TW201532610A - Novel use of a dimethyl sulphoxide (DMSO) extract or fraction from graptopetalum sp - Google Patents

Abstract

The preset invention relates to a new approach for treating a metabolic disease, such as diabetes, obesity or fatty liver disease using a dimethyl sulfoxide (DMSO) extract or fraction from Graptopetalum sp. or Rhodiola sp., or an active compound isolated from the DMSO extract or fraction. Also provided in the present invention is the use of the dimethyl sulfoxide (DMSO) extract or fraction from Graptopetalum sp. or Rhodiola sp., or the active compound isolated from the DMSO extract or fraction in activating AMPK pathway and autophagy pathway, which can be used in prevention or treatment of a neurodegenerative disease or amyloid-related disease, such as Alzheimer's disease.

Description

Novel use of dimethyl sulfoxide extract or fraction

The present invention relates to a novel method and composition for treating a metabolic disease or a neurodegenerative disease. In particular, the present invention relates to a method and composition for treating a metabolic disease or a neurodegenerative disease using an extract or fraction derived from Graptopetalum sp.

Graptopetalum paraguayense (GP) is a traditional Chinese herbal medicine with a variety of health benefits. According to the ancient Chinese prescription, GP is considered to have potential beneficial effects in alleviating liver disease, lowering blood pressure, whitening skin, relieving pain and infection, inhibiting inflammation, and improving brain function.

Studies have shown that the GP leaf extract can inhibit the activity of tyrosinase and angiotensin-converting enzyme and scavenge free radicals in vitro (Chen, et al., Studies on the inhibitory effect of Graptopetalum paraguayense E. Walther extracts on the angiotensin converting enzyme. Food Chemistry 100: 1032-1036, 2007; Chung, et al, Studies on the antioxidative activity of Graptopetalum paraguayense E. Walther. Food Chemistry 91: 419-424, 2005; Huang, et al, Studies on the inhibitory effect of Graptopetalum paraguayense E. Walther extracts on mushroom tyrosinase. Food Chemistry 89: 583-587, 2005). It has been found in the past that the water extract of GP stem and the stem extract of 50% ethanol extract and 95% ethanol have antioxidant activity, which has been confirmed for human hepatocellular carcinoma (HCC) cell line (HepG2). Proliferation has an inhibitory effect (Chen et al, In vitro antioxidant and antiproliferative activity of the stem extracts from Graptopetalum paraguayense. Am J Chin Med 2008; 36: 369-383). In vivo studies have confirmed that GP leaf extract can inhibit microglia activation, oxidative stress and iNOS expression to reduce ischemic brain damage (Kao et al, Graptopetalum paraguayense E. Walther leaf extracts protect against brain injury in Ischemic rats. Am J Chin Med 2010; 38: 495-516).

It has been disclosed in U.S. Patent No. 7,364,758, issued to Hsu in 2004, which is hereby incorporated by reference in its entirety in U.S. Patent No. 7,364,758, which is incorporated herein by reference. Then, part of its continuous application US Patent No. 7,588,776 was filed in 2008 and certified in 2009, which states that the water-soluble fraction of the genus is effective in treating diseases or symptoms of the liver, such as inflammation and steatosis. Fibrosis.

Filed in 2011 and US Patent October 11, 2012 Publication No. 20120259004 also revealed by the use of dimethyl sulfoxide (DMSO) extract is selected from the stripe do genus Rhodiola (Rhodiola sp New extracts and fractions prepared from plants of the group consisting of Echeveria sp., and new compounds isolated from the new extract are effective in treating cancer or fibrosis, such as liver cancer or liver Fibrosis.

The present invention relates to a dimethyl sulfoxide (DMSO) extract or fraction of Graptopetalum sp. or Rhodiola sp. and an active compound isolated from the DMSO extract or fraction. For the treatment of metabolic diseases, such as diabetes or fatty liver disease, or new uses of neurodegenerative diseases.

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

In another aspect, the invention provides the use of a composition for the manufacture of a medicament for the treatment of a metabolic disorder; wherein the composition comprises a dimethyl hydrazine (DMSO) extract or fraction from the genus Rhododendron or Rhodiola Or an active compound isolated from the DMSO extract or fraction.

In still another aspect, the present invention provides a pharmaceutical composition for treating a metabolic disease; wherein the composition comprises a dimethyl hydrazine (DMSO) extract or fraction, or a separation from the genus Rhododendron or Rhodiola The active compound from the DMSO extract or fraction.

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

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

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

In still another aspect, the present invention provides a method, use, or composition for treating a hepatitis B virus (HBV)-related liver-related disease (eg, HCC, liver fibrosis, or cirrhosis) using a composition, the combination The composition comprises a dimethyl hydrazine (DMSO) extract or fraction from the genus Rhodiola or Rhodiola, 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 a diabetes-related liver-related disease (eg, HCC) using a composition comprising dimethyl hydrazine from the genus Rhododendron or Rhodiola ( DMSO) extract or fraction, or active compound isolated from the DMSO extract or fraction.

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

In still another aspect, the present invention provides a method, use or composition for treating obesity-induced hypercholesterolemia or high triglyceride using a composition comprising dimethyl or erythromycin from the genus Rhodiola A guanidine (DMSO) extract or fraction, or an active compound isolated from the DMSO extract or fraction.

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

In still a further aspect, the present invention provides a synergistic method or composition for preventing or treating cancer using a pharmaceutical composition comprising dimethyl hydrazine (DMSO) extract from the genus Rhododendron or Rhodiola or Fractions, or active compounds isolated from the DMSO extract or fraction, are combined with an anti-cancer drug.

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

The invention also provides a method, use or composition for activating an AMPK pathway or an autophagy pathway, wherein the composition is a dimethyl hydrazine (DMSO) extract or fraction, or isolated from the genus Rhodiola or Rhodiola The active compound from the DMSO extract or fraction.

Further, the present invention also provides a method or composition for preventing or treating a neurodegenerative disease using a composition comprising a dimethyl hydrazine (DMSO) extract or fraction derived from the genus Rhododendron or Rhodiola, Or an active compound isolated from the DMSO extract or fraction.

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

Further, the present invention provides a method, use or composition for preventing or treating an amyloid-related disease using a composition comprising dimethyl hydrazine (DMSO) extract from the genus Rhododendron or Rhodiola or Fractions, or active compounds isolated from the DMSO extract or fraction.

