CN105813646A - Novel use of a dimethyl sulfoxide extract or fraction from Trionyx - Google Patents

Abstract

The present invention relates to a novel method for the treatment of metabolic diseases, such as diabetes, obesity or fatty liver disease, using dimethyl sulfoxide ( DMSO) extract or fraction or an active compound isolated from the DMSO extract or fraction. Also provided in the present invention are dimethyl sulfoxide (DMSO) extracts or fractions from the genus Rhodiola or Rhodiola, or active compounds isolated from the DMSO extracts or fractions in the activation of the AMPK pathway and the autophagy pathway. Use, which can be used for the prevention or treatment of neurodegenerative diseases or amyloid-related diseases, such as Alzheimer's disease.

Description

A novel use of the genus dimethyl sulfoxide extract or fraction

technical field

The present invention relates to a new method and composition for the treatment of metabolic or neurodegenerative diseases. In particular, the present invention relates to a method and composition for the treatment of metabolic or neurodegenerative diseases using extracts or fractions from the genus Graptopetalum sp.

Background technique

Stone lotus (Graptopetalum paraguayense, GP) is a traditional Chinese herbal medicine with various health benefits. According to ancient Chinese prescriptions, GP is believed to have potentially beneficial effects in alleviating liver disorders, lowering blood pressure, whitening skin, relieving pain and infection, suppressing inflammation, and improving brain function.

Studies have shown that the GP leaf extract can inhibit the activity of tyrosinase (tyrosinase) and angiotensin-converting enzyme (angiotensin-convertingenzyme) and scavenge free radicals in vitro (Chen, et al., Studies on the inhibitory effect of GraptopetalumparaguayenseE.Waltherextractsontheangiotensinconvertingenzyme.FoodChemistry100:1032 -1036, 2007; Chung, et al., Studies on the antioxidative activity of Graptopetalumparaguayense E. Walther. Food Chemistry 91:419-424, 2005; and Huang, et al., Studies on the inhibitory effect of Graptopetalumparaguayense E. Walther extractson mushroom tyrosinase. Food Chemistry 85, 830-85:5. It has been found in the past that the water extract and 50% ethanol extract and 95% ethanol extract of GP stems have antioxidant activity, which has been confirmed for the proliferation of human hepatocellular carcinoma (Hepatocellularcarcinoma, HCC) cell line (HepG2) Has an inhibitory effect (Chen et al., Invitroantioxidant and antiproliferative activity of the stem extracts from Graptopetalum paraguayense. AmJ ChinMed2008; 36:369-383). In addition, in vivo cell experiments have confirmed that the leaf extract of GP can inhibit the activation of microglial cells, oxidative stress and iNOS expression, so as to reduce ischemic brain injury (Kao et al., Gratopetalum paraguayenseE.Waltherleafextractsprotectagainstbraininjuryinischemicrats.AmJChinMed2010; 495-516).

US Patent No. 7,364,758, filed by Hsu in 2004 and certified in 2008, has revealed that ethanol extracts from the spp. have anti-fibrotic and anti-inflammatory effects in vivo and in vitro. Then, its continuation-in-part application, U.S. Patent No. 7,588,776, filed in 2008 and certified in 2009, states that the water-soluble fractions of the syringum genus are effective in treating diseases or symptoms of the liver, such as inflammation, steatosis, and fibrosis.

U.S. Patent Publication No. 20120259004, which was filed in 2011 and published on October 11, 2012, also discloses that by using dimethyl sulfoxide (DMSO) to extract the selected species from the genus Rhodiola and Rhodiola (Rhodiolasp. ) and plants of the group consisting of Echeveria sp., and new compounds isolated from the new extracts are effective in the treatment of cancer or fibrosis, such as liver cancer or liver fibrosis .

Contents of the invention

The present invention relates to a dimethyl sulfoxide (DMSO) extract or fraction of Graptopetalum sp. or Rhodiola sp. and active compounds isolated from the DMSO extract or fraction for use in New uses for treating metabolic diseases, such as diabetes or fatty liver disease, or neurodegenerative diseases.

In one aspect, the present invention provides a method for treating a metabolic disease comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of dimethyl sulfide from the genus Rhodiola or Rhodiola. A sulfone (DMSO) extract or fraction, or an active compound isolated from the DMSO extract or fraction.

In another aspect, the present invention provides a use of a composition for the preparation of a medicament for the treatment of metabolic diseases; wherein the composition comprises dimethyl sulfoxide (DMSO) extract or grade Fraction, or the active compound isolated from the DMSO extract or fraction.

In yet another aspect, the present invention provides a pharmaceutical composition for the treatment of metabolic diseases; wherein the composition comprises a dimethyl sulfoxide (DMSO) extract or fraction from the genus Rhodiola or Rhodiola, or Active compound isolated from the DMSO extract or fraction.

In a specific embodiment of the present invention, the metabolic disease is diabetes.

In another embodiment of the present invention, the metabolic disease is obesity.

In the third embodiment of the present invention, the metabolic disease is fatty liver disease, including alcoholic fatty liver disease (AFLD) or nonalcoholic fatty liver disease (nonalcoholic fatty liver disease, NAFLD).

In yet another aspect, the present invention provides a method, use or composition for treating hepatitis B virus (HepatitisBvirus, HBV)-related liver-related diseases (such as HCC, liver fibrosis or cirrhosis) using a composition, the composition Comprising an active compound from, or isolated from, a dimethyl sulfoxide (DMSO) extract or fraction of the spp. or Rhodiola.

In yet another aspect, the present invention provides a method, use or composition for treating diabetes-associated liver-related diseases (e.g. HCC) using a composition comprising dimethyl sulfoxide from the genus Rhodiola or Rhodiola (DMSO) extracts or fractions, or active compounds isolated from such DMSO extracts or fractions.

In yet a further aspect, the present invention provides a method, use or composition for treating obesity-related liver-related diseases using a composition comprising dimethyl sulfoxide (DMSO) from the genus Rhodiola or Rhodiola An extract or fraction, or an active compound isolated from the DMSO extract or fraction.

In yet another aspect, the present invention provides a method, use or composition for treating obesity-induced high blood cholesterol or high triglycerides using a composition comprising dimethyl sulfide from the genus Rhodiola or Rhodiola. A sulfone (DMSO) extract or fraction, or an active compound isolated from the DMSO extract or fraction.

In yet a further aspect, the present invention provides a method, use or composition for preventing or treating metabolic syndrome using a composition comprising a dimethyl sulfoxide (DMSO) extract from the genus Rhodiola or Rhodiola or fraction, or the active compound isolated from the DMSO extract or fraction.

In yet a further aspect, the present invention provides a synergistic method or composition for the prevention or treatment of cancer using a pharmaceutical composition comprising a dimethyl sulfoxide (DMSO) extract from the genus Rhodiola or Rhodiola or fraction, or the active compound isolated from the DMSO extract or fraction, and combined with an anticancer drug.

In a specific embodiment of the present invention, the cancer is liver cancer, such as hepatocellular carcinoma (Hepatocellular carcinoma, HCC), and the anti-cancer drug is an anti-liver cancer drug, such as Sorafenib.

The present invention also provides a method, use or composition for activating AMPK pathway or autophagy pathway, wherein the composition is a dimethyl sulfoxide (DMSO) extract or fraction, or Active compound isolated from the DMSO extract or fraction.

In addition, the present invention also provides a method or composition for preventing or treating neurodegenerative diseases using a composition comprising a dimethyl sulfoxide (DMSO) extract or fraction from the genus Rhodiola or Rhodiola , or an active compound isolated from the DMSO extract or fraction.

In a specific embodiment of the present invention, the neurodegenerative disease is Parkinson's disease, Alzheimer's disease, or Huntington's disease.

In addition, the present invention also provides a method, use or composition for preventing or treating amyloid-related diseases using a composition comprising a dimethyl sulfoxide (DMSO) extract from the genus Rhodiola or Rhodiola or fraction, or the active compound isolated from the DMSO extract or fraction.

In a specific embodiment of the present invention, the amyloid-related disease is Alzheimer's disease, type 2 diabetes, Parkinson's disease, Huntington's disease, fatal familial insomnia (Fatal Familial Insomnia), or similar Rheumatoid arthritis.

