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

Abstract

The present invention discloses a ginsenoside composition and its application in the preparation of a multi-target adipocyte development, differentiation and metabolic regulator, belonging to the field of biomedicine. The ginsenoside composition is composed of any one of protopanaxadiol PPD and protopanaxatriol PPT, ginsenoside Rg ₅ or ginsenoside 20(S)-Rg _3. The present invention proves through pharmacological experiments that the ginsenoside composition activates Hspa4 and PERK while antagonizing IGFR, FGFR2 and MAPK, promotes the nuclear translocation of RELA and the expression of CHOP, thereby inducing apoptosis of preadipocytes, inhibiting the differentiation of preadipocytes into mature adipocytes, and inhibiting the synthesis and storage of triglycerides. The precise target effect is more direct and effective, and the generation of toxic and side effects is effectively reduced, which provides a theoretical basis for its application in the prevention, treatment and alleviation of obesity and related diseases.

Description

A ginsenoside composition and its application in preparing multi-target adipocyte development, differentiation and metabolism regulator

Technical Field

The present invention relates to the field of biomedicine, and in particular to a ginsenoside composition and application thereof in the preparation of a multi-target adipocyte development, differentiation and metabolism regulator.

Background technique

Obesity is a chronic metabolic disease, and more than 95% of obese patients suffer from simple obesity (hereinafter referred to as obesity). The problem of obesity is rapidly becoming widespread and sustained, but it has not received enough attention. The main characteristics of obesity are the increase in the number of fat cells and the expansion of fat cells caused by the proliferation and differentiation of pre-adipocytes, which leads to excessive accumulation of adipose tissue. Mature adipocytes can secrete a variety of adipose cell factors and inflammatory factors. Excessive factors can cause the body to develop a variety of metabolic diseases, such as hypertension, hyperlipidemia, insulin resistance, fatty liver, diabetes, as well as cardiovascular and cerebrovascular diseases and cancer.

Adipocytes come from mesenchymal stem cells that exist in adipose tissue, which are the same as bone marrow stroma. These stem cells are called adipose-derived stem cells (ADSCs), which have the characteristics of lasting vitality, self-renewal and multidirectional differentiation. ADSCs can differentiate into adipocyte precursors, also called preadipocytes, under the stimulation of adipogenic signal factors while maintaining the active proliferation characteristics of stem cells. After repeated contact inhibition and cell fusion stages, adipocyte precursors begin to differentiate into immature adipocytes under adipogenic induction conditions, and eventually complete the full differentiation into mature adipocytes. Mature adipocytes can express and secrete a variety of adipose cell factors and inflammatory factors, such as leptin, adiponectin, interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), etc., which act on different tissues and organs, induce inflammation, and then cause a variety of other metabolic diseases.

Drug intervention is the best choice for obese people. Due to the potential and serious side effects and high costs of chemical drugs currently used for weight loss, chemical components from natural plants are increasingly becoming the best choice for developing safe and low-cost anti-obesity drugs. Studies have shown that ginsenosides have an effect on fat differentiation, but there are still contradictions in many literature reports. It has been reported that total saponins from ginseng stems and leaves can inhibit the abnormal increase of PPARγ, FAS and aP2 protein and gene levels in the liver and/or adipose tissue of mice induced by a high-fat diet. The literature also reported that ginsenosides Rb1, Rg1, Re, Rd (20μM), Rh2 (20 and 40μM), Rg3 (20 and 40μM), Rd (80μM), Rh1 (50 and 100μM), Rg5:Rk1 (100μg/mL) inhibited the differentiation process of 3T3-L1 adipocytes. There are also reports that ginsenoside Rh2 (0.01-1μM) promotes adipocyte differentiation by activating adipocyte glucocorticoid receptors; ginsenoside Rg1 can accelerate the paracrine activity and adipocyte differentiation of human breast adipose-derived stem cells. There are also reports that ginsenoside Rg1 inhibits the early development of adipocytes by activating C/EBP homologous protein 10 in 3T3-L1.

Promoting apoptosis of adipocyte precursor cells is a new strategy for fighting obesity-related diseases, but there are few related reports. Inhibiting the differentiation and metabolism of adipocyte precursor cells is another important research direction for fighting obesity-related diseases. Therefore, in-depth research on the inhibitory effects of ginsenosides on the proliferation, differentiation and metabolism of adipocyte precursor cells is of great significance for the development of multi-target drugs for fighting obesity-related diseases.

Summary of the invention

The purpose of the present invention is to provide a ginsenoside composition and its use in the preparation of a multi-target adipocyte development, differentiation and metabolism regulator to solve the problems of the above-mentioned prior art. The ginsenoside composition provided by the present invention induces apoptosis of preadipocytes, inhibits differentiation of preadipocytes into mature adipocytes, and inhibits triglyceride synthesis and storage through precise multi-target effects; and is further used to prevent, treat and alleviate obesity and related diseases.

To achieve the above object, the present invention provides the following solutions:

The present invention provides a ginsenoside composition, which is composed of the following components:

Protopanaxadiol PPD; and,

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

Furthermore, it is composed of protopanaxadiol PPD and protopanaxadiol PPT, and the molar ratio of protopanaxadiol PPD to protopanaxadiol PPT is (1-9):(9-1).

Furthermore, the molar ratio of protopanaxadiol PPD to protopanaxatriol PPT is 1:1.

The present invention also provides an application of the ginsenoside composition in preparing a multi-target adipocyte development, differentiation and metabolism regulator.

Furthermore, the multi-target adipocyte development, differentiation and metabolism regulator can inhibit IGFR, FGFR2 and MAPK while activating Hspa4 and PERK, thereby promoting the nuclear translocation of RELA and the expression of CHOP.

The present invention also provides an application of the ginsenoside composition in preparing a medicine for treating obesity or obesity-related diseases.

Furthermore, the ginsenoside composition can induce apoptosis of preadipocytes, inhibit differentiation of preadipocytes into mature adipocytes, and inhibit synthesis and storage of triglycerides.

Furthermore, the ginsenoside composition inhibits cell growth factors IGFR and FGFR2, thereby inhibiting the expression of downstream protein MAPK, thereby inducing apoptosis of preadipocytes.

Furthermore, the ginsenoside composition activates Hspa4 and PERK, promotes the nuclear translocation of RELA and the expression of CHOP, thereby inhibiting the differentiation of adipocyte precursor cells into mature adipocytes and inhibiting the synthesis and storage of triglycerides.