In a specific embodiment of the present invention, the amyloid-related diseases are Alzheimer's disease, type 2 diabetes, Parkinson's disease, Huntington's disease, Fatal Familial Insomnia, Or rheumatoid arthritis (Rheumatoid arthritis).

Further, the present invention also provides a method, use or composition for using the composition against aging, comprising dimethyl hydrazine (DMSO) extract or fraction from genus Rhodiola or Rhodiola, or isolated from The DMSO extract or fraction of the active compound.

In one embodiment of the invention, the genus is Graptopetalum paraguayense .

In a specific embodiment of the invention, the Rhodiola rosea is Rhodiola rosea .

The foregoing summary, as well as the following embodiments of the present invention, For the purpose of illustrating the invention, the present preferred embodiments are shown in the drawings. However, it should be understood that the invention is not limited to the specific embodiments shown in the drawings.

In the drawings: Figures 1A-1B show the effect of HH-F3 on inhibition of 8-Br-cAMP/dexamethasone-induced gluconeogenic enzyme gene expression in Hep3B/T2 cells. Figure 1A provides the results of treatment of cultured human liver tumor Hep3B/T2 cells with 8-bromo-cAMP (8-Br-cAMP), decortisol (Dex) alone or simultaneously for 30 minutes; mRNAs of gluconeogenesis genes, glucose-6-phosphatase ( G6Pase ) and phosphoenol pyruvate carboxykinase ( PEPCK ) were determined using β-actin as a standard. Insulin was used as a positive control group. Figure 1B provides treatment of cultured human liver tumor Hep3B/T2 cells with 8-Br-cAMP (8-Br-cAMP), decortisol (Dex) alone or simultaneously for 30 minutes, followed by different concentrations of HH-F3 and insulin. The results of the treatment; wherein mRNAs of the gluconeogenesis gene, 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 in inhibiting the expression activity of 8-Br-cAMP/Dex-induced gluconeogenesis co-activator PGC-1α in Hep3B/T2 cells. Figure 2A provides the results of treatment of cultured human liver tumor Hep3B/T2 cells with 8-Br-cAMP, decortisol (Dex) alone or simultaneously in serum-free DMEM medium, and 24 hours, in combination with HH-F3, Among them, PGC-1α mRNAs were measured by real-time quantitative PCR and normalized to β-actin, and insulin was used as a positive control group. Figure 2B shows the levels of PGC-1α and HNF-4α protein expressed in Hep3B/T2 cells treated with nuclear extracts as determined by Western blotting analysis, in which B23 is used as a standard; Dex: dicortisol (Dexamethasone); 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; these are representative data from 3 independent experiments. .

3A-3B show the effect of HH-F3 on the inhibition of gluconeogenesis gene expression by activating AMPK in cultured human liver tumor Hep3B/T2 cells. Figure 3A shows the results of pre-treatment 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 dicorticol for 30 minutes, followed by treatment with different concentrations of HH-F3 for 24 hours in serum-free DMEM; The luciferase reporter plastid driven by the glucose-6-phosphatase promoter was transfected into Hep3B/T2 cells, and the luciferase activity was co-transfected with pCMV-β-half compared to the control group. The lactobionase plastid is normalized by the activity of β-galactosidase. Figure 3C shows the results of cyclic AMP/DEX-stimulated 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. Analysis of luciferase activity; in which insulin was used as a positive control; and metformin was used as a positive control for AMPK.

4A-4B show the effect of HH-F3 in inhibiting 8-Br-cAMP/Dex-induced HBV core promoter activity in Hep3B/T2 cells. Figure 4A shows the results of a promoter activity assay; The luciferase reporter plastids driven by the HBV core promoter (CP) and the HBV X promoter (X promoter, XP) were transfected into Hep3B/T2 cells. Figure 4B shows the results after treatment with 8-Br-cAMP/Dex for 1 day in serum-free DMEM in the absence or presence of different concentrations of HH-F3; wherein cell lysates were prepared for luciferase activity Analysis; wherein the activity of the luciferase was normalized by β-galactosidase activity from the co-transfected pCMV-β-galactosidase plastid; and the insulin system was used as a positive control group.

Figures 5A-5E show the effect of HH-F3 on inhibition of HBV surface antigen, gene expression, gluconeogenesis performance, and HBV DNA levels in Hep3B/T2 or 1.3 ES2 cells. Figure 5A provides images showing cultured human liver tumor Hep3B/T2 cells treated with different concentrations 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 group. Figure 5B provides the results of treatment of cultured 1.3 ES2 cells with different concentrations of GP and HH-F3 in serum-free DMEM medium for 48 hours, wherein gluconeogenesis genes, G6Pase, PEPCK , and PGC were measured by real-time quantitative PCR. The expression of -1α mRNAs was normalized to β-actin; HE-145 was used as a positive control group. Figure 5C provides the results of treatment of cultured 1.3 ES2 cells with different concentrations of GP and HH-F3 in serum-free DMEM medium for 48 hours, in which the performance of HBV mRNA was measured by real-time quantitative PCR and β-muscle movement was used. Protein was used as a standard; HE-145 was used as a positive control group. Figure 5D provides the results of Western blot analysis using 1.3H2 cells treated with HH-F3 followed by antibodies against nuclear proteins; these are representative data from 3 independent experiments. HE-145 was used as a positive control group. Figure 5E provides the results of treatment of cultured 1.3 ES2 cells with different concentrations of GP and HH-F3 in serum-free DMEM medium for 48 hours, wherein wild-type HBV DNA in the medium was measured by real-time quantitative PCR and β- was used. Actin was used as a standard; HE-145 was used as a positive control group.

6A-6B show the effect of GP extract and HH-F3 in 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, phosphorylation-ACC (Ser ⁷⁹ ) (P-ACC), ACC, The antibodies of FASN and GAPDH were analyzed by Western blotting analysis (n=3). Figure 6B shows the results of Western blot analysis using cells treated with HH-F3 followed by antibodies against P-AMPK, AMPK, SREBP2, P-ACC, ACC, FASN, GAPD and β-actin, with BNL cells To produce subcutaneous tumors, the mice were divided into two groups: one group as an untreated control group (3 different mice), and the other four mice used HH-F3 (20 mg/day) via the oral (PO) route every day. Days) 3 weeks; after 3 weeks, the mice were sacrificed and the tumors were homogenized, followed by Western blotting and quantification.

