WO2017201994A1 - 牛樟芝萃取纯化物的用途 - Google Patents

牛樟芝萃取纯化物的用途 Download PDF

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WO2017201994A1
WO2017201994A1 PCT/CN2016/107285 CN2016107285W WO2017201994A1 WO 2017201994 A1 WO2017201994 A1 WO 2017201994A1 CN 2016107285 W CN2016107285 W CN 2016107285W WO 2017201994 A1 WO2017201994 A1 WO 2017201994A1
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purified
extract
antrodia camphorata
group
liver
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WO2017201994A9 (zh
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施纯青
郭悦雄
林正修
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施纯青
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/54—Lauraceae (Laurel family), e.g. cinnamon or sassafras
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones

Definitions

  • the invention relates to an extract of Antrodia camphorata, in particular to a purified dehydroeburicoic acid (TT) extracted from the mycelium of Antrodia camphorata for preparing anti-type 2 diabetes and anti-dyslipidemia and reducing liver fat
  • TT dehydroeburicoic acid
  • Diabetes is the main cause of multiple causes such as chronic hyperglycemia and metabolic cardiovascular disease due to insulin resistance or insufficient insulin secretion.
  • type 2 diabetes accounts for about 90% of all cases of diabetes. %-95%, characterized by insulin resistance.
  • the pathogenesis of type 2 diabetes involves insulin resistance, and 5% of the patients are due to defects in beta cell function in the pancreas.
  • the insulin resistance occurs when insulin loses its ability to exhibit diverse biochemical reactions.
  • causes symptoms including: dyslipidemia, obesity, and high blood pressure.
  • the pathology of insulin resistance comes from factors such as genetics and lifestyle, and the food intake is the main cause of these metabolic disorders, and the proportion of fat in the food intake is particularly important.
  • the pancreas secretes insulin and maintains a normal homeostasis of blood glucose, helping glucose uptake and regulating carbohydrate and lipid metabolism.
  • Research data show that glucose transporter 4 (GLUT4) has been seen as a role in regulating the steady state of glucose concentration in the blood; insulin stimulates glucose uptake, which promotes GLUT4 translocation from intracellular sites. Point to the cell membrane.
  • GLUT4 glucose transporter 4
  • the amount of expression of the membrane GLUT4 protein is considered to be an indicator of insulin reactivity, and the pathological defect that often occurs in insulin resistance is that skeletal muscle is stored from the intracellular storage site to the cell membrane.
  • the Akt/PKB message pathway plays a central role in the uptake of insulin-stimulated glucose on skeletal muscle and adipose tissue, and the action of insulin on the uptake of grapevines in surrounding tissues by Akt/PKB translocation to the cell membrane via GLUTs
  • the ability to promote glucose uptake by glucose-operating protein 4 (GLUT4) Decreased performance of GLUT4, GLUT4 translocation, and/or insulin pathway will result in insulin resistance, and hyperglycemia. Therefore, improving the content of the cell membrane GLUT4 or protein will be a therapeutic method.
  • AMP-activated protein kinase is responsible for the regulation of energy in cells and in vivo.
  • Glucose uptake efficiency including activation of Akt/PKB by insulin-dependent machine turnover, stimulation by muscle contraction induction, or AMPK stimulation by hypoxia.
  • the regulation mechanism of AMPK phosphorylation pathway is different from the regulation mechanism of GLUT4 translocation, but it is related to the metabolic disorder of lipid and glucose caused by insulin resistance. Accordingly, the therapeutic effect of AMPK activators on diabetes and related diseases is expected.
  • Metformin (Metf) is a commonly used anti-diabetic drug in clinical practice. Metformin (Metf) is a less potent compound that is usually administered at high doses, but results in the greatest efficacy of only moderate net worth; and, in addition, will cause significant side effects.
  • Antrodia camphorata (A. camphorata) (Polyporaceae, Pleurotus ostreatus) is a traditional Chinese medicinal material in Taiwan, which is extremely rare and precious because it grows only in the evergreen burdock Cinnamomum kanehirai tree. Material section. A. camphora has a variety of physiological functions.
  • Antrodia camphorata fruiting bodies include terpenoids such as arsenic acid A, B, C, E, F, and K, and zhankuic acids A, B, C, D, and E, 15 ⁇ acetyl dehydrothiochromic acid (15 ⁇ -acetyl-dehydrosulphurenic acid), dehydroeburicoic acid (chemical structure shown in Figure 1), and dehydrosulphurenic acid (TR4), methyl antcinate G, H, And a purified product such as eburicoic acid (TR1).
  • Antcin K (AnK) (chemical structural formula shown in Figure 1B) is the most important active ingredient of the body of A. niger.
  • the identified compounds of Antrodia camphorata include antroquinonol, 4-acetylantroquinonol B, succinic and maleic derivatives.
  • the solid state cultivation and the leaching culture filtrate of the fruit body were confirmed to exhibit hepatoprotective and antioxidant activities.
  • Dehydroeburicoic acid (TT) can be extracted via Poria cocos and dehydroeburicoic acid (TT).
  • mice When C57BL/6J mice were fed a high-fat diet (HFD), they induced early type 2 diabetes, significantly increased adipose tissue weight, created insulin resistance, and increased blood glucose levels. And increase the concentration of triglyceride (TG) and total cholesterol (TC) in blood. Phosphorylation at the Thr 172 position on the alpha subunit is critical for AMPK activity. Skeletal muscle and adipose tissue play a unique role in regulating the homeostasis of insulin-dependent glucose. Skeletal muscle is the most important part of glucose uptake by systemic insulin mediators. Adipose tissue accounts for a small portion of postprandial glucose placement, while skeletal muscle plays the most important part of glucose uptake.
  • HFD high-fat diet
  • this study evaluated the performance of GMUT crude extract (CruE) and AnK (the main component of this mushroom) in GLUT4 and phosphorylated Akt in vitro. Further investigation was conducted to investigate the role and mechanism of dehydrogenated and hypolipidemic effects induced by hydrogen perforate (TT) in high-fat diet-induced diabetic mice. This animal model induces type 2 diabetes, a current study investigating whether dehydrogenated tonic acid (TT) increases cell membrane glucose uptake and phosphorylation of AMPK.
  • TT dehydrogenated tonic acid
  • PPAR ⁇ peroxidase-activated peroxisome proliferator-activated receptor alpha
  • FAS fatty acid synthase
  • glycoprotein glycoprotein
  • the invention relates to a medicine for extracting and purifying the extract of Antrodia camphorata, which is used for preparing a medicine compound for treating type 2 diabetes and lowering blood sugar, the compound is a purified product extracted from the mycelium of Antrodia camphorata, and the purified substance is dehydroperporic acid (dehydroeburicoic) Acid, C 31 H 48 O 3 ).
  • the invention relates to a method for extracting purified extract of Antrodia camphorata, wherein the purified product is a purified hydrolysate (dehydroeburicoic acid) extracted from the mycelium of Antrodia camphorata; and is used for preparing a pharmaceutical compound for reducing dyslipidemia.
  • the purified product is a purified hydrolysate (dehydroeburicoic acid) extracted from the mycelium of Antrodia camphorata
  • the invention relates to a method for extracting purified extract of Antrodia camphorata, wherein the purified product is dehydroeburicoic acid purified from the mycelium of Antrodia camphorata; and is used for preparing blood fat lowering including total cholesterol (TC) or triglyceride Pharmaceutical compound of ester (TG).
  • TC total cholesterol
  • TG triglyceride
  • TC total cholesterol
  • TG triglyceride
  • the invention relates to a method for extracting purified extract of Antrodia camphorata, wherein the purified product is a pharmaceutical compound of dehydroeburicoic acid purified from the mycelium of Antrodia camphorata; the effect of lowering blood sugar of the purified product is to increase skeletal muscle
  • GLUT4 membrane glucose carrier protein 4
  • mRNA expression of G6Pase and 11 ⁇ hydroxysteroid dehydrogenase 11beta-HSD1
  • phospho-AMPK protein kinases
  • the invention relates to a method for extracting purified extract of Antrodia camphorata, wherein the purified product is a pharmaceutical compound of dehydroeburicoic acid purified from the mycelium of Antrodia camphorata; the anti-type 2 diabetes use of the purified product is used for preparation Increases phosphorylation of skeletal muscle protein kinase B (Akt) and increases islet sensitivity.
  • the purified product is a pharmaceutical compound of dehydroeburicoic acid purified from the mycelium of Antrodia camphorata
  • the anti-type 2 diabetes use of the purified product is used for preparation Increases phosphorylation of skeletal muscle protein kinase B (Akt) and increases islet sensitivity.
  • Akt skeletal muscle protein kinase B
  • the invention relates to the use of extracting and purifying the extract of Antrodia camphorata, wherein the purified product is a purified substance extracted from the mycelium of Antrodia camphorata A pharmaceutical compound of dehydroeburicoic acid; the use of the purified substance for lowering blood fat and liver is to increase phosphorylated adenosine monophosphate-activated protein kinase (phospho-AMPK) in the liver.
  • phospho-AMPK phosphorylated adenosine monophosphate-activated protein kinase
  • the invention relates to the use of extracting and purifying the extract of Antrodia camphorata, wherein the purified product is a purified compound of dehydroeburicoic acid extracted from the mycelium of Antrodia camphorata; the use of the purified product for lowering liver lipid is reduced by The content of total lipid and triacylglycerol in the liver, as well as reducing the phenomenon of ballooning degeneration in the liver.
  • the invention relates to a method for extracting and purifying the extract of Antrodia camphorata, wherein the purified product is a pharmaceutical compound of dehydroeburicoic acid purified from the mycelium of Antrodia camphorata; the use of the purified compound for treating high triglyceride is transparent Increased liver fatty acid oxidase (PPAR ⁇ ) and decreased protein expression of fatty acid synthase (FAS) in the liver, and decreased cholesterol regulatory element binding protein 1c (Stere regulatory element binding protein, SREBP-1c), glycerol- 3-phosphate acyltransferase (GPAT), adipocyte fatty acid binding protein 2 (aP2), and increased mRNA expression of CPT1a and uncoupling protein 3 (UCP3).
  • PPAR ⁇ liver fatty acid oxidase
  • FAS fatty acid synthase
  • SREBP-1c glycerol- 3-phosphate acyltransferase
  • GPAT gly
  • the invention relates to a method for extracting purified extract of Antrodia camphorata, wherein the purified product is a pharmaceutical compound of dehydroeburicoic acid purified from the mycelium of A. angustifolia; the purified product is used for preparing blood cholesterol lowering, It is because the amount of mRNA expressed by SREBP-2 is lowered, and the total cholesterol (TC) value in the blood is lowered.