The present invention also provides a medicine for treating obesity or obesity-related diseases, the medicine comprises the ginsenoside composition and a pharmaceutically acceptable carrier or excipient, and the dosage form of the medicine comprises tablets, suspension injections and hydrogel patches.

The present invention discloses the following technical effects:

In the ginsenoside composition provided by the present invention, all ingredients are natural products, are safe and easily available, and have low production costs.

The present invention proves through pharmacological experiments that the above ginsenoside composition, through precise multi-target action, antagonizes IGFR, FGFR2, and MAPK, while activating Hspa4 and PERK, promoting the nuclear translocation of RELA and the expression of CHOP; inducing apoptosis of preadipocytes, inhibiting the differentiation of preadipocytes into mature adipocytes, inhibiting the synthesis and storage of triglycerides, with a clear mechanism of action, more direct and effective precise target action, and effectively reducing the generation of toxic and side effects. It provides a theoretical basis for its application in the prevention, treatment and relief of obesity and its related diseases such as hypertension, hyperlipidemia, insulin resistance, fatty liver, diabetes and other metabolic diseases, as well as cardiovascular and cerebrovascular diseases and cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

FIG1 is a graph showing the effect of ginsenosides on the cell viability of 3T3-L1 preadipocytes in an embodiment of the present invention;

FIG2 is a graph showing the effect of ginsenosides on the cell viability of mature adipocytes in an embodiment of the present invention;

FIG3 shows the effects of PPD and PD on the morphology of 3T3-L1 preadipocytes in an embodiment of the present invention;

FIG4 is the effect of PPD and PD on apoptosis of 3T3-L1 preadipocytes in an embodiment of the present invention, wherein A is a flow cytometer to detect apoptosis, and B is a statistical chart of apoptosis rate;

FIG5 is the effect of PPD and PD on cell cycle arrest of 3T3-L1 preadipocytes in an embodiment of the present invention, wherein A is the cell cycle detected by flow cytometry, and B is a statistical diagram of the ratio of each cell cycle;

FIG6 is a diagram of differential gene pathway enrichment in PPD transcriptome sequencing according to an embodiment of the present invention, wherein A is a diagram of differential gene enrichment pathways for the top 20, and B is a volcano diagram of differential genes;

FIG7 shows the effect of PPD on the expression of apoptosis-related proteins IGFR (A), FGFR2 (B), MAPK (C), Bcl-2 and Bax (D) in 3T3-L1 preadipocytes in an embodiment of the present invention;

FIG8 is the effect of PPD on apoptosis-related genes IGFR (A) and FGFR2 (B) in 3T3-L1 preadipocytes according to an embodiment of the present invention;

FIG9 shows the effect of ginsenosides on lipid accumulation in adipocytes according to an embodiment of the present invention;

FIG10 is the Oil Red O staining result of ginsenosides on lipid accumulation in adipocytes according to an embodiment of the present invention;

FIG11 shows the improving effects of PPT, Rg ₅ and 20(S)-Rg ₃ on insulin resistance in mature adipocytes in an embodiment of the present invention;

FIG12 shows the effects of PPT, Rg ₅ and 20(S)-Rg ₃ on triglyceride consumption in cell supernatant in an embodiment of the present invention;

FIG13 is the effect of PPT, Rg ₅ and 20(S)-Rg ₃ on the consumption of free fatty acids in the cell supernatant in the embodiment of the present invention;

FIG14 shows the effect of PPT, Rg ₅ and 20(S)-Rg ₃ on glucose consumption in cell supernatant in an embodiment of the present invention;

Figure 15 shows the effects of PPT, Rg ₅ and 20(S)-Rg ₃ on the secretion of fat-related factors resistin (A), leptin (B), adiponectin (C) and TNF-α (D) in an embodiment of the present invention;

Figure 16 is a diagram of differential gene pathway enrichment in PPT transcriptome sequencing according to an embodiment of the present invention, wherein A is a diagram of differential gene enrichment pathways for the top 20, and B is a volcano diagram of differential genes;

FIG17 is the effect of PPT on the expression of differentiation-related proteins Hspa4 (A), RELA (B), PPARγ (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 regulating the expression of differentiation-related genes in adipocytes in an example of the present invention.

Detailed ways

Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

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

The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

Example 1 Protopanaxadiol induces apoptosis of preadipocytes

1. Materials and Instruments

1.1 Ginsenoside (yuan)

105 triterpenes and their glycosides, including ginsenoside Rb1, Rb2, Rb3, Rc, Rd, 20(S)-Rg ₃ , 20(R)-Rg ₃ , Rk ₁ , Rg ₅ , Rh ₂ , Re, Rg ₁ , Rf, Rg ₆ , F ₄ , Rk ₃ , Rh 4, Rg 2, Rh 1, Ro, ginsenoside Rb1, Rb2, Rb3, Rc, Rd, 20(S)-Rg 3, 20(R)-Rg 3, Rk 1, Rg 5, Rh ₂ , Re, Rg 1, Rf, Rg 6, F 4, Rk ₃ , Rh 4, Rg 2, Rh ₁ , Ro, ginsenoside Rb1, Rb2, Rb3, Rc, Rd, 20(S)-Rg 3, 20(R)-Rg 3, Rk 1, Rf, Rg 6, F 4, Rk 3, Rh 4, Rg 2, Rh 1, Ro, ginsenoside Rb1, Rb2, Rb3, Rc, Rd, 20(S)-Rg 3, 20(R)-Rg 3, Rk 1, Rf, Rg 6, F 4, Rk 3, Rh 4, Rg 2, Rh 1, Ro, ginsenoside Rb1, Rb2, Rb3, Rc, Rd, 20(S)-Rg 3, 20(R)-Rg

1.2 Materials and reagents

DMEM culture medium (HaiClone Biotechnology); fetal bovine serum (Clark, Australia); double antibody, trypsin, trypsin (EDTA-free), PBS (Beijing Solebao); dexamethasone (DEX) (Shanghai Jiuding); recombinant human insulin (Wuhan Punosai); 3-isobutyl-1-methylxanthine (IBMX) (Shanghai McLean); CCK-8 (GLPBO, USA); RIPA lysis buffer, BCA kit, ECL chemiluminescence kit (Shanghai Biyuntian); IGFR, FGFR2, MAPK, Bcl-2, BAX kits (Shanghai Keaibo); ready-to-use PI staining solution (Beijing Coolbo); AnnexinV-FITC/PI double staining cell apoptosis detection kit (Guangzhou Beibo).

