the COL-I in the kidney tissue of the rat in the BGE administration group is obviously lower than that of the ADR group (P < 0.05). BGE has a significant intervention effect on the activation of the TGF-beta 1/Smads signaling pathway, thereby inhibiting the fibrotic process through the TGF-beta 1/Smad signaling pathway. BGE dose-dependently improves ADR-induced RIF progression, and has protective effects on renal function, which is related to the regulation of TGF-beta 1/Smad3 signaling pathway.

Drawings

FIG. 1 is a standard curve of rare saponins ginsenoside Rg3, Rg5, Rk1, FIG. 1A-ginsenoside Rg 3; FIG. 1B-ginsenoside Rg 5; figure 1C-ginsenoside Rk 1;

FIG. 2 is a HPLC chart of the mixed reference substance and test solution; a-mixed reference substance, B-test substance; 1-Rg3, 2-Rk1, 3-Rg 5;

FIG. 3 is an HPLC chromatogram of blank, reference and test solutions; FIG. 3A is a blank set HPLC chromatogram; FIG. 3B is a control HPLC chromatogram; FIG. 3C is an HPLC chromatogram of the sample solution;

fig. 4 shows rat 24h urinary protein excretion (n ═ 10),. P <0.05,. P <0.01,. P <0.001VS Control; # P <0.05, # P <0.01, # P <0.001 VSModel;

fig. 5 shows the change in the content of Scr and BUN in rat serum (n ═ 10); # P <0.01 VSControl; p <0.05, P <0.01VS Model;

FIG. 6 shows pathological changes of kidney (200X), a-Control group, b-ADR-stimulated group, c-ADR + BGE 300group, d-ADR + BGE 600group, e-ADR + BGE 1200group, f-ADR + HKSX 70group, g-ADR + LOS 5.25 group;

FIG. 7 shows collagen deposition (200X) in various groups of rats; a-Control group, b-ADR-stimulated group, c-ADR + BGE 300group, d-ADR + BGE 600group, e-ADR + BGE 1200group, f-ADR + HKSX 70group, g-ADR + LOS 5.25 group;

FIG. 8 is the effect of BGE on α -SMA expression in renal tissue (200 ×); a-Control group, b-ADR-stimulated group, c-ADR + BGE 300group, d-ADR + BGE 600group, e-ADR + BGE 1200group, f-ADR + HKSX 70group, g-ADR + LOS 5.25 group;

FIG. 9 is a graph of the effect of BGE on COL-I expression in renal tissue (200X); a-Control group, b-ADR-stimulated group, c-ADR + BGE 300group, d-ADR + BGE 600group, e-ADR + BGE 1200group, f-ADR + HKSX 70group, g-ADR + LOS 5.25 group;

FIG. 10 is a graph of the effect of BGE on the TGF- β 1/Smads signaling pathway in renal tissue.

Detailed Description

mixing black ginseng powder with 50-70% ethanol water solution by volume fraction, carrying out ultrasonic extraction for 20-50 min under the condition of 40-80 KHz for 3-4 times, combining extracting solutions, filtering to obtain filtrate, concentrating, and freeze-drying to obtain a black ginseng extract; the volume ratio of the black ginseng powder to the ethanol aqueous solution is 1 g: (10-20) mL.

In the present invention, the volume fraction of the ethanol aqueous solution is preferably 55% to 65%; the ratio of the mass of the black ginseng powder to the volume of the ethanol aqueous solution is preferably 1 g: (15-20) mL; the frequency of the ultrasonic wave is preferably 60-80 KHZ; the time of single ultrasonic is preferably 35-48 min; more preferably, the volume fraction of the ethanol aqueous solution is 65%, and the ratio of the mass of the black ginseng powder to the volume of the ethanol aqueous solution is 1 g: (10-20) mL, extracting for 3 times, wherein the ultrasonic time is 48min each time, and the ultrasonic frequency is 80 KHz.

In the present invention, the black ginseng preferably includes black ginseng obtained by processing raw ginseng for 4 to 5 years; the black ginseng preferably includes black ginseng having beard and black ginseng not having beard. The extraction amount of ginsenoside Rg3, Rg5 and Rk1 of black ginseng is high. The 5-year-old black ginseng has high extraction amount of the ginsenosides Rg3, Rg5 and Rk1 compared with the 4-year-old black ginseng. The production area of the black ginseng is autonomous state of Korean along Yanbian of Jilin province. The processing method of black ginseng is not particularly limited, and the processing method known in the field can be adopted.

In the invention, the content of Rg3, Rg5 and Rk1 in the black ginseng extract is preferably determined by chromatography. The chromatographic determination method comprises the steps of preparation of a reference substance, preparation of a test sample and chromatographic analysis; the reference substance is prepared by mixing ginsenoside Rg₃、Rg₅、Rk₁The control sample is prepared into 0.52, 0.47, 0.51 mg/mL^-1The control methanol solution of (4). Diluting the obtained black ginseng extract with ethanol to 250mL volumetric flask, shaking up, and filtering with 0.22 μm filter membrane to obtain the final product. The chromatographic parameters are preferably as follows: a chromatographic column: thermo C18(250 mm. times.4.6 mm, 5 μm); mobile phase: a is acetonitrile, B is water, and gradient elution conditions are as follows: 0-5 min (25% -45% A), 6-10 min (45% -60% A), 11-20 min (60% -73% A), 21-25 min (73% -75% A), 26-35 min (75% -25% A); flow rate: 0.8 mL/min^-1(ii) a Detection wavelength: 203 nm; column temperature: 30 ℃; sample introduction amount: 20 μ L. Compared with other chromatographic conditions, the chromatographic column has good column efficiency under the liquid phase condition, the separation degree of the rare ginsenosides Rg3, Rk1 and Rg5 to be detected and adjacent unknown peaks is more than 1.0, and the components to be detected can be better separated. The result shows that the method for detecting the ginsenoside has good reproducibility and stability; the sample adding and recovering experiment shows that the accuracy is good.

The invention provides a black ginseng quick-release pellet for treating renal interstitial fibrosis, which comprises the following components in percentage by mass: 15-35% of black ginseng extract, 25-47% of microcrystalline cellulose, 23-26% of lactose, 5-8% of cross-linked sodium carboxymethyl starch (CMC-Na), 5-8% of low-substituted hydroxypropyl cellulose (L-HPC) and the balance of 50-70% of ethanol aqueous solution by volume fraction.

In the invention, the black ginseng quick-release pellet takes the black ginseng extract prepared as above as a medicinal active ingredient, and the mass of the black ginseng extract is preferably 20-30%, most preferably 25% of the total mass of the black ginseng quick-release pellet through a material loading test.

In the invention, microcrystalline cellulose and lactose are used as diluents together in the black ginseng quick-release pellets, and the comprehensive score (96.7) of the pellets prepared by using MCC + lactose as the diluents is superior to that of other two diluents (microcrystalline cellulose 84.1 or lactose 81.7). Meanwhile, experiments show that the MCC: the mass ratio of lactose is 3:2, the mass of the microcrystalline cellulose preferably accounts for 30-40% and 36% of the total mass of the quick-release black ginseng pellet. The mass of the lactose accounts for 24-25% of the total mass of the black ginseng quick-release pellet, and the most preferable mass is 24%.