Figure 7A provides images showing Huh7 cells treated with GP/HH-F3 to reduce lipid accumulation induced by oleic acid (OA) and palmitic acid (PA); wherein GP/HH-F3 is reduced in Huh7 Lipid accumulation induced by oleic acid (OA) and palmitic acid (PA); Huh7 cells were incubated with oleic acid (1 mM) and palmitic acid (2 mM) for 24 hours and 48 hours, resulting in oil red O (oil red O) Significant intracellular lipid accumulation visible by staining and cell viability by MTT assay; wherein the original magnification was 400-fold, which was treated with GP/HH-F3 for 48 hours (OA+PA/GP: oleic acid and palmitic acid) Co-treatment with GP; OA/HH-F3: oleic acid and palmitic acid co-treated with HH-F3).

Figure 7B provides the results of Huh7 cell counts (oleic acid and palmitic acid) in which the control group was treated with oleic acid and palmitic acid (OA + PA) and the cells were separately treated with oleic acid and palm The acid is co-treated with GP extract (GP) or HH-F3 for 24 or 48 hours to reduce fat load and quantify by staining intracellular lipid droplets with Oil Red O (OA+PA/GP: oleic acid and Palmitic acid was co-treated with GP; OA/HH-F3: oleic acid and palmitic acid 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 treatment of Huh7 cells with various concentrations of GP extract in the presence or absence of Lysawa (5 μM and 10 μM); cell viability was determined by SRB assay; data were expressed as mean ± three The standard deviation (SD) of an independent experiment. Figure 8B provides the results of treatment of Huh7 cells with various concentrations of HH-F3 extract in the presence or absence of Lysawa (5 μM and 10 μM); cell viability was determined by SRB assay; data was expressed as mean ± standard deviation (SD) of three independent experiments. Figure 8C provides the results of an isobologram analysis demonstrating a synergistic interaction between Lysawa and various concentrations of GP extract at 72 hours in Huh7 cells. Figure 8D provides the results of an isobologram analysis demonstrating a synergistic interaction between Lysawa and various concentrations of HH-F3 at 72 hours in Huh7 cells.

Figure 9 shows the effect of HH-F3 on Aβ _25-35- induced neurotoxicity in primary cortical neurons; pre-treatment of primary cortical neurons with vehicle (0.1% DMSO) or HH-F3 for 2 hours followed by exposure to 10 μM Aβ _{25-35 for} 40 hours; and after incubation, cell viability was determined by MTT assay; data were mean ± SD from at least three independent experiments and expressed relative to control cells, with * indicating use of Aβ and Significant differences between HH-F3 plus A[beta] treated cells (p<0.05).

Figures 10A-10C show the effect of HH-F3 on extracellular Aβ ₄₀ and Aβ ₄₂ levels in swAPP ⁶⁹⁵ -SH-SY5Y cell culture; in which APP ⁶⁹⁵ -SHSY-5Y- was incubated with vector or HH-F3 (using human APP695) Dyeed SHSY-5Y neuroblastoma cells) for 24 hours. Figure 10A shows cell viability of cells as determined by MTT assay. Figure 10B shows the level of extracellular A[beta] ₄₀ as determined by ELISA. Figure 1OC shows the level of extracellular A[beta] ₄₂ as determined by ELISA.

Figures 11A-11D show the effect of HH-F3 on APP treatment and Aβ clearance. Figure 11A provides the results of treating SH-SY5Y-APP695 cells with HH-F3 or γSI for 24 hours. The levels of APP (anti-Aβ1-16 antibody), APPX-F (anti-Aβ1-40 antibody), APP-CTF (anti-APPct antibody) and intracellular Aβ degrading enzyme were measured by immunotransfer method and APP-CTF was displayed. / Relative level of actin. Figure 11B provides treatment of SH-SY5Y-APP695 cells with HH-F3 for 24 hours, followed by Western blot analysis using antibodies against insulin-degrading enzyme (IDE) and enkephalinase (Neprilysin, NEP) Results; these are representative data from 3 independent experiments. Figure 11C provides the results of culturing SH-SY5Y-APP695 conditioned medium for 24 hours at the indicated concentrations of HH-F3. The remaining Aβ1-40 was measured by ELISA. Figure 11D provides the results of culturing SH-SY5Y-APP695 conditioned medium for 24 hours at the indicated concentrations of HH-F3. The levels of NEP and IDE in the conditioned medium were measured by immunotransfer.

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

Figures 13A-13B show the effect of HH-F3 on inhibition of A[beta] aggregation. Figure 13A provides the results of HH-F3 not incubated with Aβ for 24 hours. Fluorescence of Th-T was detected (Ex 440/Em 482). Figure 13B provides the results of HH-F3 incubated with Aβ1-40 for 24 hours. Fluorescence of Th-T was detected (Ex 440/Em 482). C = PBS; V = vector (Aβ1-40). Figure 13C provides the results of HH-F3 incubated with A? 1-42 for 24 hours. Fluorescence of Th-T was detected (Ex 440/Em 482). C=PBS; V=Load Body (Aβ 1-42).

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 μM of Aβ _{25-35 for} 46 hours. Cell viability was measured by MTT assay.

Figure 15 shows the effect of HH-F3 on A[beta] plaque load 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 cerebral amyloidosis in transgenic mice reveals early and robust pathology. EMBO 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 evaluated by BSB staining of A[beta] plaque load in the cerebral hemisphere of APP/PS1 mice, and then the number of A[beta] plaques was manually counted.

Figures 16A-16B show the effect of HH-F3 on A[beta] in the cerebral cortex of APP/PS1 mice. Figure 16A provides quantitative analysis of SDS soluble A?1-40 and A? 1-42 concentrations in cerebral cortex homogenates as determined by ELISA (vector n = 3, HH-F3n = 6). Figure 16B provides quantitative analysis of the concentration of Aβ1-40 and Aβ 1-42 of the insoluble fraction (formic acid soluble) in the cerebral cortex homogenate as determined by ELISA (vector n=3, HH-F3 n=6).

Figures 17A-17B show that HH-F3 up-regulates Aβ clearance-associated proteins in the cerebral cortex of APP/PS1 mice. Figure 17A provides the results of detecting Aβ clearance-related proteins of cortical lysates by immunotransfer. Figure 17B provides the results of a quantitative analysis of the protein between the determined WT and APP/PS1 or vector and APP/PS1.