1.3 Main instruments

Avanti TM J-3OI refrigerated centrifuge (Hainan Hermin); Spectra MAX 190 microplate reader (Shanghai Meigu); flow cytometer (Betcon Dickinson, USA); inverted fluorescence microscope (Leica, Germany); ChemStudio SA2 developer (Analytik JenaAG, Germany).

2 Experimental methods

2.1 Experimental cell lines

3T3-L1 preadipocytes were purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences (GNM25).

2.23T3-L1 adipocyte differentiation

After cell recovery and passage, the culture medium (i.e., complete culture medium containing 5% fetal bovine serum and 1% double antibody) was replaced every 48 hours. After the cell confluence reached 100%, the culture medium was replaced for another 48 hours. The culture medium was then removed, and differentiation medium (i.e., complete culture medium containing 10 μg/mL insulin, 500 μM IBMX, and 1 μM DEX) was added for continued culture. After 48 hours, the culture medium was replaced with maintenance medium (i.e., complete culture medium containing 10 μg/mL insulin) for another 48 hours. After 48 hours, the culture medium was replaced with ordinary complete culture medium, and then the complete culture medium was replaced every 48 hours. After a total of 8 days of differentiation induction, if obvious lipid droplets have been formed in the cytoplasm, the cells have differentiated into mature adipocytes under a microscope.

2.3 Effects of ginsenosides on adipocyte viability

The CCK-8 method was used to detect the effects of ginsenosides at different concentrations (0, 1, 5, 10, 25, 50, 100 μM) on the viability of 3T3-L1 preadipocytes (undifferentiated cells) and mature adipocytes (differentiated cells).

2.4DAPI fluorescence staining to observe cell morphology

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

2.5 Flow cytometric analysis of apoptosis

The cells were seeded in a 6-well plate at a density of 5×10 ⁴ cells per well, cultured for about 24 hours after attachment, and then replaced with a culture medium containing 25 μM ginsenosides to treat 3T3-L1 for 24 hours. After washing, 400 μL of Annexin V binding solution was added to each cell sample tube to suspend the cells, and 5 μL of Annexin V-FITC staining solution was added for incubation (protected from light, 15 minutes), followed by the addition of 10 μL of PI (propidium iodide) staining solution for continued incubation (protected from light, 5 minutes). The cell suspension was filtered through a 200-mesh filter into a flow tube and immediately detected by a flow cytometer. The entire detection process should be completed within one hour to avoid fluorescence quenching.

2.6 Flow cytometer analysis cycle

PI staining was used to detect the cell cycle distribution. 3T3-L was treated with 25 μM ginsenoside culture medium for 124 h, stained with PI dye (50 μg/mL), incubated in the dark for 30 min, and the cell suspension was filtered through a 200-mesh filter into a flow tube, and then the cell cycle distribution was immediately detected by flow cytometry. The entire detection process should be completed within one hour to avoid fluorescence quenching.

2.7 Transcriptomics sequencing

3T3-L1 preadipocytes were seeded in 6-well plates at a density of 1×10 ⁶ cells/well for 12 h, and then pretreated with the test drug (optimal concentration: 25 μM) for 2 h, while the control group was not given any drug. The cells were then subjected to H/R modeling and the following operations were performed.

2.7.1 RNA extraction and identification

After 2 hours of treatment with the test drugs and establishment of the H/R model, total RNA was extracted from 3T3-L1 preadipocytes 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, sequencing libraries were generated, and index codes were added to the attribute 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 Sorting

The index-encoded samples were clustered on the cBot cluster generation system using the TruSeq PE Cluster Kit v4-cBot-HS (Illumina) according to the manufacturer's instructions. After clustering, the library preparation was sequenced on the Illumina platform and paired-end reads were generated.

2.7.4 Differential expression analysis

The differential expression analysis between the two groups was performed using edgeR software. The method of Benjamini and Hochberg was used to control the false discovery rate and thus adjust the resulting p-values. Genes with adjusted p-values < 0.05 found by edgeR were called DEGs and a volcano plot was drawn.

2.7.5 Gene ontology and pathway enrichment analysis

Pathway clustering analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

2.8 Protein expression analysis

2.8.1 Extraction of total cell protein

The cells were plated in 6-well plates and transfected with lentivirus, and IGFR and FGFR2 overexpression groups (sh IGFR and sh FGFR2) were added. After 24 hours of drug treatment, the culture medium was discarded, and the cells were washed three times with pre-cooled PBS. Then 200 μL of cell lysis solution was added. After the cells were completely lysed, the cells were scraped off with a pipette tip and aspirated into a sterile 1.5 mL centrifuge tube. The cells were centrifuged at 12500 rpm for 25 minutes at 4°C, and the precipitate was removed and the supernatant was collected and stored at -80°C (the cell lysis process must be performed on an ice box).

2.8.2 Determination of protein concentration by BCA method

The protein concentration of each sample was detected using the BCA kit.

2.8.3 Kit detection of related protein expression

Protein expression was detected using IGFR, FGFR2, MAPK, Bcl-2, and BAX protein content detection kits.

2.9 RT-qPCR

Total RNA from 3T3-L1 preadipocytes was extracted with TRIzol reagent, and then the total RNA was reverse transcribed into cDNA using an RT-qPCR kit at 55°C for 5 minutes and at 90°C for 20 seconds. Then, 10 μL of green qPCR SuperMix, 0.8 μL of upstream and downstream primer mixtures, and 2 μL of cDNA template were mixed for DNA amplification. According to the cycle threshold (Ct) value, the relative expression level of the target gene was calculated.

2.10 Statistical analysis

All data were analyzed using Prism 8 (GraphPad) statistical software. The data were expressed as Mean±SD. The groups were compared using t-test combined with one-way analysis of variance. P<0.05 was considered statistically significant, *P<0.05, **P<0.01, ***P<0.001.

3 Results

3.1 Effects of ginsenosides on cell viability of 3T3-L1 preadipocytes

Among the 105 triterpenes and their glycosides tested, the more typical experimental results showed that compared with the blank group, after 3T3-L1 preadipocytes were treated with different concentrations of ginsenosides (1, 5, 10, 25, 50, 100 μM), the cell viability of the PPD-treated group was significantly inhibited within the concentration range of 10-100 μM, as shown in Figure 1, and it was concentration-dependent. (Note: Compared with the control group, *p<0.05, ***p<0.005).

