CN116999449B

CN116999449B - Ginsenoside composition and application thereof in preparation of multi-target adipose cell development differentiation and metabolism regulator

Info

Publication number: CN116999449B
Application number: CN202311035410.5A
Authority: CN
Inventors: 赵岩; 滕虹伯; 赵娅茹; 范鑫华; 于鹏程; 杜锐; 李平亚; 易星丞; 赵天一
Original assignee: Jilin Agricultural University
Current assignee: Jilin Agricultural University
Priority date: 2023-08-17
Filing date: 2023-08-17
Publication date: 2024-05-03
Anticipated expiration: 2043-08-17
Also published as: CN116999449A

Abstract

The invention discloses a ginsenoside composition and application thereof in preparing a multi-target adipose cell development differentiation and metabolism regulator, and belongs to the field of biological medicines. The ginsenoside composition comprises protopanaxadiol PPD and any one of protopanaxatriol PPT, ginsenoside Rg ₅ or ginsenoside 20 (S) -Rg ₃. Pharmacological experiments prove that the ginsenoside composition activates Hspa4 and PERK while antagonizing IGFR, FGFR2 and MAPK, promotes nuclear translocation of RELA and expression of CHOP, further induces apoptosis of fat precursor cells, inhibits differentiation of the fat precursor cells into mature fat cells, inhibits synthesis and storage of triglyceride, has more direct and effective accurate target action, effectively reduces generation of toxic and side effects, and provides theoretical basis for application of the ginsenoside composition in prevention, treatment and alleviation of obesity and related diseases thereof.

Description

Ginsenoside composition and application thereof in preparation of multi-target adipose cell development differentiation and metabolism regulator

Technical Field

The invention relates to the field of biological medicine, in particular to a ginsenoside composition and application thereof in preparing a multi-target adipose cell development differentiation and metabolism regulator.

Background

Obesity belongs to chronic metabolic diseases, and more than 95% of patients with obesity are simple obesity (hereinafter referred to as obesity). The obesity problem is rapidly and widely developed and continuously developed, but is still not paid attention to. Obesity is mainly characterized by an increase in the number of adipocytes and an enlargement of the volume of adipocytes, which are caused by proliferation and differentiation of preadipocytes, resulting in excessive accumulation of adipose tissues. Mature adipocytes can secrete a variety of adipocyte and inflammatory factors, and excessive factors can cause the body to produce a variety of metabolic diseases, such as hypertension, hyperlipidemia, insulin resistance, fatty liver, diabetes, cardiovascular and cerebrovascular diseases, cancer, and the like.

The adipose cells are derived from mesenchymal stem cells which exist in adipose tissues and are the same as bone marrow stroma, and the stem cells are called adipose-derived stem cells (adipose-DERIVED STEM CELLS, ADSCs) and have the characteristics of lasting vitality, self-renewal, multi-directional differentiation and the like. ADSCs can differentiate into adipocyte precursors, also known as preadipocytes, under stimulation of adipogenic signaling factors while maintaining the active proliferative properties of stem cells. The adipocyte precursor is subjected to the stages of repeated contact inhibition, cell fusion and the like, under the adipogenic induction condition, the adipocyte precursor starts to differentiate into immature adipocytes, and finally complete differentiation into mature adipocytes is completed. Mature adipocytes can express and secrete a variety of adipocytes and inflammatory factors, such as leptin, adiponectin, interleukin-6 (interleukin-6, IL-6), interleukin-8 (IL-8), monocyte chemotactic protein-1 (monocyte chemotactic protein-1, MCP-1), tumor necrosis factor-alpha (tumor necrosis factor-alpha, TNF-alpha) and the like, which act on different tissues and organs to cause inflammation, and thus cause other various metabolic diseases.

Pharmaceutical intervention is the most suitable choice for obese people, and due to the potential, serious side effects and high cost of current chemicals for weight loss, chemical components derived from natural plants are becoming the best choice for developing safe-to-eat, low-cost anti-obesity drugs. Studies have shown that ginsenoside has an effect on fat differentiation, but numerous literature reports still have contradictions. There are reports that ginseng stem and leaf total saponins can inhibit abnormal elevation of PPARgamma, FAS and aP2 proteins and genes in liver and/or adipose tissue of mice induced by high-fat diet. The literature also reports that ginsenoside Rb1, rg1, re, rd (20. Mu.M), rh2 (20 and 40. Mu.M), rg3 (20 and 40. Mu.M), rd (80. Mu.M), rh1 (50 and 100. Mu.M), rg5: rk1 (100. Mu.g/mL) inhibited the 3T3-L1 adipocyte differentiation process. However, ginsenoside Rh2 (0.01-1. Mu.M) has also been reported to promote adipocyte differentiation by activating the adipocyte glucocorticoid receptor; ginsenoside Rg1 can accelerate paracrine activity of human mammary gland adipose-derived stem cells and adipocyte differentiation. There is also a report that ginsenoside Rg1 inhibits early development of adipocytes by activating C/EBP homologous protein 10 in 3T 3-L1.

Promotion of fat precursor apoptosis is a new strategy for anti-obesity diseases, but there are few reports on the promotion of fat precursor apoptosis. Inhibition of lipid precursor cell differentiation and metabolism is another important research direction against obesity-related diseases. Therefore, the inhibition effect of ginsenoside substances on proliferation differentiation and metabolism of fat precursor cells is studied deeply, and the method has important significance for developing multi-target anti-obesity drugs.

Disclosure of Invention

The invention aims to provide a ginsenoside composition and application thereof in preparing a multi-target adipose cell development differentiation and metabolism regulator so as to solve the problems in the prior art. The ginsenoside composition provided by the invention induces apoptosis of fat precursor cells through the accurate multi-target effect, inhibits the differentiation of the fat precursor cells into mature fat cells, and inhibits the synthesis and storage of triglyceride; and is further useful for preventing, treating and alleviating obesity and related diseases.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides a ginsenoside composition, which consists of the following components:

Protopanoxadiol PPD; and, a step of, in the first embodiment,

Any one of protopanaxatriol PPT, ginsenoside Rg ₅ or ginsenoside 20 (S) -Rg ₃.

Further, the composition comprises protopanaxadiol PPD and protopanaxatriol PPT, wherein the mol ratio of the protopanaxadiol PPD to the protopanaxatriol PPT is (1-9): 9-1.

Further, the molar ratio of the protopanoxadiol PPD to the protopanoxatriol PPT is 1:1.

The invention also provides application of the ginsenoside composition in preparing multi-target adipose cell development, differentiation and metabolism regulators.

Further, the multi-target adipocyte development, differentiation and metabolism regulator can activate Hspa4 and PERK and promote nuclear translocation of RELA and expression of CHOP while inhibiting IGFR, FGFR2 and MAPK.

The invention also provides application of the ginsenoside composition in preparing medicines for treating obesity or diseases related to obesity.