Figure 18 shows the effect of GP extracted in different ways in inhibiting HBV surface antigen. It provides GP extraction that is shown to be extracted in different ways using serum-free DMEM medium. Images of cultured human liver tumor Hep3B/T2 cells were treated for 48 hours; (HBV surface antigen was determined by ELISA and normalized to MTT assay; insulin was used as a positive control group.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning meaning meaning meaning

The full names of the abbreviations used in this article are listed in the following table:

As used herein, the singular and " Thus, for example, reference to "a sample" includes a plurality of such samples and their equivalents as those of ordinary skill in the art.

The present invention provides a novel use of a DMSO extract of the genus Rhododendron or Rhodiola prepared by extracting a plant using DMSO, and refers to an extract treated in a metabolic disease.

According to the present invention, the extract can be prepared by extracting plants with dimethyl hydrazine (DMSO) using methods commonly used in the art or standard methods. In one embodiment of the invention, the leaves of the plant are ground and lyophilized to a powder and violently 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 in the genus, or part or parts of it. Combinations of more than one species of the genus or part thereof may also be considered. It is better to use Graptopetalum paraguayense .

In a preferred embodiment of the invention, the genus is a stone lotus.

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 of Rhodiola or parts thereof are also contemplated.

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

As used herein, the term "extract" refers to a solution obtained by soaking or mixing a substance 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 plant-derived anti-cancer components selected from the group consisting of genus, which is prepared by extracting plants using dimethyl hydrazine (DMSO). Then, it was separated by chromatography to obtain a fraction. In one embodiment of the invention, the plant is a stone lotus. This fraction was obtained by extracting plants using DMSO and separating them by chromatography to obtain fractions. In one embodiment of the invention, a Sephadex LH-20 column is used for chromatography, referred to as HH-F3. Therefore, this fraction HH-F3 is a potential therapeutic agent for the prevention or treatment of metabolic diseases.

According to an embodiment of the present invention, the stone lotus (GP) extract and the HH-F3 fraction can be prepared by the method indicated in U.S. Patent Publication No. 20120259004, the disclosure of which is incorporated herein by reference. Into this article.

It is also known that the HH-F3 fraction contains a proanthocyanidin rich in 3,4,5-trihydroxybenzylic components, the 3,4,5-three The hydroxybenzyl component has the structure of the following formula I,

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

And the structure of the former delphinidin (PD) is

As used herein, the term "metabolic disease" refers to any disease or disorder that disrupts normal metabolism. Metabolism is the process of converting food into energy at the cellular level. Metabolic diseases affect the ability of cells to undergo key biochemical reactions involving the processing or delivery of proteins (amino acids), carbohydrates (sugars and starches), or lipids (fatty acids). For example, metabolic diseases include diabetes, obesity, and fatty liver disease, whether it is 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 increase the risk of cardiovascular disease and diabetes when these medical conditions occur simultaneously.

As used herein, the term "AMPK pathway" refers to a pathway in which AMP-activated protein kinase (AMPK) plays a key role as a major regulator of cellular energy. The AMPK system 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 that responds to low ATP levels, AMPK activation is regulating signaling pathways that complement cellular ATP supply, including fatty acid oxidation and autophagy. AMPK negatively regulates the biosynthesis processes that consume ATP, including gluconeogenesis, lipids, and protein synthesis. AMPK is accomplished by direct phosphorylation of several enzymes directly involved in these processes and by transcriptional control of metabolism by phosphorylation of transcription factors, coactivators, and co-inhibitors. 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 is also involved in several species that are 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 basic catabolic mechanism involved in the degradation of cells of unwanted or abnormal cellular components by the action of lysosomes. Decomposition of this cellular component can be achieved by maintaining cellular energy levels to ensure that cells survive during starvation. If regulated, autophagy ensures the synthesis, degradation and recycling of cellular components. During this process, the cytoplasmic component of the target is isolated from the remaining cells within the autophagosomes, which are then fused to the lysosome and degraded or recycled.

The term "neurodegenerative disease" as used herein refers to the main influence on the human brain. The range of conditions of a neuron. Because neurons are the building blocks of the nervous system that include the brain and spinal nerves, and neurons are usually not copied or replaced by themselves, they cannot be replaced by the body when they are 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 a disease class involving amyloid. Amyloid is an insoluble fibrin agglomerate that shares specific structural features that occur in various improperly folded variants of proteins and polypeptides naturally present in the body. The misfolded structure changes their proper configuration, causing them to erroneously interact with each other or with other cellular components to produce insoluble filaments. It is known that amyloid is associated with pathology of severe human diseases, and abnormal accumulation of amyloid fibrils in organs may cause 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, lethal family insomnia, and rheumatoid arthritis.

It is believed that agents having the ability to inhibit A[beta] accumulation (or aggregation) may be suitable for amyloid-related diseases. In addition, protein aggregation is considered to be a common condition of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. Autophagy is a process of lysosomal degradation that recovers cellular waste and eliminates damaged organelles and protein aggregates, and obstacles in the autophagy pathway contribute to the pathogenesis of neurodegenerative diseases. Therefore, an autophagy inducing agent can be a therapeutic target for neurodegenerative diseases.

The present invention demonstrates that the GP extract or the HH-F3 fraction can inhibit the expression of the cyclic AMP/digocortisol-induced gluconeogenesis gene and the activity of the HBV core promoter in Hep3B/T2 cells. The examples also illustrate that the GP extract or HH-F3 fraction is in Hep3B/T2 or The gene expression of HBV surface antigen and gluconeogenesis enzyme was inhibited in 1.3ES2 cells; however, the GP extract/HH-F3 fraction did not affect the sugar decomposition, but shifted the metabolism of cancer cells from sugar decomposition to glucose oxidation. Inducing granulocyte-dependent apoptosis. It clarifies that the GP extract/HH-F3 fraction regulates the expression of lipid-related proteins and the expression of gluconeogenesis genes via AMPK-dependent manner; therefore, the GP extract/HH-F3 fraction can improve abnormal lipid accumulation. It also reduces the progression of tumor development, specifically in patients with type 2 diabetes who require insulin or metformin (Metformin) to inhibit liver gluconeogenesis in the body.

