Tibetan medicine supramolecular nanoparticles as well as preparation method and application thereof

Abstract

The invention provides Tibetan medicine supramolecular nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of Tibetan medicine. The preparation method of the supramolecular nanoparticles comprises the following steps: (1) weighing medicinal material powder of cortex berberidis and phyllanthus emblica, uniformly mixing, decocting with water, and filtering to obtain a cortex berberidis-phyllanthus emblica combined decoction; and (2) centrifuging the cortex berberidis-emblic leafflower fruit combined decoction, dialyzing the supernate, and drying to obtain the supramolecular nanoparticles. The supramolecular part of the cortex berberidis-emblic leafflower fruit combined decoction is prepared and separated by adopting a specific process, and the supramolecular part is supramolecular nanoparticles with good stability. The supramolecular nanoparticles can effectively improve the consumption of IR-HepG2 cell glucose and activate a PI3K / AKT signal channel, and are expected to be used for preparing medicines for treating type 2 diabetes mellitus. The invention provides a scientific basis for clinical reasonable application and modern development of the cortex berberidis-emblic leafflower fruit drug pair.

Description

Technical Field

[0001] The invention belongs to the technical field of Tibetan medicine, and specifically relates to a Tibetan medicine supramolecular nanoparticle and a preparation method and application thereof. Background Art

[0002] Type 2 diabetes is a non-insulin-dependent type of diabetes characterized by insulin resistance. Type 2 diabetes is often treated with oral biguanides, sulfonylureas and other drugs. However, long-term use of these drugs can cause side effects such as gastrointestinal discomfort, hypoglycemia, and myalgia. Therefore, finding effective drugs for the treatment of type 2 diabetes from natural medicines such as Tibetan medicine has attracted more and more attention. Decoction is the earliest and most widely used traditional classic dosage form in Tibetan medicine. During the decoction process, different types of chemical components contained in each medicinal material can self-assemble to form supramolecular systems through the influence of non-covalent bonds such as hydrogen bonds, hydrophobic interactions, electrostatic attraction, and π-π stacking. For example, alkaloids can interact with glycosides, polyphenols and other components to form supramolecular nanoparticles. These self-assembled supramolecular molecules have unique properties and functions and may be important pharmacological substances in Tibetan medicine decoctions.

[0003] Berberis bark It is a commonly used Tibetan medicinal material, first recorded in the classic Tibetan medical work "Four Medical Classics". It tastes bitter and is cold in nature. It has the effects of clearing away heat and detoxifying, and drying "yellow water". In clinical practice, Berberis bark is often used in combination with Phyllanthus emblica to treat diseases. The inventor's previous research found that after Berberis bark and Phyllanthus emblica were decocted together, the decoction was obviously turbid, while the decoction of the two herbs alone was relatively clear, indicating that the chemical components of the two herbs may interact and self-assemble to form a supramolecular system during the decoction process. How to extract this supramolecular system to obtain a substance with good stability and better efficacy requires further research. Summary of the invention

[0004] The object of the present invention is to provide a Tibetan medicine supramolecular nanoparticle and a preparation method and use thereof. The present invention provides a method for preparing Tibetan medicine supramolecular nanoparticles, which comprises the following steps:

[0005] (1) Weighing berberis bark and emblica fruit powders, mixing them evenly, decocting them with water, and filtering them to obtain a berberis bark-emblica fruit decoction;

[0006] (2) The Berberis bark-Emblica emblica decoction is centrifuged, and the supernatant is dialyzed and dried to obtain supramolecular nanoparticles.

[0007] Further,

[0008] In step (1), the mass ratio of the Berberis bark and Emblica emblica powder is 1:(1-5);

[0009] And / or, in step (1), the mass of the water is 10 to 20 times the total mass of the medicinal material powder;

[0010] And / or, in step (1), the decoction is performed 1 to 3 times;

[0011] And / or, in step (1), the decocting time is 1 to 3 hours.

[0012] Further,

[0013] In step (1), the mass ratio of the Berberis bark and Emblica emblica powder is 1:1;

[0014] And / or, in step (1), the mass of the water is 10 times the total mass of the medicinal material powder;

[0015] And / or, in step (1), the decoction is performed twice;

[0016] And / or, in step (1), the decocting time is 1 hour.