3.2 Effects of ginsenosides on cell viability of mature adipocytes

The results showed that for the induction of differentiated mature adipocytes, only PPD and PD had significant differences in cell viability at a concentration of 100 μM compared with the blank group in the administration groups of various ginsenosides, as shown in Figure 2 (Note: *p<0.05 compared with the control group), but this dose was high and had no practical significance. Therefore, only the lowest significant effective dose of PPD and PD, 25 μM, was selected for subsequent experiments to induce apoptosis of 3T3-L1 adipocyte precursors.

Effects of PPD and PD on the morphology of 3T3-L1 preadipocytes

DAPI fluorescence staining was used to observe the effect of PPD and PD treatment at the same concentration (25 μM) for 24 h on the morphology of 3T3-L1 adipocytes, and the results are shown in Figure 3. Under normal conditions, the cells were stained with uniform chromatin distribution, showing a relatively dim blue fluorescence. Compared with the blank group, after PPD and PD (25 μM) were used to treat 3T3-L1 adipocytes, the number of cells decreased, the brightness increased, the cell outline became irregular, nuclear fragmentation occurred, and apoptotic bodies and chromatin fragments could be observed (arrows in Figure 3), indicating that PPD and PD caused apoptosis of 3T3-L1 adipocytes. In addition, after the cells were treated with PPD, the number of morphologically abnormal cells increased significantly compared with the PD treatment group, and the fluorescence intensity of the PD treatment group was slightly weaker.

3.4 Effects of PPD and PD on apoptosis of 3T3-L1 preadipocytes

After 3T3-L1 preadipocytes were treated with 25 μM PPD and PD for 24 h, the cell apoptosis rate was detected by flow cytometry. The results are shown in Figure 4. Compared with the blank group, the cell apoptosis rate was increased after treatment with 25 μM PPD and PD, showing a significant difference, and the increase in the PPD group was more significant than that in the PD group (Note: compared with the control group, **p<0.01, ***p<0.005).

Effects of PPD and PD on cell cycle arrest of 3T3-L1 preadipocytes

As shown in Figure 5, compared with the blank group, the proportion of cells in the S phase after PPD treatment was significantly increased, and the difference was extremely significant, but PPD had no significant effect on the G2 phase of cells; the proportion of cells in the S phase and G2 phase after PD treatment was significantly increased (Note: compared with the control group *P<0.05, **P<0.01, ***P<0.001). This shows that both PPD and PD can induce S phase arrest in 3T3-L1 preadipocytes, and PD can also induce G2 phase arrest in cells.

3.6 PPD transcriptome sequencing differential gene analysis

DEGs in the control group and PPD group were enriched by KEGG pathways. As shown in Figure 6, the top 20 pathways with the most reliable enrichment significance (i.e., the smallest Q value) were selected to present the results. Among them, the most relevant and most significantly different pathways related to EGFR kinase inhibitor resistance were selected. Through the volcano map, it was found that the expression levels of IGFR, FGFR2, and MAPK, the related proteins that regulate cell apoptosis in this pathway, were significantly downregulated after PPT administration, which may be the main reason for PPD-induced apoptosis of fat precursor cells, and subsequent mechanism verification studies were carried out.

Effect of 3.7PPD on the expression of apoptosis-related proteins in 3T3-L1 preadipocytes

Compared with the blank group, the expression of IGFR, FGFR2 and MAPK proteins in preadipocytes was significantly inhibited after treatment with different concentrations of PPD (see Figure 7), especially at 25 and 50 μM concentrations, and the difference was extremely significant. The relative expression level of apoptotic protein Bcl-2/Bax protein was significantly reduced. However, after we overexpressed IGFR and FGFR2 in cells, the expression of the above apoptotic proteins was reversed (Note: *P<0.05, **P<0.01 compared with the control group; $$P<0.01 compared with PPD+sh IGFR/FGFR2). This shows that IGFR and FGFR2 are key targets for inducing apoptosis of preadipocytes and PPD does further regulate downstream apoptotic proteins by inhibiting IGFR, FGFR2 and MAPK proteins, thereby promoting apoptosis of preadipocytes.

3.8RT-PCR

The key protein-related genes IGFR and FGFR2 were selected for verification, as shown in Figure 8. The results showed that compared with the blank group, the PPD-treated fat precursor cell genes IGFR and FGFR2 had a significant inhibitory effect, especially at a concentration of 25 μM (Note: compared with the control group *P<0.05, **P<0.01), which was consistent with the above sequencing results and protein expression, further indicating that IGFR and FGFR2 are key targets for inducing apoptosis of fat precursor cells.

4 Conclusion

Ginsenosides PPD and PD have significant activity in inducing apoptosis of preadipocytes, among which PPD is the most significant. Its activity is achieved by simultaneously inhibiting cell growth factors IGFR and FGFR2, thereby inhibiting the expression of downstream protein MAPK, activating key apoptosis proteins, blocking the cell cycle and promoting cell apoptosis.

Example 2 Protopanaxatriol inhibits differentiation of adipocyte precursor cells

1 Materials and Instruments

1.1 Ginsenoside (yuan)

Same as “1.1” of Example 1.

1.2 Materials and reagents

The cell-related reagents are the same as "1.2" of Example 1; triglyceride (TG) determination kit, glucose (GLU) content determination kit (Nanjing Jiancheng); free fatty acid (FFA) content detection kit (Beijing Solebao); mouse tumor necrosis factor (TNF-α), mouse adiponectin (ADP), mouse resistin, mouse leptin ELISA kits (Beijing Chenglin); PERK inhibitor GSK2606414 (Sigma, USA); Hspa4, RELA, PERK, CHOP, PPARγ, C/EBPα content detection Elisa kits.

1.3 Main instruments

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 preadipocyte differentiation induction

Same as “2.2” of Example 1.

2.3 Oil Red O staining colorimetric method to detect cell differentiation

Ginsenoside solutions of different concentrations (0, 25, 50, 75, 100 μM) were prepared with a culture medium containing an inducer (i.e., a complete culture medium containing 10 μg/mL insulin, 500 μM IBMX, and 1 μM DEX). After contact inhibition, 3T3-L1 adipocytes were induced to differentiate according to the method of "2.2" in Example 1. Oil Red O staining was used, and 100 μL of isopropanol was added to dissolve the Oil Red O dye. The absorbance (520 nm) was measured, and the differentiation rate of each group of cells was calculated.