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 disruption in cellular homeostasis results in reduced response to physiological stress and organ dysfunction. Effective regulation of energy metabolism is a key requirement for cell homeostasis. AMPK is a major regulator of cellular responses to reduced ATP levels and acts as a sensor to maintain energy balance within the cell. AMPK is known to produce energy from glucose and fatty acid stimulation during stress and inhibits the energy expenditure of protein, cholesterol and glycogen synthesis. In general, activation of AMPK down-regulates synthetic pathways, such as protein, fatty acid, and cholesterol biosynthesis, but opens the catabolic pathways that produce ATP, such as fatty acid oxidation, glucose uptake, and sugar breakdown. It is achieved not only by direct phosphorylation of a variety of key metabolic enzymes, but also by altering the expression of genes in a tissue-specific manner. Currently, AMPK is considered an important molecular target because 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 present invention, it is shown that GP/HH-F3 is a AMPK activator. As a result, it is suggested that GP/HH-F3 has anti-aging ability by regulating the AMPK pathway.

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

As used herein, the term "therapeutically effective amount" refers to an amount of a medicament sufficient to achieve the desired therapeutic purpose. The therapeutically effective amount of the administered agent will vary with different factors, such as the nature of the agent, the mode of administration, the size and type of the animal receiving the agent, and the purpose for which it is administered. The therapeutically effective amount of each individual case can be determined empirically by the skilled artisan in light of the teachings herein and the methods established in the prior art.

The compositions of the present invention can 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, emulsifiers, demulcents, buffers, preservatives and the like. The final preparation step of the injection is sterilized or prepared in a sterile procedure. The compositions of the present invention may also be prepared as sterile solid preparations, for example by lyophilization, and may be employed after sterilization or dissolved in sterile injectable water or other sterile diluent immediately prior to use.

According to the invention, the composition can also be administered by oral route, wherein the composition can be in solid or liquid form. The solid compositions include tablets, pills, sachets, dispersed powders, granules, and the like. The oral compositions also include mouthwashes and sublingual tablets that can be applied to the oral cavity. The capsules include hard capsules and soft capsules. In such oral solid compositions, one The active compound or compounds may be mixed separately or mixed with a diluent, a binder, a pulverizer, a lubricant, a stabilizer, a co-solvent, and then formulated in a conventional manner. If necessary, the preparations may be coated with a coating agent or may be coated with two or more coating layers. In another aspect, the oral liquid composition comprises a pharmaceutically acceptable aqueous solution, suspension, emulsion, syrup, elixir, and the like. In such compositions, one or more of the active compounds can be dissolved, suspended or emulsified in conventional diluents (such as purified water, ethanol or mixtures thereof, etc.). In addition to such diluents, the compositions may contain wetting agents, suspending agents, emulsifying agents, sweetening agents, flavoring agents, perfuming agents, preservatives and buffers, and the like.

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

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

Example

Example 1 Preparation of GP extracts and fractions

The leaves of Stone Lotus (GP) were ground and lyophilized to a powder at -20 ° C and stored in a moisture barrier at 25 ° C prior to extraction. First, 1.5 g of GP powder was shaken vigorously with 10 ml of 100% methanol (MeOH) for 5 minutes, followed by centrifugation at 1,500 g for 5 minutes. After the supernatant was removed, 10 ml of 30% DMSO was added to each of the centrifuged precipitates to resuspend the respective extracts. The suspension was mixed by vigorous shaking for 5 minutes, centrifuged twice at 1,500 g for 5 minutes, centrifuged at 9,300 g for 5 minutes, and filtered at room temperature using a 0.45 μm filter paper. 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. The Western blotting method was used to analyze the degree of AURKA, AURKB, and FLJ10540 protein, and the active molecule was obtained in Fraction 3 and was referred to as HH-F3 fraction (data not shown). The HH-F3 fraction was further characterized by HPLC and ¹ H- and ¹³ C-NMR spectral analysis with a UV detector (Shimadzu SPD-M10A) and a normal phase HPLC column (Phenomenex Luna 5u Silica 100A, 4.6 x 250 mm). The structure of the active molecule (data not shown). The GP extract and/or HH-F3 was also dialyzed against water using a dialysis membrane (MWCO 12-14,000) (Spectrum Laboratories, Rancho Dominguez, CA) to obtain the active compound.

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

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

Cyclic AMP and decortisol (8-Br-cAMP/Dex) were used in this assay to synergistically activate key gluconeogenesis genes, glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxypyrrol (PEPCK). ) gene expression in human liver tumors Hep3B / T2 cells, which results are shown in Figure 1A. The results of the HH-F3 fraction (HH-F3) treatment are shown in Figure 1B, which indicates that treatment of the HH-F3 fraction can inhibit 8-Br-cAMP/DEX-induced G6Pase and PEPCK gene expression in Hep3B/T2 cells. .

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

Human liver tumor Hep3B/T2 cells were cultured and treated with either 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. The nuclear extract and HNF-4α protein levels of PGC-1α were determined by Western blot analysis. Use B23 as load control. Dex: Decortisol.

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

It is known that the expression of the co-activator PGC1-α drives transcription of key gluconeogenesis enzymes such as PEPCK and G6Pase, and is associated with the transcription factors HNF4-α and FoxO1, and that G6Pase and PEPCK are pathways by PGC1-α/FoxO1/Sirt1. Carry out regulation. The Hep3B/T2 cell line was treated with 8-Br-cAMP/Dex to induce the expression of gluconeogenesis genes, then treated with HH-F3, and 8-Br-cAMP and dicortisol synergistically activate genes and gluconeogenesis Protein expression of the agent PGC1-α gene. The results are shown in Figures 2A and 2B, which indicate that HH-F3 affects the pathway of PGC1-α/FoxO1/Sirt1; wherein treatment of HH-F3 inhibits 8-Br-cAMP/Dex induction in Hep3B/T2 cells PGC1-α expression levels (see Figure 2A); however, there was no effect on the expression of HNF4-α protein in human liver tumor Hep3B/T2 cells (see Figure 2B). In fact, it is known that the major metabolic regulator of the key gluconeogenesis gene and the coactivator PGC1-α have been shown to strongly co-activate FoxO1 and HNF4-α for HBV transcription. Figure 2C also indicates that treatment with HH-F3 inhibits the expression of PGC1-α, HNF4-α, and FoxO1 in Hep3B/T2 cells.

