WO2017181857A1 - 一种降脂保肝组合物及其用途 - Google Patents

一种降脂保肝组合物及其用途 Download PDF

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WO2017181857A1
WO2017181857A1 PCT/CN2017/079796 CN2017079796W WO2017181857A1 WO 2017181857 A1 WO2017181857 A1 WO 2017181857A1 CN 2017079796 W CN2017079796 W CN 2017079796W WO 2017181857 A1 WO2017181857 A1 WO 2017181857A1
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group
liver
lipid
lowering
weeks
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PCT/CN2017/079796
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French (fr)
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李卫民
朱贺年
玉荣
李铀
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北京宜生堂医药科技研究有限公司
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • 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)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/481—Astragalus (milkvetch)
    • 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)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A61K36/488—Pueraria (kudzu)
    • 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)
    • A61K36/60—Moraceae (Mulberry family), e.g. breadfruit or fig
    • A61K36/605—Morus (mulberry)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to a composition and use thereof, and more particularly to a composition having reduced obesity hyperlipidemia and improved fatty liver and uses thereof.
  • Astragalus membranaceus also known as Aphis gossypii
  • its main active ingredient is Astragaloside IV, which has the effects of inhibiting metabolic syndrome, delaying myocardial ischemia and improving cardiac function in myocardial ischemia.
  • Astragalus has enhanced immune function and liver protection. , diuretic, anti-aging, anti-stress, antihypertensive and a wide range of antibacterial effects. It can eliminate experimental nephritis proteinuria, enhance myocardial contractility, and regulate blood sugar levels.
  • Astragalus can not only expand the coronary arteries, improve myocardial blood supply, improve immune function, but also delay the process of cell aging.
  • Pueraria the dry root of the leguminous kudzu, is called kudzu.
  • the main active site in Pueraria lobata is total flavonoids, of which puerarin is one of the main active ingredients.
  • Puerarin has hypoglycemic, lipid-lowering, insulin resistance, as well as antihypertensive, dilated coronary arteries, improved myocardial ischemia, anti-hypoxia reperfusion injury, protection of endothelial function to improve hemodynamics, inhibition of platelet aggregation, etc.
  • vascular protection, so puerarin is widely used in the clinical treatment of diabetic coronary heart disease and a variety of cardiovascular diseases.
  • Mulberry white bark is the dry root bark of the mulberry plant. It contains flavonoids: mulberry, mulberry chromene, cyclosalin and the like.
  • the hypoglycemic component deoxydiamine-based glucosamine and MoranA hypoglycemic substances were isolated and confirmed in the mulberry bark. After experiments, MoranA has been shown to have a dose-dependent hypoglycemic effect on alloxan-induced hyperglycemic mice.
  • Hyperlipidemia refers to a lipid concentration in the plasma that exceeds the normal range.
  • Blood lipids are a general term for lipids contained in human plasma, including cholesterol, triglycerides, cholesterol esters, ⁇ -lipoproteins, phospholipids, and non-lipidated fatty acids.
  • serum cholesterol exceeds the normal value of 230 mg / 100 ml
  • triglyceride exceeds 140 mg / 100 ml
  • ⁇ -lipoprotein exceeds 390 mg / 100 ml or more
  • hyperlipidemia There are various reasons for inducing hyperlipidemia. First, genetically determined, especially primary hyperlipidemia.
  • Obesity-type hyperlipidemia is a second type of hyperlipidemia induced by obesity as described above.
  • Fatty liver refers to a lesion with excessive accumulation of fat in liver cells due to various reasons.
  • Fatty liver can be divided into: obesity fatty liver, alcoholic fatty liver, fast weight loss fatty liver, malnutrition fatty liver, diabetic fatty liver, drug-induced fatty liver and so on.
  • Fatty liver disease is a serious threat to the health of Chinese people and has become the second largest liver disease after viral hepatitis. It has been recognized as a common cause of occult cirrhosis.
  • puerarin and mulberry bark have been used in combination to treat diabetes and hyperlipoproteinemia in a certain proportion, but jaundice, puerarin and mulberry bark have been used to treat obesity-type hyperlipidemia and fatty liver. There have been no studies or reports yet.