2.4 Regulation of insulin resistance index in mature adipocytes

After inducing the adipocyte precursor cells into mature adipocytes, the insulin resistance model of the cells was established using 1 μM dexamethasone for 96 h, and the drug treatment was performed, and the positive drug rosiglitazone group was added. After 24 h, Oil Red O staining was used to detect the distribution of cellular lipid droplets to determine its activity against insulin resistance.

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

The extracellular triglyceride content was determined by the glycerol phosphate oxidase-peroxidase method (GPO-PAP method).

2.7 Determination of free fatty acid content

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

2.8 Determination of fat-related factors by ELISA

Mouse adiponectin, resistin, leptin and TNF-α kits (ELISA detection method) were used to determine the levels of adiponectin, resistin, leptin and TNF-α in the culture medium supernatant.

2.9 Transcriptomics sequencing

The PPT 25 μM with significant efficacy was selected for transcriptomic sequencing, and the operation was the same as "2.7" of Example 1.

2.10 Protein expression analysis

HSPA4, RELA, PPARγ, PERK, CHOP, and C/EBPα kits were used to detect the expression of related proteins, and an Hspa4 knockout group (si Hspa4) and a PERK inhibitor GSK2606414 group were added, and the operation was the same as "2.8" of Example 1.

2.11RT-PCR

The expression levels of HSPA4, RELA, PPARG1, EIF2AK3, DDIT3, and CEBPA related genes were detected, and the operation was the same as "2.9" of Example 1.

2.12 Statistical analysis

Same as “2.10” of Example 1.

3 Results

3.1 Effects of ginsenosides on lipid accumulation in adipocytes

Lipid accumulation is one of the important characteristics of mature adipocytes. Therefore, Oil Red O dye staining was selected to detect the effect of ginsenosides on cell differentiation, as shown in Figure 9. Since PPD and PD have certain cytotoxicity, they were not used in subsequent experiments to inhibit differentiation. After 3T3-L1 preadipocytes were differentiated into mature adipocytes on a 96-well plate, Oil Red O staining was performed according to method 2.3. Oil Red O dissolved in isopropanol and specifically bound to intracellular lipids was used as a differentiation marker, and the degree of cell differentiation was detected at a wavelength of 520nm using an enzyme reader. Among the 103 tested samples, typical results showed that at a dosage concentration of 10-100 μM, the lipid content of the PPT, Rg ₅ and 20(S)-Rg ₃ groups in the ginsenoside-administered groups was significantly lower than that of the control group, indicating that the ability of cellular lipid accumulation was inhibited, among which PPT performed the best, with the adipocyte differentiation rate being only 60% of that of the control at a concentration of 25 μM and only 25% of that of the control at a concentration of 100 μM (Note: compared with the control group *P<0.05, **P<0.01, ***P<0.001), much higher than that of Rg ₅ and 20(S)-Rg ₃ ; however, ginsenosides Rh ₁ , Re, Rf, and Rg ₂ promoted the differentiation of fat precursor cells, among which Rh ₁ performed the most prominently; the other ginsenosides (elements) had no obvious effect on the differentiation rate of fat precursor cells. Therefore, three ginsenosides (genus) PPT, Rg ₅ and 20(S)-Rg ₃ were selected for subsequent related experiments.

3.1.1 Oil Red O staining results

The cells were cultured on a 6-well plate. After the cells were contact inhibited, an inducer containing drugs (25 μM) was added to induce differentiation. After the differentiation was completed, the cells were stained with oil red O dye for fat staining. After staining, the lipid droplets secreted by the adipocytes were stained red, while the undifferentiated cells or the remaining structures of the differentiated cells except the lipid droplets were not stained. As shown in Figure 10, most of the adipocytes without drug intervention were stained red, and it can be seen that the cells were almost completely differentiated. In contrast, the staining of adipocytes was significantly reduced after drug treatment, which shows that the differentiation of adipocytes and the secretion of lipid droplets were inhibited, and the inhibitory effect of the PPT group was more obvious at the same drug concentration (25 μM).

3.1.2 Improvement of insulin resistance in mature adipocytes by PPT, Rg ₅ and 20(S)-Rg ₃

The results are shown in Figure 11. Control is a normal adipocyte control group, I/R is an insulin resistance model, and Ros is a positive drug rosiglitazone group. After the insulin resistance model was established, the intracellular lipid droplets were significantly larger than those in normal cells and accumulated in large quantities. After the 25μM PPT administration, the intracellular lipid droplets became significantly smaller, and the accumulation was greatly reduced and better than the positive drug rosiglitazone group. However, Rg ₅ and 20(S)-Rg ₃ did not change significantly. Therefore, PPT can not only inhibit the differentiation of preadipocytes, but also enhance the insulin sensitivity of mature adipocytes to further regulate adipocyte glucose and lipid metabolism and reduce lipid accumulation.

3.1.3 Effects of PPT, Rg ₅ and 20(S)-Rg ₃ on triglycerides in cell supernatant

After 8 days of cell differentiation, the TG released by cells in the cell culture supernatant of each group was detected using a TG kit. The results show (Figure 12) that compared with the control group, the TG content in the culture fluid of each drug-treated group at the same concentration (25 μM) was significantly reduced compared with the control group, and the PPT group had the most significant reduction in TG content compared with the other two groups (Note: **P<0.01, ***P<0.005 compared with the control group). This indicates that ginsenosides PPT, Rg ₅ and 20(S)-Rg ₃ can inhibit the differentiation of 3T3-L1 adipocytes and reduce the synthesis and release of triglycerides by cells.

3.1.4 Effects of PPT, Rg ₅ and 20(S)-Rg ₃ on free fatty acid content in cell supernatant

FFA is both a product of fat hydrolysis and a substrate for fat synthesis. The concentration of FFA in the blood is closely related to lipid metabolism, sugar metabolism, and endocrine function. The FFA content detection kit was used to detect the free FFA content in the cell supernatant. The test results showed (Figure 13) that each drug-treated group at 25μM could reduce the extracellular free FFA content, among which PPT had the best effect, and the three drug-treated groups showed extremely significant differences compared with the control group (Note: ***P<0.005 compared with the control group). This further shows that PPT, Rg ₅ and 20(S)-Rg ₃ have an inhibitory effect on fat catabolism.

