CN109731003B

CN109731003B - Application of rubrofusarin-6-O-beta-gentiobioside in preparation of lipid-lowering and weight-losing medicine

Info

Publication number: CN109731003B
Application number: CN201811349282.0A
Authority: CN
Inventors: 刘向前; 邹亲朋; 戴玲; 宋雄辉; 洪承宪; 韩耀韩
Original assignee: Changsha Broad Ocean Bio Science and Technique Co Ltd
Current assignee: Changsha Broad Ocean Bio Science and Technique Co Ltd
Priority date: 2018-11-04
Filing date: 2018-11-04
Publication date: 2021-09-10
Anticipated expiration: 2038-11-04
Also published as: CN109731003A

Abstract

The invention provides application of rubrofusarin-6-O-beta-gentiobioside (RFG) in preparation of a medicine for reducing fat and losing weight, wherein the rubrofusarin-6-O-beta-gentiobioside is a naphthopyranoside compound. RFG inhibits lipid accumulation of 3T3-L1 and hAMSCs cells by reducing the targeting effect of mTOR, PPAR gamma and C/EBP alpha; the AMPK pathway is used for regulating the expression of adipogenesis related factors, inhibiting lipid accumulation signal pathways and reducing the weight gain, the eWAT and the fatty liver; RFG inhibits adipogenic factors (PPAR γ, C/EBP α, FAS, LPL and aP2) by activating eWAT and AMPK in the liver. Therefore, it can be used as a drug for treating obesity.

Description

Application of rubrofusarin-6-O-beta-gentiobioside in preparation of lipid-lowering and weight-losing medicine

Technical Field

The invention belongs to the technical field of biological activity application of compounds, and particularly relates to a new pharmaceutical application of naphthopyrones compounds, in particular to an application of rubrofusarin-6-O-beta-gentiobioside with a formula 1 in preparation of lipid-lowering and weight-reducing medicines.

Formula 1

Background

With the improvement of living standard and the change of original eating habits of people, obesity becomes an epidemic disease in modern society, and other related diseases caused by obesity, such as diabetes, atherosclerosis, coronary heart disease, hypertension, fatty liver, other cardiovascular and cerebrovascular diseases and the like, seriously threaten the health of human beings. At present, the medical field strives to develop lipid-lowering and weight-losing medicines with small side effect and good curative effect, especially from the theory of traditional Chinese medicine, researches the causes, prepares a traditional Chinese medicine preparation through scientific and reasonable compatibility and formula, achieves the purpose of treating both symptoms and root causes, and realizes the most important research on the efficacy of lipid-lowering and weight-losing.

Semen Cassiae is dried mature seed of Cassia obtusifolia L or Cassia tora L of Leguminosae. Has effects of dispelling pathogenic wind, clearing heat, removing liver fire, improving eyesight, and loosening bowel to relieve constipation. It can be used for lowering serum cholesterol. The cassia seed contains various anthraquinone components and naphthopyrone components, and some anthraquinone glycosides and naphthopyrone glycosides have the reported effect of resisting the poison of carbon tetrachloride and galactosamine to the primary culture mouse stem cells. In numerous patent application documents, the lipid-lowering drug and the weight-reducing drug prepared by cassia seed and other traditional Chinese medicines are mentioned, and because of the compound formula, the effective components playing the key roles are difficult to understand. rubrofusarin-6-O-beta-gentiobioside (RFG) in cassia seeds has reports of regulating hyperlipidemia, but the weight-losing effect and the related action mechanism thereof are not reported yet.

This patent studies the mechanism of action of RFG to improve obesity and to regulate lipid accumulation. The adipogenesis inhibitory effect of RFG was demonstrated using 3T3-L1 and hAMSCs cell assays. RFG inhibits lipid accumulation in 3T3-L1 and hAMSCs cells by reducing the targeting effects of mTOR, PPAR γ, and C/EBP α. RFG also significantly phosphorylates AMP-activated protein kinase independently in liver kinase B. AMPK inhibitors, however, block the anti-adipogenic effects of RFG. These results indicate that RFG regulates the expression of adipogenesis-associated factors through AMPK, thereby inhibiting lipid accumulation signaling pathways. In addition, RFG reduces weight gain, eWAT and fatty liver size. These effects and mechanisms of action are similar to in vitro experiments. RFG also inhibits adipogenic factors (PPAR γ, C/EBP α, FAS, LPL and aP2) by activating eWAT and AMPK in the liver. Therefore, RFG can improve obesity and be used as a medicine for treating obesity.