Example 4 The HH-F3 action of the gluconeogenesis gene expression was inhibited by 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 the assay of promoter activity, Hep3B/T2 cells were transfected with luciferase reporter plastids driven by the glucose-6-phosphatase promoter. Luciferase activity was normalized from co-transfected pCMV-[beta]-galactosidase plastids to [beta]-galactosidase activity compared to control. Cyclic AMP/DEX stimulated G6Pase promoter activity was tested in the presence of HH-F3 or HH-F3 and AMPK inhibitor Compound C. After one day of treatment, cell lysates were prepared for luciferase activity assay. 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 activity of the G6Pase promoter and then treated with HH-F3. The results are shown in Figures 3A and 3B, which show that HH-F3 can reduce the activity of the G6 Pase promoter in a dose-dependent manner. It was previously reported that AMP-activated protein kinase (AMPK) is a cellular energy sensor that inhibits ATP depletion and stimulates ATP production in the event of energy depletion. Activation of AMPK is known to inhibit gene expression of G6Pase and PEPCK and inhibit hepatic glucose production. Therefore, testing HH-F3 regulates G6Pase via the AMPK pathway Effect. This result shows that the AMPK inhibitor compound C partially reverses the H6-F3 inhibited G6Pase promoter activity (see Fig. 3C), which indicates that HH-F3 can act through the activation of AMPK to inhibit the gene expression of gluconeogenesis.

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

The luciferase reporter plastids driven by the HBV core promoter (CP) and the HBV X promoter (XP) were transfected into Hep3B/T2 cells for promoter activity assay. Then, cells were treated with 8-Br-cAMP/Dex in serum-free DMEM in the absence or presence of different concentrations (0 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) of HH-F3. Days, cell lysates were prepared for luciferase activity assays. Luciferase activity was normalized from co-transfected pCMV-[beta]-galactosidase plastids to [beta]-galactosidase activity. Insulin was used as a positive control.

Hep3B/T2 is a hepatocyte cell line that expresses HBV. The HBV genome includes polymerase, surface, core, and HBx. This X gene encodes the X protein (HBx), which has anti-activating properties and may be important in the carcinogenesis of the liver. This core gene encodes the core nuclear sheath protein (important in viral packaging). Previous in vitro studies have suggested that mutations in the core promoter increase HBV replication. As shown in the results of Figure 4A, cyclic AMP and dicortisol synergistically activated the activity of the B liver viral core promoter, but had no effect on the X promoter. As shown in Figure 4B, the dose-dependent inhibition of 8-H-FAMP/Dex-induced HBV core promoter activity of HH-F3. This test results support that HBV infection is associated with diabetes and HCC, and provides 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 gluconeogenesis enzyme expression in Hep3B/T2 or 1.3 ES2 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 using the MTT assay. Insulin was used as a positive control. 1.3 ES2 cells were treated in serum-free DMEM medium for 48 hours using different concentrations of HH-F3. The 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 (Serum free): serum free).

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

In addition, a 1.3 ES2 cell line derived from HepG2 cells and containing an integrated replication HBV genome was used to discuss whether HH-F3 can inhibit the expression of the HBV gene via regulation of PGC1-α. As shown in Figures 5B-5E, HH-F3 inhibits key gluconeogenesis genes, G6Pase and PEPCK gene expression, and peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) gene, at 1.3 HBV gene expression in ES2 cells.

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

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

It has been recently discovered that cancer associated with abnormal lipid production is known, which shows mainly Inhibition of lipogenic enzymes can reduce tumor formation. Lipid lines are associated with a number of pathological processes, including hepatic steatosis (fatty liver), which is manifested by excessive accumulation of lipids in hepatocytes. Abnormal lipid accumulation is associated with polygenic genetic defects and hepatic malignancies in energy metabolism. Liver cancer cells require more energy to survive through the regeneration of dysregulated lipids, which may contribute to liver tumor formation and human HCC prognosis. In HCC, the extent of this abnormal lipid production is associated with clinical aggressiveness, activation of the AKT-mTOR signaling pathway, and inhibition of AMPK. Activation of hepatic AMPK, a metabolite-sensing kinase that plays a protective role in metabolic stress, stimulates fatty acid oxidation and increases phosphorylation with acetyl CoA carboxylase (ACC) and HMG-, respectively. The deactivation of CoA reductase (HMG-CoA reductase, HMGCR), a rate-limiting enzyme for the re-synthesis of fatty acids and cholesterol, sharply shuts down the synthesis of fatty acids. Furthermore, the protein expression of this fatty acid synthase (FASN) is known to be negatively regulated by AMPK. In fact, due to its tissue distribution and unusual enzymatic activity, the target FASN, which is a key enzyme in lipid production and overexpressed in HCC, offers an opportunity for therapeutic applications.

In this test, it was found that HH-F3 can inhibit the activity of 8-Br-cAMP/Dex-induced gluconeogenesis gene and HBV core promoter through the AMPK pathway and PGC1-α. Furthermore, it is known that the AMPK pathway and PGC1-alpha also regulate lipid production. Therefore, a test was performed to check whether GP/HH-F3 has a role in lipogenesis. As shown in Figure 6, treatment with GP or HH-F3 in HCC cell lines and in BALB/c mice reduced fatty acids by activating AMPK (increasing AMPK phosphorylation) and inhibiting activated AKT (reducing AKT phosphorylation) (fatty acid, FA) Synthetic activity, BALB/c mice were injected subcutaneously on the flank of BNL cells. Furthermore, treatment with GP/HH-F3 resulted in increased phosphorylation and inactivation of the AMPK downstream target ACC, as well as a decrease in FASN protein expression levels in a concentration-dependent manner. These results indicate that GP/HH-F3 processing can be in HCC Reduce the activity of fatty acid synthesis.

Example 8 GP/HH-F3 reduced the effects of 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 significant intracellular lipid accumulation visible by Oil Red O staining and cell viability as determined by MTT assay. Figure 7A shows the original magnification (x400) showing the control (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 use of 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, were able to reduce fat load 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 co-processed 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 is still 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 sharply shut down the synthesis of fatty acids via increased phosphorylation and inactivation of ACC, as well as reduced transcriptional activation of SREBP-1c and SREBP-2. In addition, SREBP-1c regulates lipid-related genes in the liver, including ACC and FAS , while SREBP-2 activates two key enzymes for cholesterol biosynthesis, Hmgcr and Hmgcs1 , which play important roles in tumor formation.