3.1.5 Effects of PPT, Rg5 and 20(S)-Rg3 on glucose consumption in cell differentiation

After 8 days of induction, 3T3-L1 preadipocytes almost differentiated into mature adipocytes, and glucose in the cell culture medium was the main nutrient in this process. The inhibitory effect of ginsenosides on cell absorption of nutrients was evaluated by measuring the glucose consumption in the culture medium. The test results are shown in Figure 14. Compared with the control group, the glucose content in the culture medium of each drug-treated group (25μM) was significantly reduced (Note: compared with the control group ***P<0.005), indicating that PPT, Rg ₅ and 20(S)-Rg ₃ can inhibit the absorption of glucose by 3T3-L1 adipocytes, and PPT has the best inhibitory effect on cell uptake of glucose.

3.2 Effects of PPT, Rg ₅ and 20(S)-Rg ₃ on the secretion of adipose-related factors

After induced differentiation and maturation, 3T3-L1 preadipocytes can secrete various adipose factors including tumor necrosis factor α (TNF-α), adiponectin, resistin and leptin, which is also one of the important characteristics of adipocyte maturation. Therefore, the ELISA method was used to determine the levels of TNF-α, adiponectin, resistin and leptin in the culture medium after different drugs (PPT, Rg ₅ and 20 (S) -Rg ₃ ) intervened in the differentiation process at a concentration of 25 μM to evaluate the inhibitory effect of drugs on adipocyte differentiation. As shown in Figure 15, after 8 days of induction of differentiation, the cells secreted a relatively high level of TNF-α, adiponectin, resistin and leptin, indicating that 3T3-L1 cells have differentiated into mature adipocytes. In addition, each drug-treated group was able to significantly reduce the secretion levels of the above hormones (Note: **P<0.01, ***P<0.005 compared with the control group), indicating that ginsenosides PPT, Rg ₅ and 20(S)-Rg ₃ can inhibit the secretion activity of adipocytes during adipogenesis, and PPT has the best inhibitory effect.

3.3 PPT transcriptome sequencing differential gene analysis

DEGs in the control group and PPT group were enriched by KEGG pathways. The top 20 pathways with the most reliable enrichment significance (i.e., the smallest Q value) were selected to present the results, as shown in Figure 16. Among them, we selected the most relevant and significantly different lipid-related pathways for atherosclerosis. The volcano plot showed that the expression levels of genes Hspa4, RELA, EIF2AK3, and DDIT3 related to the regulation of adipose precursor cell differentiation in this pathway were significantly upregulated after PPT administration, while the key genes for adipose differentiation, PPARG1 and CEBPA, were significantly downregulated, indicating that Hspa4, RELA, EIF2AK3, and DDIT3 may be the key targets of PPT in inhibiting adipose precursor cell differentiation, and subsequent mechanism verification studies were conducted.

3.4 Effects of PPT on regulatory differentiation-related proteins in adipocytes

The lipid and atherosclerosis pathway is an important pathway involved in fat regulation of energy metabolism. Hspa4 and RELA in the pathway, as inflammation-related proteins, can further regulate the adipose differentiation-related protein PPARγ. PERK and CHOP are endoplasmic reticulum stress-related regulatory proteins that can further regulate the downstream adipose differentiation-related protein C/EBPα. Therefore, in order to determine the specific mechanism by which PPT inhibits the differentiation of preadipocytes, the expression of related proteins was analyzed. The results are shown in Figure 17. Compared with the control group, the expression of Hspa4, RELA (in the nucleus), PERK, and CHOP in the PPT treatment group (25, 50 μM) was significantly increased, while the expression of PPARγ and C/EBPα was significantly inhibited. Moreover, the above situation was significantly reversed by further knocking out Hspa4 and inhibiting PERK expression (Note: *P<0.05, **P<0.01 compared with the control group; $$P<0.01 compared with PPT+si Hspa4/GSK2606414). This further illustrates that PPT promotes the nuclear translocation of the downstream inflammatory protein RELA and the expression of the endoplasmic reticulum stress protein CHOP by activating Hspa4 and PERK, thereby inhibiting the key proteins PPARγ and C/EBPα of adipocyte differentiation, thereby inhibiting the differentiation of adipocyte precursor cells.

3.5RT-PCR

Similarly, the key protein-related genes Hspa4, RELA (nuclear), PPARG1, EIF2AK3, DDIT3, and CEBPA were selected for verification. The results are shown in Figure 18. It was found that compared with the blank group, the expression levels of the fat precursor cell genes Hspa4, RELA (nuclear), EIF2AK3, and DDIT3 treated with PPT were significantly increased, while the key differentiation genes PPARG1 and CEBPA showed obvious inhibitory effects, especially at concentrations of 25 and 50 μM (Note: compared with the control group *P<0.05, **P<0.01), which is consistent with the above sequencing results and protein expression, further indicating that Hspa4 and EIF2AK3 are the key targets of PPT to inhibit the differentiation of fat precursor cells.

4 Conclusion

Ginsenosides PPT, Rg ₅ and 20(S)-Rg ₃ can significantly inhibit glucose uptake, fat catabolism and cell differentiation of adipocyte precursor cells, and reduce lipid accumulation. Among them, PPT is the most significant. Its inhibitory effect is achieved by targeting the activation of Hspa4 and PERK, promoting the nuclear translocation of the downstream inflammatory protein RELA and the expression of the endoplasmic reticulum stress protein CHOP, thereby inhibiting the key proteins PPARγ and C/EBPα of adipocyte differentiation, thereby inhibiting the differentiation of adipocyte precursor cells. It can also regulate the glucose and lipid metabolism of mature adipocytes, enhance their insulin sensitivity, and further reduce the lipid accumulation of mature adipocytes.

Example 3 Effects of protopanaxadiol and protopanaxadiol/Rg ₅ /20(S)-Rg ₃ composition on obesity and lipid metabolism in mice

1 Materials, reagents and instruments

1.1 Materials and reagents

Protopanaxadiol (PPD), protopanaxantriol (PPT), ginsenoside/Rg ₅ and 20(S)-Rg ₃ are the same as those in Example 1; free fatty acid (FFA); total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL) detection kits (Nanjing Bioengineering Co., Ltd.); mouse insulin (Insulin), mouse glucose (Glucose) and mouse leptin (Leptin) enzyme-linked immunosorbent assay ELISA kits (R&D Company, USA); other reagents are all domestically produced analytical grade.