[0017] Further,

[0018] In step (2), the centrifugal speed is 3500-7000 r / min;

[0019] And / or, in step (2), the dialysate of the dialysis is pure water;

[0020] and / or, in step (2), the molecular cut-off during dialysis is 500 to 7000 Da;

[0021] And / or, in step (2), the dialysis time is 3 to 9 hours;

[0022] And / or, in step (2), the drying method is freeze-drying.

[0023] Further,

[0024] In step (2), the centrifugal speed is 3500 r / min;

[0025] And / or, in step (2), the molecular cut-off during dialysis is 500Da;

[0026] And / or, in step (2), the dialysis time is 6 hours.

[0027] Further,

[0028] In step (2), the centrifugation time is 10 to 20 minutes;

[0029] And / or, in step (2), the pure water is replaced every 2 hours during the dialysis.

[0030] Furthermore, in step (2), during the dialysis, the dialysis bag is first boiled in a 2% by volume sodium bicarbonate aqueous solution, cooled and washed, and then the dialysis bag is placed in a 1 mmol / L EDTA-2Na aqueous solution and boiled, cooled and washed.

[0031] The invention also provides a Tibetan medicine supramolecular nanoparticle, which is prepared by the above-mentioned preparation method.

[0032] Furthermore, the components of the Tibetan medicine supramolecular nanoparticles include berberine, bamipine, magnolamine, jatrorrhizine, berberine, toad tenidine, gallic acid, ellagic acid, corilagin, cheleic acid, mucic acid-2-O-gallate, and methyl gallate.

[0033] The present invention also provides the use of the aforementioned Tibetan medicine supramolecular nanoparticles in preparing a drug for preventing and / or treating diabetes;

[0034] Preferably, the diabetes is type 2 diabetes.

[0035] The present invention has achieved the following beneficial effects:

[0036] The present invention adopts a specific process to prepare and separate the supramolecular part of the berberis bark-emblica fruit decoction, which is a supramolecular nanoparticle with good stability, mainly composed of alkaloid components represented by berberine in berberis bark and polyphenol components such as tannins, phenolic acids, etc. represented by gallic acid in emblica fruit. The supramolecular nanoparticle can effectively increase the glucose consumption of IR-HepG2 cells and activate the PI3K / AKT signaling pathway, and is expected to be used for the preparation of drugs for the treatment of type 2 diabetes. The present invention provides a scientific basis for the clinical rational application and modern development of the berberis bark-emblica fruit drug pair.

[0037] The research of the present invention shows that the supramolecular nanoparticles prepared by the optimal specific process of the present invention are superior to supramolecular nanoparticles prepared by other processes.

[0038] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0039] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1SEM images of supramolecular nanoparticles under different separation conditions: AC from left to right are centrifugal speeds of 3500r, 5000r, and 7000r; DF from left to right are dialysis bags of 500Da, 3500Da, and 7000Da; GI from left to right are dialysis times of 3h, 6h, and 9h.

[0041] Figure 2 The morphological characterization and stability of the lyophilized powder of the supramolecular part of Berberis bark-Emblica emblica prepared in Example 1: A is the result of the Tyndall effect; B is the SEM image; C is the particle size; D is the Zeta potential; E is the Tyndall effect image in different pH buffers; F is the Tyndall effect image in different NaCl solutions.

[0042] Figure 3 Total ion current diagram of positive and negative ion modes of the supramolecular part of the Berberis bark-Emblica emblica decoction prepared in Example 1: A is positive ion; B is negative ion.

[0043] Figure 4 The effects of different concentrations of each part in the Berberis bark-Emblica emblica decoction on the activity of IR-HepG2 cells; SM: supramolecular part group; CD: decoction group; TS: true solution group; SD: precipitation group.

[0044] Figure 5 To investigate the effects of different parts of Berberis bark-Emblica emblica decoction on glucose consumption in IR-HepG2 cells.