  • the invention relates to a method for extracting and purifying the extract of Antrodia camphorata, wherein the purified product is a purified compound derived from dehydroeburicoic acid extracted from the mycelium of Antrodia camphorata; the use of the purified product is for preparing a therapeutic high-fat diet The resulting high leptinemia, which reduces the concentration of leptin in the blood.
  • the invention relates to a method for extracting and purifying the extract of Antrodia camphorata, wherein the purified product is a purified compound of dehydroeburicoic acid extracted from the mycelium of Antrodia camphorata; the use of the purified product is for preparing a low-fat diet The resulting high visceral fat mass and the reduction of the hypertrophy of adipocyte.
  • the invention relates to a method for extracting and purifying the extract of Antrodia camphorata, wherein the purified product is a pharmaceutical compound of dehydroeburicoic acid purified from the mycelium of Antrodia camphorata; the use of the purified substance for reducing visceral fat is used for preparation increase
  • the protein expression level of PPAR ⁇ oxidized by fatty acids of adipose tissue reduces the amount of protein expression of FAS and PPAR ⁇ of lipid production in adipose tissue.
  • the detoxified perforate is extracted from the purified material of the extract of Antrodia camphorata, and the diabetes and other related symptoms induced by the high fat diet (HFD) are obtained by administering the purified substance to the hydrogen perforated acid.
  • HFD high fat diet
  • TG plasma triglyceride
  • TC total cholesterol
  • Dehydrogenated perforate treatment increased the expression of GLUT4 and the expression of p-AMPK in the liver and skeletal muscle of diabetic mice induced by HFD, indicating that dehydrogenated perforate has the effect of improving hyperglycemia.
  • G6-Pase glucose-6-phosphatase
  • glucose-6-phosphate glucose-6-phosphate
  • dehydrogenated perforate treatment has the effect of promoting the reduction of blood triglyceride.
  • dehydrogenated perforate treatment After dehydrogenated perforate treatment, the mRNA expression of SREBP-2 in diabetic mice was decreased by HFD and the blood cholesterol level decreased. It was shown that dehydrogenated perforating acid can increase the skeletal muscle cell membrane GLUT4 protein and regulate PPAR ⁇ and FAS. And increase the expression of p-AMPK/t-AMPK protein in skeletal muscle and liver tissue to prevent or improve the diabetes and dyslipidemia status of diabetic mice induced by HFD.
  • Figure 1 is a chemical structural formula of the purified dehydroporous acid (TT).
  • 2A to 2E are effects of in vitro administration of insulin or Astragalus membranaceus crude extract (CruE) on cell membrane GLUT4 and phosphorylated Akt/total Akt in C2C12 myyotube cells; and administration of insulin or dehydrogenated perforate (TT) and Antcin K compared to the plasma membrane GLUT4 and phosphorylated Akt / total Akt affect both (ANK), wherein FIG. 2A and 2C show produced in C 2 C 12 myotube cell culture experiments representative immunoblots; and FIG.
  • FIG. 3A and 3B show the mouse white of the CON group, the HF group, the HF+TT1 group, the HF+TT2 group, the HF+TT3 group, the HF+Feno group, and the HF+Metf group in the animal model experiment of the present invention.
  • Pathological tissue staining of HE staining of adipose tissue Fig. 3A
  • liver tissue Fig. 3B
  • Figure 4A shows the experimental animal model of the present invention in the liver tissues of mice in the CON group, the HF group, the HF+TT1 group, the HF+TT2 group, the HF+TT3 group, the HF+Feno group, and the HF+Metf group.
  • 4B to 4C show that the signals measured by G6Pase, 11 ⁇ -HSD1, SREBP1c, GPAT, aP2, UCP3, CPT1a, and SREBP2 in FIG. 4A were quantified by image analysis, and each value was normalized by GAPDH.
  • Fig. 5A shows the animal model experiment of the present invention, using Western blot analysis for the CON group, the HF group, the HF+TT1 group, the HF+TT2 group, the HF+TT3 group, the HF+Feno group, and the HF+.
  • the protein content of the cell membranes GLUT4 and phospho-AMPK (Thr 172 ) in the mouse skeletal muscle of the Metf group was measured, and the protein content of phospho-AMPK (Thr 172 ) in the liver of the mice of each group described above was measured.
  • Figure 5B shows a quantitative histogram of the protein content measured in Figure 5 (A).
  • Fig. 6A shows the animal model experiment of the present invention, using Western blot analysis for the CON group, the HF group, the HF+TT1 group, the HF+TT2 group, the HF+TT3 group, the HF+Feno group, and the HF+.
  • the protein content of PPAR ⁇ , FAS, and PPAR ⁇ in the liver of the mouse of the Metf group was measured, and the protein content of PPAR ⁇ and FAS in the mouse adipose tissue of each group described above was measured.
  • Figure 6B shows a quantitative histogram of the protein content of PPAR ⁇ , FAS, PPAR ⁇ in the liver of mice as measured in Figure 6A.
  • Figure 6C shows a quantitative histogram of the protein content of PPAR ⁇ , FAS in mouse adipose tissue measured in Figure 6A.
  • the present invention provides a purified product of Antrodia camphorata extract, dehydrogenated perforate (TT), for preparing a pharmaceutical compound for lowering blood sugar, triglyceride and total cholesterol in blood, and treating type 2 diabetes and reducing accumulation of liver lipids. new use.
  • TT dehydrogenated perforate
  • the extraction method and efficacy test of the purified extract of Antrodia camphorata will be further described below.
  • the antibody (no. sc-79838) of GLUT4 used in the present invention was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and phosphorylated AMPK (Thr 172 ), PPAR ⁇ (no.ab8934) and PPAR ⁇ (no.ab45036) was purchased from Abcam (Cambrige, MA, USA); FAS (no.3180), phosphorylated Akt (Ser 473 ) (no. 4060), total-AMPK (Thr172) and ⁇ - Actin (no. 4970) was from Cell Signaling Technology, Inc. (Danvers, MA, USA).
  • the anti-rabbit secondary antibody was from Jackson ImmunoRes Laboratories (West Grove, PA, USA).
  • the source of the mycelium of Antrodia camphorata used in the present invention is purchased from Konald Biotechnology Co., Ltd., Chiayi City, Taiwan.
  • the extract of Antrodia camphorata used in the present invention refers to a single component which is extracted and further refined from the lyophilized powder of the mycelium of Antrodia camphorata: purification Dehydroeburicoic acid (TT).
  • the specific extraction method of the dehydrogenated perforating acid (TT) of the purified product of the present invention is to extract 3.0 kg of the lyophilized powder of the mycelium of Antrodia camphorata, and extract it three times at room temperature with 12 liters of methanol, and extract once every four days for co-extraction. three times.
  • the methanol extract was evaporated in vacuo to give a brown residue which was suspended in 1 liter of purified water and then separated from a mixture of three liters of ethyl acetate.
  • the ethyl acetate was layered (200 g), chromatographed on silica gel and eluted with a mixture of hexane and ethyl acetate to increase polarity and high performance liquid chromatography (High Performance Liquid-chromatography, Further purified by abbreviated HPLC) (model Shimadzu CL 20-a, Kyoto, Japan).
  • the dehydrogenated perforating acid (TT) of the present invention is passed through a high-performance liquid chromatography method in a Hibar pre-packed column (model RT 250-10) at a ratio of The 7:1 chloroform and ethyl acetate were separated, the flow rate was 3 mL/min, and the injection volume of the sample was 100 ⁇ L.
  • the separated hydrogen perforated acid (TT) was analyzed by a refractive index detector (RI, model Knauer RI detector 2400).
  • the yield of dehydrogenated perforate (TT) produced was 0.2% (w/w) and the purity was greater than 99%.
  • the source of the body of the Antrodia camphorata used in the present invention is purchased from Balay Biotechnology Co., Ltd., Hsinchu City, Taiwan, and the (CMPC393) voucher specimen is identified by the Chinese Medical University.
  • C2C12 skeletal myoblasts are placed in a growth medium for cell culture, and the growth medium is Dulbecco's modified Eagle's medium (DMEM, Dulbecco's modified Eagle medium; from Gibco BRL), supplemented with 10% fetal bovine serum (FBS; from Hyclone), 100 U/mL penicillin/100 ⁇ g/mL streptomycin (Gibco BRL); Confluent)
  • FBS fetal bovine serum
  • streptomycin Gibco BRL
  • Confluent fetal bovine serum
  • the myoblasts were diluted and placed in a petri dish of 9 cm in diameter and cultured until the confluency reached 80%-90%, and then the medium was changed to 2% FBS/DMEM for 5-7 days. Change every 24 hours.
  • differentiated C2C12 cells were first placed in DMEM/BSA medium and serum starved for 2 hours at 37 °C. After the treatment, the crude extract of A. angustifolia methanol having a concentration of 20, 100, 200 and 500 ⁇ g/mL is subjected to cell culture, or the purified substance to be tested is cultured at a concentration of 1, 5, 10 and 25 ⁇ g/mL, or The blank group vehicle was subjected to cell culture for 25 minutes, or 100 nM of insulin was subjected to cell culture for 25 minutes.
  • the cell culture medium was centrifuged, and the precipitate produced after centrifugation was again suspended in the homogenization buffer.
  • the foregoing steps are carried out with a filter membrane provided.
  • the protein concentration was measured by a BCA protein quantification kit (BCA assay, Pierce). Take an equal amount of protein, dilute it four times in SDS sample buffer, and perform SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and Western blotting, including Akt, p Specific antibodies such as Akt Ser 473 and GLUT4 were immunostained. Density blotting analysis was performed using Alpha Easy FCsoftware.
  • the fenofibrate (Fenofibrate, hereinafter referred to as Feno) is an activator of PPAR ⁇ and has been in the treatment of hypertriglyceridemia for many years.
  • PPAR ⁇ is a key regulator of genes involved in lipid metabolism, which is involved in the regulation of many target genes such as lipid production, fatty acid oxidation, energy expenditure, etc., resulting in a decrease in blood triglycerides and fatty acids.
  • Metformin is an anti-diabetic drug that is currently quite commonly used to treat type 2 diabetes. It activates AMPK in the liver and skeletal muscle.