1.2 Instruments

ABS320-4N analytical balance (Shanghai Daohan Industrial Co., Ltd.); SpectraMax190 automatic microplate reader (Beijing Bayi Instrument Factory); CT14D desktop high-speed centrifuge (Tianmei Scientific Instrument Co., Ltd.).

2 Experimental methods

2.1 Experimental animals

5-week-old C57BL/6J mice, male, weighing 18-20 g; animal breeding environment: temperature 23±2°C, relative humidity about 55%, 12h light/dark cycle, adaptive breeding for 7 days before starting the experiment.

2.2 Animal grouping and drug administration

Mice were randomly divided into two groups, 8 in each group, including blank control group (CON group, fed with normal diet), model group (HFD group, fed with high-fat diet), PPD/PPT composition (1:1), PPD/Rg ₅ composition (1:1) and PPD/20(S)-Rg ₃ composition (1:1). The ratios in the mixtures were all molar ratios. The dosage was 100 mg/kg and the mice were fed with high-fat diet. The results showed that the PPD/PPT composition had a significant effect. Therefore, different ratios of PPD/PPT compositions (a total of 11 groups, with molar ratios of PPD and PPT 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 PPD sodium salt/PPT water of 1:1 were explored. The drug-treated group was intragastrically administered with 10 mL/kg body weight of mice every day, and the blank group and model group were intragastrically administered with an equal amount of normal saline for 12 weeks. Food intake was measured every 3 days, body weight was measured once a week, and all mice were allowed to drink water and eat freely during the entire administration period. The food efficiency ratio (FER) was calculated as follows: FER (%) = body weight gain (g/d) / food intake (g/d) × 100%.

2.3 Collection of serum and organ tissues

After administration, mice were fasted for 12 hours, blood was collected from the eyeballs, and mice were killed by cervical dislocation. Serum was separated by centrifugation at 1500 rpm and 4°C for 10 minutes. The weight and body length (the distance from the anus to the nose tip) of the mice were measured, and Lee's index was calculated as [weight (g) × 1000/body length (cm)]/3. After dissection, epididymal fat, skeletal muscle, liver and kidney were removed, washed with 0.90% (w/v) saline and weighed, and the organ index was calculated (organ index = organ weight/body weight).

2.4 Detection of biochemical indicators of serum samples

Serum insulin, glucose and leptin levels were determined by enzyme-linked immunosorbent assay according to the instructions of the ELISA kit. The insulin resistance index (IR) was calculated according to the following equation: HOMA-IR = [serum glucose (mmol/L) × serum insulin (pmol/L)]/22.50. Serum FFA, TC, TG, HDL and LDL levels were determined by the corresponding colorimetric method according to the instructions provided by the manufacturer.

2.5 Statistical methods

The experimental data were expressed as x±s and were statistically processed using SPSS21 statistical analysis software. The statistical significance of the differences between the groups was evaluated by t-test and one-way analysis of variance (ANOVAs), and P<0.05 was considered statistically significant.

3 Results

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

After 12 weeks, the body weight and weight gain of C57BL/6J mice fed with HFD containing 45% fat were significantly higher than those fed with LFD (containing 10% fat), indicating that HFD did cause obesity in mice in the free diet experiment (P<0.05). Compared with HFD mice, each combination group significantly reduced the weight gain of mice (P<0.05) and Lee's index (P<0.05), especially the PPD/PPT combination 1:1, which was the most typical. The typical results are shown in Table 1.

Table 1 Effects of each treatment group on the body weight of C57BL/6J mice (mean ± s, n = 8)

Note: ^ab values that do not share superscript letters are significantly different among the groups (P<0.05).

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

The results showed that compared with HFD mice, epididymal fat weight, daily food intake and FER of HFD-fed mice in each combination group were significantly reduced (P<0.05), especially the PPD/PPT combination 1:1 was the most typical. However, compared with CON mice, liver weight and liver index of HFD mice were significantly increased (P<0.05). Typical results are shown in Tables 2 and 3.

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

Group 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± ^0.25a PPD/PPT1:1 2.13±0.25 ^c 0.32±0.03 0.95±0.12 ^b 0.59±0.12 ^b PPD/Rg ₅ 1:1 2.54±0.21 0.31±0.01 1.13±0.11 ^b 0.88±0.13 PPD/20(S)-Rg ₃ 1:1 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 and substance 1:1 2.18±0.32 ^c 0.35±0.01 0.99±0.18 ^b 0.62±0.13 ^b

Note: Values ^abc that do not share superscript letters are significantly different among the groups (P<0.05).

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

Group Food efficiency (%) Renal 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/Rg ₅ 1:1 6.56±0.45 9.86±0.66 34.15±1.21 26.35±2.54 ^b PPD/20(S)-Rg ₃ 1:1 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 and substance 1:1 5.01±0.43 ^b 10.88±0.47 30.13±1.52 ^b 19.22±1.23 ^b

Note: Values ^abc that do not share superscript letters are significantly different among the groups (P<0.05).

3.3 Effects of the composition on serum biochemical parameters of C57BL/6J mice

The serum levels of glucose, leptin, insulin, FFA, TC, TG and LDL were measured. The results showed that all biochemical indicators of the HFD group changed significantly compared with the CON group, indicating that the model was successfully established. Compared with the HFD group, the treatment of each group of the combination can improve the fasting blood glucose level (P<0.05), reduce the levels of leptin, insulin, FFA, HOMA-IR, TC, TG and LDL, especially the PPD/PPT combination 1:1 is the most typical. Typical results are shown in Tables 4 and 5.

Table 4 Effects of each treatment group on the levels of free fatty acids, leptin, glucose and insulin in C57BL/6J mice (mean ± s, n = 8)

Note: Values ^abc that do not share superscript letters are significantly different among the groups (P<0.05).

Table 5 Effects of each treatment group on TC, TG, HDL-C and LDL-C (mean ± s, n = 8)

Group 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± ^0.05a PPD/PPT 1:1 0.81±0.09 ^b 2.83±0.65 ^b 0.07±0.01 ^b PPD/Rg ₅ 1:1 1.25±0.06 3.83±0.35 0.15±0.02 PPD/20(S)-Rg ₃ 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 and substance 1:1 0.82±0.06 ^b 2.88±0.39 ^b 0.08±0.02 ^b

Note: Values ^abc that do not share superscript letters are significantly different among the groups (P<0.05).