[0045] Figure 6 This is the effect of each group on the expression of p-PI3K and p-AKT proteins in IR-HepG2 cells. DETAILED DESCRIPTION

[0046] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0047] 1. Drugs and reagents

[0048] HepG2 cell culture medium (Cat. No. CM-0103, Wuhan Punosai Life Science Co., Ltd.); insulin resistance inducer (Cat. No. S1635) and total protein quantitative determination kit (Cat. No. P0010S) were purchased from Shanghai Bio-Tech Co., Ltd.; 0.25% trypsin (Cat. No. PYG0067) and sterile PBS (Cat. No. PYG0021) were purchased from Wuhan Bosde Biotechnology Co., Ltd.; glucose test kit (Cat. No. A154-1-1, Nanjing Jiancheng Bioengineering Institute); CCK-8 kit (Cat. No. BG0025, Chongqing Baoguang Biotechnology Co., Ltd.); dihydrochloride Metformin (catalog number: B25331, Shanghai Yuanye Biotechnology Co., Ltd.); anhydrous glucose (batch number: 2023112901, Chengdu Cologne Chemical Co., Ltd.); PI3K, AKT (catalog number: A4992, A17909,) were purchased from Wuhan Abotek Biotechnology Co., Ltd.; p-AKT antibody (catalog number: H651506050) was purchased from Hangzhou Huaan Biotechnology Co., Ltd.; p-PI3K antibody (catalog number: T40116F) was purchased from Shanghai Abimate Biotechnology Co., Ltd.; β-actin antibody (catalog number: AC240505001) was purchased from Wuhan Sevier Biotechnology Co., Ltd.

[0049] Analytical reagents such as chromatographic grade formic acid (batch number: 2023041701) and methanol (batch number: 24072801) were purchased from Chengdu Kelong Chemical Co., Ltd. The medicinal materials Berberis bark and Phyllanthus emblica were sourced from the Derge County Chinese Tibetan Hospital.

[0050] 2. Experimental instruments

[0051] Magnetic stirrer (MS5S, Qun'an Experimental Instrument Co., Ltd.); Malvern particle size analyzer (Mastersizer 2000, Malvern Instruments Ltd., UK); scanning electron microscope (JSM-IT700HR, JEOL Ltd.); freeze dryer (FDU-2110, Tokyo Rikkigi Co., Ltd.); dialysis bags (500, 3500, 7000 Da, Hunan Yibo Biotechnology Co., Ltd.); desktop high-speed microcentrifuge (D3024, Beijing Dalong Xingchuang Experimental Instrument Co., Ltd.).

[0052] Vanquish TM Ultra-high performance liquid chromatography (Thermo-Fisher, USA); Q-Exactive TM Orbitrap high-resolution mass spectrometer (Thermo-Fisher, USA); Sartorius BSA124S electronic balance (Beijing Sartorius Scientific Instrument Co., Ltd.).

[0053] Mini-PROTEAN Tetra electrophoresis tank (Bio-Rad, USA); UVP ChemStudio intelligent imaging system (Jena, Germany); microplate reader (Thermo Fisher Scientific, USA).

[0054] 3. Statistical analysis

[0055] GraphPad Prism 10.1.2 statistical software was used to perform one-way ANOVA on the data, and the experimental data were expressed as mean ± SD. P < 0.05 was considered statistically significant.

[0056] Example 1. Preparation of Tibetan medicine supramolecular nanoparticles of the present invention

[0057] 1. Preparation of Berberis bark-Emblica emblica decoction

[0058] Weigh 5 g each of berberis bark and emblica emblica powder, mix well, add water and boil twice, add 10 times the amount of water each time, boil for 1 hour each time, filter the decoction, and combine the filtrate to obtain the berberis bark-emblica emblica decoction.

[0059] 2. Treatment of dialysis bags

[0060] Cut the dialysis bag to an appropriate length, boil it in a 2% by volume sodium bicarbonate aqueous solution for 10 min, cool it, rinse it thoroughly with pure water, boil it in a 1mmol / L EDTA-2Na (pH=8, add NaOH to adjust the pH) aqueous solution for 10 min, cool it, check for leakage with pure water before use, and set it aside.

[0061] 3. Preparation of supramolecular nanoparticles

[0062] The above-mentioned Berberis bark-Emblica emblica decoction was centrifuged at 3500r / min for 15min, and the supernatant was placed in a 500Da dialysis bag, which was then placed in a beaker with pure water for dialysis. The pure water was replaced every 2h, and the supramolecular part in the decoction was obtained in the dialysis bag after 6h of dialysis, and the supramolecular part lyophilized powder (supramolecular nanoparticles) was obtained by freeze-drying.

[0063] The beneficial effects of the present invention are demonstrated below through specific test examples.