In addition, the present invention also provides an anti-aging method, use or composition using a composition comprising dimethyl sulfoxide (DMSO) extract or fraction, or isolated Active compound from the DMSO extract or fraction.

In a specific embodiment of the present invention, the genus is Graptopetalum paraguayense.

In one specific embodiment of the present invention, the Rhodiola genus is Rhodiola rosea.

Description of drawings

The foregoing summary, as well as the following embodiments of the invention, will be better understood when read in conjunction with the accompanying drawings. For purposes of illustrating the invention, a presently preferred embodiment is shown in the drawings. It should be understood, however, that the invention is not limited to the particular embodiments shown in the drawings.

In the schema:

Figures 1A-1B show the effect of HH-F3 on inhibiting 8-Br-cAMP/dexamethasone-induced gluconogenic enzyme gene expression in Hep3B/T2 cells. Figure 1A provides the results of treating cultured human liver tumor Hep3B/T2 cells with 8-bromo-cAMP (8-Br-cAMP) and Dicortisol (Dex) alone or simultaneously for 30 minutes; where measured by real-time quantitative PCR Gluconeogenesis genes, glucose-6-phosphatase (glucose-6-phosphatase, G6Pase) and phosphoenolpyruvate carboxykinase (phosphoenolpyruvatecarboxykinase, PEPCK) mRNAs and β-actin were used as the standard. Insulin was used as a positive control group. Figure 1B provides the treatment of cultured human liver tumor Hep3B/T2 cells with 8-Br-cAMP (8-Br-cAMP), Dicortisol (Dex) alone or simultaneously for 30 minutes, and then with different concentrations of HH-F3 and insulin Results of processing; wherein mRNAs of gluconeogenesis genes, glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) were measured by real-time quantitative PCR and β-actin was used as a standard.

2A-2C show the effect of HH-F3 on inhibiting the activity of 8-Br-cAMP/Dex-induced gluconeogenesis coactivator PGC-1α in Hep3B/T2 cells. Figure 2A provides the results of treatment of cultured human liver tumor Hep3B/T2 cells in serum-free DMEM medium with 8-Br-cAMP, Dicortisol (Dex) alone or simultaneously, and in combination with HH-F3 for 24 hours , in which PGC-1α mRNAs were measured by real-time quantitative PCR and normalized to β-actin, and insulin was used as a positive control. Figure 2B shows the expression of PGC-1α and HNF-4α protein levels in Hep3B/T2 cells treated with nuclear extracts, which were determined by Western blot analysis, wherein B23 was used as a control standard; Dex: Di Cortex Alcohol (Dexamethasone); where these are representative data from 3 independent experiments. Figure 2C provides the results of Western blot analysis of Hep3B/T2 cells treated with HH-F3, followed by antibodies against PGC1-α, HNF-4α, and FoxO1; where these are representative data from 3 independent experiments .

Figures 3A-3B show the effect of HH-F3 on inhibiting the expression of gluconeogenetic enzyme genes by activating AMPK in cultured human liver tumor Hep3B/T2 cells. Figure 3A shows the results of pretreatment of cultured human liver tumor Hep3B/T2 cells with 8-Br-cAMP plus Dicortisol for 30 minutes. Figure 3B shows the results of pretreatment of cultured human liver tumor Hep3B/T2 cells with 8-Br-cAMP plus Dicortisol for 30 minutes, and then using different concentrations of HH-F3 in serum-free DMEM for 24 hours; where Hep3B/T2 cells were transfected with a luciferase reporter plasmid driven by a glucose-6-phosphatase promoter, and the luciferase activity was determined by co-transfection of pCMV-β-galactoside compared with the control group Enzyme plasmids were normalized for β-galactosidase activity. Figure 3C shows the results of cyclic AMP/DEX stimulation of G6Pase promoter activity, which was tested in the presence of HH-F3 or HH-F3 and AMPK inhibitor compound C; wherein after one day of treatment, cell lysates were prepared for use with For luciferase activity analysis; where insulin was used as a positive control; and metformin was used as a positive control for AMPK.

Figures 4A-4B show the effect of HH-F3 on inhibiting 8-Br-cAMP/Dex-induced HBV core promoter activity in Hep3B/T2 cells. Figure 4A shows the results of the promoter activity test; Hep3B/T2 cells were transfected with luciferase reporter plasmids driven by HBV core promoter (corepromoter, CP) and HBVX promoter (Xpromoter, XP). Figure 4B shows the results after 1-day treatment with 8-Br-cAMP/Dex in the absence or presence of different concentrations of HH-F3 in serum-free DMEM; where cell lysates were prepared for luciferase activity assays ; Wherein the luciferase activity was normalized by co-transfected pCMV-β-galactosidase plastids with β-galactosidase activity; and insulin was used as a positive control group.

5A-5E show the effect of HH-F3 on inhibiting HBV surface antigen, gene expression, gluconeogenesis enzyme expression, and HBV DNA level in Hep3B/T2 or 1.3ES2 cells. Figure 5A provides images showing the use of different concentrations of HH-F3 in serum-free DMEM medium to treat cultured human liver tumor Hep3B/T2 cells for 48 hours; wherein HBV surface antigen was determined by ELISA and standardized by MTT assay; where Insulin was used as a positive control group. Figure 5B provides the results of treating 1.3ES2 cells cultured for 48 hours in serum-free DMEM medium with different concentrations of GP and HH-F3, wherein gluconeogenesis genes, G6Pase, PEPCK, and Expression of PGC-1α mRNAs and normalized to β-actin; HE-145 was used as a positive control. Figure 5C provides the results of treating 1.3ES2 cells cultured with different concentrations of GP and HH-F3 in serum-free DMEM medium for 48 hours, wherein the expression of HBV mRNA was measured by real-time quantitative PCR and using β-actin Protein was used as a standard; HE-145 was used as a positive control. Figure 5D presents the results of 1.3ES2 cells treated with HH-F3 followed by Western blot analysis using an antibody against nucleoprotein; where these are representative data from 3 independent experiments. HE-145 was used as a positive control group. Figure 5E provides the results of treating 1.3ES2 cells cultured for 48 hours in serum-free DMEM medium with different concentrations of GP and HH-F3, wherein the wild-type HBV DNA in the medium was measured by real-time quantitative PCR and using β - Actin was used as a standard; HE-145 was used as a positive control.

Figures 6A-6B show the effect of GP extract and HH-F3 on reducing fatty acid synthesis activity in HCC, which shows that treatment of GP extract and HH-F3 reduces fatty acid synthesis activity in HCC. Figure 6A shows treatment of Huh7 and Mahlavu cells with GP extract and HH-F3, followed by anti-phospho-AMPK (P-AMPK), AMPK, SREBP2, phospho-ACC (Ser ⁷⁹ ) (P-ACC), ACC, The results of western blot analysis (n=3) with antibodies against FASN and GAPDH. Figure 6B shows BNL cells were used to generate subcutaneous tumors, and the mice were divided into two groups: one group served as an untreated control group (3 different mice), while the other four mice were administered daily via the oral (PO) route HH-F3 (20mg/day) was treated for 3 weeks; after 3 weeks, the mice were sacrificed and the tumors were homogenized, followed by the expression of P-AMPK, AMPK, SREBP2, P-ACC, ACC, FASN, GAPD and β-actin Antibodies were subjected to western blot and quantification.

Figure 7A provides images showing Huh7 cells treated with GP/HH-F3 to reduce lipid accumulation induced by oleic acid (OleicAcid, OA) and palmitic acid (Palmiticacid, PA); wherein GP/HH-F3 decreased in Huh7 Lipid accumulation induced by oleic acid (OA) and palmitic acid (PA); Huh7 cells incubated with oleic acid (1 mM) and palmitic acid (2 mM) for 24 hours and 48 hours, visualized by oil red O (oilredO) staining Significant intracellular lipid accumulation and cell viability by MTT test; where the original magnification is 400 times, it was treated with GP/HH-F3 for 48 hours (OA+PA/GP: oleic acid and palmitic acid were combined with GP treatment; OA/HH-F3: oleic acid and palmitic acid were co-treated with HH-F3).