Further, the ginsenoside composition can induce apoptosis of fat precursor cells, inhibit differentiation of the fat precursor cells into mature fat cells, and inhibit synthesis and storage of triglyceride.

Further, the ginsenoside composition can inhibit expression of downstream protein MAPK by inhibiting cell growth factors IGFR and FGFR2, so that apoptosis of fat precursor cells is induced.

Further, the ginsenoside composition promotes nuclear translocation of RELA and expression of CHOP by activating Hspa4 and PERK, thereby inhibiting differentiation of fat precursor cells into mature fat cells and inhibiting triglyceride synthesis and storage.

The invention also provides a medicament for treating obesity or diseases related to obesity, which comprises the ginsenoside composition and a pharmaceutically acceptable carrier or auxiliary material, wherein the dosage form of the medicament comprises a tablet, a suspension injection and a hydrogel emplastrum.

The invention discloses the following technical effects:

In the ginsenoside composition provided by the invention, all components are natural products, so that the ginsenoside composition is safe and easy to obtain, and has low production cost.

Pharmacological experiments prove that the ginsenoside composition can activate Hspa4 and PERK and promote nuclear translocation of RELA and expression of CHOP while antagonizing IGFR, FGFR2 and MAPK through the accurate multi-target effect; inducing apoptosis of fat precursor cells, inhibiting differentiation of fat precursor cells into mature fat cells, inhibiting synthesis and storage of triglyceride, ensuring a definite action mechanism, ensuring more direct and effective accurate target action, and effectively reducing the generation of toxic and side effects. Provides theoretical basis for preventing, treating and relieving obesity and related diseases thereof, such as metabolic diseases of hypertension, hyperlipidemia, insulin resistance, fatty liver, diabetes and the like, cardiovascular and cerebrovascular diseases, cancers and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the effect of ginsenoside on the cell viability of 3T3-L1 fat precursor cells in an embodiment of the present invention;

FIG. 2 shows the effect of ginsenoside on the cell viability of mature adipocytes in an embodiment of the present invention;

FIG. 3 is a graph showing the effect of PPD, PD on the morphology of 3T3-L1 fat precursor cells in examples of the present invention;

FIG. 4 is a graph showing the effect of PPD and PD on apoptosis of 3T3-L1 fat precursor cells in the embodiment of the invention, wherein A is a flow cytometer for detecting apoptosis, and B is a statistical graph of apoptosis rate;

FIG. 5 is a graph showing the effect of PPD and PD on cell cycle arrest of 3T3-L1 fat precursor in the example of the present invention, wherein A is a flow cytometer for detecting cell cycle and B is a statistical chart of the ratio of each cell cycle;

FIG. 6 is a pathway enrichment map of a PPD transcriptome sequencing differential gene in an embodiment of the invention, wherein A is the pathway enrichment map of the first twenty differential genes and B is the volcanic map of the differential genes;

FIG. 7 shows the effect of PPD on expression of 3T3-L1 fat precursor apoptosis-related proteins IGFR (A), FGFR2 (B), MAPK (C), bcl-2 and Bax (D) in examples of the present invention;

FIG. 8 shows the effect of PPD on 3T3-L1 fat precursor apoptosis-related genes IGFR (A) and FGFR2 (B) in examples of the present invention;

FIG. 9 shows the effect of ginsenoside on lipid accumulation in adipocytes in an example of the present invention;

FIG. 10 shows the result of staining fat cell lipid accumulation oil red O with ginsenoside in the example of the present invention;

FIG. 11 shows the improvement effect of PPT, rg ₅ and 20 (S) -Rg ₃ on mature adipocyte insulin resistance in the examples of the present invention;

FIG. 12 is a graph showing the effect of PPT, rg ₅ and 20 (S) -Rg ₃ on triglyceride consumption of cell supernatants in examples of the invention;

FIG. 13 is a graph showing the effect of PPT, rg ₅ and 20 (S) -Rg ₃ on free fatty acid consumption of cell supernatants in examples of the invention;

FIG. 14 is a graph showing the effect of PPT, rg ₅ and 20 (S) -Rg ₃ on cell supernatant glucose consumption in examples of the present invention;

FIG. 15 shows the effect of PPT, rg ₅ and 20 (S) -Rg ₃ on secretion of fat-related factor resistin (A), leptin (B), adiponectin (C) and TNF- α (D) in examples of this invention;

FIG. 16 is a pathway enrichment map of a PPT transcriptome sequencing differential gene in an embodiment of the invention, wherein A is the pathway enrichment map of the first twenty differential genes and B is the volcanic map of the differential genes;

FIG. 17 shows the effect of PPT on the expression of the differentiation-related proteins Hspa4 (A), RELA (B), PPARgamma (C), PERK (D), CHOP (E) and C/EBPα (F) in adipocytes according to an embodiment of the present invention;

FIG. 18 shows the effect of PPT on expression of genes involved in regulating differentiation in adipocytes in examples of this invention.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

EXAMPLE 1 Protopanaxadiol induces apoptosis of fat precursor cells

1. Materials and instruments

1.1 Ginsenoside (Yuan)

Ginsenoside Rb1、Rb2、Rb3、Rc、Rd、20(S)-Rg₃、20(R)-Rg₃、Rk₁、Rg₅、Rh₂、Re、Rg₁、Rf、Rg₆、F₄、Rk₃、Rh₄、Rg₂、Rh₁、Ro、 Panaxadiol (PD), protopanaxadiol (PPD), anthropomorphic sapogenin DQ (PDQ), panaxatriol (PT), protopanaxatriol (PPT), anthropomorphic sapogenin (Ocotill), anthropomorphic saponin F11 (PF 11), oleanolic acid and other 105 triterpenes and their glycosides (part of which are donated by university of Jilin medical college, the rest of which are all purchased from Shanghai microphone) have the purity of above 98%.

1.2 Materials and reagents

DMEM medium (marine clonotype); fetal bovine serum (Clark australia); double antitrypsin, trypsin (EDTA-free), PBS (beijing soleba); dexamethasone (DEX) (shanghai jiiding); recombinant human insulin (marsupenamide); 3-isobutyl-1-methylxanthine (IBMX) (Shanghai microphone); CCK-8 (U.S. GLPBIO); RIPA lysate, BCA kit, ECL chemiluminescent kit (shanghai bi yun tian); IGFR, FGFR2, MAPK, bcl-2, BAX kit (abyssindace, abboy); instant PI staining (beijing cool stroke); annexin V-FITC/PI double-stained apoptosis assay kit (Bei Bo Guangzhou).

1.3 Major instrumentation

Avanti TM J-3OI refrigerated centrifuge (Hainan Hemin); spectra MAX 190 microplate reader (shanghai meigu); flow cytometry was purchased from (usa Betcon Dickinson); inverted fluorescence microscope (Leica, germany); chemStudio SA2 developer (Germany Analytik JenaAG).