Disclosure of Invention

The invention aims to apply the rubrofusarin-6-O-beta-gentiobioside (RFG) shown in the formula 1 to the preparation of the medicament for reducing fat and losing weight.

Formula 1

RFG and pharmaceutically acceptable medicinal adjuvants, such as starch, cyclodextrin, etc., and other adjuvants can be added according to dosage form.

The preparation formulation prepared from RFG and pharmaceutic adjuvant can be tablets, capsules, oral liquid, granules and freeze-dried powder injection.

RFG can inhibit lipid accumulation of 3T3-L1 and hAMSCs cells by reducing the targeting effect of mTOR, PPAR gamma and C/EBP alpha.

RFG can regulate the expression of adipogenesis-related factors through AMPK, and inhibit lipid accumulation signal pathways.

RFG can reduce weight gain, eWAT, and fatty liver size; adipogenic factors (PPAR γ, C/EBP α, FAS, LPL and aP2) were inhibited by activation of eWAT and AMPK in the liver.

The invention has the beneficial effects that:

(1) the invention researches the lipid-lowering and weight-losing effects of RFG and deeply researches and expounds the action mechanism of RFG;

(2) compared experiments are carried out on the RFG and the cassia seed extract, and the experimental result shows that the lipid-lowering and weight-losing effect of the RFG is obviously better than that of the cassia seed extract.

Drawings

FIG. 1(A) cytotoxicity of RFG on 3T3-L1 cells; (B) cytotoxicity of RFG on hAMSCC

FIG. 2(A) a graph of lipid droplet staining in 3T3-L1 cells after RFG treatment with oil Red O; (B) accumulation of lipid droplets in 3T3-L1 cells after RFG treatment; (C) oil red O staining of lipid droplets in RFG-treated hAMSCc cells; (D) accumulation of lipid droplets in hAMSCC cells after RFG treatment

FIG. 3(A) effect of RFG on mRNA level expression of PPAR γ and C/EBP α in 3T3-L1 cells; (B) western blot analysis of 3T3-L1 cells (GAPDH as internal control); (C) effect of RFG on PPAR γ and C/EBP α protein level expression in 3T3-L1 cells

FIG. 4(A) effect of RFG on FAS mRNA expression during differentiation of 3T3-L1 cells; (B) effect of RFG on mRNA expression of LPL in differentiation of 3T3-L1 cells; (C) effect of RFG on mRNA expression of aP2 in differentiation of 3T3-L1 cells

FIG. 5(A) Effect of RFG on the levels of p-LKB1, LKB1, p-AMPK α, AMPK α, p-mTOR, and mTOR protein expression in 3T3-L1 cells; (B) analyzing the relative ratio of pAMPK alpha/AMPK alpha, p-LKB1/LKB1 and p-mTOR/mTOR in RFG-treated 3T3-L1 cells; (C) effect of RFG on p-PI3K, PI3K, p-AKT and AKT protein level expression in 3T3-L1 cells; (D) relative ratio analysis of p-PI3K/PI3K and p-AKT/AKT in RFG-treated 3T3-L1 cells

FIG. 6(A) effects of RFG on the expression levels of p-LKB1, LKB1, p-AMPK α, AMPK α, p-mTOR, mTOR, PPAR γ, and C/EBP α proteins in hAMSC cells; (B, C) analyzing the relative ratios of pAMPK α/AMPK α, p-LKB1/LKB1, p-mTOR/mTOR, PPAR γ/GAPDH and C/EBP α/GAPDH in RFG-treated hAMSC cells; (D) effect of RFG on mRNA expression levels of PPAR γ, C/EBP α and aP2 in hAMCc cells

FIG. 7(A) RFG-treated HFD induced weight changes in obese mice over 10 weeks; (B) HFD treatment with RFG induces weight differences in obese mice from the beginning to the end of the experiment