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

Various concentrations of GP (0 μg/ml, 50 μg/ml, 150 μg/ml, 250 μg/ml, and 500 μg/ml) were used in the presence or absence of Lysa (5 μM and 10 μM), respectively. Huh7 cells were treated with HH-F3 (0 μg/ml, 5 μg/ml, 15 μg/ml, 25 μg/ml, and 50 μg/ml). Cell viability was determined by this SRB test. Data are expressed as mean ± standard deviation (SD) of three independent experiments. In addition, isobologram analysis demonstrated a synergistic interaction between Lysawa and various concentrations of GP/HH-F3 at 72 hours in Huh7 cells.

Various concentrations of GP and HH-F3 and Lysawa (the only drugs approved by the FDA for pre-HCC patients) were combined in Huh7, and the results of the cell proliferation test performed at 72 hours are shown in Figures 8A and 8B. The combination index (CI) analysis is also shown in Figures 8C and 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 by the results, both GP and HH-H3 synergistically inhibited the proliferation of Huh7 with Lysawa.

Example 10 HH-F3 completely destroys Aβ-induced neurotoxicity in primary cortical neurons and attenuates Aβ production in cell culture of swAPP ⁶⁹⁵ -SH-SY5Y

The primary cortical neuron was 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 determined by MTT assay. As shown in Figure 9, 10 μM of Aβ _25-35 fibrils detected by MTT reduction resulted in a significant decrease in cell viability by 39.4 ± 7.5%, and Aβ _25-35 induced cell death was attenuated by HH-F3 in a dose-dependent manner. .

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

Extracellular Aβ accumulation can be reduced by inhibiting the amyloid degeneration pathway or increasing Aβ clearance, including enhancing Aβ degrading enzyme activity and autophagy. Therefore, an immunotransfer method was performed to detect the levels of APP and APP-CTF (Fig. 11A). There was no significant change in the level of overall APP after 24 hours of treatment with the indicated concentrations of HH-F3. Unexpectedly, when APP was immunotransferred with an anti-Aβ 1-40 antibody, an unidentified APP species (APP-XF) was detected and the species was reduced by HH-F3. Basically, the level of the APP-CTF is increased (Fig. 11A). This γ-secretase inhibitor was used to verify the involvement of γ-secretase in APP-CTF levels (Fig. 11A). On the other hand, major Aβ degrading enzymes such as insulin degradation enzyme (IDE) and enprilysin (NEP) in cells were not affected by HH-F3 (Fig. 11B). The remaining Aβ1-40 was examined by 24 hours of incubation to detect the activity of Aβ degrading enzyme in the conditioned medium of SH-SY5Y-APP695 cells (Fig. 11C). The results showed that HH-F3 activated the activity of Aβ degrading enzyme 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. Recent reports indicate that beclin-1 (an early autophagy regulator) is down-regulated in the brains of AD patients (Pickford et al, The autophagy-related protein beclin 1 shows reduced expression in early Alzheimer disease and regulates amyloid beta accumulation in mice) , J Clin Invest 2008; 118: 2190-2199). The effect of HH-F3 on autophagy activation in swAPP ⁶⁹⁵ -SH-SY5Y cells was also tested. The APP ⁶⁹⁵ -SHSY-5Y cell line was incubated with vehicle or HH-F3 for 24 hours and the LC3 autophagy marker was detected by Western blotting. This result is shown in FIG.

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 (TH-T), a molecule that can be inserted into the β-sheet structure of Aβ, is used to detect Aβ aggregation. When combined with amyloid fibrils, the emission of Th-T shifts, 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) inhibited the formation of both Aβ _1-40 and Aβ _1-42 fibrils. HH-F3 inhibited aggregation of both Aβ _1-40 and Aβ _1-42 in a concentration-dependent manner ( FIG. 13 ).

Prevention of Aβ-mediated neurotoxicity has become an important therapeutic strategy for AD therapy. Aβ _25-35 is a synthetic peptide of 11 amino acids in the intermembrane domain of APP. It is obvious that Aβ _25-35 cannot be produced by normal APP treatment. However, the Aβ _25-35 line was selected as a model for full-length Aβ because it retains both its physical and biological properties, while its short length can 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: a general strategy to prevent amyloid formation. J Biol Chem 2000;275:25109-25115). Although it is not present in biological systems, the Aβ _25-35 fragment has been widely used or replaced by many scientists with 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 a fragment of the Amyloid precursor associated with Alzheimer's disease. Science 1989; 245: 417-420). Cortical neurons were pretreated with HH-F3 for 2 hours. Subsequently, cells were stimulated with 10 μM of Aβ _{25-35 for} 46 hours. This Aβ _25-35 -mediated loss of cell viability was significantly restored by HH-F3. 20 and 50 μg/ml of HH-F3 significantly restored cell survival rates of 19% and 33%, respectively. The concentration-dependent recovery effect of the HH-F3 on cell viability occurred, and the HH-F3 treatment was before Aβ treatment but not after treatment (Fig. 14).

HH-F3 has multiple functions in AD pathology in vitro. Therefore, the test of this animal model was performed to investigate the in vivo effect of HH-F3. The APP/PS1 transgenic mice developed A[beta] plaques in the cerebral cortex at 120 days of age (Radde et al., 2006). The 140-day-old APP/PS1 mouse was orally administered HH-F3 (300 mg/kg/day) for one month. In an in vitro assay, the present inventors have demonstrated that HH-F3 can inhibit A[beta] coagulation. Therefore, amyloid plaques in the cerebral hemisphere were stained (Fig. 15). Due to its high affinity with Aβ, the dye 1-bromo-2,5-bis(3-carboxy-4-hydroxystyryl)benzene , BSB) has been used to detect amyloid plaques. This result shows that HH-F3 only slightly reduces the load of Aβ plaques. The number of Aβ plaque loads between the vehicle group and the HH-F3 group was 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 soluble in a 2% SDS fraction which may contain monomers, dimers and oligomers. The insoluble A[beta]s are soluble in the 70% formic acid fraction. Soluble and insoluble Aβs 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 associated with Aβ clearance in the cerebral cortex of APP/PS1 mice were detected by immunotransfer method (Fig. 17A). The IDE levels of the transgenic mice were down-regulated compared to wild-type mice, while the levels of NEP and APOE were up-regulated by HH-F3. Furthermore, PPARγ and pAMPK levels were down-regulated in this transgenic mouse compared to wild-type mice, while PPARγ, pAKT and pAMPK were significantly up-regulated by HH-F3 (Fig. 17B).