  • An object of the present invention is to provide a lipid-lowering and liver-protecting composition which can be effectively used for the treatment of obesity-type hyperlipidemia and ameliorating fatty liver by overcoming the above-mentioned deficiencies of the prior art. Meanwhile, the present invention also provides a preparation method and use of the lipid-lowering and liver-protecting composition.
  • composition composed of Astragalus, Radix Puerariae and Morus alba L. in a specific ratio relationship as described above has a particularly remarkable effect in reducing obesity hyperlipidemia and improving fatty liver.
  • another object of the present invention is to provide a method for preparing a lipid-lowering and liver-protecting composition as described above, and to achieve the object, the technical solution adopted by the present invention is: a method for preparing a lipid-lowering and liver-protecting composition
  • the method comprises the following steps: mixing the astragalus, puerarin and mulberry white skin and extracting with a solvent under reflux, recovering the solvent after extraction, and extracting the extract with a macroporous resin to obtain a lipid-lowering and liver-protecting composition.
  • the present invention provides a medicament for treating obesity-type hyperlipidemia and fatty liver, which comprises the lipid-lowering liver-protecting composition as described above.
  • the present invention provides the use of the lipid-lowering and liver-protecting composition as described above for the preparation of foods, medicaments and health care products for preventing or treating obesity hyperlipidemia.
  • the present invention provides the use of the lipid-lowering liver-protecting composition as described above for the preparation of foods, medicaments and health care products for preventing or treating fatty liver.
  • the present invention provides the use of the lipid-lowering liver-protecting composition as described above for the preparation of foods, medicaments and health care products for preventing or treating obesity.
  • the present invention provides the use of the lipid-lowering and liver-protecting composition as described above for the preparation of foods, medicaments and health care products for preventing or treating hypercysteinemia.
  • the lipid-lowering liver protecting composition is used in the preparation of a medicament for the treatment of myocardial and skeletal muscle.
  • the lipid-lowering and liver-protecting composition of the invention can be effectively used for the treatment of obesity hyperlipidemia and fatty liver, and produces remarkable effects.
  • the medicine containing the lipid-lowering and liver-protecting composition can effectively treat obesity hyperlipidemia and fatty liver, and provides a new choice for the treatment of obesity hyperlipidemia and fatty liver.
  • Figure 1 is a graph comparing the fasting blood glucose of each group after administration of streptozotocin (STZ)-high fat diet (HFD) in type 2 diabetic rats for 7 weeks.
  • Fig. 2 is a graph showing the comparison of cholesterol in each group after administration of streptozotocin (STZ)-high fat diet (HFD) in type 2 diabetic rats for 7 weeks.
  • Fig. 3 is a graph showing the comparison of triglycerides in each group after administration of streptozotocin (STZ)-high fat diet (HFD) in type 2 diabetic rats for 7 weeks.
  • Fig. 4 is a comparison chart of low-density lipoproteins of each group after administration of streptozotocin (STZ)-high fat diet (HFD)-induced type 2 diabetic rats for 7 weeks.
  • Fig. 5 is a graph showing the comparison of fasting blood glucose in each group after administration of streptozotocin (STZ)-high fat diet (HFD)-induced type 2 diabetic rats for 12 weeks.
  • Fig. 6 is a graph showing the comparison of cholesterol in each group after administration of streptozotocin (STZ)-high fat diet (HFD)-induced type 2 diabetic rats for 12 weeks.
  • Fig. 7 is a comparison chart of triglycerides of each group after administration of streptozotocin (STZ)-high fat diet (HFD)-induced type 2 diabetic rats for 12 weeks.
  • Fig. 8 is a graph showing the comparison of alanine aminotransferases in each group after administration of streptozotocin (STZ)-high fat diet (HFD)-induced type 2 diabetic rats for 12 weeks.
  • Fig. 9 is a graph showing the comparison of each group of aspartate aminotransferases after administration of streptozotocin (STZ)-high fat diet (HFD)-induced type 2 diabetic rats for 12 weeks.
  • Fig. 10 is a graph showing the comparison of fasting blood glucose in each group after administration of glucocorticoid (GC) + high fat diet in type 2 diabetic rats for 6 weeks.