FIG. 8RFG treatment of HFD induces the weight of eWAT and liver in obese mice

FIG. 9(A) RFG treatment of HFD induces TG, TC, HDL, LDL levels in serum of obese mice; (B) HFD treatment with RFG induced AST and ALT levels in serum of obese mice; (C) HFD treatment with RFG induces creatinine and BUN levels in serum of obese mice

Fig. 10(a) RFG-treated HFD induced H & E staining of pathological sections of obese mouse liver tissue (. 200); (B) HFD treatment with RFG induces H & E staining epididymis adipocyte size in obese mice

FIG. 11(A, B) Effect of RFG on the PPAR γ and C/EBP α protein level expression of eWAT; (C) the effect of RFG on the mRNA level expression of PPAR γ and C/EBP α of eWAT; (D, E) the effect of RFG on PPAR γ and C/EBP α protein level expression in the liver; (F) effect of RFG on mRNA level expression of PPAR γ and C/EBP α in the liver

FIG. 12(A) Effect of RFG on the expression levels of p-LKB1, LKB1, p-AMPK α, AMPK α, p-mTOR, and mTOR proteins in eWAT; (B) analyzing the relative ratio of p AMPK alpha/AMPK alpha, p-LKB1/LKB1 and p-mTOR/mTOR in the eWAT after RFG treatment; (C) effect of RFG on the mRNA expression levels of FAS, LPL, aP2 in eWAT

FIG. 13(A) Effect of RFG on the expression levels of p-LKB1, LKB1, p-AMPK α, AMPK α, p-mTOR, and mTOR proteins in the liver; (B) analyzing the relative ratio of p AMPK alpha/AMPK alpha, p-LKB1/LKB1 and p-mTOR/mTOR in the liver after RFG treatment; (C) effect of RFG on the mRNA expression levels of FAS, LPL, aP2 in the liver

Detailed Description

The purpose of the invention is realized by the following technical scheme:

example 1 preparation of rubrofusarin-6-O-beta-gentiobioside

(1) Adding appropriate amount of methanol into semen Cassiae powder, performing flash extraction, filtering, mixing extractive solutions, and recovering methanol to obtain semen Cassiae extract.

(2) Mixing semen Cassiae extract with silica gel at a mass ratio of 1: 1, eluting with chloroform-methanol, dry loading, eluting for 4 times of column volume, removing the upper sample layer, eluting silica gel column with methanol, and tracking by TLC until no erythromycin-6-O-beta-gentiobioside is detected. Recovering the eluent to obtain a crude extract of rubrofusarin-6-O-beta-gentiobioside.

(3) Dissolving crude extract of rubrofusarin-6-O-beta-gentiobioside with 39% methanol aqueous solution by volume concentration to prepare test solution, and filtering with 0.45 μm microporous membrane; separating with high performance liquid chromatography column, performing online ultraviolet detection with 40% methanol-water solution as eluting system, collecting eluate containing rubrofusarin-6-O-beta-gentiobioside, concentrating under reduced pressure, and drying to obtain yellow powder I.

(4) Performing high performance liquid phase preparation on the yellow powder I for the second time by using an elution system of methanol water solution with the volume concentration of 42%, performing the same step (3) under other conditions, and collecting fractions with the purity of more than 98% and containing rubrofusarin-6-O-beta-gentiobioside; freeze drying the above fractions to obtain rubrofusarin-6-O-beta-gentiobioside with purity of more than 98%.

Example 2 Effect of rubrofusarin-6-O-. beta. -gentiobioside on proliferation and differentiation of 3T3-L1 cells and hAMSCs cells

First, experiment content

(1)3T3-L1 cell culture and induced differentiation

3T3-L1 cells were seeded on a plate and cultured in DMEM medium containing 1% penicillin-streptomycin and 10% newborn bovine serum (NBCS) at 37 ℃ in a 5% CO2 incubator, and two days after cell fusion, incubated in DMEM medium containing 25mM glucose, 0.5mM IBMX, 1. mu.M dexamethasone (Dex), 1. mu.g/ml insulin (MDI) and 10% Fetal Bovine Serum (FBS) for 48 hours, and then incubated in DMEM medium containing 10% FBS, 1. mu.g/ml MDI and different concentrations of REG for 48 hours. Then, the culture was continued for two days in DMEM medium containing 10% FBS and 1. mu.g/ml MDI. RFG was added at various concentrations on day 1 of 3T3-L1 cell differentiation induction until the end of the experiment.