In the invention, the cross-linked sodium carboxymethyl starch and the low-substituted hydroxypropyl cellulose are jointly used as disintegrating agents in the quick-release black ginseng pellet. Experiments show that 5% CMC-Na + 5% L-HPC is more suitable as a disintegrant for immediate release pellets than the other groups (10% CMC-Na, 10% L-HPC, 10% PPVP, 5% CMC-Na + 5% PPVP, 5% L-HPC + 5% PPVP). The addition of the composite disintegrant in the pellet is beneficial to the forming of granules and shortens the disintegration time limit of the granules, so 5% of CMC-Na + 5% of L-HPC is selected as the composite disintegrant of the quick-release pellet. The mass of the CMC-Na accounts for 7 percent of the total mass of the quick-release pellet of the black ginseng. The mass of the L-HPC accounts for 7% of the total mass of the quick-release pellet of the black ginseng.

In the invention, the quick-release pellet of black ginseng takes an ethanol water solution as a wetting agent. Experiments show that the pellet prepared by taking 50-70% ethanol water solution as the wetting agent has comprehensive score superior to that of 5% starch slurry, and the 50% ethanol water solution is preferably used as the wetting agent of the quick-release pellet from the cost saving viewpoint. And the 50% ethanol aqueous solution is used for complementing the total mass of the quick-release black ginseng pellet to 100%, and preferably accounts for 1% of the total mass of the quick-release black ginseng pellet.

In the invention, the preparation method of the quick-release black ginseng pellet is preferably prepared by adopting an extrusion-spheronization method. The extrusion-spheronization process preferably comprises the following steps:

adding disintegrating agent and diluent into the prepared ginseng extract, sieving with a 100-mesh sieve, grinding, mixing uniformly, adding wetting agent to prepare soft material, extruding into a bar column shape, rolling in a rolling machine at the rotation speed of 1500r/min for 8min to obtain pills, drying at 40 ℃ for 6h, and sieving with a 20-mesh sieve to obtain the finished pellets. The black ginseng quick-release pellet prepared by the invention is preferably a brown yellow pellet, the appearance is round, the ratio range of the maximum diameter to the minimum diameter of the pellet is 1.05-1.08, and the round degree of the pellet is qualified; the particle size is preferably 0.09-0.13 mm; the pill weight difference result meets the regulation. The bulk density had an average value of 0.762 g/ml. The fluidity is good. Disintegration experiments and dissolution experiments show that the disintegration time of the quick-release black ginseng pellet prepared by the invention is preferably 30s, the cumulative dissolution rate is preferably more than 90% within 10min, and the release of ginsenoside in a certain time has higher stability and repeatability. The average recovery rates of the ginsenosides Rg3, Rg5 and Rk1 are respectively 98.79%, RSD is 0.984% and 98.99%, RSD is 0.772% and 99.01%, and RSD is 1.342%, which indicates that the method has good accuracy and is feasible. HPLC method for determining the content of 3 rare saponins in the quick-release pellet of black ginseng, the content of ginsenoside Rg3, Rg5 and Rk1 should not be less than 0.295mg ∙ g^-1、0.294mg∙g^-1、0.142mg∙g^-1。

In the present invention, the method for preparing the black ginseng extract or the quick-release pellet of black ginseng is not particularly limited, and the method for preparing the pellet known in the art may be used. The content of the black ginseng extract in the medicine is 15-35%, and more preferably 20-30%.

In the invention, the results of physiological and biochemical index measurement show that compared with a model group, the administration group can obviously reduce the urinary protein excretion and reduce the levels of BUN and Scr; the pathological detection result of the kidney tissue shows that the administration group can obviously reduce the degree of interstitial fibrosis; meanwhile, the immunohistochemistry result shows that the administration group prevents the alpha-SMA and COL-I protein level from increasing in a dose-dependent mode, and has obvious intervention effect on the activation of a TGF-beta 1/Smads signal path, so that the fibrosis process is inhibited through the TGF-beta 1/Smad signal path. Therefore, the black ginseng extract or the black ginseng quick-release pellet improves ADR-induced RIF process in a dose-dependent manner, and has a protective effect on renal function, which is related to the regulation of TGF-beta 1/Smad3 signaling pathway.

The following examples are provided to describe the black ginseng extract, the black ginseng immediate-release pellet for treating renal interstitial fibrosis and the application thereof in detail, but they should not be construed as limiting the scope of the present invention.

The medicinal materials are as follows: the black ginseng is a processed product (provided by Yanbian black ginseng health food Limited liability company) with or without fibrous root removal of 4-year-old and 5-year-old garden ginseng, and meets the quality requirement through the laboratory identification.

The sources of the reagents and reagents are shown in Table 1.

TABLE 1 reagents, reagents

The apparatus used is shown in Table 2.

TABLE 2 Instrument Equipment

Example 1

Preparation method of black ginseng extract sample

Precisely weighing dried 4-year-old non-black ginseng powder, taking 65% ethanol as a solvent, taking the material-liquid ratio of 1:20, extracting for 3 times by adopting an ultrasonic extraction method, carrying out ultrasonic treatment for 48min each time, carrying out ultrasonic treatment at the ultrasonic frequency of 80KHz, combining the extracting solutions for 3 times, filtering, concentrating to the concentration of 1mL of black ginseng decoction pieces which is equivalent to 1g, and carrying out freeze drying to obtain a black ginseng extract sample.

Example 2

Preparation method of black ginseng extract sample

Precisely weighing dried 4-year-old fibrous black ginseng powder, extracting 3 times with ultrasonic extraction method at ultrasonic frequency of 80KHz for 48min each time by using 65% ethanol as solvent at a material-to-liquid ratio of 1:20, mixing the 3 times of extractive solutions, filtering, concentrating to 1mL of concentration equivalent to 1g of black ginseng decoction pieces, and freeze-drying to obtain black ginseng extract sample.

Example 3

Preparation method of black ginseng extract sample

Precisely weighing dried 5-year-old black-free ginseng powder, extracting 3 times with ultrasonic extraction method at ultrasonic frequency of 80KHz for 48min for 3 times by using 65% ethanol as solvent at a material-to-liquid ratio of 1:20, mixing the 3 times of extractive solutions, filtering, concentrating to 1mL of concentration equivalent to 1g of black ginseng decoction pieces, and freeze-drying to obtain a black ginseng extract sample.

Example 4

Preparation method of black ginseng extract sample

Precisely weighing dried 5-year-old fibrous black ginseng powder, extracting 3 times by using an ultrasonic extraction method with 65% ethanol as a solvent and the material-liquid ratio of 1:20 for 48min each time and the ultrasonic frequency of 80KHz, combining the extracting solutions for 3 times, filtering, concentrating to the concentration of 1mL of black ginseng decoction pieces which is equivalent to 1g, and freeze-drying to obtain a black ginseng extract sample.

Example 5

Preparation method of black ginseng extract sample

Precisely weighing dried 4-year-old non-black ginseng powder, extracting for 3 times by using an ultrasonic extraction method with 70% ethanol as a solvent and the material-liquid ratio of 1:20, performing ultrasonic treatment for 35min each time and the ultrasonic frequency of 60KHz, combining the extracting solutions for 3 times, filtering, concentrating to the concentration of 1mL of black ginseng decoction pieces which is equivalent to 1g, and performing freeze drying to obtain a black ginseng extract sample.

Example 6

Preparation method of black ginseng extract sample

Precisely weighing dried 4-year-old non-black ginseng powder, extracting for 3 times by using an ultrasonic extraction method with 55% ethanol as a solvent and the material-liquid ratio of 1:15, performing ultrasonic treatment for 35min each time and the ultrasonic frequency of 60KHz, combining the extracting solutions for 3 times, filtering, concentrating to the concentration of 1mL of black ginseng decoction pieces which is equivalent to 1g, and performing freeze drying to obtain a black ginseng extract sample.