  • fenofibrate and metformin were selected as a control drug group for the evaluation of dehydro-perforated acid (TT) against a second type of diabetes treatment and a control drug.
  • TT dehydro-perforated acid
  • the present invention induces diabetic mice as a type 2 diabetes with a high-fat diet (HFD).
  • HFD high-fat diet
  • the animal model was tested for skeletal muscle and liver tissue to investigate the effect of dehydro-perforated acid (TT) on anti-type 2 diabetes and anti-dyslipidemia, and to determine dehydro-perforated acid (TT). Resistance to type 2 diabetes, adipogenesis, and target genes in liver tissues including PPAR ⁇ and fatty acid synthase FAS.
  • the following is an experimental design of an animal model for testing the efficacy of dehydrogenated perforate (TT) of the extract of Antrodia camphorata extract for preventing or treating type 2 diabetes and dyslipidemia, and blood data and serum biochemical value analysis of the mouse. , pathological tissue analysis, hepatic lipid analysis, and RNA extraction and relative quantification of mRNA expression of mRNA.
  • TT dehydrogenated perforate
  • mice were fasted overnight and blood samples were collected from the retro-orbital sinus. After four weeks of treatment feeding, the food was removed and the mice were sacrificed after 12 hours of fasting. The desired tissue samples were collected from the mice and weighed. Some tissue samples were taken and sent to a temperature of minus 80 ° C for freezing for subsequent target gene analysis. Plasma samples are collected by centrifugation of whole blood at The plasma separation was completed within 30 minutes at 1600 x g for 15 minutes at 4 °C. A sample of the obtained partial plasma was used for the analysis of triglyceride (TG) and total cholesterol. Metabolic parameters, including body weight, weight gain, and food intake, were performed as follows. The body weight in the overall study was determined by daily measurement, and the weight gain was determined by the difference of body weight for two consecutive days; the total weight of the feed was measured every day, and the weight of the feed was weighed after 24 hours. Daily intake.
  • TG triglyceride
  • Metabolic parameters including body weight, weight gain, and food intake, were performed as follows
  • ELISA enzyme-linked immunosorbent assay
  • EWAT epidermal white adipose tissue
  • liver tissue specimens were tested, and the above specimens were immersed in formalin and neutral buffer, coated with paraffin, and some of them (8m)
  • the cells were cut out and stained with hematoxylin and eosin, and microscopic images were taken using a microscope (Olympus BX51, BX51, Olympus, Tokyo, Japan). Please refer to FIG. 3(A) and FIG. 3(B).
  • Liver fat was analyzed according to the previous procedure.
  • the extracted liver fat sample (0.375 g) was uniformly mixed with 1 ml of distilled water for 5 minutes. Finally, the dried precipitate was resuspended in ethanol (0.5 mL) and utilized.
  • the triglyceride quantification kit replaces the blood triglyceride kit as an analytical tool.
  • RNA from liver tissue was isolated using Trizol reagent according to the manufacturer's instructions (Molecular Research Center, Inc., Cincinnati, OH). The total RNA integrity extracted was quantified by 2% agarose gel electrophoresis, and the RNA concentration was determined by 2% agarose gel electrophoresis and UV absorbance at 260 and 280 nm (Spectrophotometer U-2800A, Hitachi) ). Total RNA (1 ⁇ g) was reverse transcribed into cDNA, and 5 mL of Moroni mouse leukemia virus was reverse transcribed into an enzyme as described above (Earth, Madison, WI, USA).
  • the polymerase chain reaction was carried out in the last 25 ⁇ L containing 1 U of Blend Taq-Plus (TOYOBO, Japan), 1 ⁇ L of RT first-strand cDNA product, 10 ⁇ M of each forward and reverse primer, 75 mM Tris -HCl (trishydroxymethylaminomethane hydrochloride) containing 1 mg/L of Tween 20 (tween-20, pH 8.3, also known as: polyoxyethylene sorbitan monolaurate), 2.5 mM dNTP (deoxy-ribonucleoside triphosphate, deoxyribonucleoside triphosphate) and 2 mM MgCl 2 (magnesium chloride).
  • the primers are shown in Table 1 below.
  • the product was assayed on a 2% agarose gel and stained with ethidium bromide.
  • Test results of cell culture of the present invention The results showed that the administration of Insulin and CruE (200, 500 ⁇ g/mL) in C2C12 myoblasts increased the protein expression of cell membrane GLUT4 and phospho-Akt (Ser 473 )/total-Akt (Ser 473 ) (Fig. 2A, Fig. 2A, 2B).
  • Administration of the Insulin, AnK groups (5, 10, and 25 ⁇ g/mL), and TT (1, 5, 10, and 25 ⁇ g/mL) increased the amount of protein expression of the cell membrane GLUT4.
  • mice produced by HFD induction were given the effect of dehydro-perforated acid (TT) on absolute body weight, food intake, and blood parameters of the tissues (Table 2).
  • TT dehydro-perforated acid
  • TT dehydro-perforated acid
  • TT Dehydrogenated perforate
  • TT Dehydrogenated perforate
  • CON indicates a blank control group (hereinafter referred to as CON group) cultured in DMEM
  • DMSO indicates a control group cultured in DMSO (hereinafter referred to as DMSO group)
  • Insulin indicates an experimental group cultured in insulin (hereinafter referred to as Insulin).
  • a is a statistical analysis result compared to the CON group, and indicates a P ⁇ 0.001.
  • CON control means blank control group (hereinafter referred to as CON group)
  • HFD HFD induction control group (hereinafter referred to as HF group)
  • HF+TT1 HFD+TT2
  • HF+TT3 HFD induction control group
  • the experimental group of hydrogen perforated acid (TT) (hereinafter referred to as HF+TT1 group, HF+TT2 group, HF+TT3 group), and HF+Metf indicates the experiment of oral administration of metformin (Metformin, Metf) induced by HFD.
  • Group hereinafter referred to as HF+Metf group
  • HF+Feno indicates an experimental group (hereinafter referred to as HF+Feno group) which was induced by HFD and orally administered with fenofibrate (Fenofibrate, Feno).
  • the upper reference numerals a, b, and c are statistical analysis results compared with the CON group, where a P ⁇ 0.05, b P ⁇ 0.01, c P ⁇ 0.001; and the upper labels d, e, and f are compared with HF +
  • the results of statistical analysis of the vehicle group indicated that d P ⁇ 0.05, e P ⁇ 0.01, and f P ⁇ 0.001.
  • mice in the HF+TT1 group, HF+TT2 group, HF+TT3 group, HF+Feno group, and HF+Metf group also decreased significantly.
  • EWAT epididymal white adipose tissue
  • MWAT mesente
  • mice that induced type 2 diabetes on a high-fat diet showed higher values of blood glucose, insulin, and leptin than the mice in the control group (CON).
  • the blood glucose concentrations of the HF+TT1 group, the HF+TT2 group, the HF+TT3 group, and the HF+Feno group, and the HF+Metf group of the well acid or the two control drugs were significantly lower than those of the HF group.
  • the blood insulin and leptin concentrations were significantly greater in the HF+TT1 group, the HF+TT2 group, the HF+Feno group, and the HF+Metf group compared with the HF group after administration of dehydro-perforated acid or two control drugs.
  • the ground was reduced, but the experimental mice in the HF+TT3 group exhibited the same concentration as the mice in the control group (CON).
  • mice induced by the high-fat diet induced type 2 diabetes showed higher data points than plasma triglycerides (TG) compared with the control group (CON).
  • TC total cholesterol
  • FFA free fatty acids
  • HF+TT1, HF+TT2, HF+TT3, and HF+Feno, and HF administered with dehydrocortic acid or two control drugs.
  • the +Metf group reduced the plasma triglyceride (TG), total cholesterol (TC), and free fatty acid (FFA) concentration indices compared to the HF group.
  • the experimental mice induced by the high-fat diet induced type 2 diabetes showed an increase in the total fat mass of the liver and the amount of triglyceride in the liver compared with the control group (CON), and the dehydrogenated perforation was administered.
  • the HF+TT1 group, HF+TT2 group, HF+TT3 group, HF+Feno group, and HF+Metf group of acid or two control drugs showed lower total liver fat mass and liver triglyceride than HF group.
  • the amount of ester was administered to determine the amount of ester.
  • mice in the HF group showed hypertrophy of the adipocytes compared to the control group (CON) (the fat cell area of the HF group in this experiment: 11212.6 ⁇ 485.2 ⁇ m 2 ; in the CON group: 6033.1 ⁇ 258.8 ⁇ m 2 ), the following table data is obtained: (please refer to Figure 3A)
  • G6-Pase, 11beta-HSD1, SREBP-1c, aP2, and SREBP-2 in the HF group had higher mRNA expression than the CON group, but CPT-1a. It has a lower mRNA expression than the CON group.
  • HF+TT1 group, HF+TT2 group, HF+TT3 group, HF+Feno group and HF+Metf group showed G6-Pase, 11 ⁇ -HSD1, SREBP
  • the mRNA expression levels of -1c, aP2, GPAT, and SREBP-2 were lower than those in the HF group, but the mRNA expression of CPT-1a was increased.
  • the mRNA of UCP3 in the HF+TT2 group and the HF+TT3 group was increased. The amount of performance was increased compared to the HF group.
  • GLUT4 membrane protein expression level (GLUT4) in the skeletal muscle of the HF group was lower than that of the CON group (the statistical analysis result was P ⁇ 0.01).
  • HF+TT1, HF+TT2, HF+TT3, HF+Feno, and HF+Metf significantly increased skeletal muscle.
  • GLUT4 membrane protein expression (statistical analysis results were: P ⁇ 0.001, P ⁇ 0.001, P ⁇ 0.001, P ⁇ 0.001 and P ⁇ 0.001).
  • HF+TT1, HF+TT2, HF+TT3, HF+Feno, and HF+Metf significantly increased liver p
  • the amount of -AMPK/t-AMPK protein expression (statistical analysis results were: P ⁇ 0.001, P ⁇ 0.001, P ⁇ 0.001, P ⁇ 0.001, and P ⁇ 0.001).
  • the expression of PPAR ⁇ protein in the liver of the HF group was lower than that of the CON group (statistical analysis result: P ⁇ 0.001); dehydrogenated perforate or two control drugs were administered.
  • the PPAR ⁇ protein expression in the liver was significantly increased compared with the HF group (the statistical analysis results were: P ⁇ 0.001, P ⁇ 0.001, P ⁇ 0.001, P ⁇ 0.001, and P ⁇ 0.001).