2 Experimental methods

2.1 Experimental cell lines

3T3-L1 fat precursor cells were purchased from the China academy of sciences typical culture Collection Committee cell Bank (GNM 25).

Induced differentiation of 2.23T3-L1 fat precursor cells

After resuscitating and passaging, the culture medium is replaced every 48 hours (namely, the complete culture medium containing 5% of fetal bovine serum and 1% of double antibodies), and after the cell fusion degree reaches 100%, the culture medium is replaced for continuous culture for 48 hours. The medium was then removed, the culture was continued with the addition of differentiation medium (i.e., complete medium containing 10. Mu.g/mL insulin, 500. Mu.M IBMX and 1. Mu.M DEX), and after 48 hours the culture was continued with the replacement of maintenance medium (i.e., complete medium containing 10. Mu.g/mL insulin), and after 48 hours the culture was replaced with normal complete medium, and then every 48 hours the complete medium was replaced, after co-induced differentiation for 8 days, and microscopic observation was made that if distinct lipid droplets had formed in the cytoplasm, the cells had differentiated into mature adipocytes.

2.3 Effect of ginsenoside on adipocyte viability

The effect of ginsenoside on the viability of 3T3-L1 fat precursor cells (undifferentiated) and mature adipocytes (differentiated cells) at different concentrations (0, 1, 5, 10, 25, 50, 100. Mu.M) was examined by CCK-8.

2.4 Fluorescent staining of DAPI to observe cell morphology

The 3T3-L1 cells in the logarithmic growth phase were treated with 25. Mu.M of the drug-containing medium for 24 hours. DAPI staining was performed and morphology was observed under an inverted fluorescence microscope and photographed.

2.5 Flow cytometry analysis of apoptosis

Cells were seeded in 6-well plates at a density of 5×10 ⁴ cells per well, incubated for about 24h after adherence, then 3T3-L124 h was treated with 25 μm ginsenoside-containing medium, after washing, 400 μ LAnnexin V conjugate suspension cells were added to each cell sample tube, incubated (protected from light, 15 min) with 5 μ LAnnexin V-FITC staining solution, then 10 μl PI (propidium iodide) staining solution was added for further incubation (protected from light, 5 min), the cell suspension was beaten into flow tubes through 200 mesh screens and immediately examined by flow cytometry, and the whole examination should be completed within one hour to avoid fluorescence quenching.

2.6 Flow cytometer analysis period

The periodic distribution of the cells was detected by PI staining. 3T3-L was treated with 25. Mu.M ginsenoside medium for 124h, stained with PI stain (50. Mu.g/mL), incubated in the dark for 30min, and the cell suspension was beaten into a flow tube through a 200 mesh screen, immediately followed by detection of cell cycle distribution with a flow cytometer. The whole detection process should be completed within one hour to avoid fluorescence quenching.

2.7 Transcriptomic sequencing

3T3-L1 fat precursor cells were seeded at a density of 1X 10 ⁶ cells/well in 6-well plates for 12h and then pretreated with test drug (optimal concentration: 25. Mu.M) for 2h, while the control group was not dosed. The cells were then H/R modeled and the following operations were performed.

2.7.1RNA extraction and identification

After 2 hours of treatment with the test drug and establishment of the H/R model, total RNA was extracted from 3T3-L1 fat precursor cells using TRIzol reagent. RNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo FISHER SCIENTIFIC).

2.7.2 Preparation of transcriptome sequencing library

A total RNA volume of 1 μg per sample was used as input material for RNA sample preparation, a sequencing library was generated, and an index code was added to the property sequence of each sample. PCR was performed using Phusion high fidelity DNA polymerase, universal PCR primers and index (X) primers.

2.7.3 Clustering and ranking

Index-encoded samples were clustered on a cBot cluster generation system using TruSeq PE Cluster Kit v-cBot-HS (Illumina) according to the manufacturer's instructions. After generating clusters, library preparations were sequenced on the Illumina platform and paired end reads were generated.

2.7.4 Differential expression analysis

Differential expression analysis was performed on both groups using the edge software. The methods of Benjamini and Hochberg are used to control the false discovery rate and thus adjust the p-value produced. The gene found by edder to adjust p value <0.05 was designated DEGs and volcanic mapping was performed.

2.7.5 Gene ontology and pathway enrichment analysis

Pathway cluster analysis was performed using the kyoto gene and genome encyclopedia (KEGG) as a database.

2.8 Analysis of protein expression level

2.8.1 Extraction of Total cellular proteins

Cells were plated in 6-well plates and lentivirally transfected with the addition of IGFR, FGFR2 overexpression groups (sh IGFR and sh FGFR 2) 24h after dosing. After the medium was discarded, after three washes with pre-chilled PBS, 200. Mu.L of cell lysate was added, after complete cell lysis, the cells were scraped off with a gun head, sucked into a sterile 1.5mL centrifuge tube, centrifuged at 12500rpm for 25min at 4℃and the pellet was removed and the supernatant collected and stored at-80℃where the cell lysis process was performed on an ice box.

2.8.2BCA method for determining protein concentration

The BCA kit was used to detect the protein concentration of each sample.

2.8.3 Detection of the expression level of the protein of interest by the kit

Protein expression level detection was performed using IGFR, FGFR2, MAPK, bcl-2, BAX protein content detection kit.

2.9RT-qPCR

Extracting total RNA of 3T3-L1 fat precursor cells by TRIzol reagent, then reacting for 5 minutes at 55 ℃ by using RT-qPCR kit, reacting for 20 seconds at 90 ℃, and then reversely transcribing the total RNA into cDNA. Then, 10. Mu.L of green qPCR Supermix, 0.8. Mu.L of upstream and downstream primer mix, and 2. Mu.L of cDNA template were mixed for DNA amplification. Based on the value of the cycle threshold (Ct), the relative expression level of the gene of interest is calculated.

2.10 Statistical treatments

All data were statistically analyzed using Prism 8 (GraphPad) statistical software, data were expressed as mean±sd, and comparisons between groups were performed using t-test combined with one-way anova, with P <0.05 being differential and statistically significant, P <0.05, P <0.01, P <0.001.

3 Results

3.1 Effect of ginsenoside on cell viability of 3T3-L1 fat precursor cells

Of the 105 triterpenes and their glycosides tested, the more typical experimental results showed that after 3T3-L1 fat precursor cells were treated with different concentrations of ginsenoside (aglycone) (1, 5, 10, 25, 50, 100 μm) compared to the blank, the cell viability of PPD treated groups was significantly inhibited in the concentration range of 10-100 μm, see fig. 1, and exhibited concentration dependence. (note: p <0.05, < p <0.005 compared to control).