[0064] Experimental Example 1: Determination of the supramolecular fraction separation method of Berberis bark-Emblica emblica decoction

[0065] 1. Experimental Methods

[0066] 1. Selection of centrifugal speed

[0067] According to the method described in Example 1, a berberis bark-emblica fruit decoction was prepared and a dialysis bag was processed. The decoction was centrifuged at 3500r / min, 5000r / min and 7000r / min for 15min, and the supernatant was placed in a 3500Da dialysis bag, which was then placed in a beaker with pure water for dialysis. Pure water was replaced every 2h, and supramolecular sites in the decoction were obtained in the dialysis bag after dialysis for 6h, and the supramolecular sites were freeze-dried to obtain freeze-dried powder (supramolecular nanoparticles) of the supramolecular sites. The freeze-dried powder was placed under SEM to observe the morphology and particle size, and the optimal centrifugal speed was selected.

[0068] 2. Selection of dialysis bags

[0069] The same method as in item "1" of the above-mentioned Experimental Example 1 was used to dialyze the combined decoction into 500Da, 3500Da and 7000Da dialysis bags respectively at the optimal centrifugal speed (3500 r / min). The resulting supramolecular part lyophilized powder (supramolecular nanoparticles) was observed by SEM to select the optimal dialysis bag.

[0070] 3. Choice of dialysis time

[0071] According to the same method as in the above-mentioned Experimental Example 1 under item "1", the combined decoction was dialyzed for 3h, 6h and 9h respectively (pure water was replaced every 2h) under the conditions of the optimal centrifugal speed (3500r / min) and the optimal dialysis bag (500Da). The resulting supramolecular part freeze-dried powder (supramolecular nanoparticles) was observed by SEM to select the optimal dialysis time.

[0072] 4. SEM observation method

[0073] The freeze-dried powder of supramolecular parts dispersed in deionized water was dropped on the silicon wafer, and after the water was naturally evaporated, gold was sprayed on it and the microscopic morphology of the supramolecular parts was observed under SEM.

[0074] 2. Experimental Results

[0075] SEM results are as follows Figure 1 Shown by: Figure 1 A-1C found that when centrifuged at 7000r / min, no nanoparticles were found due to the high centrifugal speed; when centrifuged at 5000r / min, the particle size was significantly reduced, the particles were clearer and more complete, but there were fewer nanoparticles; when centrifuged at 3500r / min, the nanoparticles measured were more complete and clear, and the particle size distribution was more uniform, so 3500r / min was selected as the best centrifugal speed for separation. Figure 1D-1F found that the supramolecular particles separated by dialysis bags of 3500Da and 7000Da had large differences in particle size, while the supramolecular particles separated by 500Da had more uniform particle size, rounded shape, and more dense distribution, so the 500Da dialysis bag was selected to separate the supramolecular particles. Figure 1 G-1I found that the shorter the dialysis time, the fewer nanoparticles were obtained, but as the dialysis time increased, the shape of the nanoparticles changed. When the dialysis time was 6 hours, the particle size of the supramolecular part was relatively uniform and the shape was regular, so 6 hours was selected as the optimal dialysis time.

[0076] Through the above single factor considerations, it was concluded that the optimal separation method for the supramolecular part of the Berberis bark-Emblica emblica decoction was: take Berberis bark and Emblica emblica decoction (mass ratio of 1:1), mix them evenly, add water and boil them twice, each time for 1 hour, filter the decoction, combine the filtrates, centrifuge at 3500r / min for 15 minutes, take the supernatant and put it into a 500Da dialysis bag and dialyze it in pure water for 6 hours, stir it continuously with a magnetic stirrer, repeat the operation many times, and freeze-dry the liquid in the final dialysis bag to obtain.

[0077] Experimental Example 2: Morphological Characterization and Stability Study of the Supramolecular Part of Berberis Bark-Emblica Emblica Decoction I. Experimental Methods

[0078] The samples used for morphological characterization and stability study were all freeze-dried powders of supramolecular fractions prepared according to the method described in Example 1.

[0079] 1. Morphology characterization

[0080] The lyophilized powder of supramolecular parts was reconstituted to 1 mg / mL with deionized water and transferred to a glass bottle. The Tyndall phenomenon was observed by laser irradiation, and the particle size and dispersion index (PDI) of the sample were measured using a Malvern particle size analyzer. The sample was dropped on a silicon wafer, sprayed with gold after the water evaporated naturally, and the microscopic morphology of the supramolecular system was observed under SEM.