Example 7

Preparation method of black ginseng extract sample

Precisely weighing dried 4-year-old non-black ginseng powder, extracting for 3 times by using an ultrasonic extraction method with 70% ethanol as a solvent and the material-liquid ratio of 1:15, performing ultrasonic treatment for 30min each time and the ultrasonic frequency of 80KHz, combining the extracting solutions for 3 times, filtering, concentrating to the concentration of 1mL of black ginseng decoction pieces which is equivalent to 1g, and performing freeze drying to obtain a black ginseng extract sample.

Example 8

Preparation method of black ginseng extract sample

Precisely weighing dried 4-year-old non-black ginseng powder, extracting for 3 times by using an ultrasonic extraction method with 70% ethanol as a solvent and the material-liquid ratio of 1:10, performing ultrasonic treatment for 35min each time and the ultrasonic frequency of 60KHz, combining the extracting solutions for 3 times, filtering, concentrating to the concentration of 1mL of black ginseng decoction pieces which is equivalent to 1g, and performing freeze drying to obtain a black ginseng extract sample.

Example 9

Preparation method of black ginseng extract sample

Precisely weighing dried 4-year-old non-black ginseng powder, extracting for 3 times by using an ultrasonic extraction method with 55% ethanol as a solvent and the material-liquid ratio of 1:10, performing ultrasonic treatment for 35min each time and the ultrasonic frequency of 60KHz, combining the extracting solutions for 3 times, filtering, concentrating to the concentration of 1mL of black ginseng decoction pieces which is equivalent to 1g, and performing freeze drying to obtain a black ginseng extract sample.

Example 10

Preparation method of black ginseng extract sample

Precisely weighing dried 4-year-old non-black ginseng powder, extracting for 3 times by using an ultrasonic extraction method with 70% ethanol as a solvent and the material-liquid ratio of 1:10, performing ultrasonic treatment for 35min each time and the ultrasonic frequency of 80KHz, combining the extracting solutions for 3 times, filtering, concentrating to the concentration of 1mL of black ginseng decoction pieces which is equivalent to 1g, and performing freeze drying to obtain a black ginseng extract sample.

Comparative example 1

A black ginseng extract sample was prepared according to the method of example 5, except that the solvent was a 40% ethanol aqueous solution.

Comparative example 2

A black ginseng extract sample was prepared according to the method of example 6, except that the sonication time was 20 min.

Comparative example 3

A black ginseng extract sample was prepared according to the method of example 9, except that the frequency of ultrasound was 20 KHz.

Example 11

Method for determining content of rare ginsenoside in black ginseng

Determining contents of ginsenoside Rg3, Rk1 and Rg5 in black ginseng by HPLC method, and establishing methodology

1. Preparation of Mixed control solutions

Accurately weighing ginsenoside Rg3, Rg5, Rk1 reference substances 5.200, 4.700, and 5.100mg respectively, and making into 0.52, 0.47, and 0.51 mg/mL^-1The control methanol solutions of (1) were precisely measured in the same 10mL measuring flask at 2 mL of the control solutions to give the reference solutions of 0.104, 0.094, and 0.102 mg/mL^-1The mixed control solution of (4) is filtered through a 0.22 μm filter for use.

The mixed control solutions were measured out at 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0mL, each in a 1mL volumetric flask, and made up with methanol to concentrations of 0.01, 0.02, 0.03, 0.04， 0.05，0.06，0.07，0.08，0.09，0.10mg·mL^-1. 20. mu.L of each was measured by HPLC, and the peak area was measured. Through the regression calculation of the peak area (Y) to the abscissa sample amount (X), as shown in fig. 1, the regression equations of the ginsenosides Rg3, Rg5 and Rk1 are respectively Y ═ 129.43X +0.0551 and r ═ 0.9993; Y517.13X +1.4498, r 0.9993; Y416.95X +3.6333, r 0.9992. The results show that the ginsenosides Rg3, Rk1 and Rg5 are respectively 0.0104-0.104 mg/mL^-1、0.0102～0.102 mg·mL^-1、0.0094～0.094mg·mL^-1The linear relation between the sample amount and the peak area meets the requirement.

2. Preparation of test solution

Diluting the black ginseng extract obtained in the embodiment 1-10 with ethanol to a 250mL volumetric flask, shaking up, and filtering with a 0.22 μm filter membrane to obtain the black ginseng extract.

3. Chromatographic condition and system suitability test

A chromatographic column: thermo C18(250 mm. times.4.6 mm, 5 μm); mobile phase: a is acetonitrile, B is water, and gradient elution conditions are as follows: 0-5 min (25% -45% A), 6-10 min (45% -60% A), 11-20 min (60% -73% A), 21-25 min (73% -75% A), 26-35 min (75% -25% A); flow rate: 0.8mL min-1; detection wavelength: 203 nm; column temperature: 30 ℃; sample introduction amount: 20 μ L. And (3) quantifying by an external standard method, and mixing chromatograms of the reference substance and the test substance, which is shown in figure 2. The test sample has corresponding chromatographic peak at the corresponding position of the chromatographic retention time of the reference sample. Under the liquid phase condition, the theoretical plate numbers meet the requirements, the chromatographic column has good column efficiency, the separation degree of the rare ginsenosides Rg3, Rk1 and Rg5 to be detected and adjacent unknown peaks is more than 1.0, and the components to be detected can be better separated.

Calculating the contents of ginsenoside Rg3, Rk1 and Rg5 by an external standard method. The measurement result shows that the contents and the total contents of the ginsenosides Rg3, Rk1 and Rg5 in the sample are shown in Table 3.

TABLE 3 results of content determination of various samples

4. Precision survey

4.1 the mixed reference substance is measured with intermediate precision, peak areas of the ginsenosides Rg3, Rk1 and Rg5 are detected according to the detection method, and results show that RSD values of peak areas of the ginsenosides Rg3, Rg5 and Rk1 are respectively 0.653% (n is 6), 0.112% (n is 6) and 0.349% (n is 6), which indicates that the precision of the instrument is good.

4.2 repeatability is investigated and the sample solution is precisely measured, the peak areas of the ginsenosides Rg3, Rk1 and Rg5 are detected according to the detection method, and the results show that the RSD values of the peak areas of the ginsenosides Rg3, Rg5 and Rk1 are 0.274% (n is 6), 1.021% (n is 6) and 1.136% (n is 6), which indicates that the method has good reproducibility.

4.3 stability Studies

The same test sample solution is precisely measured, the test sample is detected under the chromatographic conditions at room temperature for 0, 2, 4, 6, 8, 12 and 24 hours, and the results show that the RSD values of the peak areas of the ginsenosides Rg3, Rg5 and Rk1 are 0.777% (n-6), 0.796% (n-6) and 1.279% (n-6), respectively, which indicates that the test sample solution is relatively stable in 24 hours.

4.4 sample application recovery Rate Observation

Precisely measuring 9 parts of black ginseng extract with known content, precisely measuring and adding 3 mixed reference substances with different concentrations, and preparing 3 parts of each concentration respectively. The test sample is prepared by the method, peak area values of the ginsenosides Rg3, Rk1 and Rg5 are detected by the method, and the result shows that the average recovery rates of the ginsenosides Rg3, Rg5 and Rk1 are respectively 99.06 percent and the RSD is 0.899 percent; 98.65%, RSD 1.115%; 99.36% and RSD 1.049%, indicating that the method is good and feasible.