  • the expression of FAS and PPAR ⁇ protein in the liver of the HF group was higher than that of the CON group (statistical analysis results were: P ⁇ 0.001 and P ⁇ 0.001); after administration of dehydrogenated perforate or two control drugs, HF+ In the TT1 group, the HF+TT2 group, the HF+TT3 group, the HF+Feno group, and the HF+Metf group, the expression levels of FAS and PPAR ⁇ proteins in the liver were significantly lower than those in the HF group.
  • the expression of PPAR ⁇ and FAS protein in the adipose tissue of the HF group was higher than that of the CON group (statistical analysis results were: P ⁇ 0.001 and P ⁇ 0.001); similarly, dehydrogenated perforate or After two control drugs, the expression of PPAR ⁇ and FAS protein in the HF+TT1 group, HF+TT2 group, HF+TT3 group, HF+Feno group and HF+Metf group was significantly lower than that in the HF group.
  • the present invention has developed a purified extract of Antrodia camphorata extract, dehydrogenated perforate (TT), as a therapeutic effect on type 2 diabetes and dyslipidemia.
  • TT dehydrogenated perforate
  • High-fat diet induced mice with type 2 diabetes after oral administration of dehydrogenated perforate (TT) not only significantly reduced blood glucose and decreased insulin concentrations, but also reduced plasma triglycerides and total cholesterol.
  • Dehydroporous acid (TT) significantly increased the amount of GLUT4 membrane protein present in skeletal sarcolemma to increase glucose uptake.
  • TT dehydroporous acid
  • AMPK adenylate-activated protein kinase
  • Dehydroporate acid reduces liver 11 ⁇ -HSD1 mRNA expression resulting in attenuated insulin resistance.
  • Dehydroporous acid transmits FAS which inhibits lipid production in the liver and increases the expression of fatty acid oxidized PPAR ⁇ protein in the liver, which is accompanied by an increase in the mRNA expression of CPT1a and UCP3, thereby enhancing fatty acid oxidation;
  • the mRNA expression levels of SREBP1c, aP2 and GAPT in the liver reduce the synthesis of triglycerides in hepatocytes, which in turn reduces plasma triglycerides and fatty liver.
  • Dehydroporous acid reduces the amount of protein expression including PPAR ⁇ and FAS fat synthesis genes present in adipose tissue, which may be beneficial in reducing adipocyte differentiation and lipid storage. Further, dehydroporous acid (TT) reduces the amount of mRNA expression of SREBP2, which results in a decrease in total cholesterol in the blood. This study shows that dehydroporous acid (TT) has excellent therapeutic potential for dyslipidemia symptoms associated with type 2 diabetes.

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Abstract

一种自牛樟芝提取的纯化物去氢齿孔酸及其用于抗第二型糖尿病、抗高三酸甘油酯、及抗高胆固醇血症、以及降低肝脏脂质累积的用途。其中，该纯化物是由牛樟芝的菌丝体中提取获得的去氢齿孔酸(TT)，并透过量测骨骼肌细胞膜中的葡萄糖转运蛋白4(glucose transporter 4；GLUT4)、肝脏中过氧化物酶体增殖物活化受体α(peroxisome proliferator-activated receptor；PPARα)、以及骨骼肌及肝脏的磷酸化腺苷单磷酸活化蛋白激酶(phospho-AMP-activated protein kinase；p-AMPK)的表现量的增加，以及肝脏中脂肪酸合成酶Fatty acid synthase(FAS)及过氧化物酶体增殖物活化受体γ(PPARγ)的表现量的降低，据以证实去氢齿孔酸(TT)具有抗第二型糖尿病及抗血脂异常的医疗用途。

Description

牛樟芝萃取纯化物的用途 技术领域
本发明是关于一种牛樟芝提取物，特别是指从牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid，简称TT)用于制备抗第二型糖尿病及抗血脂异常以及降低肝脏脂质的医药化合物的用途。
背景技术
糖尿病是由于胰岛素作用障碍或胰岛素分泌不足或以上两者，所导致的慢性高血糖、代谢性心血管疾病等多重病因的主因；其中，第二型糖尿病(Type 2diabetes)约占所有糖尿病病例的90％-95％，其特征在于胰岛素阻抗。第二型糖尿病的发病机制包含胰岛素阻抗(insulin resistance)，以及其中有5％的病患是因为胰脏中β细胞功能缺陷，胰岛素阻抗的发生是当胰岛素失去呈现多样生化反应的功能时，将导致症状包含：血脂异常、肥胖、和高血压。胰岛素阻抗的病理是来自于遗传基因和生活方式等因素，而所摄取的食物是调节这些代谢紊乱的主因，所摄取的食物中的脂肪比例是特别重要。
胰脏分泌胰岛素和维持体内正常的血糖静态平衡，帮助葡萄糖的摄取、和调节碳水化合物和脂质的代谢。研究数据显示，葡萄糖转运蛋白4(glucose transporter 4，简称GLUT4)已被视为扮演一种要角色在调节血液中葡萄糖浓度稳定状态；胰岛素会刺激葡萄糖摄取，乃经由促进GLUT4转位从细胞内位点到细胞膜上。
细胞膜GLUT4蛋白的表现量被认为是评估胰岛素反应性的指标因子，在胰岛素阻抗中常常发生的病理缺陷是在于骨骼肌从细胞内储存位置到细胞膜上。Akt/PKB讯息路径扮演一个中心角色在胰岛素刺激的葡萄糖的摄取在骨骼肌及脂肪组织上，和胰岛素的作用在周边组织的葡萄萄的摄取上借着Akt/PKB乃经由GLUTs转位至细胞膜的能力，因此促进葡萄糖的摄取藉由葡萄糖运转蛋白4(GLUT4)。GLUT4的表现量降低、GLUT4转位、和/或胰岛素路径将导致胰岛素阻抗、和高血糖。因此，改善细胞膜GLUT4或蛋白质的含量将会是一个治疗的方法。
AMP-activated protein kinase(AMPK)负责细胞和体内能量的调节。研究建议，周边的葡萄糖摄取进入骨骼肌(此为葡萄糖最主要的置放位置)可经由两条路径促进葡 萄糖的摄取效率，其中包括：胰岛素依赖的机转所造成的Akt/PKB的活化作用、和肌肉收缩诱导致的刺激、或缺氧导致的AMPK刺激作用。其中，AMPK磷酸化的路径的调节机制不同于GLUT4转位的调节机制，但皆与胰岛素阻抗所引起的脂质和葡萄糖的代谢障碍有关。缘此，AMPK活化剂作用于糖尿病和相关疾病的治疗效果是可预期的。
二甲双胍(Metformin；Metf)为一般临床常用的抗糖尿病药物。二甲双胍(Metf)是一药效强度较低的化合物，通常给药时采取高剂量，但是就会造成仅中度净值的最大药效；还有，且将造成明显的副作用发生。
牛樟芝(Antrodia camphorata；A.camphorata)(多孔菌科，无褶菌目)，在台湾是为一种传统中药材，其极为罕见以及珍贵，因为仅生长在常绿的牛樟Cinnamomum kanehirai树材内心材部分。牛樟芝(A.camphorata)具有多种的生理功能。樟芝子实体包括萜类化合物，如樟芝酸A、B、C、E、F、以及K、和zhankuic acids A、B、C、D、以及E、15α乙酰基去氢硫色多孔菌酸(15□-acetyl-dehydrosulphurenic acid)、去氢齿孔酸(dehydroeburicoic acid)(化学结构式如图1所示)、和去氢硫色多孔菌酸(dehydrosulphurenic acid；TR4)、methyl antcinate G、H、以及齿孔酸(eburicoic acid；TR1)等纯化物。Antcin K(AnK)(化学结构式如图1B所示)是牛樟芝子实体最主要的活性成分。牛樟芝菌体经鉴定的化合物包含有antroquinonol、4-acetylantroquinonol B、succinic和maleic衍生物。子实体的固态栽培和浸出培养的滤液经证实显现出具有保肝和抗氧化活性。去氢齿孔酸(dehydroeburicoic acid；TT)可以经由Poria cocos和牛樟芝dehydroeburicoic acid(TT)抽提出来。