Conclusion: The combination of protopanaxadiol and protopanaxantriol (1:1) can significantly reduce the body weight, organ index and epididymal fat weight of obese mice, and significantly improve the biochemical indexes such as fasting blood glucose level, glucose, leptin, insulin, FFA, TC, TG and LDL, as well as the levels of fatty acids, leptin, glucose and insulin. This shows that the combination is effective in preventing and treating obesity and other related diseases caused by abnormal adipocyte differentiation and metabolism, and has great development prospects.

Example 4 Tablet Preparation

Reagents: Starch (pharmaceutical grade, Tianjin Jindong Tianzheng Fine Chemical Reagent Factory); citric acid (Shanghai McLean Biochemical Technology Co., Ltd.); magnesium stearate (Shanghai McLean Biochemical Technology Co., Ltd.).

Preparation:

① Preparation of 10% starch slurry: Dissolve 0.25g of citric acid in 25mL of pure water, add 2.5g of starch and disperse evenly, heat to gelatinize, and obtain 10% starch slurry.

② Granulation: Take an appropriate amount of protopanaxadiol and protopanaxadiol composition (protopanaxadiol and protopanaxadiol molar ratio of 1:1) and mix evenly with starch, add an appropriate amount of 10% starch slurry, mix and grind evenly, make a soft material, granulate through a 16-mesh sieve, and dry at 50-60°C for 1h. After granulation through a 16-mesh sieve, add an appropriate amount of lubricant magnesium stearate and press into tablets with a shallow punch with a diameter of 10mm.

Results: The tablets were off-white in color, uniform in color, uniform in thickness, moderate in hardness, and the tablet weight and disintegration time met the requirements.

Conclusion: The obtained protopanaxadiol and protopanaxadiol composite tablets meet the requirements and can be used as tablets.

Example 5 Preparation of Suspension Injection

Reagents: polylactic acid (PLA, Shanghai Zhenzhun Biotechnology Co., Ltd.); polylactic acid-glycolic acid copolymer (PLGA, Shanghai Yuanye Biotechnology Co., Ltd.); poloxamer 188 (Xi'an Tianzheng Pharmaceutical Excipients Co., Ltd.); dichloromethane, methanol, acetonitrile, etc. (Tianjin Tiantai Chemical Co., Ltd.).

Preparation:

① Preparation of polymer microparticles: Weigh an appropriate amount of protopanaxadiol and protopanaxadiol composition (molar ratio of protopanaxadiol to protopanaxadiol is 1:1) and a carrier (PLA/PLGA) and place them in a 50 mL round-bottom flask, add 5 mL of dichloromethane to dissolve, remove most of the organic solvent by reduced pressure distillation at 28 ° C, and then vacuum dry at 40 ° C for 24 h until the solvent is completely removed, grind, and pass through a sieve with a pore size of 150 μm to obtain polymer microparticles of the concatenated protopanaxadiol and protopanaxadiol composition.

② Preparation of protopanaxadiol and protopanaxantriol composite suspension injection: 4.0 g of the above product was dispersed in 250 mL of an aqueous solution containing 10 g/L of poloxamer 188 stabilizer under continuous stirring to achieve complete dispersion. The drug dispersion was ground to the desired particle size, and the protopanaxadiol and protopanaxantriol composite microparticle suspension was obtained, centrifuged at 5000 rpm for 2 min, and dispersed with 10 mL of stabilizer aqueous solution to concentrate the preparation to about 25 g/L.

Results: The obtained protopanaxadiol and protopanaxadiol composite suspension injection had uniform particle size, and the water content and surface particle size of the preparation met the requirements. The in vitro sustained release effect was good and the stability was good.

Conclusion: The obtained protopanaxadiol and protopanaxadiol composite suspension injection meets the requirements and can be used as a suspension injection.

Example 6 Preparation of hydrogel patch

Reagents: sodium polyacrylate (Shanghai Yuanye Biotechnology Co., Ltd.), aluminum glycolate (Shanghai Yuanye Biotechnology Co., Ltd.), sodium carboxymethyl cellulose (Shanghai Yuanye Biotechnology Co., Ltd.), soybean lecithin (Shanghai Yuanye Biotechnology Co., Ltd.), cholesterol (Shanghai Yuanye Biotechnology Co., Ltd.), chloroform (Tianjin Tiantai Chemical Co., Ltd.), azone (Tianjin Tiantai Chemical Co., Ltd.), propylene glycol (Tianjin Tiantai Chemical Co., Ltd.).

Preparation:

① Preparation of colloid: Accurately weigh sodium polyacrylate, aluminum glycolate, and sodium carboxymethyl cellulose into a mortar, measure glycerol and add it to the mortar for grinding and stirring, mix it thoroughly and set aside, take 5% azone and 3.75% propylene glycol by volume, dissolve azone in propylene glycol solution in a certain proportion as a penetration enhancer, vortex it to mix evenly and then add it to the mortar.

② Preparation of protopanaxadiol and protopanaxadiol composition liposomes: soybean lecithin, cholesterol, and PPD/PPT composition (PPD and PPT molar ratio 1:1) are mixed, chloroform is added, and ultrasonication is performed to dissolve the liposomes; the organic solvent is removed by rotary evaporation under reduced pressure to form a uniform dry film on the bottle wall; hydration is performed by ultrasonication at room temperature, and freeze-drying is performed.

③ Preparation of protopanaxadiol and protopanaxadiol composition hydrogel patch: Accurately weigh protopanaxadiol and protopanaxadiol composition liposomes, sodium benzoate, and citric acid, add them into a mortar, add purified water to mix, and concentrate to prepare a high-molecular milky white hydrogel paste. The high-molecular milky white hydrogel paste is applied on a backing layer to solidify and form, and then divided. A polyethylene film is covered on the sticky surface of the hydrogel paste and pressed to obtain a protopanaxadiol and protopanaxadiol composition hydrogel patch.

Results: The obtained protopanaxadiol and protopanaxadiol composition hydrogel patch had good adhesion and other indicators, no film residue and skin residue, no irritation, good uniform penetration, and the drug loading was within the specified range.

Conclusion: The obtained protopanaxadiol and protopanaxadiol composite hydrogel patch meets the requirements and can be used as a hydrogel patch.

The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims (4)

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.