3.2 Effect of ginsenoside on cell viability of mature adipocytes

The results showed that the cell viability of PPD and PD alone at a concentration of 100 μm was significantly different in the individual ginsenoside-dosed groups than in the blank group for the induced differentiation of mature adipocytes, see fig. 2 (note: p <0.05 compared to the control group), but this dose was higher and not practically meaningful. Thus, only the lowest significant effective dose of PPD with PD, 25. Mu.M, was selected for subsequent experiments to induce apoptosis of 3T3-L1 fat precursor cells.

3.3 Effects of PPD, PD on 3T3-L1 fat precursor cell morphology

DAPI fluorescent staining the effect of PPD and PD treatment for 24h at the same concentration (25 μm) on the morphology of 3T3-L1 fat precursor cells was observed and the results are shown in fig. 3. The cells are stained under normal conditions to see uniform distribution of chromatin and to exhibit a darker blue fluorescence. Compared to the blank, PPD and PD (25 μm) treated 3T3-L1 fat precursor cells, the number of cells became smaller, the brightness became larger, the cell profile became irregular, nuclear fragmentation occurred, and apoptotic bodies and chromatin fragments (arrows in fig. 3) could be observed, indicating that PPD and PD caused apoptosis of 3T3-L1 fat precursor cells. In addition, after cells are treated by PPD, the number of morphologically abnormal cells is obviously increased compared with that of PD-treated groups, and the fluorescence intensity of the PD-treated groups is slightly weaker.

3.4 Effects of PPD, PD on apoptosis of 3T3-L1 fat precursor cells

After 24h treatment of 3T3-L1 fat precursor cells with 25 μm PPD and PD, apoptosis rate was examined with a flow cytometer, and the results are shown in fig. 4, in which the apoptosis rate was up-regulated by both 25 μm PPD and PD treatment compared to the blank group, showing significant differences, and the up-regulation amplitude of PPD administration group was more significant than that of PD group (note: p <0.01, < p < 0.005) compared to the control group.

3.5 Effects of PPD, PD on 3T3-L1 fat precursor cell cycle arrest

As shown in fig. 5, the proportion of cells in S phase of PPD-treated cells was significantly increased, and the difference was extremely significant, compared to the blank group, but PPD had no significant effect on G2 phase of cells; the proportion of cells in both S and G2 phases after PD treatment was significantly increased (note: P <0.05, P <0.01, P <0.001 compared to control). It was demonstrated that both PPD and PD could induce S phase arrest in 3T3-L1 fat precursor cells, while PD could also induce further G2 phase arrest in cells.

3.6PPD transcriptome sequencing differential Gene analysis

DEGs in the control and PPD groups were enriched by the KEGG pathway. The first 20 pathways with the most reliable enrichment significance (i.e., minimum Q value) were selected to present the results as shown in fig. 6. Among these, the relevant pathways with the most relevant and most distinct EGFR kinase inhibitor resistance were selected. Through volcanic image, the expression level of the related proteins IGFR, FGFR2 and MAPK for regulating apoptosis of the cell is obviously reduced after PPT administration treatment, which is probably the main reason why PPD induces apoptosis of fat precursor cells, and subsequent mechanism verification research is carried out.

3.7 Influence of PPD on expression of 3T3-L1 fat precursor cell apoptosis-related protein

Compared with the blank group, after PPD treatment at different concentrations, the expression amounts of IGFR, FGFR2 and MAPK proteins in the fat precursor cells were all significantly inhibited, see fig. 7, and the differences were very significant, especially at 25, 50 μm concentrations. The relative expression level of apoptosis protein Bcl-2/Bax protein is obviously reduced. However, after we overexpressed IGFR and FGFR2 in the cells, the expression of the apoptotic proteins was reversed (note: P <0.05 compared to control, P <0.01; P <0.01 compared to PPD+sh IGFR/FGFR 2). It is demonstrated that IGFR and FGFR2 are key targets for inducing apoptosis of fat precursor cells and PPD does promote apoptosis of fat precursor cells by inhibiting IGFR, FGFR2 and MAPK proteins to further regulate downstream apoptosis proteins.

3.8RT-PCR

Relevant genes IGFR and FGFR2 for key proteins were selected for validation, see fig. 8. As a result, it was found that both IGFR and FGFR2, a PPD-treated adipocyte, produced a significant inhibition effect, especially at a concentration of 25 μm, relative to the blank (note: P <0.05, P <0.01 compared to the control), which was consistent with the sequencing results and protein expression described above, further demonstrating that IGFR and FGFR2 are key targets for inducing apoptosis of adipocyte precursor cells.

Conclusion 4

Ginsenoside PPD and PD have remarkable activity of inducing apoptosis of fat precursor cells, wherein PPD is the most remarkable, and the activity is that cell growth factors IGFR and FGFR2 are simultaneously inhibited, so that expression of downstream protein MAPK is inhibited, apoptosis key proteins are activated, cell cycle is retarded, and apoptosis is promoted.

EXAMPLE 2 Protopanaxatriol inhibits fat precursor cell differentiation

1 Materials and instruments

1.1 Ginsenoside (Yuan)

Same as "1.1" of example 1.

1.2 Materials and reagents

The cell-associated reagent is the same as "1.2" of example 1; triglyceride (TG) assay kit, glucose (GLU) content assay kit (nanjing build); free Fatty Acid (FFA) content detection kit (beijing solibao); mouse tumor necrosis factor (TNF-alpha), mouse Adiponectin (ADP), mouse resistin (resistin), mouse leptin (leptin) ELISA kit (Beijing City forest); PERK inhibitor GSK2606414 (Sigma, USA); hspa4, RELA, PERK, CHOP, PPAR γ and C/EBPα content detection Elisa kit.

1.3 Major instrumentation

Same as "1.3" of example 1.

2 Experimental methods

2.1 Experimental cell lines

Same as "2.1" of example 1.

2.23T3-L1 fat precursor cells induce differentiation

"2.2" As in example 1.

2.3 Detection of the degree of differentiation of cells by oil Red O staining colorimetry

Ginsenoside was prepared into ginsenoside solutions (0, 25, 50, 75, 100. Mu.M) with different concentrations using a medium containing an inducer (i.e., complete medium containing 10. Mu.g/mL insulin, 500. Mu.M IBMX, and 1. Mu. MDEX), after contact inhibition, 3T3-L1 fat precursor cells were induced to differentiate according to the method "2.2" of example 1, stained with oil red O, and 100. Mu.L of isopropanol was added to dissolve the oil red O dye, and absorbance (520 nm) was measured to calculate the differentiation rate of each group of cells.

2.4 Modulation of mature adipocyte insulin resistance index

After the fat precursor cells are induced into mature fat cells, the cells are subjected to insulin resistance model establishment by using 1 mu M dexamethasone for 96 hours, are subjected to administration treatment, are added with a positive medicine rosiglitazone group, and are subjected to detection of lipid drop distribution by using oil red O staining after 24 hours, so that the activity of the cells on insulin resistance is judged.