Example 12

1. Study on physical and chemical properties of black ginseng extract

BGE preparation: taking 4-year-old non-black ginseng, preparing a black ginseng extracting solution according to the optimized extraction process, concentrating under reduced pressure until the material-liquid ratio is 1:1, placing the concentrated solution in a vacuum freeze dryer, freeze-drying to obtain freeze-dried powder, crushing, and sieving with a 100-mesh sieve to obtain the BGE.

1) Moisture-wicking property

Precisely weighing 1.000g of black ginseng extract, 3 parts in total, drying at 105 ℃ to constant weight, standing for 24 hours in a saturated ammonium chloride solution humidity environment, and measuring the moisture-wicking rate for 24 hours. The result shows that the moisture absorption rate of the extract is 17% within 24h, which indicates that the extract is extremely moisture-absorbing.

2) Solubility in water

Precisely weighing 1.000g of black ginseng extract, 3 parts in total, respectively placing in 5mL of 25 + -2 deg.C distilled water, swirling for 30s every 2min, observing the dissolution state of the extract, and determining complete dissolution without visible particles or liquid drops. The observation shows that the product is completely dissolved within 5min, which indicates that the product is easy to dissolve in water.

Example 13

1. Process for preparing micropill by extrusion-spheronization method

Adding auxiliary materials into the BGE prepared in the embodiment 12, sieving with a 100-mesh sieve, grinding and uniformly mixing, adding a wetting agent to prepare a soft material, extruding into a similar strip column shape by an extruder (with the aperture of 1.0mm), then adjusting the rotating speed of a spheronizer to 1500r/min and the spheronization time to 8min according to the preparation process parameters determined by a pre-experiment, completely spheronizing the strip-shaped object by friction force to obtain pills, drying the pills at 40 ℃ for 6h, and sieving with a 20-mesh sieve to complete the pills, thereby obtaining the pellets.

2. Comprehensive score evaluation index and measuring method

According to the characteristics of the quick-release pellets, the yield and the disintegration time are determined as investigation indexes, and the weights in the comprehensive score are respectively 40% and 60%;

composite score ═ 40 × sample yield/maximum yield for each group of samples) + (60 × minimum disintegration time for each group of samples/sample disintegration time).

(1) And (3) yield determination: weighing the prepared pellets, collecting the pellets with the screening diameter of-18 to +24 meshes, and weighing; the pellet yield is the mass after screening/total mass of the pellets multiplied by 100%, the experiment is carried out for 3 times, and the average value is calculated.

(2) Determination of disintegration time: weighing 6 parts of 0.500g of qualified pellets, respectively placing the qualified pellets in hanging baskets of a disintegration tester, measuring the time required for all pellets to pass through a screen (constant temperature of 37 ℃ and screen diameter of 0.425mm), namely the disintegration time, repeating the experiment for 3 times, and calculating the average value.

3. Prescription single factor investigation of pellet preparation

Pellets were prepared according to the method of item 1, and the pellet loading, diluent type (MCC, lactose), disintegrant type (CMS-Na, L-HPC, PPVP), and binder (wetting) type (5% starch slurry, 60% ethanol) were determined by single factor investigation.

(1) Amount of material carried

BGE is a hygroscopic material and its amount in the formulation determines the quality of the pellets. MCC is selected as an auxiliary material and the dosage of the MCC is considered, the mass ratio of BGE to MCC is respectively 2:1, 3:2, 1:1, 2:3 and 1:2, namely the mass fraction of BGE in the prescription is respectively 66.7%, 60.0%, 50.0%, 40.0% and 33.3%, and the optimal loading amount is obtained by adopting a comprehensive evaluation method.

(2) Kind of diluent

Weighing 3 parts of BGE, respectively and uniformly mixing with MCC, lactose and MCC + lactose in proportion, preparing a soft material by using 60% ethanol as a wetting agent, extruding and rolling, drying and screening, and evaluating the pellets by adopting a comprehensive grading method.

(3) Kinds of disintegrating agents

Weighing 6 parts of BGE, adding MCC and lactose in corresponding proportion into each part according to the diluent investigation result, then respectively and uniformly mixing with 10% of CMS-Na, 10% of L-HPC, 10% of PPVP, 5% of CMS-Na + 5% of L-HPC, 5% of CMS-Na + 5% of PPVP and 5% of L-HPC + 5% of PPVP, making a soft material by using 60% of ethanol as a wetting agent, extruding, rolling, drying, screening, and evaluating the pellets by adopting a comprehensive evaluation method.

(4) Classes of adhesive (wetting) agents

Weighing 2 parts of BGE, adding MCC and lactose, 5% of CMS-Na + 5% of L-HPC and 5% of starch slurry and 60% of ethanol in corresponding proportions into each part according to the investigation result of a diluent and a disintegrant, preparing a soft material by respectively adding the 5% of starch slurry and the 60% of ethanol, extruding, rolling, drying, screening, and evaluating the pellets by adopting a comprehensive grading method.

(5) Optimized preparation prescription for orthogonal design experiment

According to the types and the dosage of the auxiliary materials screened by the single-factor test, L9 (3) is adopted for three factors of lactose-MCC ratio (A, g/g), disintegrant dosage (B, percent) and humectant ethanol concentration (C, percent)⁴) And (4) designing an experimental optimization prescription in an orthogonal mode. And obtaining the optimal prescription according to the comprehensive grading result. Each factor and eachThe levels are shown in Table 4 and the test design is shown in Table 5.

TABLE 4 factors and levels

TABLE 5 test design

Prescription investigation result of quick-release pellet

(1) Investigation result of material loading

The results show that the total score of the prepared pellets is higher than that of other groups when the mass ratio of BGE to MCC is 1:2, so that the mass ratio of BGE to MCC is 1:2 for the optimal loading amount, and the table 6 shows.

TABLE 6 results of loading

(2) Examination result of diluent species

The results show that when the diluent is MCC + lactose, the prepared pellets have a higher comprehensive score than other groups, so the MCC + lactose is selected as the diluent of the quick-release pellets, and the table 7 shows.

TABLE 7 Diluent species screening results

(3) The results of examining the types of disintegrants

The results show that when 5% CMS-Na + 5% L-HPC is used as disintegrant, the pellets produced have a higher overall score than the other groups. The compound disintegrant not only affects the forming effect of the pellet, but also significantly shortens the disintegration time of the pellet, so 5% CMS-Na + 5% L-HPC is selected as the compound disintegrant of the quick-release pellet, see Table 8.

TABLE 8 disintegrating agent species screening results

(4) Adhesion (wetting) species investigation

The results show that the pellets prepared by using 60% ethanol as the wetting agent have a comprehensive score better than that of 5% starch slurry, so 60% ethanol is selected as the wetting agent for the quick-release pellets, and the table 9 shows.

TABLE 9 wetting agent species screening results

Results of orthogonal design experiments

1. Experimental design and results

The experimental design and results are shown in Table 10, the analysis of variance is shown in Table 11, and the results show that the effect of each factor is A > B > C. The factor A has the most obvious influence on the result, the factor A3 level is selected through visual analysis, the factor B has the obvious influence on the result, the factor B3 level is selected through visual analysis, the factor C has no obvious influence on the result, and the level C1 is selected for reducing the cost, so that the optimal preparation formula is A3B3C 1. Namely, the ratio of MCC to lactose is 3:2, the dosage of the disintegrating agent is 7 percent, and the wetting agent is 50 percent ethanol.