(2) hAMSCs cell culture and induced differentiation

hAMSCs cells were seeded on a culture plate, cultured in DMEM medium containing 1% penicillin-streptomycin and 10% newborn bovine serum (NBCS) at 37 ℃ in a 5% CO2 incubator, and two days after cell fusion, cells were induced to differentiate for 6 days with 10% FBS/DMEM containing 0.5Mm IBMX, 1 μm Dex, 1 μm insulin and 100 μm indomethacin, and fresh medium was changed every three days during the culture. On the sixth day, cells were incubated with 10% FBS/DMEM containing 1. mu.g/ml insulin, with fresh medium being changed every two days until day 14.

(3) Cytotoxicity evaluation of 3T3-L1 and hAMSCs

3T3-L1 cells and hAMSCs cells were seeded at 5 × 103/well in 96-well plates and cultured in DMEM medium containing 10% NBCS. After 24 hours, different concentrations of RFG were replaced. After 48 hours, 20 μ L of MTS solution was added to each well, incubation was continued for 4 hours, the culture was terminated, the 490nm wavelength was selected, and the absorbance values (formazan concentration, which is proportional to the number of viable cells) were determined for each well on a verse plate reader.

(4) Oil red O staining experiment

3T3-L1 preadipocytes and hAMSCs cells were treated with RFG and then fixed in 10% formaldehyde for 2 hours. After washing with 60% isopropanol, staining was performed with 0.3% oil red O solution at room temperature for 30 minutes, and unstained cells were washed with water to remove the oil red O solution. Cells were visualized using an EVOS XL core imaging system. The dye was extracted from the cells with 100% isopropanol (3 ml/well). The dissolved dye was measured at 500nm using a VERSA max microplate reader to calculate absorbance.

(5) Statistical method

Results are expressed as mean ± SD of independent experiments, statistical methods using t-test. All statistical results were analyzed with SPSS statistical software. Significant differences were shown when p < 0.05.

Second, experimental results

(1) Results of cytotoxicity experiments

As shown in fig. 1(a), RFG was not cytotoxic to 3T3-L1 cells below 200 μ M, and RFG at 400 μ M significantly reduced cell viability of 3T3-L1 cells. RFG was therefore selected at 50, 100 and 200 μ M as the experimental concentration.

As shown in FIG. 1(B), although RFG up to 400. mu.M showed no cytotoxicity to hAMSCs cells, 50, 100 and 200. mu.M were selected as experimental concentrations in the following experiments in order to keep agreement with the concentrations in the 3T3-L1 cell experiment.

(2) Oil red O staining test results

As shown in fig. 2(a) and (B), lipid droplet accumulation in 3T3-L1 cells gradually decreased in a dose-dependent manner as RFG concentration increased.

As shown in fig. 2(C) and (D), lipid droplet accumulation in hAMSCs cells gradually decreased in a dose-dependent manner with increasing RFG concentration.

Example 3 study of the regulatory mechanism of rubrofusarin-6-O-beta-gentiobioside to inhibit adipocyte differentiation

First, experiment content

(1) RNA extraction and real-time quantitative PCR

Total RNA was extracted from 3T3-L1 cells and hAMSCs cells using the QIAzol lysis kit protocol. The extracted total RNA is used as a template for synthesizing a first strand of cDNA, and reverse transcription is carried out by using a power cDNA synthesis kit according to the using method described by the kit. PCR products were detected using a Step One Plus real-time fluorescent quantitative RT-PCR system and the relative expression levels of PPAR γ and C/EBP α were determined by comparative CT using Step One v2.1 software with GAPGH and h36B4 as endogenous controls. The forward and reverse primers for amplification of the target cDNA are shown in Table 1.