经先前的研究已证实，于体内代谢方面，牛樟芝中的13种萜类化合物的测定是透过液相层析串联式质谱仪(LC/MS/MS)测定经口服试验的小鼠的血浆，发现经血浆测定后的ergostanoids的血浆浓度比lanostanoids高得多，且当ergostanoids可于生物体内进行还原和羟化反应时，其平均滞留时间范围是3～6小时；而当lanostanoids于代谢反应中失去活性时，其平均滞留时间将减缓至9～16小时。
当给予C57BL/6J小鼠喂予高脂肪饮食(high-fat diet；HFD)会诱导小鼠导致早期的第二型糖尿病、显著增加脂肪组织重量，制造出对胰岛素的阻抗，和增高血糖值、以及增加血中三酸甘油酯(triglyceride，简称TG)和总胆固醇(total cholesterol，简称TC)浓度。α亚基上的Thr172位置的磷酸化是影响AMPK活性的关键。骨骼肌和脂肪组织扮演独特的角色在调节胰岛素依赖的葡萄糖的体内平衡上。骨骼肌是全身胰岛素 媒介的葡萄糖摄取的最主要部位。脂肪组织说明一小部分的饭后葡萄糖的置放，而骨骼肌扮演葡萄糖摄取的最主要部分。因此，本研究首次在体外试验评估牛樟芝(A.camphorata)的MeOH粗提取物(CruE)和AnK(此为此蕈菇类最主要成分)在GLUT4和磷酸化Akt的表现量。进行进一步的调查去氢齿孔酸(TT)在高脂访饮食诱导致糖尿病小鼠的降血和降血脂的作用和机转探讨。此动物模式会诱导致第二型糖尿病，这目前的研究调查是否去氢齿孔酸(TT)会增加细胞膜葡萄糖摄取和AMPK的磷酸化。此外，对标靶基因的表达进行测定，该标靶基因和脂肪酸氧化作用有关联的过氧化物酶体增殖物激活受体α(PPARα)、脂肪酸合酶(fatty acid synthase；FAS)、糖质新生的glucose6-phosphoatase(G6Pase)、和carnitine palmitoyl transferase Ia(CPT-1a)。
发明内容
一种牛樟芝萃取纯化物的用途，其是用于制备治疗第二型糖尿病、降低血糖的医药化合物，该化合物是自牛樟芝菌丝体提取的纯化物，该纯化物为去氢齿孔酸(dehydroeburicoic acid，C31H48O3)。
一种牛樟芝萃取纯化物的用途，其中该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)；是用于制备降低血脂异常的医药化合物。
一种牛樟芝萃取纯化物的用途，其中该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)；是用于制备降低血脂中包括总胆固醇(TC)或三酸甘油酯(TG)的医药化合物。
一种牛樟芝萃取纯化物的用途，其中该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)的医药化合物；该纯化物降血糖的作用是透过增加骨骼肌的膜葡萄糖载体蛋白4(GLUT4)蛋白的显现量而增加血液葡萄糖的摄取，以及抑制肝脏葡萄糖生成的G6Pase与11βhydroxysteroid dehydrogenase(11beta-HSD1)的mRNA表现，另增加骨骼肌的磷酸化腺苷单磷酸活化蛋白激酶(phospho-AMPK)的总和作用而达成。
一种牛樟芝萃取纯化物的用途，其中该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)的医药化合物；该纯化物的抗第二型糖尿病用途是用于制备增加骨骼肌蛋白质激酶B(Akt)的磷酸化，并提升胰岛性敏感性。
一种牛樟芝萃取纯化物的用途，其中该纯化物是自牛樟芝菌丝体提取的纯化物去氢 齿孔酸(dehydroeburicoic acid)的医药化合物；该纯化物的降血脂和肝质的用途是因增加肝脏的磷酸化腺苷单磷酸活化蛋白激酶(phospho-AMPK)。
一种牛樟芝萃取纯化物的用途，其中该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)的医药化合物；该纯化物的降低肝脏脂质的用途是透过降低肝脏中的总脂质(total lipid)及三酸甘油酯(triacylglycerol)的含量，以及降低肝脏空泡样变(ballooning degeneration)现象。
一种牛樟芝萃取纯化物的用途，其中该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)的医药化合物；该纯化物的治疗高三酸甘油酯症用途，是透过增加肝脏的脂肪酸氧化酶(PPARα)和降低肝脏内的脂肪酸合成酶(FAS)的蛋白的显现量、以及降低胆固醇调节组件结合蛋白1c(Sterol regulatory element binding protein，简称SREBP-1c)、glycerol-3-phosphate acyltransferase(GPAT)、adipocyte fatty acid binding protein 2(aP2)，及增加CPT1a及uncoupling protein3(UCP3)的mRNA显现量。
一种牛樟芝萃取纯化物的用途，其中该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)的医药化合物；该纯化物的用途是用于制备降低血中胆固醇，是因透过SREBP-2的mRNA表现量降低，而使血液中总胆固醇(TC)数值降低。
一种牛樟芝萃取纯化物的用途，其中该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)的医药化合物；该纯化物的用途是用于制备治疗因高脂肪饮食所引起的高瘦体素血症，亦即降低血中瘦体素(leptin)浓度。
一种牛樟芝萃取纯化物的用途，其中该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)的医药化合物；该纯化物的用途是用于制备降低因高脂肪饮食所引起的高内脏脂肪的重量(visceral fat mass)，以及降低内脏脂肪细胞大小(hypertrophy of adipocyte)。
一种牛樟芝萃取纯化物的用途，其中该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)的医药化合物；该纯化物的降低内脏脂肪的用途是用于制备增加脂肪组织的脂肪酸氧化的PPARα的蛋白质显现量，降低脂肪组织的脂质生成的FAS和PPARγ的蛋白质显现量。
藉此，透过该由牛樟芝提取物中的纯化物去氢齿孔酸，使经高脂饮食(HFD)诱导引起的糖尿病及其他相关症状，经施予纯化物去氢齿孔酸后，达到以下功效：
施予剂量10、20、40mg/kg/day的去氢齿孔酸治疗的小鼠的血糖数值明显地下降，具有改善糖尿病的功效。
透过去氢齿孔酸治疗，使HF诱导糖尿病小鼠的血中三酸甘油酯(plasma triglyceride，TG)及总胆固醇(total cholesterol，TC)浓度下降，显示去氢齿孔酸具有改善血脂异常的功效。
透过去氢齿孔酸治疗，增加HFD诱导糖尿病小鼠的肝脏及骨骼肌中的GLUT4的表现量以及p-AMPK的表现量，显示去氢齿孔酸具有改善高血糖症的功效。
在透过去氢齿孔酸治疗改善血糖异常症状的过程中，G6Pase的mRNA表现量会随的降低，达到抑制肝脏葡萄糖生成以及减弱糖尿病状态的功效。(G6-Pase，glucose-6-phosphatase，6-磷酸葡萄糖)
透过增加PPARα的肝脏蛋白表现量以及CPT-1a的mRNA表现量，显示去氢齿孔酸治疗具有降低脂肪生成的效果。
透过降低FAS蛋白表现量、降低SREBP1c、aP2以及GPAT的mRNA表现量，去氢齿孔酸治疗具有促进血液三酸甘油酯降低的效果。
透过去氢齿孔酸治疗，经HFD诱导糖尿病小鼠的SREBP-2的mRNA表现量降低且血液中胆固醇数值减少，显示去氢齿孔酸能够透过增加骨骼肌细胞膜GLUT4蛋白，调控PPARα、FAS以及增加骨骼肌及肝脏组织中p-AMPK/t-AMPK蛋白表现量，达到防止或改善经HFD诱导糖尿病小鼠的糖尿病及血脂异常状态。
附图说明
图1是纯化物去氢齿孔酸(TT)的化学结构式。
图2A～2E是体外试验在C2C12myotube细胞中给予胰岛素或牛樟芝甲醇粗提取物(CruE)对于细胞膜GLUT4和磷酸化Akt/总Akt的影响；以及给予胰岛素或去氢齿孔酸(TT)与Antcin K(AnK)两者的相较对于细胞膜GLUT4和磷酸化Akt/总Akt的影响，其中图2A及2C显示于C2C12myotube细胞培养实验中产生的代表性的免疫印迹；而图2B，2D及2E显示细胞膜GLUT4蛋白的显现量以及磷酸化Akt/总Akt的比值；C2C12细胞试验中的骨骼成肌细胞(myotube)经牛樟芝甲醇粗提取物或给予化合物如去氢齿孔酸(TT)或Antcin K(AnK)，以及相同数量的藉由SDS-PAGE溶出的溶胞物质以及GLUT4，total-Akt(Ser473)，与磷酸化Akt(Ser473)的印迹；所有数量为均值±SE(n＝6)，ap＜0.001。
图3A、图3B显示本发明动物模型实验中，CON组、HF组、HF+TT1组、HF+TT2组、HF+TT3组、HF+Feno组、及HF+Metf组的小鼠附睪白色脂肪组织(图3A)及肝组织(图3B)的经H-E染色法的病理组织染色图。
图4A显示本发明实验动物模型实验中，对CON组、HF组、HF+TT1组、HF+TT2组、HF+TT3组、HF+Feno组、及HF+Metf组的小鼠肝脏组织中的G6Pase，11β-HSD1，SREBP1c，GPAT，aP2，UCP3，CPT1a，和SREBP2于肝脏组织中的mRNA表现进行半定量RT-PCR的代表图。
图4B至图4C显示将图4A中的G6Pase，11β-HSD1，SREBP1c，GPAT，aP2，UCP3，CPT1a，和SREBP2所测得的信号经图像分析定量，且透过GAPDH使每个值标准化。
图5A显示本发明动物模型实验中，以西方点墨分析法(Western blot)对CON组、HF组、HF+TT1组、HF+TT2组、HF+TT3组、HF+Feno组、及HF+Metf组的小鼠骨骼肌中的细胞膜GLUT4及phospho-AMPK(Thr172)的蛋白质含量进行测定，以及对前述各组的小鼠肝脏中的phospho-AMPK(Thr172)的蛋白质含量进行测定。