TABLE 10 test design and results

TABLE 11 results of ANOVA

Note: f0.05(2, 2) ═ 19.00

2 validation test

Pellets were prepared according to the above orthogonal design experimental results according to the following formulation: the content of BGE in the pellet was 25%, MCC 36%, lactose 24% (MCC: lactose ratio 3:2), croscarmellose sodium (CMS-Na) 7%, low-substituted hydroxypropylcellulose 7%, humectant 50% ethanol) 1%. The prepared pellet is subjected to 3 verification tests, and the results show that the optimal pellet preparation has stable prescription and good repeatability, and the optimal pellet preparation is shown in table 12.

Table 12 verifies the test results

The pellet prepared by adopting the optimized optimal prescription has good appearance, proper hardness and high yield, can be disintegrated within 30s, can meet the requirement of the quick-release pellet, and has good repeatability of drug release.

Example 14

The samples of the quick-release black ginseng pellet are prepared according to the pellet formula obtained in example 13, and in order to control the product quality, the items of the pellet such as appearance, roundness, particle size distribution, pellet weight difference, dissolution rate and the like are checked, and a quality standard draft of the quick-release black ginseng pellet is established.

1 form of appearance

20 pellets are randomly selected and spread on A4 paper, and the pellets are round in appearance, uniform in size and color and free of adhesion when observed by naked eyes under a daylight lamp light source.

2 bulk density

100.0g of each of 3 batches of pellet samples was weighed, poured into a 500mL graduated cylinder through a glass funnel, the loose volume was measured, and the bulk density pb was calculated as w/v (where w is the weighed mass and v is the volume poured into the graduated cylinder), the average of which was found to be 0.762 g/mL.

3 fluidity (angle of repose)

Weighing 100.0g of pellet sample, taking a glass funnel, horizontally placing a piece of white paper at the bottom, slowly adding the sample, measuring the inclination angle of a formed accumulation body by using a protractor, repeating for 3 times, and measuring the inclination angle, namely the repose angle, to be less than 30 degrees, thereby indicating that the fluidity is good.

4 degree of roundness

The pellet roundness can reflect the quality of pellet formation or molding. Taking 3 batches of samples, measuring the diameter of each batch of 30 pellets by adopting an objective ruler, and calculating the ratio of the maximum diameter to the minimum diameter, wherein the range of the maximum diameter to the minimum diameter is qualified between 0.6 mm and 1.2 mm. The results show that the ratio range of the maximum diameter to the minimum diameter of the pellets is between 1.05 and 1.08, and the pellet roundness is qualified.

5 difference in pill weight

Taking 3 batches of pellet samples, 20 pellets in each batch, precisely weighing the pellets respectively, and calculating the average pellet weight of the pellets in each batch. The pellet weight difference results of 3 batches of samples meet the regulations.

6 particle size distribution

Weighing 100.0g of pellet samples, respectively sieving with 60-mesh, 40-mesh, 20-mesh, 18-mesh and 16-mesh standard test sieves, and determining the mass of the screened pellets. The results show that 87.68 percent of the pellets have the particle size distribution in the range of-18 to +24 meshes.

7 in vitro dissolution test

7.1 preparation of the solution

7.1.1 preparation of control solutions

Accurately weighing ginsenoside Rg3, Rg5, Rk1 reference substances 5.200, 4.700, and 5.100mg, respectively, and making into 0.52, 0.47, and 0.51 mg/mL^-1The control methanol solutions of (1) were precisely measured in the same 10mL measuring flask at 2 mL of the control solutions to give the reference solutions of 0.104, 0.094, and 0.102 mg/mL^-1The mixed control solution of (4) is filtered through a 0.22 μm filter for use.

7.1.2 preparation of test solutions

Precisely weighing 4.000g of pellet, adding 200mL of degassed H under 37 + -0.5 deg.C water bath condition₂Dissolving O to obtain the final product.

7.1.3 preparation of blank solution

Precisely weighing 1.440g MCC, 1.000g lactose and 0.2 g80g CMS-Na, 0.280g L-HPC, 200mL degassed H was added in a 37. + -. 0.5 ℃ water bath₂Dissolving O to obtain the final product.

7.2 pellet dissolution measurement

Dissolution is an important indicator of quality control in immediate release formulations. The dissolution conditions of the marked components of the ginsenoside Rg3, Rk1 and Rg5 in the product are determined by an HPLC method.

Taking 6 batches of pellet samples, taking the contents of the ginsenoside Rg3, Rg5 and Rk1 in the quick-release black ginseng pellets as indexes, and determining the dissolution conditions of the three marked components according to the conditions. Adopting a small cup method, rotating at 100r ∙ min^-1Degassing H at 37 +/-0.5 deg.C₂O is a dissolution medium. Precisely weighing 6 batches of pellets with the main drug content equivalent to 1.000g, respectively placing the pellets in dissolution cups filled with 200mL of dissolution media, respectively taking 1mL of samples at fixed time positions in 1, 2, 3, 5, 7, 8, 10, 12, 15, 20 and 30min, and simultaneously supplementing 1mL of degassed H₂O (37 +/-0.5 ℃), filtering with a 0.22 mu m filter membrane, sucking 20 mu L filtrate, injecting into a liquid phase instrument, measuring the contents of the three ginsenosides, calculating the concentration and the simultaneous cumulative release degree of the medicines, and drawing a time-dissolution rate curve.

7.3 examination of dissolution component determination methodology

7.3.1 chromatographic Condition and System suitability test

The chromatogram conditions are the same as above, and the chromatogram of blank, reference and test sample is shown in FIG. 3. The result shows that the theoretical plate number meets the requirement, under the condition, the separation degree of the rare ginsenosides Rg3, Rg5 and Rk1 to be detected and adjacent unknown peaks is more than 1.0, and the components to be detected can be well separated; at the same retention time as the reference solution, the test solution has a corresponding chromatographic peak, and the blank solution has no chromatographic peak.

7.3.2 repeatability test

The test solution to be tested is precisely measured, peak area values of the ginsenosides Rg3, Rg5 and Rk1 are detected according to the method, and results show that the RSD values of the peak areas of the ginsenosides Rg3, Rg5 and Rk1 are respectively 1.047% (n is 6), 1.125% (n is 6) and 0.963% (n is 6), which indicates that the method has good reproducibility.

7.3.3 stability Studies

The same test solution is precisely measured, the peak area values of the ginsenosides Rg3, Rg5 and Rk1 in the test solution are detected at room temperature for 0, 2, 4, 6, 8, 12 and 24 hours, and the results show that the RSD values of the peak areas of the ginsenosides Rg3, Rg5 and Rk1 are 1.361% (n is 6), 1.367% (n is 6) and 1.482% (n is 6), respectively, which indicates that the test solution is relatively stable in 24 hours.

7.3.4 sample recovery Studies

The method comprises the steps of precisely measuring 9 parts of a sample solution of the quick-release black ginseng pellet with a known content, precisely measuring and adding 3 mixed reference substance solutions with different concentrations, preparing a test sample according to the conditions, and detecting peak area values of the ginsenosides Rg3, Rg5 and Rk1 according to the conditions, wherein the results show that the average recovery rates of the ginsenosides Rg3, Rg5 and Rk1 are 98.79%, respectively, and the RSD is 0.984% and 98.99%, respectively, 0.772% and 99.01%, and 1.342%, which indicates that the method has good and feasible accuracy.