Figure 7B provides the results of enumeration (oleic acid and palmitic acid) of Huh7 cells, in which the control group was treated with oleic acid and palmitic acid (OA+PA), and the cells were treated with oleic acid and palmitic acid and GP extract (GP) Or HH-F3 co-treatment for 24 h or 48 h to reduce fat load and quantified by staining intracellular lipid droplets with Oil Red O (OA+PA/GP: co-treatment of oleic acid and palmitic acid with GP; OA/HH-F3: Oleic acid and palmitic acid were co-treated with HH-F3).

Figures 8A-8D show the synergistic effect of the combination of GP/HH-F3 and Sorafenib on cell viability of Huh7. Figure 8A provides the results of treating Huh7 cells with various concentrations of GP extracts in the presence or absence of Lesava (5 μM and 10 μM); wherein the cell viability was determined by SRB assay; and the data are expressed as mean ± Standard deviation (SD) of three independent experiments. Figure 8B provides the results of treating Huh7 cells with various concentrations of HH-F3 extracts in the presence or absence of Lesava (5 μM and 10 μM); wherein cell viability was determined by SRB assay; and data are expressed as mean Values ± standard deviation (SD) of three independent experiments. Figure 8C provides the results of an isobologram analysis demonstrating a synergistic interaction between Resava and various concentrations of GP extracts in Huh7 cells for 72 hr. Figure 8D provides the results of an isobologram analysis demonstrating a synergistic interaction between Resava and various concentrations of HH-F3 in Huh7 cells for 72 hr.

Figure 9 shows the effect of HH-F3 on Aβ _25-35 -induced neurotoxicity in primary cortical neurons; primary cortical neurons were pretreated with vehicle (0.1% DMSO) or HH-F3 for 2 hours and subsequently exposed to 10 μM Aβ _25-35 for 40 hours; and after incubation, the cell viability was detected by MTT assay; where the data are mean ± SD from at least three independent experiments, and expressed relative to control cells, and * indicates the use of Aβ and Significant difference (p<0.05) between HH-F3 plus Aβ treated cells.

Figures 10A-10C show the effect of HH-F3 on extracellular Aβ40 and Aβ42 levels in _swAPP ⁶⁹⁵ -SH-SY5Y cell culture; where APP ⁶⁹⁵ _-SHSY -5Y- (transformed with human APP695) was incubated with vector or HH-F3 SHSY-5Y neuroblastoma cells) for 24 hours. Figure 10A shows the cell viability of cells, which was detected by MTT assay. Figure 10B shows the levels of extracellular A[beta] ₄₀ as determined by ELISA. Figure 10C shows the levels of extracellular A[beta] ₄₂ as determined by ELISA.

Figures 11A-11D show the effect of HH-F3 on APP processing and A[beta] clearance. Figure 11A provides the results of SH-SY5Y-APP695 cells treated with HH-F3 or γSI for 24 hours. The levels of APP (anti-Aβ1-16 antibody), APPX-F (anti-Aβ1-40 antibody), and APP-CTF (anti-APPct antibody) and intracellular Aβ-degrading enzymes were measured by immunoblotting and APP-CTF was displayed /Relative levels of actin. Figure 11B provides the results of treating SH-SY5Y-APP695 cells with HH-F3 for 24 hours, and then using antibodies against insulin-degrading enzyme (insulin-degradingenzyme, IDE) and neprilysin (Neprilysin, NEP) for western blot analysis ; where these are representative data from 3 independent experiments. Figure 11C provides the results of incubation of SH-SY5Y-APP695 conditioned media with the indicated concentrations of HH-F3 for 24 hours. The remaining Aβ1-40 was measured by ELISA. Figure 1 ID provides the results of incubation of SH-SY5Y-APP695 conditioned media with the indicated concentrations of HH-F3 for 24 hours. NEP and IDE levels in the conditioned medium were measured by immunoblotting.

Figure 12 shows the effect of HH-F3 on autophagy activity in swAPP ⁶⁹⁵ -SH-SY5Y cells; wherein APP ⁶⁹⁵ -SHSY-5Y was incubated with vector or HH-F3 for 24 hours. The autophagy marker LC3 was detected by Western blot.

Figures 13A-13B show the effect of HH-F3 on inhibiting A[beta] aggregation. Figure 13A provides the results of HH-F3 not incubated with Aβ for 24 hours. Detection of Th-T fluorescence (Ex440/Em482). Figure 13B provides the results of HH-F3 incubated with Aβ1-40 for 24 hours. Detection of Th-T fluorescence (Ex440/Em482). C = PBS; V = vehicle (Aβ1-40). Figure 13C provides the results of HH-F3 incubated with Aβ1-42 for 24 hours. Detection of Th-T fluorescence (Ex440/Em482). C = PBS; V = vehicle (Aβ1-42).

Figure 14 shows the protective effect of HH-F3 on Aβ25-35-induced cytotoxicity. Primary cortical neurons were pretreated with the indicated concentrations of HH-F3 for 2 hours. Subsequently, cells were stimulated with 10 μΜ Aβ _25-35 for 46 hours. Cell viability was measured by MTT assay.

Figure 15 shows the effect of HH-F3 on Aβ plaque burden in APP/PS1 mice. The APP/PS1 transgenic mice developed Aβ plaques in the cerebral cortex at 120 days of age (Radde et al., Abeta42-driven rebralamyloidosis in transgenic mice reveals early and robust pathology. EMBOR Rep 2006; 7:940-946). HH-F3 (300 mg/kg/day) was orally administered to 140-day-old APP/PS1 mice for one month. This representative image was assessed by BSB staining for Aβ plaque burden in the brain hemispheres of APP/PS1 mice, followed by manual counting of Aβ plaque numbers.

Figures 16A-16B show the effect of HH-F3 on Aβ in the cerebral cortex of APP/PS1 mice. Figure 16A provides the results of quantitative analysis of SDS-soluble Aβ1-40 and Aβ1-42 concentrations in cerebral cortex homogenates determined by ELISA (vehicle n=3, HH-F3n=6). Figure 16B provides the results of quantification of Aβ1-40 and Aβ1-42 concentrations in the insoluble fraction (formic acid soluble) of cerebral cortex homogenates determined by ELISA (vehicle n=3, HH-F3n=6).

Figures 17A-17B show that HH-F3 upregulates Aβ clearance-related proteins in the cerebral cortex of APP/PS1 mice. Figure 17A provides the results of detection of Aβ clearance-related proteins in cortical lysates by immunoblotting. Figure 17B provides the results of assayed protein quantitation ratios between WT and APP/PS1 or vehicle and APP/PS1.

Figure 18 shows the effect of GPs extracted in different ways in inhibiting HBV surface antigens. It provides images showing cultured human liver tumor Hep3B/T2 cells treated for 48 hours with differently extracted GP extracts (200 μg/ml) in serum-free DMEM medium; where HBV surface antigen was determined by ELISA and Normalization was the MTT test; where insulin was used as a positive control.

detailed description

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary knowledge in the art to which this invention belongs.

List the full names of the abbreviations used in this article in the table below:

As used herein, the singular forms "a" and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a sample" includes a plurality of such samples and equivalents thereof that are known to those of ordinary skill in the art.

The present invention provides a new use of the DMSO extract of the genus Rosacea or Rhodiola rosea prepared by extracting plants with DMSO, referring to the extract in the treatment of metabolic diseases.

According to the present invention, the extract can be prepared by extracting plants with dimethyl sulfoxide (DMSO) using methods commonly used or standard in the art. In one embodiment of the present invention, the leaves of the plant are ground and lyophilized into powder, and vigorously shaken with DMSO, preferably 30% DMSO. Further extraction with methanol (MeOH) may be included prior to extraction with DMSO.

As used herein, the term "Graptopetalum sp." refers to any plant of the genus Graptopetalum sp., or a part or parts thereof. Combinations of more than one species in the genus or parts thereof may also be considered. Preferably, the genus is Stone Lotus (Graptopetalum paraguayense.).

In a preferred embodiment of the present invention, the genus is Echeveria.

As used herein, the term "Rhodiola sp." refers to any plant in the genus Rhodiola, or a part or parts thereof. Combinations of more than one species in the genus Rhodiola or parts thereof are also contemplated.

In a preferred embodiment of the present invention, the Rhodiola genus is Rhodiola rosea.

The term "extract" as used herein refers to a solution obtained by soaking or mixing substances to be extracted with a solvent. In the present invention, the extract is a DMSO extract.