TABLE 1 real-time quantitative PCR primer sequences

(2) Western blot analysis

The induced differentiation of 3T3-L1 cells and hAMSCs cells was performed by lysis with a protein extract, and the protein concentration was measured by the Bradford method. Adding a sample buffer into a protein sample, boiling for denaturation, performing 10% SDS-PAGE electrophoresis, transferring a mold, sealing with 5% skimmed milk powder TBST for 2h, adding a primary antibody respectively, standing overnight at 4 ℃, washing the membrane for 3 times with 0.1% TBST, treating with a proper secondary antibody, and performing ECL development with a chemiluminescence gel imaging system. The band density was measured with Image-J software.

(3) Immunofluorescence staining method

hAMSCs (5 × 103 cells/well) were seeded into 8-well chamber slides and differentiated. Differentiated cells were fixed and permeabilized with 0.1% Triton X-100. After incubation in blocking buffer containing 3% BSA and 0.1% Triton X-100 in PBS, the primary antibody was reacted overnight at 4 ℃ in a dark room. After 1 hour of treatment of Alexa488 and Alexa 555-bound secondary antibody, slides were washed with PBS and counterstained with DAPI. After washing with PBS, slides were captured using the EVIS (TM) FL imaging system.

(4) Statistical method

Second, experimental results

(1) Effect of RFG on mRNA and protein level expression of PPAR γ and C/EBP α in 3T3-L1 cells

As shown in FIG. 3, 3T3-L1 cells after RFG treatment both significantly inhibited the expression of mRNA and protein levels of PPAR γ and C/EBP α.

(2) RFG activates AMPK pathway and inhibits lipogenesis in 3T3-L1 cells

To determine the molecular mechanism of lipid lowering action of RFG, the upstream and downstream factors of PPAR γ and C/EBP α were determined. The mRNA expression levels of FAS, LPL and aP2, downstream factors of PPAR γ and C/EBP α, were first determined. As shown in figure 4, mRNA expression levels of FAS, LPL, and aP2 were all significantly reduced after concentration-dependent RFG treatment. To further determine the pathway by which RFG inhibits adipocyte expression, phosphorylation of LKB1, AMPK, and mTOR was studied. As shown in fig. 5, phosphorylation of AMPK was dose-dependently increased by RFG treatment, and activated AMPK inhibited phosphorylation of mTOR, promoting PPAR γ expression. However, IKB 1, which is an upstream factor of AMPK, is not activated by RFG, and phosphorylation of PI3K and AKT is not inhibited by RFG. The above results indicate that RFG may inhibit adipogenesis through phosphorylation of AMPK.

(3) RFG activates AMPK pathway, inhibits hAMSCs from differentiating into mature fat cells

To confirm whether RFG inhibits adipogenesis by AMPK, the AMPK inhibitor Compound C (CC) was used. As shown in fig. 2(C, D), the combined use of RFG and CC significantly inhibited the lipid-lowering effect of RFG compared to the RFG group. As shown in fig. 6, in agreement with the results of 3T3-L1 cells, RFG can activate AMPK pathway, phosphorylation of mTOR, and significantly inhibit protein expression of PPAR γ and C/ebpa. However, RFG did not activate LKB1 phosphorylation. In addition, RFG activated phosphorylation of mTOR, significantly inhibiting protein expression of PPAR γ and C/ebpa after CC treatment. At the mRNA level, RFG inhibited the expression of PPAR γ and C/EBP α, while CC treatment did not. The downstream factor aP2 of PPAR γ and C/EBP α also showed similar trends. Immunofluorescent staining demonstrated that RFG inhibited PPAR γ and C/ebpa in a concentration-dependent manner, but CC treatment also blocked this effect. The above results indicate that RFG can inhibit the differentiation of hAMSCs into mature adipocytes by activating AMPK pathway.