7.4 pellet dissolution rate determination results

The result shows that the cumulative dissolution of the quick-release pellet of the black ginseng is 60 percent in 5min, the cumulative dissolution of the quick-release pellet of the black ginseng exceeds 90 percent in 10min, the cumulative dissolution of the quick-release pellet of the black ginseng reaches 100 percent in 30min, and the requirement of the quick-release pellet can be met. The method has the advantages of strong specificity, easy operation, high sensitivity, good repeatability and no interference of auxiliary materials, and can effectively control the product quality.

Determination of contents of ginsenosides Rg3, Rg5 and Rk1 in 8-black ginseng quick-release pellet

8.1 preparation of the solution

8.1.1 preparation of Mixed control solutions

The same as the above-mentioned mixed reference solution.

8.1.2 preparation of test solutions

Precisely weighing 1.000g of quick-release pellet of radix scrophulariae, adding 15mL of methanol, performing ultrasonic treatment for 3 times, each time for 20min, mixing extractive solutions, volatilizing solvent, and dissolving with 5mL of methanol.

8.2 determination of ginsenoside content in sample

Precisely weighing 1.000g of 3 batches of the quick-release black ginseng pellets, preparing samples according to the method, and detecting the contents of the ginsenosides Rg3, Rg5 and Rk1 in the samples according to the recording method, wherein the results are shown in Table 13. Therefore, the product contains ginsenoside Rg3, Rg5 and Rk1 not less than 0.295mg∙g^-1、0.294mg∙g^-1、0.142mg ∙g^-1(average content. times.90%).

TABLE 13 determination results of 3 ginsenoside contents in quick-release pellet

The black ginseng quick-release pellet is a brownish yellow pellet, has round and uniform appearance, particle size of 0.09-0.13 mm, disintegration time of 30s and cumulative dissolution rate of more than 90% after 10 min. HPLC method is used for determining the content of 3 rare saponins in the black ginseng quick-release pellets, and the product contains ginsenoside Rg₃、Rg₅、Rk₁Not less than 0.295mg ∙ g^-1、0.294mg∙g^-1、0.142mg∙g^-1。

Example 15

1 laboratory animal

70 SPF SD male rats with weight (200 +/-20 g) (provided by Changyz animal technology, Inc., license number: SCXK Gi 2012-0002) are adapted to be fed with 1W rats, are numbered, are fed with drinking water freely and are fed in a single cage, and the cages are periodically sterilized. Good indoor ventilation and relative humidity of 65-75%.

2 method of experiment

2.1 animal grouping and administration

After the experimental animal is fed with 1W, collecting the metabolite urine for 24h, measuring the urine protein for 24h, and bringing the urine protein into the experiment after all the urine protein is negative. The test pieces were randomly divided into a blank group (Control), a model group (ADR), a model administration group and a positive Control group, and 10 pieces were added to each group. Except for Control group, the rest groups were injected with 6.2 mg/kg of doxorubicin through tail vein^-1Molding; beginning with molding 1d, rats were administered the corresponding dose of drug by gavage every morning, and the Control and ADR groups were gavaged with the same volume of saline for 9 consecutive weeks. After the experiment is finished, taking rat serum and left and right kidneys, and detecting each index. Experimental groups and dosing concentrations are shown in table 14.

TABLE 14 Experimental groups and concentrations administered (mg. kg)^-1·day^-1)

2.2 general examination

In the experimental period, the weight, mental status, diet and drinking water, behavior and activity, fur color and the like of the animals are observed every day.

2.3 index detection

2.3.124 h urinary protein excretion

In the experimental period, collecting 24h urine of rats in a metabolism cage periodically every week, detecting the 24h urine protein concentration of each group of rats by adopting a biuret method after the urine is pretreated, repeating the step for 3 times to obtain an average value, and calculating the 24h urine protein excretion according to a formula (1).

24h urine protein excretion (mg) to 24h urine protein concentration (mg. mL)^-1) X 24h urine volume (mL) equation (1)

2.3.2 detection of Kidney function indicators in serum

After the experiment, ether anesthesia is carried out, blood is taken from eyeball, the blood is placed for 30min at room temperature, and the speed is 3000 rpm.min^-1Centrifuging for 10min, collecting supernatant in 1.5mL tube, storing at-80 deg.C, and determining the content of urea nitrogen (BUN) and Serum Creatinine (SCR) with kit.

2.4 histopathological examination

2.4.1 Paraffin section

(1) Fixing rat kidney tissue with 4% paraformaldehyde for 24h, vertically cutting kidney tissue with thickness of about 3mm, loading into embedding box, and washing with running water for more than 30 min;

(2) and (3) dehydrating, and then, transparent and wax-dipping embedding: placing the tissue in 40% and 70% ethanol for 20min respectively, 80% ethanol I, ethanol II, anhydrous ethanol I and anhydrous ethanol II for 10min respectively, xylene I and xylene II for 5min respectively, soft wax at 56-58 deg.C for 30min, and hard wax embedding for 40 min;

(3) embedding: the tissue is placed in the center of the embedding box, so that bubbles are prevented from being generated;

(4) slicing: cutting kidney tissue into 3 μm thickness after embedding;

(5) fishing out the slices, unfolding the slices and baking the slices: spreading in 50 deg.C water bath, baking for 1 hr on a baking machine to prevent flaking.

2.4.2 HE staining

(1) Slice dewaxing to water: washing with flowing water for 5min in xylene I for 10min, respectively with xylene II, anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 90% ethanol, and 85% ethanol for 5 min;

(2) HE staining: after 2min of hematoxylin liquid staining, washing with small water flow for 5min, returning blue with 1 XPBS for 2min, washing with running water for 5min, staining with eosin for 6min, and washing with running water for 5 min;

(3) dehydrating, transparent and sealing: slicing under ethanol concentration of 85%, 90%, 95% I, 95% II, 100% I, and 100% II respectively 10, xylene I and xylene II respectively 3min, and sealing with neutral gum.

2.4.3 MASSON staining

(1) Slice dewaxing to water: the same as above;

(2) soaking the slices in a Bouin fixing solution overnight at room temperature, and washing with water until the slices are yellow;

(3) dyeing the azure stone blue for 5min, and slightly washing with distilled water;

(4) staining with hematoxylin for 5min, and slightly washing with distilled water;

(5) differentiating with 5% glacial acetic acid water solution for 15s, and washing with running water;

(6) acid re-reddening and phosphomolybdic acid water solutions are respectively dip-dyed for 10 min;

(7) differentiating 5% phosphotungstic acid for 10-20min, and slightly sucking with filter paper;

(8) dyeing with benzene glue blue liquid for 15min, and rapidly passing through 95% alcohol;

(9) and (3) dehydrating, and then, transparent and sealing a sealing sheet: under the condition of absolute ethyl alcohol I, II and III for 5-10 min respectively; i, II xylene for 3-5 min each; the neutral gum was encapsulated and observed under a mirror.