The present invention also provides a fraction enriched in anti-cancer components from a plant selected from the group consisting of the genus Thyme, which fraction is prepared by extracting the plant with dimethyl sulfoxide (DMSO). , and then separated by chromatography to obtain fractions. In one embodiment of the present invention, the plant is Echeveria. The fraction was obtained by extracting the plant using DMSO and separating by chromatography to obtain fractions. In one embodiment of the present invention, a Sephadex LH-20 column is used for chromatography, called HH-F3. Therefore, this fraction HH-F3 is a potential therapeutic agent for the prevention or treatment of metabolic diseases.

According to one embodiment of the present invention, Echeveria flower (GP) extract and HH-F3 fraction can be prepared by the method indicated in U.S. Patent Publication No. 20120259004, the disclosure of which is incorporated by reference in its entirety. into this article.

It is also known that the HH-F3 fraction contains a proanthocyanidin rich in 3,4,5-trihydroxybenzyl (3,4,5-trihydroxybenzylic) The benzyl component has the following formula I structure,

One of the Rs is H or a precyanidin (prucyanidin, PC) unit; and the other R is an OH or a predelphindine (prodelphindine, PD) unit; n is a number ranging from 21 to 38; and a PC unit: PD unit <1:20. The structure of the proanthocyanidin (PC) unit is

And the structure of prodelphinidin (PD) is

As used herein, the term "metabolic disease" refers to any disease or disorder that disrupts normal metabolism, the process that converts food into energy at the cellular level. Metabolic diseases affect the ability of cells to carry out key biochemical reactions involving the processing or transport of proteins (amino acids), carbohydrates (sugars and starches), or lipids (fatty acids). For example, metabolic diseases include diabetes, obesity, and fatty liver disease, whether alcoholic fatty liver disease (AFLD) or nonalcoholic fatty liver disease (NAFLD).

As used herein, the term "metabolic syndrome" refers to a combination of medical conditions that, when co-occurring, increase the risk of cardiovascular disease and diabetes.

The term "AMPK pathway" as used herein refers to a pathway in which AMP-activated protein kinase (AMPK) plays a key role as a master regulator of cellular energy homeostasis. AMPK is activated in response to stresses that deplete the cellular ATP supply, such as hypoglycemia, hypoxia, ischemia and heat shock. As a cellular energy sensor in response to low ATP levels, AMPK activation positively regulates signaling pathways that replenish cellular ATP supply, including fatty acid oxidation and autophagy. AMPK negatively regulates ATP-consuming biosynthetic processes, including gluconeogenesis, lipid and protein synthesis. AMPK does this through direct phosphorylation of several enzymes directly involved in these processes and through transcriptional control of metabolism by phosphorylation of transcription factors, co-activators, and co-repressors. Because of its role as a central regulator of both lipid and glucose metabolism, AMPK is considered a potential therapeutic target for type II diabetes, obesity and cancer treatment. AMPK has also been implicated in several species as key modulators of aging through its interaction with mTOR and sirtuins.

As used herein, "autophagy" or "autophagy pathway", also known as autophagy, refers to a fundamental catabolic mechanism involving the cellular degradation of unnecessary or abnormal cellular components through the action of lysosomes. This breakdown of cellular components can ensure cell survival during starvation by maintaining cellular energy levels. If regulated, autophagy ensures the synthesis, degradation and recycling of cellular components. During this process, the cytoplasmic components of the target are separated from the rest of the cell within autophagosomes, which then fuse with lysosomes and are degraded or recycled.

As used herein, the term "neurodegenerative disease" refers to a range of conditions that primarily affect neurons in the human brain. Because neurons are the building blocks of the nervous system, which includes nerves in the brain and spinal cord, and because neurons don't usually replicate or replace themselves, they cannot be replaced by the body when they get damaged or die. Some typical examples of neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, and Huntington's disease.

As used herein, the term "amyloid-related disease" refers to the class of diseases involving amyloid. Amyloids are insoluble fibrin aggregates sharing specific structural features that occur in various improperly folded variants of proteins and polypeptides naturally occurring in the body. The misfolded structures alter their proper configuration, causing them to incorrectly interact with each other or with other cellular components to generate insoluble fibrils. Amyloid is known to be associated with the pathology of serious human diseases, abnormal accumulation of amyloid fibrils in organs may lead to amyloidosis, and may play a role in various neurodegenerative diseases. The amyloid-related diseases include, but are not limited to, Alzheimer's disease, type 2 diabetes, Parkinson's disease, Huntington's disease, fatal familial insomnia, and rheumatoid arthritis.

It is believed that agents with the ability to inhibit A[beta] accumulation (or aggregation) may be useful in amyloid-related diseases. Additionally, protein aggregation is thought to be a common pathology in neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's. Autophagy is a lysosomal degradation process that recycles cellular waste and eliminates damaged organelles and protein aggregates, and disturbances in the autophagy pathway contribute to the pathogenesis of neurodegenerative diseases. Therefore, inducers of autophagy may be therapeutic targets for neurodegenerative diseases.

The present invention confirms that the GP extract or the HH-F3 fraction can inhibit the expression of gluconeogenetic enzyme genes induced by cyclic AMP/dicortisol and the expression activity of HBV core promoter in Hep3B/T2 cells. Examples also illustrate that GP extracts or HH-F3 fractions suppress the gene expression of HBV surface antigens and gluconeogenetic enzymes in Hep3B/T2 or 1.3ES2 cells; however, the GP extracts/HH-F3 fractions do not affect Instead, it shifts the metabolism of cancer cells from glycolysis to glucose oxidation and induces mitochondria-dependent apoptosis. It explained that the GP extract/HH-F3 fraction regulated the expression of lipogenesis-related proteins and the expression of gluconeogenesis enzyme genes in an AMPK-dependent manner; therefore, the GP extract/HH-F3 fraction could improve abnormal lipid Accumulation of glycosylation and reduction of progression of tumor development, specifically in patients with type 2 diabetes who require the drug insulin or metformin to suppress hepatic gluconeogenesis in vivo.

It was also demonstrated in Example 7 and Figure 8 that the combination of GP/HH-F3 and Sorafenib provided a synergistic effect on the cell viability of Huh7. Accordingly, the prophylactic invention provides a synergistic method or composition for preventing or treating cancer.

Aging is a universal process that affects all organs. Age-related disruptions in cellular homeostasis lead to reduced responsiveness to physiological stress and organ dysfunction. Efficient regulation of energy metabolism is a key requirement for cellular homeostasis. AMPK is a master regulator of the cellular response to reduced ATP levels and acts as a sensor to maintain energy balance within the cell. AMPK is known to stimulate energy production from glucose and fatty acids and to inhibit energy expenditure for protein, cholesterol and glycogen synthesis during stress. In general, AMPK activation downregulates synthetic pathways, such as protein, fatty acid, and cholesterol biosynthesis, but turns on ATP-producing catabolic pathways, such as fatty acid oxidation, glucose uptake, and sugar breakdown. It does so not only by direct phosphorylation of several key metabolic enzymes, but also by altering gene expression in a tissue-specific manner. Currently, AMPK is considered an important molecular target, as AMPK activators are believed to be useful in the treatment of metabolic and neurodegenerative diseases, and as anti-aging drugs.

Therefore, in view of the results shown in the present invention that GP/HH-F3 is an AMPK activator, it is suggested that GP/HH-F3 has anti-aging ability by regulating the AMPK pathway.

The present invention also provides a use, method or composition using the extract, fraction or compound of formula I of the present invention.

As used herein, the term "therapeutically effective amount" refers to an amount of drug sufficient to achieve the desired therapeutic purpose. The therapeutically effective amount of the administered agent will vary depending on factors such as the nature of the agent, the mode of administration, the size and type of animal receiving the agent, and the purpose of the administration. The therapeutically effective amount for each individual case can be determined empirically by a skilled person based on the disclosure herein and methods established in the prior art.