  • Figure 11 is a graph showing the comparison of cholesterol in each group after administration of glucocorticoid (GC) + high fat diet in type 2 diabetic rats for 6 weeks.
  • Fig. 12 is a comparison chart of triglycerides of each group after administration of glucocorticoid (GC) + high fat diet in a type 2 diabetic rat for 6 weeks.
  • GC glucocorticoid
  • Fig. 13 is a graph showing the comparison of fasting blood glucose in each group after administration of glucocorticoid (GC) + high fat diet in a type 2 diabetic rat for 10 weeks.
  • GC glucocorticoid
  • Fig. 14 is a graph showing the comparison of cholesterol in each group after administration of glucocorticoid (GC) + high fat diet in a type 2 diabetic rat for 10 weeks.
  • GC glucocorticoid
  • Fig. 15 is a graph showing the comparison of triglycerides in each group after administration of glucocorticoid (GC) + high fat diet in a type 2 diabetic rat for 10 weeks.
  • GC glucocorticoid
  • Fig. 16 is a diagram showing the morphology of hepatocytes in the SD group after 12 weeks of administration to a type 2 diabetic rat induced by glucocorticoid (GC) + high fat diet.
  • GC glucocorticoid
  • Figure 17 is a diagram showing the morphology of hepatocytes in the SD+GC group after 12 weeks of administration to a type 2 diabetic rat induced by glucocorticoid (GC) + high fat diet.
  • Figure 18 is a diagram showing the morphology of hepatocytes in the HFD group after 12 weeks of administration to glucocorticoid (GC) + high fat diet-induced type 2 diabetic rats.
  • Figure 19 is a diagram showing the morphology of hepatocytes in the HFD+GC group after 12 weeks of administration to glucocorticoid (GC) + high fat diet-induced type 2 diabetic rats.
  • Figure 20 is a diagram showing the morphology of hepatocytes in the rosiglitazone group after 12 weeks of administration to glucocorticoid (GC) + high fat diet-induced type 2 diabetic rats.
  • GC glucocorticoid
  • Figure 21 shows the administration of glucocorticoid (GC) + high-fat diet combined with type 2 diabetic rats for 12 weeks.
  • Skin 2:1:1 group liver cell morphology.
  • GC glucocorticoid
  • Figure 23 is a diagram showing the morphology of hepatocytes in the group of jaundice: Pueraria lobata: mulberry bark 1: 2:1 after administration of glucocorticoid (GC) + high fat diet for 12 weeks.
  • GC glucocorticoid
  • Fig. 24 is a graph showing the body weight and FPG, TC, TG, and LDL-C levels of a model group rat (HFD group) and a normal group rat (SD group) obtained after feeding for 7 weeks of high fat diet.
  • HFD group model group rat
  • SD group normal group rat
  • Fig. 25 is a graph showing the comparison of the dietary dose of each group of rats during prophylactic administration and therapeutic administration.
  • Fig. 26 is a graph showing changes in the levels of FPG, TC, TG, and LDL-C in each group of rats after 7 weeks of prophylactic administration.
  • Figure 27 is a graph showing changes in the levels of FPG, TC, TG, and LDL-C in each group of rats after 9 weeks of therapeutic administration.
  • Fig. 28 is a graph showing changes in the levels of FPG, TC, TG, and LDL-C in each group of rats after 12 weeks of prophylactic administration.
  • Fig. 29 is a graph showing changes in the levels of FPG, TC, TG, and LDL-C in each group of rats after 14 weeks of therapeutic administration.
  • Fig. 30 is a graph showing the level of homocysteine in the liver of each group of rats obtained by homogenizing the liver after 15 weeks of prophylactic administration and 15 weeks of therapeutic administration.
  • Fig. 31 is a graph showing the levels of glutamyl transpeptidase in the liver of each group of rats obtained by homogenizing the liver after 15 weeks of prophylactic administration and 15 weeks of therapeutic administration.
  • Fig. 32 is a graph showing the levels of lipid (TC, TG) in the liver of each group of rats obtained by homogenizing the liver after 15 weeks of prophylactic administration and 15 weeks of therapeutic administration.