[0081] 2. Stability study

[0082] 1 mg of lyophilized powder of the supramolecular fraction of Berberis chinensis-Emblica emblica was weighed and placed in phosphate buffer solutions with pH values ​​of 1, 3, 6, 7.4, and 8 and 100, 200, 300, 400, and 500 mM sodium chloride aqueous solutions, respectively. The colloidal solutions were irradiated with an infrared laser pen to observe the changes in the intensity of the Tyndall effect, analyze its pH stability and ion stability, and take photos for record.

[0083] 2. Experimental Results

[0084] Depend on Figure 2 It can be seen that after the supramolecular part is irradiated by the laser pen, the light path is uniform and obvious ( Figure 2A), indicating that the supramolecular part is obviously different from the solution, and is uniform and stable as a whole. SEM results show that the supramolecular part is spherical particles with relatively uniform size and round shape ( Figure 2 B). The supramolecular part was characterized by Malvern particle size analyzer, and the results showed that ( Figure 2 C) The supramolecular part is a nanoparticle with a particle size of about 514 nm, a polydispersity index (PDI) of 0.398, and a zeta potential of -14.1 mV.

[0085] The stability of the supramolecular part of the Berberis bark-Emblica emblica decoction in different pH buffer solutions was investigated by observing the change in the intensity of the Tyndall effect. Figure 2 As shown in E, when pH < 8, a Tyndall effect with small scattering and strong light path is generated, which indicates that the supramolecular part of Berberis bark-Emblica emblica is stable and not easily degraded when pH < 8. Therefore, when the drug is in the stomach (pH 1-3), it is not easily consumed by gastric acid and can exist stably; as the drug enters the small intestine (pH 7.6), the supramolecular part can also exist relatively stably. In addition, neutral salt ions can change the electrostatic interaction of nanoparticles through electrostatic shielding, thereby promoting the aggregation and precipitation of particles. Figure 2 As shown in Figure F, the red light intensity of the Tyndall effect is obvious in the supramolecular part in NaCl aqueous solution of different concentrations, and the particle size and dispersibility of the particles can be kept stable in the ion concentration range of 0-500mM. This also suggests that the supramolecular part of Berberis bark-Emblica emblica can stably exist in the normal osmotic pressure environment of the human body (NaCl concentration is 150mM).

[0086] Experimental Example 3: Analysis of chemical components of supramolecular fraction of Berberis bark-Emblica emblica decoction

[0087] 1. Experimental Methods

[0088] 1. Preparation of test solution

[0089] 1.00 mg of lyophilized powder of the supramolecular fraction of the Berberis bark-Emblica emblica decoction prepared in Example 1 was accurately weighed, diluted to 100 μg / mL with chromatographic grade methanol, sonicated for 30 min, and filtered through a 0.22 μm microporous membrane for testing.

[0090] 2. Chromatographic and mass spectrometry acquisition conditions

[0091] Chromatographic conditions: Chromatographic column ZORBAX SB-C 18 Column (150 mm × 2.1 mm, 1.8 μm); mobile phase: methanol (A)-0.1% formic acid aqueous solution (B); gradient elution program: 0-40 min, 2%-80% A; flow rate 0.3 mL·min -1 ; Column temperature: 30℃; Injection volume: 2μL.

[0092] Mass spectrometry conditions: HESI ion source, positive ion detection mode, electrospray voltage of +3500V; negative ion detection mode, electrospray voltage of -3000V; sheath gas flow rate of 40arb; auxiliary gas flow rate of 15arb; capillary temperature of 350℃; secondary mass spectrometry using Full MS / dd-MS 2 Scan mode, Full MS resolution 70000, dd-MS 2 The resolution is 17500 and the scanning range is m / z100~1500.

[0093] 3. Data Analysis

[0094] The raw data were imported into Compound Discoverer 3.0 software for extraction, retention time correction and other preprocessing. A workflow retrieval database was established, and the molecular formula was fitted using Xcalibar 2.0 software according to the precise molecular weight provided by the primary mass spectrometer, with an error range of ±5.0ppm. The sample mass spectrometry data information was matched with the component data in the databases ChemSpider, mzCloud and mzVault, and the components were preliminarily attributed and identified by the exact relative molecular mass and secondary mass spectrometry fragment ions in the database. At the same time, online databases such as ChemSpider and Pubchem and related literature were consulted to retrieve the possible chemical structures and pyrolysis fragments of the components, and the identified components were further compared and verified.