The compositions of the present invention may be administered by any suitable route including, but not limited to, parenteral or oral administration. The compositions for parenteral administration include solutions, suspensions, emulsions, and solid injectable compositions, which can be dissolved or suspended in a solvent immediately before use. The injection can be made by dissolving, suspending, or emulsifying one or more active ingredients in a diluent. Examples of the diluent are distilled water for injection, physiological saline, vegetable oil, alcohol and combinations thereof. Furthermore, the injection may contain stabilizers, solubilizers, suspending agents, emulsifying agents, emollients, buffers, preservatives and the like. The final preparation step for the injection is sterilized, or made using aseptic procedures. The composition of the present invention can also be prepared as a sterile solid preparation, such as by freeze-drying, and can be used after sterilization, or immediately dissolved in sterile injectable water or other sterile diluents immediately before use. use.

According to the present invention, the composition may also be administered by oral route, wherein the composition may be in solid or liquid form. The solid compositions include tablets, pills, sachets, dispersible powders, granules and the like. The oral compositions also include mouthwashes and sublingual tablets that can be adhered to the oral cavity. The capsules include hard capsules and soft capsules. In these solid compositions for oral use, one or more active compounds may be mixed alone or with diluents, binders, pulverizers, lubricants, stabilizers, solubilizers, and then mixed in a conventional manner. Formulated into preparations. These formulations may be coated with a coating agent, or may be coated with two or more coating layers, as necessary. In another aspect, the liquid compositions for oral administration include pharmaceutically acceptable aqueous solutions, suspensions, emulsions, syrups, elixirs and the like. In these compositions, one or more active compounds can be dissolved, suspended or emulsified in common diluents (such as purified water, ethanol or their mixtures, etc.). Besides such diluents, the composition may also contain wetting agents, suspending agents, emulsifying agents, sweetening agents, flavoring agents, perfuming agents, preservatives and buffers, and the like.

In addition, the present invention provides the use of the GP extract, HH-F3 fraction or the compound of formula I of the present invention in the manufacture of medicines for treating metabolic diseases, specifically diabetes, obesity or fatty liver disease. In another aspect, the present invention provides a method for preventing or treating metabolic diseases, in particular diabetes, obesity or fatty liver disease, comprising administering a therapeutically effective amount of the extract of the present invention to an individual in need of such prevention or treatment , fraction or compound, in particular diabetes, obesity or fatty liver disease.

The following examples are provided to illustrate the invention and are not to be construed in any way as limiting the scope of the invention.

Example

Example 1 Preparation of GP extracts and fractions

The leaves of Echinacea (GP) were ground and freeze-dried at -20°C to a powder, and stored in a moisture-proof box at 25°C before extraction. First, 1.5 g of GP powder was vigorously shaken with 10 ml of 100% methanol (MeOH) for 5 minutes, followed by centrifugation at 1,500 g for 5 minutes. After removing the supernatant, 10 ml of 30% DMSO was added to each pellet to resuspend each extract. The suspension was mixed by vigorous shaking for 5 minutes and centrifuged twice at 1,500 g for 5 minutes, then at 9,300 g for 5 minutes, and filtered using 0.45 μm filter paper at room temperature. The 30% DMSO supernatant was stored at -20°C as a 150 mg/ml stock solution (referred to as GP extract) or separated into four fractions (F1-F4) by a Sephadex LH-20 column. Active molecules were obtained in fraction 3 by Western blot analysis using AURKA, AURKB, and FLJ10540 protein levels and referred to as the HH-F3 fraction (data not shown). The HH-F3 fraction was further analyzed by HPLC with UV detector (Shimadzu SPD-M10A) and normal phase HPLC column (Phenomenex Luna5uSilica100A, 4.6×250 mm) and ¹ H- and ¹³ C-NMR spectroscopy to identify the structure of the active molecule (data not shown). The GP extract and/or HH-F3 were also dialyzed against water with a dialysis membrane (MWCO 12-14,000) (Spectrum Laboratories, Rancho Dominguez, CA) to obtain the active compounds.

Example 2 Effect of HH-F3 on 8-Br-cAMP/dicortisol-induced gluconeogenesis gene expression in Hep3B/T2 cells

Human liver tumor Hep3B/T2 cells were cultured and treated with either or both of 0.5 mM 8-Br-8-Br-cAMP (8-Br-cAMP) and 0.5 μM Dicortisol (Dex) for 30 minutes. Then, HH-F3 fractions at different concentrations (5 μg/ml, 10 μg/ml, and 20 μg/ml) were added to serum-free DMEM for 24 hours. The mRNAs of gluconeogenesis genes, glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) were measured by real-time quantitative PCR and normalized to β-actin. Insulin was used as a positive control.

Cyclic AMP and Dicortisol (8-Br-cAMP/Dex) were used in this test to synergistically activate key gluconeogenic genes, glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK ) gene expression in human liver tumor Hep3B/T2 cells, the results are shown in Figure 1A. The results of the treatment of the HH-F3 fraction (HH-F3) are shown in Figure 1B, which indicated that the treatment of the HH-F3 fraction could inhibit the expression of G6Pase and PEPCK genes induced by 8-Br-cAMP/DEX in Hep3B/T2 cells .

Example 3 Effect of HH-F3 fraction on the expression activity of gluconeogenesis coactivator PGC-1α induced by 8-Br-cAMP/Dex.

Human liver tumor Hep3B/T2 cells were cultured and treated with either or both 0.5 mM 8-Br-8-Br-cAMP (8-Br-cAMP) and 0.5 μM Dicortisol (Dex) for 30 minutes, and Co-treatment with 10 nM insulin or 20 μg/ml HH-F3 fraction (HH-F3) in serum-free DMEM medium for 24 hours. PGC-1α mRNAs were measured by real-time quantitative PCR and normalized to β-actin. Insulin was used as a positive control group. Nuclear extracts of PGC-1α and HNF-4α protein levels were determined by Western blot analysis. Use B23 as load control. Dex: Dicortisol.

Mahlavu cell line was treated with different concentrations (0μg/ml, 5μg/ml, 25μg/ml, and 50μg/ml) of HH-F3, and then analyzed by Western blot using antibodies to PGC1-α, SIRT1, PPARγ, and FoxO1 . These are representative data from 3 independent experiments.

It is known that the expression of the coactivator PGC1-α drives the transcription of key gluconeogenic enzymes such as PEPCK and G6Pase, and is associated with the transcription factors HNF4-α and FoxO1, and G6Pase and PEPCK are through the PGC1-α/FoxO1/Sirt1 pathway To regulate. The Hep3B/T2 cell line was treated with 8-Br-cAMP/Dex to induce the expression of gluconeogenesis genes, and then treated with HH-F3, and 8-Br-cAMP and Dicortisol synergistically activated genes and co-activated gluconeogenesis Protein expression of agent PGC1-α gene expression. The results are shown in Figure 2A and Figure 2B, which pointed out that HH-F3 affects the pathway of PGC1-α/FoxO1/Sirt1; wherein the treatment of HH-F3 inhibited the 8-Br-cAMP/Dex-induced PGC1-α expression levels (see FIG. 2A ); however, there was no effect on HNF4-α protein expression in human liver tumor Hep3B/T2 cells (see FIG. 2B ). In fact, it is known that PGC1-α, a master metabolic regulator and coactivator of key gluconeogenesis genes, has been shown to strongly coactivate FoxO1 and HNF4-α for HBV transcription of key gluconeogenesis genes. It has been proved that the regulation is obviously regulated by the co-activator PGC1-α and the co-activator FoxO1 and HNF4-α, and this pathway is the same as that of HBV transcription. Figure 2C also indicated that HH-F3 treatment inhibited the expression of PGC1-α, HNF4-α, and FoxO1 in Hep3B/T2 cells.

Example 4 The effect of HH-F3 on inhibiting the expression of gluconeogenic enzyme genes through the activation of AMPK.

Human liver tumor Hep3B/T2 cells were cultured and pretreated with 0.5 mM 8-Br-cAMP and 0.5 μM Dicortisol (Dex) for 30 minutes. Then, different concentrations (0 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) of HH-F3 were added to serum-free DMEM for 24 hours. For testing of promoter activity, Hep3B/T2 cells were transfected with a luciferase reporter plasmid driven by the glucose-6-phosphatase promoter. Luciferase activity was normalized from co-transfected pCMV-β-galactosidase plasmids to that of β-galactosidase compared to control. Cyclic AMP/DEX stimulated G6Pase promoter activity was examined in the presence of HH-F3 or HH-F3 with the AMPK inhibitor compoundC. One day after treatment, cell lysates were prepared for luciferase activity assays. Insulin was used as a positive control group. Metformin was used as a positive control for AMPK.