图5B显示将图5(A)测得的蛋白质含量的定量直方图。
图6A显示本发明动物模型实验中，以西方点墨分析法(Western blot)对CON组、HF组、HF+TT1组、HF+TT2组、HF+TT3组、HF+Feno组、及HF+Metf组的小鼠肝脏中的PPARα、FAS、PPARγ的蛋白质含量进行测定，以及对前述各组的小鼠脂肪组织中的PPARγ、FAS的蛋白质含量进行测定。
图6B显示将图6A测得的小鼠肝脏中的PPARα、FAS、PPARγ的蛋白质含量的定量直方图。
图6C显示将图6A测得的小鼠脂肪组织中的PPARγ、FAS的蛋白质含量的定量直方图。
具体实施方式
本发明特征与优点的一些典型实施例将在以下说明中详细叙述。应理解的是本发明能够在不同的态样上具有各种的变化，然其皆不脱离本发明的范围，且其中的说明及图式在本质上是当作说明之用，而非用于限制本发明。
本发明提供了牛樟芝提取物的纯化物去氢齿孔酸(TT)用于制备降低血糖、血中三酸甘油酯和总胆固醇、以及治疗第二型糖尿病、减少肝脏脂质的累积的医药化合物的新 用途。以下将进一步说明牛樟芝纯化物的提取方法及其功效试验。
(一)材料与方法
本发明所使用的GLUT4的抗体(no.sc-79838)购自圣克鲁斯生物技术(圣克鲁斯，CA，USA)、以及磷酸化AMPK(Thr172)，PPARα(no.ab8934)和PPARγ(no.ab45036)是购自Abcam公司(Cambrige，MA，USA)；而FAS(no.3180)，磷酸化Akt(Ser473)(no.4060号)，总-AMPK(Thr172)和β-肌动蛋白(no.4970)是来自Cell Signaling Technology公司(Danvers，MA，USA)。抗兔次级抗体是从Jackson ImmunoRes实验室公司(West Grove，PA，USA)。
本发明所使用的牛樟芝菌丝体来源，是向台湾嘉义市Konald生物技术公司购买，本发明所使用的牛樟芝提取物是指从牛樟芝菌丝体的冻干粉提取并进一步精制的单一成分：纯化物去氢齿孔酸(dehydroeburicoic acid，简称TT)。本发明纯化物去氢齿孔酸(TT)的具体提取方法，是将3.0kg的牛樟芝菌丝体的冻干粉，以12公升的甲醇在室温下萃取三次，每四天萃取一次，共萃取三次。该甲醇萃取液在真空中蒸发，得到棕色残留物，将该棕色残留物悬浮于1公升的纯水中，然后用1公升的乙酸乙酯分三次将之分离出一混合物。将乙酸乙酯分层(200克)，在硅胶上进行层析，并用己烷与乙酸乙酯的混合物洗提，藉以增加极性并以高效能液相层析仪(High Performance Liquid-chromatography，简称HPLC)(型号Shimadzu CL 20-a，Kyoto，Japan)进一步纯化。于本实施例中，本发明的去氢齿孔酸(TT)是在一高压预填柱(Hibar pre-packed column，型号RT 250-10)中透过高效能液相层析法以比例为7∶1的氯仿及乙酸乙酯进行分离，其流率为3mL/min，样品的注射体积为100μL。分离后的氢齿孔酸(TT)并以折射率检测器(Refractive index，RI，型号Knauer RI detector 2400)分析组成。制得的去氢齿孔酸(TT)的产率为0.2％(w/w)，纯度大于99％。
再者，本发明所使用的牛樟芝子实体来源，是向台湾新竹市Balay生物技术公司购买，且其(CMPC393)凭证标本是由中国医学大学所鉴定。
其中，3.0公斤的子实体用甲醇萃取三次，之后用50％乙酸乙酯和50％己烷层析。该程序如前段所描述。纯度99％以上的Antcin K(AnK)其分析仪器是高效液相层析仪(HPLC，Shimadzu CL 20-A，Kyoto，Japan)，HPLC管柱(TOSOH TSKgel DS-80Ts)，并利用100％的MeOH(甲醇)作为冲洗填充HPLC管柱的有机溶剂。
1.1细胞培养实验
以下测试在培养的C2C12肌纤维细胞(C2C12myotube cells)中，给予胰岛素或牛樟芝甲醇粗提取物(CruE)测试能否调节细胞膜GLUT4或影响磷酸化Akt的蛋白表现，或测试给予胰岛素或Antcin K(AnK)或去氢齿孔酸(TT)调节细胞膜GLUT4或影响磷酸化Akt的蛋白表现的比较效果。
本发明取C2C12骨骼成肌细胞(ATCC，CRL-1772)置于生长培养基中进行细胞培养，该生长培养基为达尔伯克(氏)改良伊格尔(氏)培养基(DMEM，Dulbecco’s modified Eagle medium；来自Gibco BRL公司)，其添加了10％胎牛血清(fetal bovine serum，FBS；来自Hyclone公司)、100U/mL青霉素/100μg/mL链霉素(Gibco BRL公司)；当细胞融合(confluent)达到80％时，用0.05％的胰蛋白酶分裂(split)为1∶4。将成肌细胞稀释并放置在直径9cm的培养皿中，并培养至其融合状态(confluency)达到80％-90％，接着将培养基更换为2％的FBS/DMEM，为时5-7天，每隔24小时更换一次。
在试管中测定GLUT4及p-Akt(Ser473)/t-Akt蛋白的实验中，先将分化的C2C12细胞置于DMEM/BSA培养基中，37℃下持续2小时进行血清饥饿(serum Starved)处理之后，再分组对浓度为20、100、200及500μg/mL的牛樟芝甲醇粗提取物进行细胞培养，或对浓度为1、5、10及25μg/mL的待测纯化物进行细胞培养，或对空白组载体(vehicle)进行25分钟的细胞培养，或对100nM的胰岛素进行25分钟的细胞培养。
并将上述经细胞培养的培养液进行离心处理，并将离心后所产生的沉淀物，用匀浆缓冲液再次使沉淀物悬浮。前述步骤是在设有过滤膜的情况下进行。再者其中，蛋白质浓度是透过BCA蛋白质定量试剂盒(BCA assay，Pierce)测得。取等量的蛋白质，在SDS样品缓冲液中稀释四次，并进行SDS-PAGE(十二烷基硫酸钠聚丙烯酰胺凝胶电泳)以及西方墨点法(Western blotting)，利用包括Akt、p-Akt Ser473以及GLUT4等特异性抗体进行免疫染色。使用Alpha Easy FCsoftware进行密度墨点(density blotting)分析。
1.2动物模型实验
药品非诺贝特(Fenofibrate，以下简称为Feno)是一种PPARα的活化剂，并在高三酸甘油酯血症的治疗上行之多年。PPARα是与脂质代谢相关的基因的关键调节因子，其通过参与调节脂质生成、脂肪酸氧化、能量消耗等许多靶基因，导致血液三酸甘油酯和脂肪酸的减少。
二甲双胍(Metformin)是目前相当普遍用于治疗第二型糖尿病的抗糖尿病药品， 其能够激活肝脏及骨骼肌中的AMPK。
因此，非诺贝特及二甲双胍被选为对照药物组，用于评估去氢齿孔酸(TT)的抗第二型糖尿病疗与作用的对照药物。为了评估体内GLUT4和phospho-AMPK进行血糖调节的表现是否改变，以及α次元Thr172的磷酸化对AMPK活性是必要的，故本发明以高脂肪饮食(HFD)诱导糖尿病小鼠作为第二型糖尿病的动物模型并对其骨骼肌及肝脏组织进行了检测，藉以探讨去氢齿孔酸(TT)对于抗第二型糖尿病及抗血脂异常方面的效果，并测定去氢齿孔酸(TT)在抗第二型糖尿病、脂肪形成以及肝组织中标靶基因包括PPARα及脂肪酸合成酶FAS等的调控表现。
以下说明本发明于测试牛樟芝提取物的纯化物去氢齿孔酸(TT)于预防或治疗第二型糖尿病及血脂异常的功效的动物模型实验设计，以及小鼠的血液数据、血清生化值分析、病理组织分析、肝脂质分析、以及RNA的萃取及mRNA的相对定量标的基因表现量。
从国家实验动物繁育研究中心购买四周龄的雄鼠C57BL/6J(总量＝63只)，并将其区分为控制组/低脂饮食组(low-fat-diet；CON(CD；Diet 12450B，Research Diets，Inc.，New Brunswick，NJ，USA)，总量：9只小鼠)和高脂饮食组(HFD(Diet 12451，Research Diets，Inc.)，总量：54只小鼠)，其中，低脂饮食组(CON)的饮食组成为20％蛋白质，70％碳水化合物，以及10％的脂肪；而高脂饮食组(high-fat-diet；HFD)的饮食组成为20％蛋白质，35％碳水化合物，以及45％的脂肪。如是两组相异饮食组成的实验小鼠，于本发明实验中将经过12周的疗程喂养。
控制组以及高脂饮食组的脂肪摄取分别为10％以及45％；又HFD组小鼠经8周诱导后次分为6组(每组9只小鼠)，并将这6组小鼠于实验八周诱导之后的再给予四周的纯化物或药物治疗，施喂6种不同种类以及剂量的药物进行治疗，包括：(1)TT1＝去氢齿孔酸10mg/kg/day、(2)TT2＝去氢齿孔酸20mg/kg/day、(3)TT3＝去氢齿孔酸40mg/kg/day、(4)Feno(Sigma Chemical Co.，St Louis，MO，USA)＝非诺贝特fenofibrate0.25g/kg/day、(5)Metf(Sigma Chemical Co.，St Louis，MO，USA)＝二甲双胍metformn0.3g/kg/day、以及(6)相当剂量的蒸馏水(vehicle)，所述蒸馏水、去氢齿孔酸(TT)、二甲双胍或非诺贝特是在最后28天(四周)内，每天口服灌胃一次。在实验期间，所有小鼠禁食过夜后从眶后窦收集血液样本。经四周治疗喂养后，移走食物使小鼠禁食12小时后将小鼠牺牲。从小鼠收集所需的组织样本并称重，部分组织样本取得后立即送入零下80℃的环境进行冷冻，以用于后续标靶基因分析。血浆样品通过将全血离心收集在 1600×g下在4℃15分钟，血浆的分离在30分钟之内完成。得到的部分血浆的样品用于甘油三酯(TG)和总胆固醇的分析。其中代谢参数，包括体重，体重增加量和食物摄取量，以如下实验进程进行。整体研究中的体重是经由每日的测定，体重增加量，是藉由连续两天的体重的差额而认定；每天测定饲料的总重，经24小时再秤饲料重量，其显示的差额是表示每日摄取量。
1.3空腹血糖及生化指标测定
将前述从禁食12小时候的小鼠眶后窦收集的血液样本的一部分立即用于葡萄糖数值的分析，另一部分用于血液中三酸甘油酯(TG)、总胆固醇(TC)及游离脂肪酸(free fatty acids)、血液胰岛素、瘦体素、以及脂联素数值的分析，是采用葡萄糖分析仪(Model1500sidekick glucose analyzer；YSI)分析血液葡萄糖，血浆甘油三酯(Plasma triglycerides，TG)、总胆固醇(total cholesterol，TC)、游离脂肪酸(free fatty acids，FFA)是使用市售试剂盒根据制造商指示进行测定(甘油三酯-E测试/Triglycerides-E test、胆固醇-E测试/Cholesterol-E test及游离脂肪酸-C测试/FFA-C test；和光纯药/Wako Pure Chemical，日本大阪)。而胰岛素和瘦体素浓度是通过酶联免疫吸附试验(ELISA)(mouse insulin ELISA kit，Mercodia，Uppsala，Sweden；mouse leptin ELISA kit，Morinaga，Yokohama，Japan)。