[0095] 2. Experimental Results

[0096] After the test solution was prepared according to the above method, the total ion current diagrams under positive and negative ion modes were obtained respectively. Figure 3 According to the above data analysis, the supramolecular part is mainly composed of alkaloid components represented by berberine in Berberis bark and polyphenol components such as tannins and phenolic acids represented by gallic acid in Phyllanthus emblica. The specific contents are shown in Table 1.

[0097] Table 1. Chemical composition analysis results of the supramolecular fraction of Berberis bark-Emblica emblica

[0098]

[0099]

[0100] Experimental Example 4: Evaluation of the efficacy of different parts of Berberis bark-Emblica emblica decoction on IR-HepG2 cells and study of its mechanism of action

[0101] 1. Experimental Methods

[0102] 1. Preparation of drugs

[0103] Preparation of different parts of Berberis bark-Emblica emblica decoction: Berberis bark-Emblica emblica decoction (CD) was prepared according to the method described in Example 1, and freeze-dried for later use; the supramolecular part freeze-dried powder (SM) of Berberis bark-Emblica emblica was prepared according to the method described in Example 1; when preparing the freeze-dried powder in Example 1, the solution outside the dialysis bag after dialysis was the true solution part (TS), which was freeze-dried for later use; when preparing the freeze-dried powder in Example 1, the precipitate obtained by centrifugation was the precipitate part (SD), which was freeze-dried for later use.

[0104] CD, SM, TS and SD were dissolved in complete culture medium to prepare 5000 μg / ml stock solutions, which were fully dissolved for use in subsequent cell experiments.

[0105] Preparation of glucosamine (GlcN) solution: Weigh 0.215 g of GlcN powder and dissolve it in 1 mL of PBS to prepare a 1 mol / L GlcN solution.

[0106] Preparation of metformin hydrochloride solution: weigh 0.063 g of metformin hydrochloride powder, fully dissolve it in 10 mL of PBS, shake and mix to make a 50 mmol / L metformin hydrochloride stock solution, filter it with a 0.22 μm microporous filter membrane, store it at 4°C after aliquoting, and dilute it to a concentration of 6 mmol / L with culture medium before use.

[0107] 2. Establishment of IR-HepG2 cell model

[0108] After HepG2 cells were revived, they were cultured in a constant temperature incubator at 37°C and 5% CO2 using DMEM high glucose medium containing 10% fetal bovine serum. When the cells were attached to the wall and grew to 90% full, they were digested and passaged with 0.25% trypsin. HepG2 cells in the logarithmic growth phase were taken and plated at 5×10 5 / mL density was inoculated in a 96-well cell culture plate, 100μL / well, and a control group and a model group were set up, with 6 replicates in each group, and cultured in an incubator at 37°C and 5% CO2 for 24 hours. After the cells adhered to the wall, the culture supernatant in the 96-well plate was discarded, and normal culture medium was added to the control group, and complete culture medium with a GlcN concentration of 9mmol / L was added to the model group. The cells were incubated in the incubator for 24 hours, and high-glucose culture medium was added to continue culturing for 24 hours to establish the IR-HepG2 cell model. The cell culture medium was taken, and the glucose content was detected by the grape peroxidase method, and the cell survival rate was detected by the CCK-8 method.

[0109] 3. Screening of concentrations of berberis bark-emblica fruit decoction in different parts

[0110] After incubation of HepG2 cells for 24 hours, a blank group, a berberis bark-emblica emblica decoction group (CD) at a concentration of 25, 50, 100, 200, 400, 800, 1600, 3200 μg / mL, a supramolecular fraction group (SM) and a true solution group (TS), and a precipitation group (SD) at a concentration of 25, 50, 100, 200, 400, 800, 1600, 3200 μg / mL were set up, with 6 replicates in each well. After 24 hours of culture, the culture medium in the well was discarded and 110 μL of complete culture medium containing CCK-8 (complete culture medium: CCK-8 = 10:1, volume ratio) was added, and the cells were placed in a cell culture incubator for 2 hours, and the OD value was detected at a wavelength of 450 nm on an ELISA reader to calculate the cell viability.