The Hep3B/T2 cell line was treated with 8-Br-cAMP/Dex to induce the activity of the G6Pase promoter, and then treated with HH-F3. The results are shown in Figure 3A and Figure 3B, which shows that HH-F3 can reduce the activity of G6Pase promoter in a dose-dependent manner. It was previously reported that AMP-activated protein kinase (AMPK) is a cellular energy sensor that inhibits ATP consumption and stimulates ATP production under conditions of energy depletion. Activation of AMPK is known to repress the gene expression of G6Pase and PEPCK and suppress hepatic glucose production. Therefore, the effect of HH-F3 on regulating G6Pase via the AMPK pathway was tested. The results showed that AMPK inhibitor compound C could partially reverse the G6Pase promoter activity inhibited by HH-F3 (see FIG. 3C ), which indicated that HH-F3 could inhibit the gene expression of gluconeogenic enzymes through the activation of AMPK.

Example 5 Effect of HH-F3 on 8-Br-cAMP/Dex-induced HBV core promoter activity in Hep3B/T2 cells.

Hep3B/T2 cells were transfected with luciferase reporter plasmids driven by HBV core promoter (CP) and HBVX promoter (XP) to test the promoter activity. Cells were then treated with 8-Br-cAMP/Dex in the absence or presence of different concentrations (0 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) of HH-F3 in serum-free DMEM1 On the next day, cell lysates were prepared for luciferase activity assays. Luciferase activity was normalized to β-galactosidase activity from co-transfected pCMV-β-galactosidase plasmids. Insulin was used as a positive control.

Hep3B/T2 is a hepatic cell line expressing HBV. The HBV genome includes polymerase, surface, core, and HBx. The X gene encodes protein X (HBx), which has inactivating properties and may be important in hepatic carcinogenesis. The core gene encodes the core nucleosheath protein (important in viral packaging). Mutations in the core promoter have been suggested in previous in vitro studies to increase HBV replication. As shown in the results in Figure 4A, cyclic AMP and Dicortisol synergistically activated the activity of the core promoter of hepatitis B virus, but had no effect on the X promoter. As shown in Figure 4B, dose-dependent inhibition of HH-F3 inhibited 8-Br-cAMP/Dex-induced HBV core promoter activity. The test results can support the association of HBV infection with diabetes and HCC, and provide a possible explanation for the association of hepatitis B virus (HBV) infection with diabetes and HCC.

Example 6 Effect of HH-F3 on HBV surface antigen, gene expression and gluconeogenic enzyme expression in Hep3B/T2 or 1.3ES2 cells.

Human liver tumor Hep3B/T2 cells were cultured and treated with different concentrations (0 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) of HH-F3 in serum-free DMEM medium for 48 hours. HBV surface antigen was determined by ELISA and normalized by MTT assay. Insulin was used as a positive control. 1.3ES2 cells were treated with different concentrations of HH-F3 in serum-free DMEM medium for 48 hours. Expression of gluconeogenesis genes, G6Pase, PEPCK, and PGC-1α mRNAs and HBV mRNA was measured by real-time quantitative PCR and normalized to β-actin. HE-145 was used as a positive control (SF (Serumfree): no serum).

As shown in Figure 5A, HH-F3 dose-dependently suppressed the expression of HBV surface antigen in Hep3B/T2 cells.

In addition, the 1.3ES2 cell line derived from HepG2 cells and containing an integrally replicated HBV genome was used to investigate whether HH-F3 could suppress HBV gene expression through regulation of PGC1-α. As shown in Figures 5B-5E, HH-F3 inhibited key gluconeogenesis genes, G6Pase and PEPCK gene expression, and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) gene, at 1.3 HBV gene expression in ES2 cells.

Effect of Example 7GP and HH-F3 on the Activity of Fatty Acid Synthesis in HCC

Huh7 and Mahlavu cell lines were treated with GP and HH-F3, and then westernized with antibodies against phospho-AMPK, phospho-AKT, phospho-ACC (Ser ⁷⁹ ), AMPK, SREBP2, AKT, ACC, and FASN. Ink blot analysis (n=3). BNL cells were used to generate subcutaneous tumors. Divide the mice into two groups. One group served as an untreated control group (3 different mice), while the other four mice were treated daily with HH-F3 (20 mg/day) via the oral (PO) route for 3 weeks. After 3 weeks, mice were sacrificed and tumors were homogenized, followed by western blot and quantification.

Aberrant lipogenesis is known to be associated with cancer from recent findings showing that inhibition of major lipogenic enzymes can reduce tumor formation. The lipid system has been linked to a number of pathological processes, including hepatic steatosis (fatty liver), which is manifested by excessive accumulation of lipids in liver cells. Abnormal lipid accumulation is associated with polygenic genetic defects in energy metabolism and hepatic malignancies. HCC cells require more energy to survive through dysregulated lipid regeneration, which may contribute to liver tumorigenesis and human HCC prognosis. In HCC, the degree of this abnormal lipid production correlates with clinical aggressiveness, activation of the AKT-mTOR signaling pathway, and inhibition of AMPK. Activation of hepatic AMPK, a metabolite-sensing kinase, plays a protective role under conditions of metabolic stress, stimulating fatty acid oxidation and reduction via increased phosphorylation and acetyl-CoA carboxylase (ACC) and HMG-CoA, respectively Deactivation of the enzyme (HMG-CoAreductase, HMGCR), the rate-limiting enzyme for the de novo synthesis of fatty acids and cholesterol, acutely shuts down fatty acid synthesis. In addition, the protein expression of the fatty acid synthase (FASN) is known to be negatively regulated by AMPK. Indeed, targeting FASN, a key enzyme of lipogenesis and overexpressed in HCC, offers an opportunity for therapeutic application due to its tissue distribution and unusual enzymatic activity.

In this test, it was found that HH-F3 could inhibit the activities of 8-Br-cAMP/Dex-induced gluconeogenesis enzyme gene and HBV core promoter through AMPK pathway and PGC1-α. Furthermore, it is known that the AMPK pathway and PGC1-α also regulate lipogenesis. Therefore, a test was performed to examine whether GP/HH-F3 has a role in adipogenesis. As shown in Figure 6, treatment with GP or HH-F3 in HCC cell lines and in BALB/c mice decreased fatty acid by activating AMPK (increasing AMPK phosphorylation) and inhibiting activated AKT (decreasing AKT phosphorylation). (fattyacid, FA) synthetic activity, BALB/c mice were injected subcutaneously on the flank with BNL cells. Furthermore, treatment with GP/HH-F3 resulted in increased phosphorylation and consequent inactivation of the ACC target under AMPK, as well as decreased protein expression levels of FASN in a concentration-dependent manner. These results indicate that GP/HH-F3 treatment can reduce the activity of fatty acid synthesis in HCC.

Example 8 Effect of GP/HH-F3 on reducing oleic acid (OA) and palmitic acid (PA)-induced lipid accumulation in Huh7.

The Huh7 cell line was incubated with oleic acid (1 mM) and palmitic acid (2 mM) for 24 hours and 48 hours, respectively, which resulted in a marked accumulation of intracellular lipids visible by Oil Red O staining and cell viability assayed by MTT. Figure 7A shows the original magnification (×400) showing the control group (no treatment), the group treated with OA and PA (OA+PA), the group treated with OA and PA and GP for 48 hours, and the group treated with OA and PA Results of the group treated with HH-F3 for 48 hours. In Figure 7, it was found that Huh7 cells treated with GP and HH-F3, respectively, could reduce the fat load as quantified by Oil Red O staining of intracellular lipid droplets (OA+PA/GP: oleic acid and palmitic acid co-treated with GP ; OA/HH-F3: oleic acid and palmitic acid were co-treated with HH-F3).

It can be concluded that GP/HH-F3 activates AMPK and inhibits activated AKT, thereby reducing lipid synthesis (see Figure 7), although it remains unclear whether GP/HH-F3 directly or indirectly regulates AMPK and AKT. Furthermore, it is known that activation of hepatic AMPK and inhibition of AKT can acutely shut down fatty acid synthesis via increased phosphorylation and inactivation of ACC, and decreased transcriptional activation of SREBP-1c and SREBP-2. In addition, SREBP-1c regulates lipid production-related genes in the liver, including ACC and FAS, while SREBP-2 activates two key enzymes in cholesterol biosynthesis, Hmgcr and Hmgcs1, which play important roles in tumor formation.