2.5 immunohistochemical method for detecting expression of alpha-SMA and COL-I proteins in renal tissues

(1) Slice dewaxing to water: the same as 2.4.2;

(2) placing the slices in 0.01M sodium citrate, heating to boil, heating at low temperature for 20min, and cooling for 20 min;

(3) washing with PBS for 5min × 2 times, and discarding PBS;

(4) 50 μ L of 3% H was added dropwise at room temperature ₂0₂Wetting the box for 10min, blocking the activity of endogenous peroxidase, and washing with PBS for 5min × 2 times;

(5) dripping into 50 μ L5% coat Serum wet box for 30 min;

(6) dripping 50 μ L primary antibody (diluted by rabbit anti-mouse alpha-SMA 1:100, diluted by rabbit anti-mouse COL-I1: 100), standing overnight at 4 deg.C in a wet box, and washing with PBS for 5min × 3 times;

(7) dripping 50 μ L secondary antibody, standing at room temperature for 30min, and washing with PBS for 5min × 3 times;

(8) adding 50 μ L HRP anti-Rabbit IgG dropwise, incubating at room temperature for 20min, and washing with PBS for 5min × 3 times;

(9) DAB solution color development;

(10) counter-staining with hematoxylin for 10s, washing with water for 5min, and returning blue with PBS for 10 s;

(11) dehydrating, transparent and sealing: under the condition of absolute ethyl alcohol I, II and III for 5-10 min respectively; i, II xylene for 3-5 min each; sealing the slices with neutral gum for a long time, and observing under a mirror;

(12) under 200-fold microscopy, 5 to 10 non-overlapping fields were randomly selected for each sample, and the percentage immunohistochemistry that each section was positive in the entire field was calculated, taking 3 averages.

2.6 Westernblotting detection of protein expression in Kidney tissue

2.6.1 Kidney tissue protein extraction

(1) Taking 10-15 mg of kidney tissue, adding lysis solution to grind under the low temperature condition, cracking until no obvious blocky tissue exists, collecting in a 1.5mL tube, cracking in a refrigerator at 4 ℃ for 30min, performing ultrasonic treatment in ice bath for 10min, and storing at-80 ℃ overnight;

(2) taking out the tissue homogenate the next day, centrifuging at 4 deg.C 10000rpm/min for 30min, and taking the upper layer;

(3) taking a little homogenate to determine the protein concentration (BCA method), adding a Loading Buffer to the rest, mixing uniformly, boiling in boiling water for 8min, and storing at-80 ℃.

2.6.2 BCA assay for protein concentration

(1) Preparing a BCA working solution according to the number of samples, wherein the ratio of reagents A and B is 50: 1, mixing the components completely and uniformly, and using the mixture as it is;

(2) preparation of BSA standard: with 1 XPBS and 1 mg/mL^-1BSA solution was prepared at a concentration of 0, 0.05 mg/mL^-1，0.1mg·mL^-1，0.2mg·mL^-1，0.4mg·mL^-1，0.6mg·mL^-1，0.8 mg·mL^-1And 1 mg. mL^-1The protein standard of (1);

(3) dilution of the protein sample: total protein was removed from-80 ℃ and diluted 10-fold after thawing by nature (2 μ L protein sample +18 μ L1 × PBS);

(4) after the preparation of earlier stage work, respectively adding a standard substance and a sample to be detected into a 96-well plate, wherein each sample is provided with 3 multiple wells, and 2 mu L of the sample and 200 mu L of working solution are added into each well; the well plate was placed in an oven and incubated for 30min (37 ℃), the absorbance of the sample at 562nm was measured, and the protein concentration of the sample was determined according to the standard curve.

2.6.3 Westernblotting detection of protein expression

(1) Preparing glue: taking clean glass, wiping residual stains and paper scraps on the glass with 75% alcohol and a piece of mirror wiping paper, and putting the glass on a rubber frame; selecting gel preparation concentration according to the size of the prepared protein, preparing lower layer gel, namely separation gel according to the proportion of each reagent in the table 15, quickly adding the mixture into a space between two pieces of glass on a gel frame after uniformly mixing in a vortex manner, wherein each volume is about 5mL, adding 1mL of saturated n-butyl alcohol to the uppermost layer to level the liquid surface, and discharging bubbles and sealing the liquid; after 1h, solidifying the lower layer glue, pouring out saturated n-butyl alcohol, washing the liquid level with double-distilled water, washing the residual n-butyl alcohol, sucking dry with filter paper, preparing the upper layer glue, namely 5% concentrated glue according to the proportion of each reagent in the table 16, quickly adding the mixture after vortex mixing, and inserting a comb selected in advance according to the number of samples; after 30min, the upper layer is solidified, the comb is pulled out, bubbles in the hole and residual upper layer glue are washed away by double distilled water, and the machine is assembled.

TABLE 15 preparation of the separation gels

TABLE 16 preparation of concentrated gums

(2) Preparing a sample: calculating the amounts of a protein Sample, 1-PBS and a Sample Buffer solution (Sample Loading Buffer) according to the measured protein concentration of the Sample according to the target Loading amount, preparing the Sample according to the proportion, placing the Sample into a metal bath at 95 ℃ for 5min after centrifugation-vortex-centrifugation to denature the Sample, cooling the Sample, centrifuging-vortex-centrifugation, placing the Sample into-20 ℃ for temporary storage, and waiting for Loading;

(3) sample loading and glue running: pouring a proper amount of Buffer solution, starting sample Loading, filling 2 mu L of Marker on the left side of the first sample, starting glue running at 80V voltage, and stopping running until the sample runs to the bottom of the separation glue after the sample is completely concentrated in the concentrated glue, wherein the sample Loading solution in the rest holes is 30 mu L;

(4) transfer printing: soaking a PVDF membrane in methanol in advance, stripping off concentrated gel, only reserving separation gel for standby, putting sponge-filter paper-PVDF membrane-separation gel-filter paper-sponge in a transfer buffer solution in sequence, fastening a transfer clamp, transferring in an ice bath transfer tank, and determining transfer time according to the size of protein;

(5) blocking: carefully remove the PVDF membrane and seal it in 5% skim milk (1g skim milk +20mLPBST) for 1h, then wash the membrane with PBST for possible residual skim milk;

(6) primary antibody incubation: adding rabbit anti-mouse TGF-beta 1, Smad3, p-Smad3 and Smad7(1:2500) primary antibodies for incubation; placing on a shaking table at 4 ℃ overnight, and washing the membrane by PBST;

(7) and (3) secondary antibody incubation: adding secondary antibody (1:2500) corresponding to the primary antibody, incubating at room temperature for 1h, and repeatedly washing the membrane by PBST;

(8) and fully soaking in ECL solution for color development, and exposing in an imaging system.

3 statistical analysis

Data are expressed as Mean ± SD, statistical analysis was compared using Student's t test or one-way ANOVA, and Tukey's comparison was examined. Statistical analysis was performed with GraphPad prism6.0 Software (GraphPad Software, inc. la Jolla, CA, USA), with P <0.05 indicating a statistical difference.

4 results of the experiment

4.1 general case

The weight of the Control component is increased, and the color and luster are bright; ADR group weight increase is slow, hair color is dark, edema, diarrhea and other conditions appear, about 90% ADR group tails appear different degree swelling, rot, it is caused by the side effect of adriamycin, regularly sterilize rat tail iodine tincture; after administration, each administration group relieves each symptom of ADR group to different extent, and the tail rot condition is improved.

4.2 rat body weight changes

The body weight results of the groups showed that the ADR group had a decrease in body weight and the rats had an increase in body weight after administration, but there was no significant difference, compared to the Control group.