1.4病理组织分析测定
针对收集到的一部分EWAT(附睾白色脂肪组织)和肝组织标本进行检测，将上述标本浸泡于福尔马林与中性缓冲液中，再以石蜡包覆之，并将其中的部分(8m)切下并用苏木精和伊红染色，再使用显微镜(Olympus BX51，BX51，Olympus，Tokyo，Japan)拍摄显微图像。请配合参照图3(A)、图3(B)所示。
肝脏脂质的分析
肝脏脂肪是按照先前程序进行分析，将经萃取的肝脏脂肪样品(0.375g)与1毫升的蒸馏水均匀混和5分钟，最后，将经干燥的沉淀物再次悬浮于乙醇(0.5mL)中，并利用三酸甘油酯定量套组代替血液三酸甘油酯套组作为分析工具。
1.5mRNA的相对定量显示基因表现分析与西方墨点法测定
根据制造商的指示，利用Trizol试剂(Trizol reagent)分离来自肝脏组织的总RNA(分子研究中心公司，辛辛那提，美国俄亥俄州)。透过2％琼脂糖凝胶电泳对前述提取的总RNA完整性进行量化测定，并通过2％琼脂糖凝胶电泳以及260和280nm的紫外 线吸亮度测定RNA浓度(分光亮度计U-2800A，日立)。总RNA(1μg)反转录为cDNA，以及5mL的莫罗尼鼠白血病病毒如前述方案反转录为酶(震中，麦迪逊，WI，USA)。聚合酶链反应是在最后的25μL中进行，其含有1U的Blend Taq-Plus(TOYOBO公司，日本)、1μL的RT第一链cDNA产物、各10μM的正向引子和反向引子、75mM的Tris-HCl(三羟甲基氨基甲烷盐酸盐)其中含有1mg/L的吐温20(tween-20，pH值为8.3，又称：聚氧乙烯山梨糖醇酐单月桂酸酯)、2.5mM的dNTP(deoxy-ribonucleoside triphosphate，脱氧核糖核苷三磷酸)以及2mM的MgCl2(氯化镁)。所述引子如下表1所示。该产物是在2％琼脂糖凝胶上进行测定，并用溴化乙锭(ethidium bromide)染色。
Figure PCTCN2016107285-appb-000001
测定骨骼肌细胞膜GLUT4、和肝脏及骨骼肌的p-AMPK(Thr172)采用免疫墨点法(immunoblot)方法，我们测定肝脏组织的PPARα及FAS的蛋白质显现量，也测定脂肪组织PPARγ和FAS的蛋白质显现量，以及测定骨骼肌细胞膜GLUT4细胞膜，且总细胞 膜部份(total membrane fraction)和缓冲液一起被收集并以前述方法进行测定。细胞膜GLUT4、p-AMPK以及总AMPK的蛋白质含量以前述西方墨点法测定。
(二)试验结果
本发明细胞培养的试验结果。实验结果显示在C2C12成肌细胞中给予Insulin、CruE(200、500μg/mL)中会增加细胞膜GLUT4和phospho-Akt(Ser473)/total-Akt(Ser473)的蛋白质显现量(图2A、图2B)。给予Insulin、AnK组(5、10、和25μg/mL)、以及TT(1、5、10、和25μg/mL)会增加细胞膜GLUT4的蛋白质显现量。给予Insulin、AnK组(10、25μg/mL)、以及TT(10和25μg/mL)会增加phospho-Akt(Ser473)/total-Akt(Ser473)的蛋白质显现量(图2C、图2D、图2E)。在MTT(3-(4，5-dimethylthiazol-2-yl)-2，5-diphenyltetrazolium bromide)试验中显示给予TT(剂量在1至25μg/mL)对C2C12成肌细胞是没有毒性的。
以下配合表2说明前述测试中，本发明在动物模型实验中，经高脂饮食诱导诱导第二型糖尿病小鼠的试验结果。动物模型实验中，经HFD诱导所生成的患糖尿病小鼠给予去氢齿孔酸(TT)在组织的绝对体重、摄食量、和血液参数的影响(表2)。
给予去氢齿孔酸(TT)的病理组织学的影响在附睾白色脂肪组织/肝脏组织(图3A、图3B)。
给予去氢齿孔酸(TT)对经高脂肪饮食诱导致糖尿病小鼠的肝脏组织G6-Pase、11beta-HSD1、SREBP1c、GPAT、aP2、UCP3、CPT1a、SREBP-2、及beta-actin表现的半定量RT-PCR的影响(图4A、图4B、图4C)。
给予去氢齿孔酸(TT)对经高脂肪饮食诱导致糖尿病小鼠骨骼肌的细胞膜GLUT4、骨骼肌phospho-AMPK(Thr172)、total-AMPK(Thr172)、GAPDH的蛋白质显现量测定；肝脏phospho-AMPK(Thr172)、total-AMPK(Thr172)、β肌动蛋白、GAPDH的蛋白质显现量测定(图5A、图5B)
对经高脂肪饮食诱导致糖尿病小鼠肝脏的PPARα、FAS、PPARγ、β肌动蛋白的蛋白质含量测定(图6A、图6B、图6C)、小鼠脂肪细胞的PPARγ、FAS、β肌动蛋白的蛋白质显现量测定(图6A、图6B、图6C)。
2.1体外试验中细胞膜中的GLUT4蛋白与磷酸化Akt的表现量
请配合参示图2A、图2B，是显示在本发明细胞培养实验中，以不同培养基对C2C12骨骼成肌细胞进行细胞培养后的蛋白质显现量分析结果。其中，CON(control)表示以DMEM培养的空白对照组(以下简称CON组)，DMSO表示以DMSO进行培养的对照组(以下简称DMSO组)，Insulin表示以胰岛素进行培养的实验组(以下简称Insulin组)，在CON组、DMSO组、Insulin组、CruE组(20、100、200、和500μg/mL)中的细胞膜GLUT4、phospho-Akt(Ser473)、total-Akt(Ser473)及β肌动蛋白的蛋白质含量(图2A、图2B)。在CON组、DMSO组、Insulin组、与AnK组(1、5、10、25μg/mL)、及TT组(1、5、10、25μg/mL)中的细胞膜GLUT4、phospho-Akt(Ser473)、total-Akt(Ser473)及β肌动蛋白的蛋白质显现量(图2C、图2D、图2E)。其中，于图2A～2E中，a是相较于CON组的统计分析结果表示，是表示aP＜0.001。
2.2代谢参数分析
再请配合参阅表2所示，是显示在本发明动物模型实验中，喂食不同剂量的纯化物去氢齿孔酸(TT)、二甲双胍(Metformin)或非诺贝特(Fenofibrate)对于HFD诱导第二型糖尿病小鼠的组织及血液数值分析结果。其中，CON(control)表示空白对照组(以下简称CON组)，HFD表示HFD诱导对照组(以下简称HF组)，HF+TT1、HF+TT2、HF+TT3分别表示服用不同剂量的纯化物去氢齿孔酸(TT)的实验组(以下依序简称HF+TT1组、HF+TT2组、HF+TT3组)，而HF+Metf表示经HFD诱导并口服药品二甲双胍(Metformin，Metf)的实验组(以下简称HF+Metf组)，HF+Feno表示经HFD诱导并口服药品非诺贝特(Fenofibrate，Feno)的实验组(以下简称HF+Feno组)。上标号a、b、c是相较于CON组的统计分析结果表示，其中，表示aP＜0.05、bP＜0.01、cP＜0.001；而上标号d、e、f是相较于HF+水(vehicle)组的统计分析结果表示，其中，表示dP＜0.05、eP＜0.01、fP＜0.001。
【1】表2.
表2.去氢齿孔酸(TT)对于组织重量、摄食量、肝脏脂肪以及血液参数值的影响
Figure PCTCN2016107285-appb-000002
Figure PCTCN2016107285-appb-000003
如是表2的数据结果，实验终止时，所有高脂肪喂食的小鼠的最后体重和体重增加量是显著增加相对于控制组(CON)；再来比对经药物治疗的组群，其中，HF+TT2、HF+TT3、HF+Feno、及HF+Metf组的小鼠明显下降最终体重(final body weight)(相对于HF组)。同时，HF+TT1组、HF+TT2组、HF+TT3组、HF+Feno组、及HF+Metf组的小鼠亦明显降 低体重增加量(bodyweight gain)(相对于HF组)。食物摄取量的部分，HF组的实验小鼠小于控制组(CON)，但HF+TT3组，HF+Feno组食物摄取量比较少相对于HF组；而经施喂高脂肪饮食后，显示出会造成附睾白色脂肪组织(Epididymal white adipose tissue；EWAT)、肠系膜白色脂肪组织(Mesenteric white adipose tissue；MWAT)，腹膜后脂肪组织(Retroperitoneal white adipose tissue；RWAT)和内脏脂肪(visceral fat)及棕色脂肪(brown adipose tissue；BAT)的绝对重量的增加相较于控制组(CON)，而当经给予去氢齿孔酸(包括HF+TT3组)及HF+Feno组、和HF+Metf组后相较于HF组，会显著降低附睾白色脂肪组织(EWAT)及腹膜后脂肪组织(RWAT)重量。当经给予去氢齿孔酸(包括HF+TT2组、HF+TT3组)、HF+Feno组、和HF+Metf组后相较于HF组，显著降低内脏脂肪、以及棕色脂肪的重量，当经给予去氢齿孔酸(包括HF+TT1组，HF+TT2组，HF+TT3组)相较于HF组，显示出降低了肝脏组织重量，但是，HF+Metf组小鼠明显地增加肝脏组织重量。
2.3血糖、胰岛素以及瘦素指标分析
请配合参阅表2数据数据所示，经高脂饮食诱发第二型糖尿病的小鼠比起控制组(CON)的小鼠呈现高数值的血糖、胰岛素以及瘦素，而经施喂去氢齿孔酸或两对照药物的HF+TT1组、HF+TT2组、HF+TT3组以及HF+Feno组、和HF+Metf组相较于HF组，其血糖浓度呈现大大地降低。经施喂去氢齿孔酸或两对照药物的HF+TT1组、HF+TT2组、以及HF+Feno组、和HF+Metf组相较于HF组，其血液胰岛素与瘦素浓度示现大大地减低，但HF+TT3组的实验小鼠与控制组(CON)的小鼠呈现浓度一样。
2.4血液中三酸甘油脂、总胆固醇、以及肝脏脂肪指标分析
请配合参阅表2数据数据所示，经高脂饮食诱发第二型糖尿病的实验小鼠，比起控制组(CON)的实验小鼠，呈现更高数据指针于血浆三酸甘油脂(TG)、总胆固醇(TC)、以及游离脂肪酸(FFA)，而经施喂去氢齿孔酸或两对照药物的HF+TT1组、HF+TT2组、HF+TT3组以及HF+Feno组、和HF+Metf组相较于HF组降低血浆三酸甘油脂(TG)、总胆固醇(TC)、以及游离脂肪酸(FFA)的浓度指数。经高脂饮食诱发第二型糖尿病的实验小鼠，比起控制组(CON)的实验小鼠，呈现增加肝脏的总脂质量和肝脏的三酸甘油酯量，而经施喂去氢齿孔酸或两对照药物的HF+TT1组、HF+TT2组、HF+TT3组以及HF+Feno组、和HF+Metf组相较于HF组，呈现降低肝脏的总脂质量和肝脏的三酸甘油酯量。