2.5 Glucose consumption detection

The degree of extracellular glucose consumption was measured by the glucose oxidase-peroxidase method (GOD-POD method).

2.6 Determination of extracellular Triglycerides

Extracellular triglyceride content was determined by phosphoglycerate oxidase-peroxidase method (GPO-PAP method).

2.7 Determination of free fatty acid content

The free fatty acid content of the culture supernatant was determined using a free fatty acid detection kit.

2.8 Determination of fat-related factors by ELISA

The adiponectin, resistin, leptin, and TNF- α content of the culture supernatant was determined using a mouse adiponectin, resistin, leptin, and TNF- α kit (ELISA assay).

2.9 Transcriptomic sequencing

PPT 25. Mu.M with significant efficacy was selected for transcriptomic sequencing, and "2.7" of example 1 was performed.

2.10 Analysis of protein expression level

The HSPA4, RELA, pparγ, PERK, CHOP, C/ebpα kit was used to detect the related protein expression levels, and HSPA4 knockout group (si HSPA 4), PERK inhibitor GSK2606414 group were added, and the same procedure as in "2.8" of example 1 was followed.

2.11RT-PCR

The expression levels of HSPA4, RELA, PPARG1, EIF2AK3, DDIT3 and CEBPA-related genes were examined, and the procedure of "2.9" in example 1 was followed.

2.12 Statistical treatments

Same as "2.10" of example 1.

3 Results

3.1 Effect of ginsenoside on lipid accumulation in adipocytes

Lipid accumulation is one of the important features of mature adipocytes, and therefore, oil red O dye was selected to examine the effect of ginsenoside on cell differentiation, see fig. 9, and PPD and PD were not used in subsequent experiments of inhibition differentiation studies because they have some cytotoxicity. After 3T3-L1 fat precursor cells are differentiated into mature fat cells on a 96-well plate, oil red O staining is carried out according to a 2.3 method, the oil red O specifically combined with intracellular lipid is dissolved by isopropanol to be used as a differentiation marker, and an enzyme-labeled instrument is used for detecting the cell differentiation degree at a wavelength of 520 nm. Typical results in 103 samples showed that the lipid levels in the ginsenoside-dosed groups were significantly lower in PPT, rg ₅ and 20 (S) -Rg ₃ compared to the control group, indicating that the ability of the cells to accumulate lipid was inhibited, with the best performance of PPT, with adipocyte differentiation rates of only 60% of control at 25 μm and only 25% of control at 100 μm (note: P <0.05, P <0.01, P <0.001 compared to the control group, much higher than Rg ₅ and 20 (S) -Rg ₃); however, ginsenoside Rh ₁、Re、Rf、Rg₂ promotes differentiation of fat precursor cells, with Rh ₁ being the most prominent; the rest ginsenoside (aglycone) has no obvious influence on the differentiation rate of fat precursor cells. Thus, three ginsenosides (aglycones) PPT, rg ₅ and 20 (S) -Rg ₃ were selected for subsequent correlation experiments.

3.1.1 Results of oil Red O staining

Cells were cultured on 6-well plates, and after the cells were inhibited by contact, differentiation was induced by adding an inducer containing a drug (25. Mu.M), and after completion of differentiation, fat staining was performed with an oil red O dye solution, and after staining, lipid droplets secreted by adipocytes were stained red, while undifferentiated cells or the remaining structures of differentiated cells except lipid droplets were not stained. As shown in fig. 10, the fat cells without drug intervention were mostly stained red, and almost all the cells were seen to differentiate. In contrast, after the adipocytes are treated by the drug, the staining sites are obviously reduced, the differentiation of the adipocytes and the secretion of lipid droplets are inhibited, and the inhibition effect of the PPT group (25 mu M) is more obvious at the same administration concentration.

3.1.2PPT, rg ₅ and 20 (S) -Rg ₃ effect on improving insulin resistance of mature adipocytes

As shown in fig. 11, control is a normal adipocyte Control group, I/R is an insulin resistance model, ros is a positive drug rosiglitazone group, intracellular lipid droplets are significantly larger than normal intracellular lipid droplets and accumulate in large amounts after the insulin resistance model is established, and intracellular lipid droplets are significantly smaller after 25 μm PPT administration treatment, and the accumulation amount is greatly reduced and superior to that of the positive drug rosiglitazone group. Whereas Rg ₅ and 20 (S) -Rg ₃ were not significantly changed. Therefore, the PPT can not only inhibit the differentiation of fat precursor cells, but also enhance the insulin sensitivity of mature fat cells to further regulate the glycolipid metabolism of the fat cells and reduce the accumulation of lipid.

3.1.3PPT, rg ₅ and 20 (S) -Rg ₃ effects on cell supernatant triglycerides

Cell release TG in each group of cell culture supernatants was detected using TG kit 8 days after cell differentiation. As can be seen from the results (fig. 12), TG content in each of the dosing group cultures at the same concentration (25 μm) was significantly reduced compared to the control group, and the PPT group had the most significant reduction in TG content compared to the other two groups (note: P <0.01, P <0.005 compared to the control group). It is demonstrated that ginsenoside PPT, rg ₅ and 20 (S) -Rg ₃ can inhibit 3T3-L1 fat precursor cell differentiation and reduce cell synthesis and release triglyceride.

3.1.4PPT, rg ₅ and 20 (S) -Rg ₃ effects on free fatty acid content of cell supernatants

FFA is both a product of fat hydrolysis and a substrate for fat synthesis. FFA concentration in blood is closely related to lipid metabolism, glycometabolism, and endocrine function. The FFA content detection kit is selected to detect the free FFA content in the cell supernatant, and the detection result shows that each administration group at 25 μm can reduce the free FFA content in the cell, wherein PPT has the best effect, and all three administration groups show very significant differences compared with the control group (note: P <0.005 compared with the control group). This further suggests that PPT, rg ₅ and 20 (S) -Rg ₃ have an inhibitory effect on catabolism of fat.

3.1.5PPT, rg5 and 20 (S) -Rg3 effects on cell differentiation glucose consumption

After 8 days of induction, 3T3-L1 preadipocytes almost differentiated into mature adipocytes, and glucose in the cell culture medium is the main nutrient for this process. The inhibition of nutrient uptake by the cells by ginsenoside was evaluated by measuring the glucose consumption in the medium. As shown in FIG. 14, the glucose content in the culture medium was significantly reduced in each of the administration groups (25. Mu.M) compared with the control group (note: P <0.005 compared with the control group), indicating that PPT, rg ₅ and 20 (S) -Rg ₃ each inhibited glucose uptake by 3T3-L1 adipocytes, and that PPT inhibited glucose uptake by cells best.