Example 4 study of weight and lipid reducing effects of rubrofusarin-6-O-beta-gentiobioside on HFD-induced obesity in mice

First, experiment content

(1) HFD-induced obese mouse modeling and experimental grouping

C57BL/6J male mice (4 weeks, 16-18 g) were kept under stable experimental conditions for one week for 12h light/dark cycle. To induce obesity, mice were fed 60% kcal high fat Diet (HFD, american Research Diet experimental animal feed model D12492). Mice were randomized into four groups (8 mice per group), namely, normal diet group, HFD + RFG (50 mg/kg/day) group and HFD + RFG (100 mg/kg/day) group. Normal groups were fed standard diet, HFD and HFD + RFG groups were fed HFD and distilled water or RFG for 10 weeks, weighed once per week, and changes in body weight of mice were recorded. All procedures used in the animal experiments were performed according to procedures approved by the institutional review board of university of round lights animal Care and use Committee (confirmation No.: wk u 17-128).

(2) Observation indicator and method

After 4 weeks, fasting is not forbidden for 6 hours, the mice are anesthetized by intraperitoneal injection of 5% chloral hydrate with the volume of 0.2ml/100g after weighing, the abdominal aorta is subjected to blood collection, the blood is kept for 1 hour, and supernatant serum is sucked by a pipette after centrifugation, and the blood is stored at the temperature of 20 ℃ below zero. Serum was analyzed for Total Cholesterol (TC), High Density Lipoprotein (HDL), Low Density Lipoprotein (LDL) Triglyceride (TG), aspartate Aminotransferase (AST), alanine Aminotransferase (ALT), Blood Urea Nitrogen (BUN) and creatinine content.

Mice, eWAT and liver were harvested and weighed. The right lobe tissue of the liver was cut, washed with physiological saline, dried by blotting with filter paper, fixed in 10% neutral formalin, and 5 μm paraffin sections were prepared and stained with hematoxylin-eosin. Microscopic images were captured by the EVOS XL core imaging system. Adipose tissue size was analyzed using Image-J software.

(3) Statistical method

Second, experimental results

(1) RFG reduces HFD-induced body weight, epididymal fat and liver weight in obese mice

The body weight, epididymal fat and liver weight were significantly reduced in the HFD + RFG (50 mg/kg/day) group and the HFD + RFG (100 mg/kg/day) group, compared to the HFD group. As shown in fig. 7, the body weight of the HFD + RFG group mice was significantly lower than that of the HFD group. The weight of ND group is 8.27 plus or minus 1.09g, and the weight of HFD group is 25.41 plus or minus 0.86 g. While the HFD + RFG (100 mg/KG/day) group gained 13.17. + -. 1.53 g. The results show that dose-dependent reduction of body weight gain is possible with RFG. As shown in figure 8, RFG-treated mice reduced eWAT and liver weight dose-dependently.

(2) RFG significantly ameliorates HFD-induced lipid metabolism disorders in obese mice

As shown in fig. 9, the serum levels of TC, HDL, and LDL were significantly higher in the HFD group than in the normal group, while the TG level was significantly lower than in the normal group, indicating that the fat metabolism of HFD-induced obese mice was disturbed. After RFG medicament treatment, the blood fat detection result shows that: RFG is effective in improving HFD-induced lipid metabolism disorder in obese mice, and reducing TC and LDL levels (p < 0.05). Compared with the HFD group, oral administration of RFG10 did not cause hepatorenal toxicity for weeks.

(3) RFG reduces HFD-induced liver lipoid lesions in obese mice

The H & E staining results showed that, as shown in fig. 10, the HFD group had significantly increased adipocytes compared to the normal group, while the HFD + RFG group was able to significantly inhibit the adipocyte increase. The size of adipocytes in the HFD group was 5944+414 μm2, while the size of adipocytes in the HFD + RFG (50 mg/kg/day) and HFD + RFG (100 mg/kg/day) groups was 4079. + -. 305 μm2 and 2813. + -. 169 μm2, respectively. Thus, RFG can improve liver lipoid lesions.

Example 5 study of the mechanism of action of rubrofusarin-6-O-beta-gentiobioside on weight and lipid reduction in HFD-induced obese mice

First, experiment content

(1) RNA extraction and PCR

Total RNA was extracted from liver and eWAT using the QIAzol lysis kit protocol. The extracted total RNA is used as a template for synthesizing a first strand of cDNA, and reverse transcription is carried out by using a power cDNA synthesis kit according to the using method described by the kit. PCR products were detected using a Step One Plus real-time fluorescent quantitative RT-PCR system and the relative expression levels of PPAR γ and C/EBP α were determined by comparative CT using Step One v2.1 software with GAPGH and h36B4 as endogenous controls. The forward and reverse primers for amplification of the target cDNA are shown in Table 1.