2.5组织病理检测
经过12周的高脂肪饮食诱导，于HF组别的小鼠，比起控制组(CON)的实验小鼠，呈现其脂肪细胞hypertrophy(本实验中的HF组小鼠的脂肪细胞面积：11212.6±485.2μm2；CON组小鼠则为：6033.1±258.8μm2)，得到下述表格数据：(请配合参照图3A)
实验组别 脂肪细胞面积(μm2)
HF+TT1组 6872.5±160.8
HF+TT2组 6530.2±148.2
HF+TT3组 5972.4±279.5
HF+Feno组 6270.5±165.4
HF+Metf组 6919.5±195.1
高脂肪饮食诱导导致肝细胞显著地空泡状变性(ballooning degeneration)，本研究发现显示该肝细胞的空泡状变性是发生在HF组的小鼠身上，其导致了肝细胞死亡并且于肝糖(糖原质)堆积于细胞中间，且于图3中细胞核仁因而被挤压到另一边，此情形被称为肝脏的空泡状变性(ballooning degeneration)(如图箭头所示)。
2.6肝脏组织标靶基因表现量
如图4A、图4B、图4C所示，HF组的G6-Pase、11beta-HSD1、SREBP-1c、aP2、SREBP-2相较于CON组具有较高的mRNA表现量，然CPT-1a则相较于CON组具有较低的mRNA表现量。经施喂去氢齿孔酸或两对照药物后，HF+TT1组，HF+TT2组，HF+TT3组，HF+Feno组以及HF+Metf组显示出其G6-Pase、11β-HSD1、SREBP-1c、aP2、GPAT、SREBP-2的mRNA表现量相较于HF组是降低的，但是CPT-1a的mRNA表现量是增加的；其中，HF+TT2组，HF+TT3组的UCP3的mRNA表现量相较于HF组是增加的。
2.7不同组织中的标靶基因表现量
如图5A、图5B所示，在实验终止后，HF组的骨骼肌中GLUT4膜蛋白表现量(membrane expressions levels of GLUT4)相较低于CON组(统计分析结果为P＜0.01)。经施喂去氢齿孔酸或两对照药物后，HF+TT1组，HF+TT2组，HF+TT3组，HF+Feno组以及HF+Metf组(相较于HF组)显著增加骨骼肌中GLUT4膜蛋白表现量(统计分析结果依序为：P＜0.001、P＜0.001、P＜0.001、P＜0.001及P＜0.001)。HF组的骨骼肌及肝脏中 phospho-AMPK/total-AMPK蛋白表现量相较低于CON组(统计分析结果依序为：P＜0.05、P＜0.01)。经施喂去氢齿孔酸或两对照药物后，HF+TT1组，HF+TT2组，HF+TT3组，HF+Feno组以及HF+Metf组(相较于HF组)显著增加骨骼肌中phospho-AMPK/total-AMPK蛋白表现量(统计分析结果依序为：P＜0.01、P＜0.01、P＜0.001、P＜0.01及P＜0.01)。经施喂去氢齿孔酸或两对照药物后，HF+TT1组，HF+TT2组，HF+TT3组，HF+Feno组以及HF+Metf组(相较于HF组)显著增加肝脏中p-AMPK/t-AMPK蛋白的表现量(统计分析结果依序为：P＜0.001、P＜0.001、P＜0.001、P＜0.001及P＜0.001)。
如图6A、图6B、图6C所示，HF组的肝脏中PPARα蛋白表现量相较低于CON组(统计分析结果为：P＜0.001)；经施喂去氢齿孔酸或两对照药物后，HF+TT1组，HF+TT2组，HF+TT3组，HF+Feno组以及HF+Metf组相较于HF组的肝脏中PPARα蛋白表现量显著增加了(统计分析结果依序为：P＜0.001、P＜0.001、P＜0.001、P＜0.001及P＜0.001)。HF组的肝脏中FAS及PPARγ蛋白表现量相较高于CON组(统计分析结果依序为：P＜0.001及P＜0.001)；经过施喂去氢齿孔酸或两对照药物后，HF+TT1组，HF+TT2组，HF+TT3组，HF+Feno组以及HF+Metf组相较于HF组的肝脏中FAS及PPARγ蛋白表现量显著降低了。再者，HF组的脂肪组织中PPARγ及FAS蛋白表现量相较高于CON组(统计分析结果依序为：P＜0.001及P＜0.001)；同理，经施喂去氢齿孔酸或两对照药物后，HF+TT1组，HF+TT2组，HF+TT3组，HF+Feno组以及HF+Metf组相较于HF组的脂肪组织中PPARγ及FAS蛋白表现量显著被降低了。
综上所述，本发明开发了牛樟芝提取物的纯化物去氢齿孔酸(TT)作为对于第二型糖尿病及血脂异常的治疗效果。高脂肪饮食诱导致第二型糖尿病小鼠在口服纯化物去氢齿孔酸(TT)后，不仅显著地降低血糖及降低胰岛素浓度，也降低了血浆三酸甘油酯及总胆固醇。去氢齿孔酸(TT)显著地增加了骨骼肌膜中的GLUT4膜蛋白表现量以提升葡萄糖摄取。此外，透过给予去氢齿孔酸(TT)至高脂肪饮食导致的糖尿病小鼠，将增加骨骼肌及肝脏组织中腺苷酸活化蛋白激酶(AMPK)的磷酸化，且AMPK磷酸化的比率与该去氢齿孔酸(TT)的施喂量呈正相关。再者，给予去氢齿孔酸(TT)会抑制肝脏葡萄糖生成作用与降低G6Pase mRNA表现是相关的。给予去氢齿孔酸(TT)到糖尿病小鼠，透过增强骨骼肌中GLUT4膜蛋白以及降低肝脏中葡萄糖生成之结合，导致降低血液中葡萄糖浓度。给予 去氢齿孔酸(TT)会降低肝脏11β-HSD1mRNA表现导致于减弱胰岛素阻抗作用。去氢齿孔酸(TT)透过抑制肝脏的脂质生成的FAS以及增加肝脏中脂肪酸氧化PPARα蛋白的表现量，伴随着增加CPT1a及UCP3的mRNA表现量，进而增进脂肪酸氧化作用；此外，降低肝脏SREBP1c、aP2以及GAPT的mRNA表现量，降低肝细胞内三酸甘油酯的合成，进而降低了血浆中三酸甘油酯、及脂肪肝。去氢齿孔酸(TT)减少了包括存在于脂肪组织中PPARγ及FAS脂肪合成基因的蛋白表现量，其可能有利于减少脂肪细胞分化及脂质贮存。进一步地，去氢齿孔酸(TT)减少了SREBP2的mRNA表现量，这导致了血液中总胆固醇降低。本研究显示去氢齿孔酸(TT)对于第二型糖尿病有关的血脂异常症状有优异的治疗潜力。

Claims (11)

  1. 一种牛樟芝萃取纯化物的用途，其特征在于，用于制备治疗第二型糖尿病、降低血糖的医药化合物，该化合物是自牛樟芝菌丝体提取的纯化物，该纯化物为去氢齿孔酸(dehydroeburicoic acid，C31H48O3)。
  2. 如权利要求1所述的牛樟芝萃取纯化物的用途，其特征在于，该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)；是用于制备降低血脂异常包括总胆固醇(TC)或三酸甘油酯(TG)的医药化合物。
  3. 如权利要求1所述的牛樟芝萃取纯化物的用途，其特征在于，该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)；是用于制备增加骨骼肌与肝脏的磷酸化腺苷单磷酸活化蛋白激酶(phospho-AMPK)、以及增加骨骼肌细胞膜的葡萄糖载体蛋白4(GLUT4)的医药化合物。
  4. 如权利要求1所述的牛樟芝萃取纯化物的用途，其特征在于，该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)的医药化合物；该纯化物降血糖的作用是透过增加骨骼肌的膜葡萄糖载体蛋白4(GLUT4)蛋白的显现量而增加血液葡萄糖的摄取，以及抑制肝脏葡萄糖生成的G6 Pase与11β-HSD1的mRNA表现，另增加骨骼肌的磷酸化腺苷单磷酸活化蛋白激酶(phospho-AMPK)的总和作用而达成。
  5. 如权利要求1所述的牛樟芝萃取纯化物的用途，其特征在于，该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)的医药化合物；该纯化物的抗第二型糖尿病用途是用于制备增加骨骼肌蛋白质激酶B(Akt)的磷酸化，并提升胰岛性敏感性。
  6. 如权利要求1所述的牛樟芝萃取纯化物的用途，其特征在于，该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)的医药化合物；该纯化物的降低肝脏脂质的用途是包括降低肝脏中的总脂质(total lipid)及三酸甘油酯(tracylglyxerol)的含量，以及降低肝脏空泡样变(ballooning degeneration)现象。
  7. 如权利要求1所述的牛樟芝萃取纯化物的用途，其特征在于，该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)的医药化合物；该纯化物的治疗高三酸甘油酯症及降低肝脏脂质累积的用途，是透过增加肝脏的磷酸化腺苷单磷酸活化蛋白激酶(phospho-AMPK)，以及增加肝脏的脂肪酸氧化酶(PPARα)和降低 肝脏内的脂肪酸合成酶(FAS)的蛋白的显现量、以及降低胆固醇调节组件结合蛋白1c(SREBP-1c)、GPAT、aP2，及增加CPT1a以及UCP3的mRNA显现量的总和净效应的作用而达成。
  8. 如权利要求1所述的牛樟芝萃取纯化物的用途，其特征在于，该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)的医药化合物；该纯化物的用途是用于制备降低血中胆固醇，是因透过SREBP-2的mRNA表现量降低，而使血液中总胆固醇(TC)数值降低。
  9. 如权利要求1所述的牛樟芝萃取纯化物的用途，其特征在于，该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)的医药化合物；该纯化物的用途是用于制备治疗因高脂肪饮食所引起的高瘦体素血症，亦即降低血中瘦体素(leptin)浓度。
  10. 如权利要求1所述的牛樟芝萃取纯化物的用途，其特征在于，该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)的医药化合物；该纯化物的用途是用于制备降低因高脂肪饮食所引起的高内脏脂肪的重量(visceral fat mass)，以及降低内脏脂肪细胞大小(hypertrophy of adipocyte)。
  11. 如权利要求1所述的牛樟芝萃取纯化物的用途，其特征在于，该纯化物是自牛樟芝菌丝体提取的纯化物去氢齿孔酸(dehydroeburicoic acid)的医药化合物；该纯化物的降低内脏脂肪的用途是用于制备增加脂肪组织的脂肪酸氧化的PPARα的蛋白质显现量，降低脂肪组织的脂质生成的FAS和PPARγ的蛋白质显现量。
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