3.2 Effect of PPT, rg ₅ and 20 (S) -Rg ₃ on secretion of fat-related factors

After 3T3-L1 fat precursor cells are induced to differentiate and mature, various fat factors including tumor necrosis factor alpha (TNF-alpha), adiponectin (adiponectin), resistin (restistin) and leptin (leptin) can be secreted, which is also one of important characteristics of fat cell maturation. Thus, ELISA was used to determine the levels of TNF- α, adiponectin, resistin, and leptin in the medium after intervention of different drugs (PPT, rg ₅, and 20 (S) -Rg ₃) at 25. Mu.M concentration to evaluate the inhibition of adipocyte differentiation by the drugs. As shown in FIG. 15, cells secreted relatively high levels of TNF- α, adiponectin, resistin, and leptin 8 days after induction of differentiation, indicating that 3T3-L1 cells had differentiated into mature adipocytes. In addition, each administration group significantly reduced the secretion level of the above hormone (note: P <0.01, P < 0.005) compared with the control group, indicating that ginsenoside PPT, rg ₅ and 20 (S) -Rg ₃ inhibit the secretory activity of adipocytes during adipogenesis, and that PPT has the best inhibitory effect.

3.3PPT transcriptome sequencing differential Gene analysis

DEGs in the control and PPT groups were enriched by the KEGG pathway. The first 20 pathways with the most reliable enrichment significance (i.e., minimum Q value) were selected to present the results, as shown in fig. 16. Among these, we selected the most relevant and significantly different pathways associated with atherosclerosis. Through volcanic images, the expression levels of related genes Hspa4, RELA, EIF2AK3 and DDIT3 for regulating and controlling the differentiation of the fat precursor cells are obviously up-regulated after the PPT administration treatment, while the expression levels of the fat differentiation key genes PPARG1 and CEBPA are obviously down-regulated, which indicates that Hspa4, RELA, EIF2AK3 and DDIT3 can be key targets of the PPT for inhibiting the differentiation of the fat precursor cells, and subsequent mechanism verification research is carried out.

3.4 Effect of PPT on differentiation-controlling related proteins in adipocytes

Lipid and atherosclerosis pathways are important pathways involved in fat regulation of energy metabolism, wherein Hspa4 and RELA in the pathways are used as inflammatory syndrome related proteins, and can further regulate fat differentiation related protein pparγ. And PERK and CHOP are endoplasmic reticulum stress related regulatory proteins which can further regulate downstream fat differentiation related protein C/EBP alpha. Therefore, in order to determine a specific mechanism of the PPT to inhibit the differentiation of fat precursor cells, the expression level analysis of the related proteins was performed. The results are shown in FIG. 17, in which the PPT-treated groups (25, 50. Mu.M) had significantly increased expression levels of Hspa4, RELA (in-core), PERK, CHOP, and PPARgamma and C/EBPalpha were significantly inhibited compared to the control group, and the above was significantly reversed by further knocking out Hspa4 and inhibiting PERK expression (note: P <0.05, P <0.01 compared to the control group; and $P <0.01 compared to PPT+siHspa 4/GSK 2606414). This further demonstrates that PPT promotes nuclear translocation of the downstream inflammatory protein RELA and expression of the endoplasmic reticulum stress protein CHOP by activating Hspa4 and PERK, thereby inhibiting the adipocyte differentiation key proteins pparγ and C/ebpα, thereby inhibiting adipocyte differentiation.

3.5RT-PCR

The related genes Hspa4, RELA (nuclear), PPARG1, EIF2AK3, DDIT3 and CEBPA of the key proteins are also selected for verification. As shown in fig. 18, the expression levels of the PPT-treated fat precursor cell genes Hspa4, RELA (intranuclear), EIF2AK3 and DDIT3 were significantly improved, whereas the differentiation-critical genes ppavg 1 and CEBPA showed significant inhibition, especially at 25 and 50 μm concentrations (note: P <0.05 and P <0.01 compared with the control group), which is consistent with the above-described sequencing results and protein expression conditions, further demonstrating that Hspa4 and EIF2AK3 are key targets for PPT inhibition of fat precursor cell differentiation.

Conclusion 4

Ginsenoside PPT, rg ₅ and 20 (S) -Rg ₃ can significantly inhibit glucose uptake, fat catabolism and cell differentiation of fat precursor cells and reduce lipid accumulation, wherein PPT is most significant, and its inhibition effect is to promote nuclear translocation of downstream inflammatory proteins RELA and expression of endoplasmic reticulum stress protein CHOP by targeted activation of Hspa4 and PERK, thereby inhibiting differentiation key proteins pparγ and C/ebpα of fat cells, thereby inhibiting differentiation of fat precursor cells, regulating glycolipid metabolism of mature fat cells, enhancing insulin sensitivity of mature fat cells, and further reducing lipid accumulation of mature fat cells.

Example 3 Effect of protopanaxadiol and protopanaxatriol/Rg ₅/20(S)-Rg₃ composition on mouse obesity and lipid metabolism

1 Materials, reagents and instruments

1.1 Materials and reagents

Protopanaxadiol (PPD), protopanaxatriol (PPT), ginsenoside/Rg ₅ and 20 (S) -Rg ₃ are as in example 1; free Fatty Acids (FFA); total Cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL) detection kit (south-ky bioengineering limited); mouse Insulin (ins), mouse Glucose (Glucose) and mouse Leptin (Leptin) enzyme-linked immunoassay ELISA kits (R & D company in the united states); other reagents are all of domestic analytical purity.

1.2 Instruments

ABS320-4N type analytical balance (kokumi, shanghai); spectraMax190 automatic enzyme labelling machine (eight instrument works in Beijing); CT14D bench top high-speed centrifuge (Tianmei science instruments Co., ltd.).

2 Experimental methods

2.1 Laboratory animals

C57BL/6J mice, males, body weight 18-20g at 5 weeks of age; animal feeding environment: the temperature is 23+/-2 ℃, the relative humidity is about 55 percent, the light/dark cycle is 12 hours, and the feed is adaptively fed for 7 days before the experiment is started.

2.2 Grouping and administration of animals

Mice were randomized, and 8 mice per group were grouped into a control group (CON group, fed with normal diet), a model group (HFD group, fed with high fat diet), a PPD/PPT composition (1:1), a PPD/Rg ₅ composition (1:1), and a PPD/20 (S) -Rg ₃ composition (1:1), each in a molar ratio of 100mg/kg, fed with high fat diet, and as a result, the PPD/PPT composition was found to be significantly effective, and thus, gastric administration was performed on mice in a daily basis in a 10mL/kg body weight, and in a physiological equivalent manner for the PPD/PPT composition (11 groups total, PPD and PPT molar ratios of 0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0), respectively, and for the gastric administration in a gastric administration model of 10mL/kg, respectively, and for 12 weeks. Food intake was measured every 3 days, body weight was measured every week, and all mice were allowed free to drink and eat throughout the dosing period. The Food Efficiency Ratio (FER) is calculated as follows: FER (%) =body weight gain (g/d)/food intake (g/d) ×100%.