(2) Western blot analysis

The induced differentiation of liver and eWAT tissues was lysed with protein extracts and the protein concentration was determined by Bradford method. Adding a sample buffer into a protein sample, boiling for denaturation, performing 10% SDS-PAGE electrophoresis, performing brick mold, sealing with 5% skimmed milk powder TBST for 2h, adding a primary antibody, standing overnight at 4 ℃, washing the membrane for 3 times with 0.1% TBST, treating with a proper secondary antibody, and performing ECL development with a chemiluminescence gel imaging system. The band density was measured with Image-J software.

(3) Statistical method

Second, experimental results

(1) Mechanism for regulating PPAR gamma and C/EBP alpha by RFG

As shown in fig. 11, RFG treatment significantly inhibited PPAR γ and C/ebpa protein expression and mRNA expression in eWAT and liver tissues. This result suggests that RFG may inhibit adipocyte expansion by inhibiting PPAR γ and C/ebpa in eWAT and liver tissues.

As shown in fig. 10, the liver of the HFD group with hepatic steatosis had more lipid droplets than the ND group. However, by RFG administration, accumulation of lipid droplets was alleviated.

(3) Inhibition of adipogenesis by RFG activation of the AMPK pathway

As shown in figure 12, RFG treatment increased AMPK phosphorylation in eWAT and inhibited mTOR phosphorylation, activating LKB1 phosphorylation compared to HFD group. Furthermore, RFG significantly down-regulated the mRNA expression of FAS, LPL and aP 2. As shown in figure 13, consistent with the results of eWAT, treatment of liver with RFG also modulated phosphorylation of AMPK, LKB and mTOR and inhibited mRNA expression of FAS, LPL and aP2 in liver, thereby inhibiting adipocyte production.

Example 6 comparative experiments on Fusarium rubrum-6-O-beta-gentiobioside and Cassia tora extract for lipid lowering and weight loss

The lipid-lowering and weight-losing effects of rubrofusarin-6-O-beta-gentiobioside and extract of cassia seed (effects on body weight, liver and epididymal tissue weight, and blood lipid of HFD-induced obese mice) were compared at animal level according to the method of example 4, and the data are as follows:

TABLE 2 influence of rubrofusarin-6-O-beta-gentiobioside and cassia seed extract on body weight, liver and epididymal tissue weight of experimental mice

TABLE 3 influence of rubrofusarin-6-O-beta-gentiobioside and cassia seed extract on blood lipid of experimental mice

Unit: mg/dl

The results show that the RFG with the same dosage has the fat-reducing and weight-losing effects obviously superior to the fat-reducing and weight-losing effects of the cassia seed extract.

The foregoing embodiments illustrate and describe the principles and general features of the present invention and its advantages. It will be understood by those skilled in the art that the present invention is not limited by the embodiments described above, which are given by way of illustration of the principles of the invention and are not to be taken as limiting the scope of the invention in any way, and that various changes and modifications may be made therein without departing from the scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. Application of naphthopyranoketose glycoside compound rubrofusarin-6-O-beta-gentiobioside shown in formula 1 in preparation of weight-reducing medicine

2. The use of claim 1, wherein said rubrofusarin-6-O- β -gentiobioside inhibits lipid accumulation in 3T3-L1 and hAMSCs cells by reducing mTOR, PPAR γ and C/ebpa targeting.

3. The use of claim 1, wherein rubrofusarin-6-O- β -gentiobioside inhibits lipid accumulation signaling pathway by modulating adipogenesis associated factor expression via AMPK.

4. The use of claim 1, wherein said rubrofusarin-6-O- β -gentiobioside reduces weight gain, eWAT and fatty liver size; adipogenic factors, PPAR γ, C/ebpa, FAS, LPL and aP2, are inhibited by activation of eWAT and AMPK in the liver.