  • Fig. 33 is a graph showing the comparison of steatosis of liver cells of each group after oil-red O staining for 15 weeks after prophylactic administration and 15 weeks after therapeutic administration.
  • Figure 34 is a graph comparing the ratio of brown fat to epididymal fat in each group of rats after 15 weeks of prophylactic administration and 15 weeks of therapeutic administration.
  • Figure 35 is a graph comparing the ratio of brown fat to perirenal fat in each group of rats after 15 weeks of prophylactic administration and 15 weeks of therapeutic administration.
  • Fig. 36 is a graph showing the ratio of bone weight coefficient of each group of rats after 15 weeks of therapeutic administration.
  • Figure 37 is a micro-CT scan of a whole body scan of a living rat using Latheta LCT200.
  • Figure 38 is a graph showing the comparison of white fat content in living groups of each group of rats after 14 weeks of prophylactic prophylactic administration
  • Figure 39 is a graph showing the fat content in the liver of each group of rats after 14 weeks of prophylactic prophylaxis
  • Figure 40 is a graph showing the comparison of femur cortical bone density, cancellous bone density, and total bone density in each group after 14 weeks of prophylactic administration;
  • Figure 41 is a graph showing the pathological results of pancreatic tissue of each group of rats after 16 weeks of prophylactic administration and 16 weeks of therapeutic administration.
  • Figure 42 is a graph showing the pathological results of myocardial tissue of each group of rats after 16 weeks of prophylactic administration and 16 weeks of therapeutic administration.
  • Figure 43 is a graph showing the pathological results of skeletal muscle tissue of each group after 16 weeks of prophylactic administration and 16 weeks of therapeutic administration.
  • Figure 44 is a graph showing the results of myocardial collagen fiber morphology after Masison staining in each group after 16 weeks of prophylactic administration and 16 weeks of therapeutic administration.
  • Figure 45 is a graph showing the relative content of collagen in each group of myocardial tissue after 16 weeks of prophylactic administration and 16 weeks of therapeutic administration.
  • Fig. 46 is a graph showing the results of morphology of skeletal muscle collagen fibers after Masison staining in each group after 16 weeks of prophylactic administration and 16 weeks of therapeutic administration.
  • Figure 47 is a graph showing the relative content of collagen in skeletal muscle tissue of each group after 16 weeks of prophylactic administration and 16 weeks of therapeutic administration.
  • Fig. 48 is a graph showing the contents of GLU, TG, and TC in each group of diabetic rats after 16 weeks of therapeutic administration.
  • Figure 49 is a graph showing the comparison of Hcy and GGT1 contents in homogenized liver and diabetic rats in each group after 16 weeks of therapeutic administration.
  • Figure 50 is a graph showing the expression levels of UCP-1 in diabetic rats of each group after 12 weeks of therapeutic administration.
  • Figure 51 is a graph showing the amount of Cidea expression in each group of diabetic rats after 12 weeks of therapeutic administration.
  • Figure 52 is a graph showing the expression levels of MCP-1 in each group of diabetic rats after 12 weeks of therapeutic administration.
  • Figure 53 is a graph showing the expression levels of PRDM16 transcription factor mRNA in each group of diabetic rats after 12 weeks of therapeutic administration.
  • STZ streptozotocin
  • the injection volume was 5ml/Kg
  • the injection was 10
  • blood was taken from the fundus venous plexus
  • fasting blood glucose FBG was determined
  • rats with FBG>7.2 mmo/L were selected as type 2 diabetes model rats and fed with high fat diet.
  • mice in the normal group were taken out as the A-normal group, and then the above-mentioned type 2 diabetic model rats were randomly divided into 6 groups, 8 in each group, 6 groups were respectively: B-model group; G-test group 1 H-test group 2; I-test group 3; J-test group 4; K-test group 5.
  • the dose of the composition of the present invention is 60 g of crude drug per day, and the clinical equivalent dose of the animal is calculated according to the BioS's method (animal kilogram weight coefficient coefficient algorithm): 7 g of crude drug/kg of the composition of the present invention.