[0111] 4. Determination of glucose consumption of IR-HepG2 cells by different parts of Berberis bark-Emblica emblica decoction

[0112] Complete medium was added to the normal group. According to the drug concentration screened under the above "3", the model group, positive drug group (6mmol / L, Metformin), CD, SM, TS and SD groups were set up respectively. The drug groups were set up as high (200μg / mL), medium (100μg / mL) and low (50μg / mL) dose groups respectively. The culture medium was high-glucose medium. After 24h of drug administration, the glucose consumption of each group was calculated.

[0113] 5. Western blotting (WB) to detect the expression of proteins related to the PI3K / AKT signaling pathway

[0114] HepG2 cells in logarithmic growth phase were cultured at 6×10 5 / well inoculated in 6-well plates, after the cells were completely attached to the wall, the IR-HepG2 model was established, and a normal control group was set up with a high-glucose medium. 24 hours after modeling, a model group, a positive drug group (6mmol / L, Metformin), a high-dose group of supramolecular parts (SM, 200μg / mL), a high-dose group of Berberis bark-Emblica emblica decoction (CD, 200μg / mL) and a blank control group set up with a high-glucose medium were set up, and 6 replicates were set up in each group. After 24 hours of culture, the cells were collected. The cells were collected, and RIPA lysis buffer was added to extract the protein, and the protein concentration was determined by the BCA method. The protein samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to PVDF membrane, added with 5% BSA, blocked at room temperature for 1 h, and washed with TBST, and then PI3K, AKT, p-PI3K, p-AKT, and β-actin antibodies were added respectively, and incubated at 4°C overnight; after washing the membrane, the secondary antibody was added, incubated at room temperature for 1 h, and finally developed with ECL luminescent solution, and the grayscale analysis of the bands was performed using Image J software.

[0115] 2. Experimental Results

[0116] 1. Evaluation of the efficacy of different parts of Berberis bark-Emblica emblica decoction on IR-HepG2 cells and study of its mechanism of action

[0117] 1.1 Evaluation of IR-HepG2 cell model

[0118] Compared with the control group (glucose consumption was 4.23±0.04mmol / mL), when the GlcN concentration was 9mmol / mL, the glucose consumption (1.80±0.14mmol / mL) decreased significantly (P<0.001), and the cell survival rate (81.85±3.84%) was greater than 80%, and the cell lethality was relatively small. The above results indicate that the IR-HepG2 model was successfully constructed.

[0119] 1.2 Screening of concentrations of berberis bark-emblica fruit decoction in different parts

[0120] like Figure 4 As shown in the data, when the drug concentration in each site was 25-200 μg / mL, the viability of HepG2 cells was not significantly different from that of the normal group. When the concentration increased to 400 μg / mL, the viability of HepG2 cells in the SM, CD and SD groups was significantly different from that in the normal group (P < 0.001). When the concentration of the true solution site (TS) in the combined decoction increased to 800 μg / mL, the cell viability decreased significantly (P < 0.001). Therefore, it can be considered that the SM, CD and SD sites will not affect the viability of HepG2 cells at a concentration of 200 μg / mL, and the TS site will not affect the viability of HepG2 cells at a concentration of 400 μg / mL.

[0121] 1.3 Effects of different parts of Berberis bark-Emblica emblica decoction on glucose consumption in IR-HepG2 cells

[0122] In order to evaluate the effects of different parts of Berberis bark-Emblica emblica decoction on the glucose consumption of IR-HepG2 cells, the concentrations (200, 100, 50 μg / mL) at which each part had no significant effect on the activity of HepG2 cells were selected as the administration concentrations for evaluating the glucose consumption of IR-HepG2 cells. Figure 5 As shown. Compared with the normal group, the glucose consumption in the model group was significantly reduced (P < 0.001), indicating that the IR-HepG2 cell model was successfully established. Compared with the model group, the glucose consumption increased to varying degrees after administration of different doses in various sites. The glucose consumption in the metformin hydrochloride group, CD high (200 μg / mL), medium (100 μg / mL) dose group, and SM high and medium dose groups increased significantly (P < 0.01). The glucose consumption of cells in other sites also showed an upward trend, but there was no statistical difference.