4.3 index detection results

4.3.124 h quantitative results of urine protein

Experiments are carried out at 0-1W, and the UP of rats in different groups has no obvious difference (P is more than 0.05); 2W after the experiment, the 24h urine protein excretion of the rat in the ADR group is obviously increased (P is less than 0.05) compared with that in the Control group; at 9W after the experiment, the urine protein output of rats in the administration group at 24h is reduced compared with that in the ADR group (P <0.05), and the BGE high-dose group is most remarkable, and is shown in figure 4.

4.3.2 Biochemical index detection results

The serum BUN content of the rats in the ADR group is obviously increased compared with that in the Control group (P <0.05), and the BUN level of each administration group is obviously reduced compared with that in the ADR group (P < 0.05). The level of Scr in the ADR group was significantly higher than that in the Control group (P <0.05), and the amount of Scr in each administration group was significantly lower than that in the ADR group (P <0.05), as shown in FIG. 5.

4.4 pathological examination of Kidney tissue

4.4.1 HE staining results

Under an optical microscope, the kidney of the rat in the Control group is not abnormally changed, the shape of the renal tubule is intact, the arrangement is compact, and the interstitium is not abnormally changed; the renal tubular morphology of the ADR group is lost, and meanwhile, obvious renal tubular expansion, epithelial cell apoptosis necrosis, cell loss and renal interstitium abnormal change can be seen; after different dosages of BGE are given, various pathological changes are improved to different degrees, and the degree of interstitial fibrosis is reduced; the BGE high dose group improved significantly more than the HKSX, LOS group, see FIG. 6.

4.4.2 Masson staining results

Under an optical microscope, the kidney of the rat in the Control group has no collagen production or has little filamentous deposition; the kidney of the rat in the ADR group obviously has a large amount of collagen fibers which are enriched around the renal tubules, the renal interstitium is widened, the collagen fibers are increased, a large amount of protein tubes are formed, and the renal interstitium is distributed in a diffuse manner; the collagen deposition and fibrosis area of each administration group was significantly lower than that of the ADR group, and the BGE high dose group had only a small amount of filamentous fibers deposited, and the improvement was most significant, as shown in fig. 7.

4.5 immunohistochemical results

4.5.1 expression results of alpha-SMA protein

From the immunohistochemistry and semi-quantitative data results, it can be seen that only slight brownish yellow particles appeared in the Control group rats, indicating that there was little expression of α -SMA; the increase in α -SMA expression was very significant in the ADR group (P < 0.05); the BGE-administered group prevented elevated α -SMA protein levels in a dose-dependent manner compared to the ADR group, and the conversion of the BGE high-dose group to protein was most significant compared to the other administered groups (P < 0.05). The semi-quantitative analysis result shows that the average positive area expression rate of the alpha-SMA in the kidney tissue of the rat in the ADR group is obviously higher than that of the Control group, while the average positive area expression rate of the alpha-SMA in the kidney tissue of the rat in the BGE administration group is obviously lower than that of the ADR group (P <0.05), and the result is shown in figure 8.

4.5.2 COL-I protein expression results

The expression of COL-I in kidney tissue of rats in each group is shown in FIG. 9. As can be seen from the figure, the kidney tissue COL-I of the rats in the Control group is only expressed in a small amount; compared with the Control group, a large amount of brown yellow particles are deposited in the kidney tissues of the rats of the ADR model group, and the expression of COL-I is obviously increased (P < 0.05); compared with the ADR group, the BGE administration group can inhibit the expression of COL-I protein in rat kidney tissues, wherein the change of the high dose group is most remarkable (P < 0.05). The semi-quantitative analysis result shows that the average positive area expression rate of COL-I in the kidney tissues of the rats in the ADR group is obviously higher than that of the Control group, and the average positive area expression rate of COL-I in the kidney tissues of the rats in the BGE administration group is obviously lower than that of the ADR group (P < 0.05).

4.6 BGE balances TGF-. beta.1/Smad 3 signals in renal tissue

This experiment examined the effect of BGE on the TGF-. beta.1/Smad 3 signaling pathway, which has been shown to be significant for RIF regulation. Compared with the ADR group, the BGE administration group down-regulates the expression level of TGF-beta 1 in a dose-dependent manner, reduces the ratio of P-Smad3 to Smad3, up-regulates the expression of the negative regulator Smad7 (P <0.05), and the change of the BGE high dose group is very obvious, as shown in FIG. 10. The results show that BGE has obvious interference effect on the activation of TGF-beta 1/Smads signal path, thereby inhibiting the fibrosis process through the TGF-beta 1/Smads signal path.

In conclusion, BGE dose-dependently improved ADR-induced RIF progression with protective effects on renal function, which is associated with its regulation of the TGF-. beta.1/Smad 3 signaling pathway.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a black ginseng extract for treating renal interstitial fibrosis is characterized by comprising the following steps:

mixing black ginseng powder with 50-70% ethanol water solution by volume fraction, carrying out ultrasonic extraction for 20-50 min under the condition of 40-80 KHz for 3-4 times, combining extracting solutions, filtering to obtain a filtrate, concentrating the filtrate, and carrying out freeze drying to obtain a black ginseng extract;

2. The preparation method according to claim 1, wherein the volume fraction of the ethanol aqueous solution is 55-65%; the volume ratio of the black ginseng powder to the ethanol aqueous solution is 1 g: (15-20) mL; the frequency of the ultrasonic wave is 60-80 KHz; the time of single ultrasonic extraction is 35-48 min.

3. The method according to claim 1, wherein the ethanol aqueous solution has a volume fraction of 65%, and the ratio of the mass of the black ginseng powder to the volume of the ethanol aqueous solution is 1 g: 20mL, 3 times of extraction, 48min of single ultrasonic extraction time, and 80KHz of ultrasonic frequency.

4. The method according to claim 1 or 2, wherein the black ginseng is processed from ginseng having a 4-year old or older; the black ginseng comprises the black ginseng with fibrous root and the black ginseng without fibrous root.

5. The black ginseng extract prepared by the preparation method of any one of claims 1 to 4, which comprises the ginsenosides Rg3, Rg5 and Rk 1; the content of the Rg3 is 1.14-7.62 mg ∙ g^-1(ii) a The content of the Rg5 is 1.22-8.38 mg ∙ g^-1(ii) a The Rk1 content is 0.52-8.26 mg ∙ g^-1(ii) a The total content of the ginsenoside is 2.92-24.26 mg ∙ g^-1。

6. The quick-release black ginseng pellet for treating renal interstitial fibrosis is characterized by comprising the following components in percentage by mass: 15-35% of black ginseng extract, 25-47% of microcrystalline cellulose, 23-26% of lactose, 5-8% of cross-linked sodium carboxymethyl starch, 5-8% of low-substituted hydroxypropyl cellulose and the balance of 50-70% of ethanol aqueous solution by volume fraction.

7. The quick-release black ginseng pellet of claim 6, which comprises the following components in percentage: 25% of black ginseng extract, 36% of microcrystalline cellulose, 24% of lactose, 7% of cross-linked sodium carboxymethyl starch, 7% of low-substituted hydroxypropyl cellulose and the balance of 50% ethanol water solution.

8. The quick-release black ginseng pellet as claimed in claim 6 or 7, wherein the quick-release black ginseng pellet is a brownish yellow pellet, has a round and uniform appearance, a particle size of 0.09-0.13 mm, a disintegration time of less than 30s and an accumulated dissolution rate of more than 90% after 10 min.

9. Use of the black ginseng extract according to claim 5 or the quick-release pellet of black ginseng according to any one of claims 6 to 8 in the preparation of a medicament for treating renal interstitial fibrosis.