2.3 Collection of serum and visceral tissue

After administration, mice were fasted for 12h, and after blood was collected from the eyeballs, the mice were sacrificed by cervical dislocation and serum was isolated by centrifugation at 1500 rpm for 10min at 4 ℃. The body weight, body length (distance from anus to tip of nose) of the mice were measured, and Lee's index = [ body weight (g) ×1000/body length (cm) ]/3 was calculated. Dissecting, taking out epididymal fat, skeletal muscle, liver and kidney, washing with 0.90% (w/v) physiological saline and weighing, and calculating organ index (organ index=organ mass/body mass).

2.4 Detection of biochemical indicators of serum samples

The serum Insulin, glucose and Leptin content were both determined by enzyme-linked immunosorbent assay according to the ELISA kit instructions. Insulin resistance Index (IR) is calculated according to the following equation: HOMA-IR= [ serum glucose (mmol/L). Times.serum insulin (pmol/L) ]/22.50. The serum FFA, TC, TG, HDL and LDL levels were determined by corresponding chromogenic methods according to the manufacturer's instructions.

2.5 Statistical methods

Experimental data are expressed as x±s and were statistically processed using SPSS21 statistical analysis software. The statistical significance of the differences between the groups was assessed by t-test and one-way analysis of variance (anova), with P <0.05 being significant and statistically significant.

3 Results

3.1 Effect of composition on the weight of C57BL/6J mice

After 12 weeks, C57BL/6J mice fed a HFD diet containing 45% fat had significantly higher body weight and weight gain than mice fed LFD (containing 10% fat), indicating that HFD did lead to obesity (P < 0.05) in the free diet experiment. Each group of compositions significantly reduced the weight gain of mice (P < 0.05), the Lee's index (P < 0.05), especially with PPD/PPT composition 1:1 most typically, as shown in table 1.

Table 1 influence of the treatments of the groups on the body weight of C57BL/6J mice (mean+ -s, n=8)

Note that: ^ab Values that do not share superscript letters differ significantly between groups (P < 0.05).

3.2 Effect of the composition on the organs of C57BL/6J mice

The results show that HFD fed mice of each composition group had significantly reduced epididymal fat weight, daily food intake and FER (P < 0.05), especially with PPD/PPT composition 1:1 most typical, compared to HFD mice. But HFD mice had significantly increased liver weight and liver index (P < 0.05) compared to CON mice. Typical results are shown in tables 2 and 3.

Table 2 effects of each group treatment on food intake and organ weight in C57BL/6J mice (mean+ -s, n=8)

Group of	Food intake (g/day)	Kidney weight (g)	Liver weight (g)	Fat weight (g)
					CON	3.82±0.33^a	0.31±0.02	0.92±0.07^b	0.54±0.05^b
HFD	2.71±0.28^b	0.33±0.02	1.45±0.23^a	1.46±0.25^a
					PPD/PPT1:1	2.13±0.25^c	0.32±0.03	0.95±0.12^b	0.59±0.12^b
PPD/Rg₅1:1	2.54±0.21	0.31±0.01	1.13±0.11^b	0.88±0.13
					PPD/20(S)-Rg₃1:1	2.66±0.23	0.33±0.05	1.15±0.13^b	0.84±0.15
PPD/PPT1:9	2.23±0.31^c	0.34±0.04	1.02±0.15^b	0.75±0.22^b
					PPD/PPT9:1	2.21±0.34^c	0.33±0.02	0.98±0.11^b	0.64±0.28^b
PPD sodium salt/PPT water sum 1:1	2.18±0.32^c	0.35±0.01	0.99±0.18^b	0.62±0.13^b

Note that: ^abc Values that do not share superscript letters differ significantly between groups (P < 0.05).

Table 3 effects of each group treatment on food efficiency ratio and organ index of C57BL/6J mice (mean+ -s, n=8)

Group of	Food efficiency (%)	Kidney index (mg/g)	Liver index (mg/g)	Fat index (mg/g)
					CON	3.45±0.76^b	10.33±0.69	30.12±2.04^b	16.98±1.46^b
HFD	9.29±0.98^a	8.98±0.51	36.68±2.55^a	35.45±2.63^a
					PPD/PPT1:1	4.63±0.42^b	10.15±0.67	30.19±1.88^b	17.82±1.76^b
PPD/Rg₅1:1	6.56±0.45	9.86±0.66	34.15±1.21	26.35±2.54^b
					PPD/20(S)-Rg₃1:1	6.24±0.55^b	9.85±0.57	33.86±1.69^b	25.24±2.55^b
PPD/PPT1:9	5.11±0.63^b	10.38±0.66	30.65±2.01^b	23.15±1.28^b
					PPD/PPT9:1	5.23±0.54^b	10.24±0.57	30.33±1.89^b	20.35±2.36^b
PPD sodium salt/PPT water sum 1:1	5.01±0.43^b	10.88±0.47	30.13±1.52^b	19.22±1.23^b

3.3 Effect of the composition on the serum Biochemical index of C57BL/6J mice

Serum levels of Glucose, leptin, insulin, FFA, TC, TG and LDL were determined. The results showed that all biochemical indexes of HFD group were significantly changed compared to CON group, indicating successful modeling. Compositions each treatment improved fasting blood glucose levels (P < 0.05), reduced Leptin, insulin, FFA, HOMA-IR, TC, TG and LDL levels compared to HFD groups, especially with PPD/PPT compositions 1:1 being most typical. Typical results are shown in tables 4 and 5.

Table 4 influence of the treatments of the groups on the levels of free fatty acids, leptin, glucose, insulin in C57BL/6J mice (mean+ -s, n=8)

TABLE 5 influence of the treatments of the groups on TC, TG, HDL-C and LDL-C (mean+ -s, n=8)

Group of	TG(mmol/l)	TC(mmol/l)	LDL-C(mmol/l)
				CON	0.82±0.07^b	2.52±0.18^c	0.07±0.02^b
HFD	1.43±0.18^a	4.85±0.71^a	0.20±0.05^a
				PPD/PPT 1:1	0.81±0.09^b	2.83±0.65^b	0.07±0.01^b
PPD/Rg₅ 1:1	1.25±0.06	3.83±0.35	0.15±0.02
				PPD/20(S)-Rg₃ 1:1	1.18±0.09	3.54±0.75	0.12±0.01^b
PPD/PPT 1:9	0.94±0.04^b	3.22±0.88^b	0.11±0.03^b
				PPD/PPT 9:1	0.91±0.08^b	3.05±0.98^b	0.09±0.01^b
PPD sodium salt/PPT water sum 1:1	0.82±0.06^b	2.88±0.39^b	0.08±0.02^b

Conclusion: the protopanaxadiol and protopanaxatriol composition (1:1) can obviously reduce the weight, organ index and epididymal fat weight of obese mice, can obviously improve the biochemical indexes such as fasting blood glucose level, glucose, leptin, insulin, FFA, TC, TG and LDL and the levels of fatty acid, leptin, glucose and insulin, shows that the composition has obvious effects on preventing and treating relevant diseases such as obesity caused by abnormal differentiation and metabolism of fat cells, and has a great development prospect.