[0123] 1.4 Effects of Berberis bark-Emblica emblica decoction and supramolecular fractions on PI3K / AKT protein expression in IR-HepG2 cells

[0124] like Figure 6 As shown in the results, compared with the blank control group, the expression of p-PI3K protein in the model group was significantly decreased (P<0.05), and the expression of p-AKT protein was significantly decreased (P<0.01), indicating that PI3K / AKT signaling was inhibited during IR; compared with the model group, the expression of p-PI3K protein in the SM group was significantly increased after administration (P<0.05), and the expression of p-AKT protein was significantly increased (P<0.001), indicating that the supramolecular part of the berberis bark-emblica fruit decoction may improve IR by activating the PI3K / AKT signaling pathway, thereby slowing down the progression of diabetes. At the same time, it can be found that its expression is comparable to that of the positive drug metformin and the decoction, and it is an important pharmacological substance of the berberis bark-emblica fruit decoction.

[0125] In summary, the present invention adopts a specific process to prepare and separate the supramolecular part of the berberis bark-emblica fruit decoction, which is a supramolecular nanoparticle with good stability, mainly composed of alkaloid components represented by berberine in berberis bark and polyphenol components such as tannins, phenolic acids, etc. represented by gallic acid in emblica fruit. The supramolecular nanoparticle can effectively increase the glucose consumption of IR-HepG2 cells and activate the PI3K / AKT signaling pathway, and is expected to be used for the preparation of drugs for the treatment of type 2 diabetes. The present invention provides a scientific basis for the clinical rational application and modern development of the berberis bark-emblica fruit drug pair.

Claims

1. A method for preparing Tibetan medicine supramolecular nanoparticles, characterized in that: It includes the following steps: (1) Weighing berberis bark and emblica fruit powders, mixing them evenly, decocting them with water, and filtering them to obtain a berberis bark-emblica fruit decoction; (2) The Berberis bark-Emblica emblica decoction is centrifuged, and the supernatant is dialyzed and dried to obtain supramolecular nanoparticles.

2. The preparation method according to claim 1, characterized in that: In step (1), the mass ratio of the Berberis bark and Emblica emblica powder is 1:(1-5); And / or, in step (1), the mass of the water is 10 to 20 times the total mass of the medicinal material powder; And / or, in step (1), the decoction is performed 1 to 3 times; And / or, in step (1), the decocting time is 1 to 3 hours.

3. The preparation method according to claim 2, characterized in that: In step (1), the mass ratio of the Berberis bark and Emblica emblica powder is 1:1; And / or, in step (1), the mass of the water is 10 times the total mass of the medicinal material powder; And / or, in step (1), the decoction is performed twice; And / or, in step (1), the decocting time is 1 hour.

4. The preparation method according to claim 1, characterized in that: In step (2), the centrifugal speed is 3500-7000 r / min; And / or, in step (2), the dialysate of the dialysis is pure water; And / or, in step (2), the molecular cut-off during dialysis is 500 to 7000 Da; And / or, in step (2), the dialysis time is 3 to 9 hours; And / or, in step (2), the drying method is freeze-drying.

5. The preparation method according to claim 4, characterized in that: In step (2), the centrifugal speed is 3500 r / min; And / or, in step (2), the molecular cut-off during dialysis is 500Da; And / or, in step (2), the dialysis time is 6 hours.

6. The preparation method according to claim 5, characterized in that: In step (2), the centrifugation time is 10 to 20 minutes; And / or, in step (2), the pure water is replaced every 2 hours during the dialysis.

7. The preparation method according to claim 1, characterized in that: In step (2), during the dialysis, the dialysis bag is first boiled in a 2% by volume sodium bicarbonate aqueous solution, cooled and washed, and then the dialysis bag is placed in a 1mmol / L EDTA-2Na aqueous solution and boiled, cooled and washed.

8. A Tibetan medicine supramolecular nanoparticle, characterized in that: The invention is prepared by the preparation method described in any one of claims 1 to 7.

9. The Tibetan medicine supramolecular nanoparticles according to claim 8, characterized in that: The components of the Tibetan medicine supramolecular nanoparticles include berberine, palmatine, magnolamine, jatrorrhizine, berberine, toad tenidine, gallic acid, ellagic acid, corilagin, cheleic acid, mucic acid-2-O-gallate and methyl gallate.

10. Use of the Tibetan medicine supramolecular nanoparticles according to claim 8 or 9 in preparing a drug for preventing and / or treating diabetes; Preferably, the diabetes is type 2 diabetes.

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