The crude extract of GP (H ₂ O, methanol, 100% ethanol, 70% ethanol, or 50% ethanol) did not significantly affect HBsAg levels in Hep3B/T2. GP extracts in DMSO, 30% DMSO, and 80% acetone partially reduced HBsAg levels in Hep3B/T2 (Figure 18).

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

In conclusion, the 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 high blood caused by obesity Cholesterol or high triglyceride levels. It is suggested that GP extract or HH-F3 fraction can be used to treat or prevent metabolic syndrome.

Furthermore, it is indicated in the present invention that the GP extract or HH-F3 fraction is capable of activating the AMPK pathway and the autophagy pathway. Therefore, it is recommended that the GP extract or the HH-F3 fraction be developed into a drug for preventing or treating a neurodegenerative disease or an amyloid-related disease, such as Parkinson's disease, Alzheimer's disease, and Huntington. And even anti-aging drugs.

It is believed that one of ordinary skill in the art to which the present invention pertains can utilize the invention to the broadest scope and no further description. The scope of the invention is to be construed as being limited by the scope of the invention.

Claims (29)

Use of a composition for the preparation of a medicament for treating a metabolic disease, wherein the composition comprises a dimethyl sulfoxide (DMSO) from Graptopetalum sp. or Rhodiola sp. An extract or fraction, or an active compound isolated from the DMSO extract or fraction. For example, the use of the patent scope of the first aspect, wherein the metabolic disease is diabetes, obesity, fatty liver disease, hepatitis B virus (HBV) related liver related diseases, diabetes related liver related diseases, obesity-related Hepatic-related disease, or obesity-induced high blood cholesterol or high triglyceride levels. The use of the second aspect of the patent application, wherein the metabolic disease is diabetes. The use of the second aspect of the patent application, wherein the metabolic disease is obesity. The use of the second aspect of the patent application, wherein the metabolic disease is fatty liver disease. The use of the fifth aspect of the patent application, wherein the fatty liver disease is alcoholic fatty liver disease (AFLD) or nonalcoholic fatty liver disease (NAFLD). The use of the second aspect of the patent application, wherein the metabolic disease is a hepatitis B virus (HBV) related liver related disease. The use of the second aspect of the patent application, wherein the metabolic disease is a liver-related disease associated with diabetes. The use of the second aspect of the patent application, wherein the metabolic disease is a liver-related disease associated with obesity. The use of any one of the claims 7-9, wherein the liver related disease is hepatocyte Hepatocellular carcinoma (HCC), liver fibrosis or cirrhosis. The use of the scope of claim 10, wherein the liver related disease is hepatocellular carcinoma (HCC). For example, the use of the hepatitis B virus (HBV)-related liver-related disease is HBV-induced liver fibrosis and cirrhosis. Use of a composition for the preparation of a medicament for preventing or treating metabolic syndrome, wherein the composition comprises a dimethyl hydrazine (DMSO) extract or fraction from the genus Rhodiola or Rhodiola, or a DMSO extract or fraction of active compound. Use of a composition for the preparation of a medicament for preventing or treating cancer, wherein the composition comprises a dimethyl hydrazine (DMSO) extract or fraction from the genus Rhodiola or Rhodiola, or an isolated from the DMSO The active compound of the extract or fraction is combined with a synergistic ratio of an anticancer drug in preventing or treating cancer. For example, the application of the scope of claim 14 wherein the cancer is liver cancer, and the anticancer drug is an anti-hepatocarcinoma drug. For example, the application of the scope of claim 15 wherein the anticancer drug is Sorafenib. The use of the scope of claim 15 wherein the cancer is HCC and the anticancer drug is Lysawa. Use of a composition for the manufacture of a medicament for activating an AMPK pathway or an autophagy pathway, wherein the composition comprises a dimethyl hydrazine (DMSO) extract or fraction, or a separation from the genus Rhodiola or Rhodiola The active compound from the DMSO extract or fraction. Use of a composition for the preparation of a medicament for preventing or treating a neurodegenerative disease, wherein the composition comprises a dimethyl hydrazine (DMSO) extract or fraction from the genus Rhodiola or Rhodiola, or isolated from The DMSO extract or fraction of the active compound. The use of claim 19, wherein the neurodegenerative disease is Parkinson's disease, Alzheimer's disease, and Huntington's disease. Use of a composition for the preparation of a medicament for preventing or treating amyloid-related diseases, wherein the composition comprises a dimethyl hydrazine (DMSO) extract or fraction from the genus Rhododendron or Rhodiola Or an active compound isolated from the DMSO extract or fraction. The use of the scope of claim 21, wherein the amyloid-related diseases are Alzheimer's disease, type 2 diabetes, Parkinson's disease, Huntington's disease, and fatal family insomnia (Fatal Familial Insomnia) Or rheumatoid arthritis. Use of a composition for the preparation of an anti-aging agent, wherein the composition comprises a dimethyl hydrazine (DMSO) extract or fraction from the genus Rhodiola or Rhodiola, or is isolated from the DMSO extract or grade The active compound of the fraction. The use of any one of the claims 1 to 23 , wherein the genus is Graptopetalum paraguayense . The use of any one of claims 1 to 23, wherein the Rhodiola rosea is Rhodiola rosea . The use of any one of the preceding claims, wherein the fraction is enriched in an active ingredient by extracting the plant with dimethyl hydrazine (DMSO), followed by chromatographic separation. A fraction called HH-F3 was obtained. For example, the use of the Sephadex LH-20 column is used for chromatography. The use of any one of the preceding claims, wherein the active compound is a compound having the structure of Formula I Wherein one of R is H or a procucyanidin (PC) unit, and the other R is OH or a prodelphindine (PD) unit; n is a number ranging from 21 to 38; and the PC unit is PD The ratio of units does not exceed 1:20. A compound as claimed in claim 28 for the preparation of a medicament for the treatment of a metabolic disease, prevention or treatment of metabolic syndrome, cancer, or neurodegenerative diseases, amyloid-related diseases; and activating an AMPK pathway or an autophagy pathway Or the use of anti-aging drugs.