Example 4 tablet preparation

Reagent: starch (pharmaceutical grade, tianjin, east-day fine chemical reagent plant); citric acid (Shanghai microphone Biochemical technologies Co., ltd.); magnesium stearate (Shanghai microphone Biochemical technologies Co., ltd.).

The preparation method comprises the following steps:

① Preparation of 10% starch slurry: 0.25g of citric acid is dissolved in 25mL of pure water, 2.5g of starch is added for uniform dispersion, and the mixture is heated to gelatinize the starch, thus obtaining 10% starch slurry.

② Granulating: mixing proper amount of protopanaxadiol and protopanaxatriol composition (molar ratio of protopanaxadiol to protopanaxatriol 1:1) with starch, adding proper amount of 10% starch slurry, mixing, grinding, making soft material, sieving with 16 mesh sieve, granulating, and drying at 50-60deg.C for 1 hr. After the 16 mesh sieve is sized, a proper amount of lubricant magnesium stearate is added, and the mixture is pressed into tablets by a shallow punch with the diameter of 10 mm.

Results: the obtained tablet has the advantages of off-white color, uniform thickness and moderate hardness. Tablet weight and disintegration time meet the requirements.

Conclusion: the obtained protopanaxadiol and protopanaxatriol composition tablet meets the requirements, and can be used as tablet.

EXAMPLE 5 preparation of suspension injection

Reagent: polylactic acid (PLA, shanghai screening quasi-biotechnology limited); polylactic-co-glycolic acid (PLGA, shanghai-derived biotechnology limited); poloxamer 188 (sienna n pharmaceutical excipients limited); dichloromethane, methanol, acetonitrile, etc. (Tianjin Tiantai chemical Co., ltd.).

The preparation method comprises the following steps:

① Preparation of polymer microparticles: weighing a proper amount of the protopanaxadiol and protopanaxatriol composition (the molar ratio of the protopanaxadiol to the protopanaxatriol is 1:1) and a carrier (PLA/PLGA) and placing the composition and the carrier into a 50mL round bottom flask, adding 5mL of dichloromethane for dissolution, distilling at 28 ℃ under reduced pressure to remove most of organic solvents, then drying in vacuum at 40 ℃ for 24 hours until the solvents are completely removed, crushing, and sieving with a screen with the aperture of 150 mu m to obtain polymer particles of the interlinked protopanaxadiol and protopanaxatriol composition.

② Preparation of a suspension injection of a combination of protopanaxadiol and protopanaxatriol: 4.0g of the above product was dispersed in 250mL of an aqueous solution containing 10g/L poloxamer 188 stabilizer with continuous stirring to complete the dispersion. Grinding the medicinal dispersion to desired particle size, taking out to obtain a particle suspension of the protopanaxadiol and protopanaxatriol composition, centrifuging at 5000rpm for 2min, dispersing with 10mL of stabilizer aqueous solution, and concentrating to about 25g/L.

Results: the obtained protopanaxadiol and protopanaxatriol composition suspension injection has uniform particle size, and the water content and the surface particle size of the preparation meet the regulations. The in vitro slow release effect is better, and the stability is better.

Conclusion: the obtained protopanaxadiol and protopanaxatriol composition suspension injection meets the requirements, and can be used as suspension injection.

EXAMPLE 6 hydrogel patch preparation

Reagent: sodium polyacrylate (Shanghai Seiyaku Biotechnology Co., ltd.), aluminum glycidate (Shanghai Seiyaku Biotechnology Co., ltd.), sodium carboxymethylcellulose (Shanghai Seiyaku Biotechnology Co., ltd.), soybean lecithin (Shanghai Seiyaku Biotechnology Co., ltd.), cholesterol (Shanghai Seiyaku Biotechnology Co., ltd.), chloroform (Tianjin Tiantai chemical Co., ltd.), azone (Tianjin Tiantai chemical Co., ltd.), and propylene glycol (Tianjin Tiantai chemical Co., ltd.).

The preparation method comprises the following steps:

① Preparing colloid: precisely weighing sodium polyacrylate, aluminum glycollate and sodium carboxymethylcellulose into a mortar, measuring glycerol, adding the glycerol into the mortar, grinding and stirring, fully mixing the glycerol and the propylene glycol, standing for standby, dissolving the azone into the propylene glycol solution according to a certain proportion as a penetration enhancer, and adding the azone into the mortar after vortex to uniformly mix the azone and the propylene glycol.

② Preparation of protopanaxadiol and protopanaxatriol composition liposomes: mixing soybean phospholipid, cholesterol, PPD/PPT composition (PPD and PPT molar ratio 1:1), adding chloroform, and dissolving by ultrasonic treatment; removing the organic solvent by reduced pressure rotary evaporation to form a uniform dry film on the bottle wall; hydrating at normal temperature, performing ultrasonic treatment, and freeze drying.

③ Preparing a hydrogel plaster of a protopanaxadiol and protopanaxatriol composition: precisely weighing protopanaxadiol and protopanaxatriol composition liposome, sodium benzoate, and citric acid, adding into mortar, adding purified water, mixing, concentrating to obtain polymer milky hydrogel paste, coating the polymer milky hydrogel paste on a backing layer, solidifying, cutting, covering polyethylene film on the adhesive surface of the hydrogel paste, and pressing to obtain protopanaxadiol and protopanaxatriol composition hydrogel patch.

Results: the obtained protopanaxadiol and protopanaxatriol composition hydrogel plaster has good indexes such as adhesion, no film residue and skin residue, no irritation, good uniform permeation, and drug loading meeting the specified range.

Conclusion: the obtained protopanaxadiol and protopanaxatriol composition hydrogel plaster meets the requirements, and can be used as hydrogel plaster.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

1. The application of a ginsenoside composition in preparing a medicament for treating obesity is characterized in that the ginsenoside composition consists of protopanoxadiol PPD and protopanaxatriol PPT; the molar ratio of the protopanoxadiol PPD to the protopanoxatriol PPT is 1:1.

2. The use according to claim 1, wherein the ginsenoside composition is capable of inducing apoptosis of fat precursor cells, inhibiting differentiation of fat precursor cells into mature fat cells, inhibiting triglyceride synthesis and storage.

3. The use according to claim 2, wherein the ginsenoside composition induces fat precursor apoptosis by inhibiting cell growth factors IGFR and FGFR2, thereby inhibiting expression of downstream protein MAPK.

4. The use according to claim 2, wherein the ginsenoside composition promotes nuclear translocation of RELA and expression of CHOP by activating Hspa4 and PERK, thereby inhibiting differentiation of fat precursor cells into mature adipocytes, inhibiting triglyceride synthesis and storage.