Example 9 The synergistic effect of the combination of GP/HH-F3 and Sorafenib on the cell viability of Huh7.

Various concentrations of GP (0 μg/ml, 50 μg/ml, 150 μg/ml, 250 μg/ml, and 500 μg/ml) and HH-F3 ( 0 μg/ml, 5 μg/ml, 15 μg/ml, 25 μg/ml, and 50 μg/ml) to treat Huh7 cells. Cell viability was determined by the SRB assay. Data are presented as mean ± standard deviation (SD) of three independent experiments. Furthermore, isobologram analysis demonstrated a synergistic interaction between Resava and various concentrations of GP/HH-F3 in Huh7 cells for 72 hours.

The results of combining various concentrations of GP and HH-F3 with Resava (the only FDA-approved drug for pre-HCC patients) in Huh7, followed by cell proliferation assay at 72 hours are shown in Figure 8A and Figure 8B. The combination index (combination index, CI) analysis is also shown in Figure 8C and Figure 8D, which uses the Chou-Talalay method to assess the interaction between these potential therapies as synergistic (CI<1), additive (CI= 1) or antagonistic (CI>1). As shown in the results, both GP and HH-H3 can cooperate with Lesava to inhibit the proliferation of Huh7.

Example 10 Effects of HH-F3 Treatment on Complete Abolition of Aβ-Induced Neurotoxicity in Primary Cortical Neurons and Attenuation of Aβ Production in Cell Culture of swAPP ⁶⁹⁵ -SH-SY5Y

The primary cortical neurons were pretreated with vehicle (0.1% DMSO) or HH-F3 for 2 hours and then exposed to 10 μM Aβ _25-35 for 40 hours. After incubation, cell viability was detected by MTT assay. As shown in Figure 9, 10 μM of _Aβ25-35 fibrils resulted in a marked decrease in cell viability by 39.4±7.5% as measured by MTT reduction, and _Aβ25-35 -induced cell death was abrogated by HH-F3 treatment in a dose-dependent manner .

Currently, the failure of monotherapy strategies for Alzheimer's disease indicates that more than one pathway contributes to the pathogenesis of Alzheimer's disease. Drugs with more than one target may be more promising. Therefore, HH-F3 was also tested to check whether it could reduce Aβ production in swAPP ⁶⁹⁵ -SH-SY5Y cell culture. The extracellular Aβ levels in the SH-SY5Y cells were below the detectable limit, but the Aβ ₄₀ and Aβ ₄₂ levels in the swAPP ⁶⁹⁵ -SH-SY5Y cells were 1.2 ng/ml and 57.5 pg/ml, respectively. After HH-F3 treatment, no toxicity was found on SH-SY5Y cells (see Figure 10A). Extracellular _Aβ40 and _Aβ42 levels were determined by ELISA. The results are shown in Figure 10B and Figure 10C, which indicated that 50 μg/ml of HH-F3 had the ability to reduce the production of _Aβ40 and _Aβ42 by 30.0±11.2% and 36.8±16.0%, respectively.

Extracellular Aβ accumulation can be reduced by inhibiting the amyloidogenic pathway or increasing Aβ clearance, including enhancing the activity of Aβ-degrading enzymes and autophagy. Therefore, immunoblotting was performed to detect the levels of APP and APP-CTF (Fig. 11A). After 24 hours of treatment with the indicated concentrations of HH-F3, the level of overall APP was not significantly changed. Unexpectedly, when the APP line was immunoblotted with anti-Aβ1-40 antibody, an unidentified APP species (APP-XF) was detected and reduced by HH-F3. Essentially, the levels of the APP-CTF were increased (Fig. 11A). The γ-secretase inhibitor was used to verify the involvement of γ-secretase on APP-CTF levels (Fig. 11A). On the other hand, the expression of major Aβ-degrading enzymes, such as insulin degradation enzyme (insulindegradationenzyme, IDE) and neprilysin (neprilysin, NEP) in cells was not affected by HH-F3 ( FIG. 11B ). The activity of Aβ-degrading enzymes in the conditioned medium of SH-SY5Y-APP695 cells was detected by examining the remaining Aβ1-40 after 24 hours of incubation ( FIG. 11C ). The results showed that HH-F3 activated the activity of Aβ degrading enzymes to promote the clearance of Aβ1-40 in the conditioned medium, because the levels of IDE and NEP in the conditioned medium were not affected by HH-F3 ( FIG. 11D ).

Autophagy is involved in the clearance of Aβ. A recent report indicated downregulation of beclin-1, an early regulator of autophagy, in the brain of AD patients (Pickford et al., The autophagy-related protein beclin1 shows reduced expression in early Alzheimer disease and regulates samyloid beta accumulation in mice, J Clin Invest 2008; 118:2190-2199). The effect of HH-F3 on autophagy activation in ^swAPP695 -SH-SY5Y cells was also tested. The APP ⁶⁹⁵ -SHSY-5Y cell line was incubated with the vehicle or HH-F3 for 24 hours, and the LC3 autophagy marker was detected by Western blot. The results are shown in FIG. 12 .

HH-F3 contains polyphenolic compounds such as gallic acid and tannic acid. Polyphenolic compounds have been reported to inhibit Aβ aggregation. In this patent, Thioflavin T (ThioflavinT, TH-T), a molecule that can insert into the β-sheet structure of Aβ, is used to detect Aβ aggregation. Th-T emission is shifted when bound to amyloid fibrils, causing a dense specific emission band (482 nm) in its fluorescence spectrum. The results show that tannic acid (10 μM) and Congo red (10 μM) inhibit the formation of both Aβ _1-40 and Aβ _1-42 fibrils. HH-F3 inhibited the aggregation of both A[beta] _1-40 and A[beta] _1-42 in a concentration-dependent manner (Figure 13).

Prevention of Aβ-mediated neurotoxicity has become an important therapeutic strategy for AD treatment. A[beta] _25-35 is a synthetic peptide of 11 amino acids in the intermembrane domain of APP. It is evident that Aβ _25-35 cannot be produced by normal APP processing. However, the Aβ _25-35 line was chosen as a model for full-length Aβ because it retains both its physical and biological properties, while its short length may easily allow derivatives to be synthesized and studied (Hughes et al., Inhibition of toxicity in the beta-amyloid peptide fragment beta- (25-35) using N-methylated derivatives: general strategy to prevent tamyloid formation. J Biol Chem 2000; 275: 25109-25115). Although not present in biological systems, the Aβ _25-35 fragment is widely used by many scientists in conjunction with or in place of the endogenous fragment Aβ ₄₂ , and has been found to be at least as toxic as the full-length fragment (Yankner et al., Neurotoxicity of fragment of the amyloid precursor associated with Alzheimer's disease. Science 1989 ; 245:417-420). Cortical neurons were pretreated with HH-F3 for 2 hr. Subsequently, cells were stimulated with 10 μΜ Aβ _25-35 for 46 hours. This A[beta] _25-35 -mediated loss of cell viability was significantly restored by HH-F3. HH-F3 at 20 and 50 μg/ml significantly restored cell viability by 19% and 33%, respectively. The concentration-dependent restoration effect of HH-F3 on cell viability occurred while the HH-F3 treatment was pre-Aβ-treatment but not post-treatment ( FIG. 14 ).

HH-F3 has multiple functions in AD pathology in vitro. Therefore, experiments in this animal model were performed to investigate the in vivo effects of HH-F3. The APP/PS1 transgenic mice developed Aβ plaques in the cerebral cortex at 120 days of age (Radde et al., 2006). 140-day-old APP/PS1 mice were orally administered with HH-F3 (300 mg/kg/day) for one month. In vitro tests, the present invention has confirmed that HH-F3 can inhibit Aβ aggregation. Accordingly, amyloid plaques in the cerebral hemispheres were stained (Fig. 15). Due to its high affinity for Aβ, the dye 1-bromo-2,5-bis(3-carboxy-4-hydroxystyryl)benzene (1-Bromo-2,5-bis(3-carboxy-4-hydroxystyryl)benzene , BSB) have been used to detect amyloid plaques. This result showed that HH-F3 only slightly reduced the burden of Aβ plaques. The Aβ plaque burden numbers between the vehicle group and the HH-F3 group were 43.3±23.1 and 22.3±7.5 ( FIG. 15 ).

The present invention uses two steps to separate soluble and insoluble A[beta]s. The soluble A[beta]s are dissolved in a 2% SDS fraction which may contain monomers, dimers and oligomers. The insoluble Aβs were dissolved in the 70% formic acid fraction. Soluble and insoluble A[beta]s were then measured by ELISA. HH-F3 reduced soluble and insoluble Aβ in the cerebral cortex of APP/PS1 mice (Fig. 16).

HH-F3 affects Aβ clearance in SH-SY5Y-APP695 cells. Therefore, proteins related to Aβ clearance were detected in the cerebral cortex of APP/PS1 mice by immunoblotting ( FIG. 17A ). Compared with wild-type mice, the IDE level of this transgenic mouse was down-regulated, while the levels of NEP and APOE were up-regulated by HH-F3. In addition, compared with wild-type mice, the levels of PPARγ and pAMPK were down-regulated in this transgenic mouse, while PPARγ, pAKT and pAMPK were significantly up-regulated by HH-F3 ( FIG. 17B ).

Crude extracts of GP (H ₂ O, methanol, 100% ethanol, 70% ethanol, or 50% ethanol) did not significantly affect HBsAg levels in Hep3B/T2. GP extracts of DMSO, 30% DMSO and 80% acetone could partially reduce HBsAg levels in Hep3B/T2 ( FIG. 18 ).

In view of the above results, HH-F3 is expected to be used in the treatment of Alzheimer's disease.

In conclusion, this GP extract or HH-F3 fraction has potential as a novel therapy for patients with metabolic diseases such as diabetes, obesity, fatty liver disease, including alcoholic fatty liver disease ( AFLD), nonalcoholic fatty liver disease (NAFLD), HBV-related liver-related diseases (including HCC, liver fibrosis or cirrhosis), diabetes-related liver-related diseases, obesity-related liver-related diseases, and obesity-induced hypertrophy Blood cholesterol or high triglyceride levels. It suggests that GP extract or HH-F3 fraction can be used to treat or prevent metabolic syndrome.

Furthermore, it is pointed out in the present invention that the GP extract or HH-F3 fraction is able to activate AMPK pathway and autophagy pathway. Therefore, it is suggested that GP extract or HH-F3 fraction can be developed into a drug for the prevention or treatment of neurodegenerative diseases or amyloid-related diseases, such as Parkinson's disease, Alzheimer's disease, and Huntington's syndrome, and even anti-aging drugs.

It is believed that one having ordinary knowledge in the art to which this invention pertains can, based on the description herein, utilize the present invention to its widest scope without further elaboration. Accordingly, the description and claims provided are to be understood for purposes of illustration and are not intended to limit the scope of the invention in any way.

Claims (29)

1. A composition is used for the preparation of the purposes of the medicine for the treatment of metabolic diseases, wherein the composition comprises a dimethyl sulfoxide (dimethylsulfoxide, DMSO) from Graptopetalumsp. or Rhodiolasp. ) extract or fraction, or an active compound isolated from the DMSO extract or fraction. 2. purposes as claimed in claim 1, wherein this metabolic disease is diabetes, obesity, fatty liver disease, hepatitis B virus (HepatitisBvirus, HBV) related liver-related disease, diabetes-related liver-related disease, obesity-related Liver-related disease, or obesity-induced high blood cholesterol or triglyceride levels. 3. The use as claimed in claim 2, wherein the metabolic disease is diabetes. 4. The use as claimed in claim 2, wherein the metabolic disease is obesity. 5. The use as claimed in claim 2, wherein the metabolic disease is fatty liver disease. 6. The use according to claim 5, wherein the fatty liver disease is alcoholic fatty liver disease (alcoholic fatty liver disease, AFLD) or nonalcoholic fatty liver disease (nonalcoholic fatty liver disease, NAFLD). 7. The use as claimed in claim 2, wherein the metabolic disease is hepatitis B virus (HBV)-associated liver-related disease. 8. The use as claimed in claim 2, wherein the metabolic disease is diabetes-related liver-related disease. 9. The use according to claim 2, wherein the metabolic disease is obesity-related liver-related disease. 10. The use according to any one of claims 7-9, wherein the liver-related disease is hepatocellular carcinoma (Hepatocellular carcinoma, HCC), liver fibrosis or liver cirrhosis. 11. The use according to claim 10, wherein the liver-related disease is hepatocellular carcinoma (HCC). 12. The use according to claim 7, wherein the hepatitis B virus (HBV)-related liver-related diseases are HBV-induced liver fibrosis and liver cirrhosis. 13. Use of a composition for the preparation of a medicament for the prevention or treatment of metabolic syndrome, wherein the composition comprises a dimethyl sulfoxide (DMSO) extract or fraction, or a Active compound isolated from the DMSO extract or fraction. 14. Use of a composition for the preparation of a medicament for the prevention or treatment of cancer, wherein the composition comprises a dimethyl sulfoxide (DMSO) extract or fraction, or an isolated The active compound from the DMSO extract or fraction is combined with an anticancer drug in a ratio that has a synergistic effect on preventing or treating cancer. 15. The use according to claim 14, wherein the cancer is liver cancer, and the anti-cancer drug is an anti-liver cancer drug. 16. The use according to claim 15, wherein the anticancer drug is Sorafenib. 17. The use of claim 15, wherein the cancer is HCC, and the anticancer drug is Rezava. 18. Use of a composition for the preparation of a medicament for activating the AMPK pathway or the autophagy pathway, wherein the composition comprises a dimethyl sulfoxide (DMSO) extract or fraction from the genus Rhodiola or Rhodiola, or an active compound isolated from the DMSO extract or fraction. 19. Use of a composition for the preparation of a medicament for the prevention or treatment of neurodegenerative diseases, wherein the composition comprises a dimethyl sulfoxide (DMSO) extract or fraction from the genus Rhodiola or Rhodiola, Or the active compound isolated from the DMSO extract or fraction. 20. The use according to claim 19, wherein the neurodegenerative disease is Parkinson's disease, Alzheimer's disease, and Huntington's disease. 21. Use of a composition for the preparation of a medicament for the prevention or treatment of amyloid (amyloid)-related diseases, wherein the composition comprises a dimethyl sulfoxide (DMSO) extract from the genus Rhodiola or Rhodiola or a fraction, or an active compound isolated from the DMSO extract or fraction. 22. The use as claimed in claim 21, wherein the amyloid-related diseases are Alzheimer's disease, type 2 diabetes, Parkinson's disease, Huntington's disease, fatal family insomnia (Fatal Familial Insomnia), or rheumatoid arthritis. 23. Use of a composition for the preparation of an anti-aging medicament, wherein the composition comprises a dimethyl sulfoxide (DMSO) extract or fraction from the genus Rhodiola or Rhodiola, or is isolated from the DMSO extract Active compounds of substances or fractions. 24. The use according to any one of claims 1-23, wherein the genus is Graptopetalum paraguayense. 25. The use according to any one of claims 1-23, wherein the Rhodiola species is Rhodiola rosea. 26. The use according to any one of claims 1-23, wherein the fraction enriched in active ingredient is prepared by extracting the plant with dimethylsulfoxide (DMSO), followed by chromatographic separation A fraction called HH-F3 was obtained. 27. The use according to claim 26, wherein a Sephadex LH-20 column is used for chromatography. 28. The use according to any one of claims 1-23, wherein the active compound is a compound having a formula I structure Wherein one of R is H or proanthocyanidin (prucyanidin, PC) unit, and another R is OH or former delphinidin (prodelphindine, PD) unit; n is a number ranging from 21 to 38; and PC unit pair The ratio of PD units does not exceed 1:20. 29. A compound as proposed in claim 28 is used for the preparation of treatment of metabolic diseases, prevention or treatment of metabolic syndrome, cancer, or neurodegenerative diseases, amyloid-related diseases; and the drug for activating AMPK pathway or autophagy pathway ; or the use of anti-aging drugs.