  • the composition of the present invention (Astragalus: Pueraria: Mulberry) test group 1 (2:1:1); test group 2 (1:2:1); test group 3 (1:1:2); test 4 (1) : 1:1); test group 5 (2:1:0).
  • the normal group and the model group were respectively administered with 10% gum arabic (solvent), G-test group 1, H-test group 2, I-test group 3, J-test group 4, and K-test group 5 respectively.
  • the pharmaceutical compositions of Examples 1 to 5 (the pharmaceutical compositions of Examples 1 to 5 were mixed, and the extracts of Astragalus, Radix, and Mulberry were extracted with a solvent under reflux, and the solvent was recovered after extraction, and the extract was extracted with a macroporous resin.
  • each group of animals was intragastrically administered once a day, and administered intragastrically at 10 ml/Kg/d, respectively, and the fasting blood glucose of each group of animals after 7 weeks of administration and 12 weeks after administration was tested.
  • Glu triglyceride
  • TG cholesterol
  • LDL-C low density lipoprotein
  • the cholesterol of the model group was significantly higher than that of the normal group (P ⁇ 0.05); compared with the cholesterol of the model group, the different proportions of the Huangqisan administration group had Significant difference (P ⁇ 0.05); K group (2:1:0) was significantly different from other drug-administered groups (P ⁇ 0.05); each drug group had a lowering effect on cholesterol, but K group (2:1:0) The effect of lowering cholesterol was more pronounced than in the other groups.
  • the model group had a significant increase in triglyceride compared to the normal group (P ⁇ 0.05); G (2: 1: 1) compared to the model group. There was a significant difference (P ⁇ 0.05) between H (1:2:1) and J (1:1:1), which could significantly reduce triglyceride levels. There was no significant difference between group I and model group (P>0.05), and there was no effect of reducing triglyceride.
  • the low-density lipoprotein in the model group was significantly increased compared with the normal group (P ⁇ 0.05); compared with the model group, each drug-administered group had Significant difference (P ⁇ 0.05); K group was significantly different from other drug-administered groups (P ⁇ 0.05); different proportions of Huangqisan group could reduce LDL levels, but K group was better than other groups. The effect of lowering LDL levels was more pronounced.
  • G (2:1:1); H (1:2:1); J (1:1:1) has a significant reduction in blood sugar, cholesterol, triglycerides, and low-density lipoprotein.
  • I (1:1:2), K (2:1:0) can lower the levels of cholesterol, low-density lipoprotein, and blood sugar, but does not reduce the effect of triglyceride levels.
  • test group was the same as above, and the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of each group of animals were tested after 12 weeks of administration.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • the model group had a significantly higher trend of aspartate aminotransferase (AST) than the normal group, but there was no significant difference (P>0.05); however, compared with the model group.
  • the aspartate aminotransferase of each administration group has a tendency to decrease.
  • the alanine aminotransferase in the model group was significantly increased, there was a significant difference (P ⁇ 0.05), and the liver function of the type 2 diabetic rats was impaired after 12 weeks.
  • each administration group significantly reduced the level of aspartate aminotransferase (P ⁇ 0.05) and improved liver function.
  • there was no significant difference in alanine aminotransferase between the two groups (P>0.05). From this, it can be concluded that different proportions of the composition of the present invention can prevent the condition of liver dysfunction caused by type 2 diabetes.
  • Example 7 Test of lipid-lowering and liver-protecting effect of the lipid-lowering and liver-protecting composition of the invention
  • the trial was divided into 9 groups, each of which was as follows:
  • SPF-grade SD rats were conditioned at the normal level animal house for two weeks (about 200-220 g), and the basal level was determined by fasting blood sampling. After 3 days, except for the normal basal diet group, the other groups began to administer prednisone acetate 3.5 mg/kg once a day, and then intragastric administration was started 1 hour later, and the high-fat diet was fed for 12 weeks. After 6 weeks of administration and 10 weeks of administration, they were fasted for 12 hours respectively. The parameters of fasting blood glucose, cholesterol and triglyceride in each group were measured by plasma. The fasting blood glucose, cholesterol and each group were observed after 6 weeks of administration and 10 weeks after administration. Changes in triglycerides.
  • the blood glucose of the model HFD+GC group was higher than that of the normal basic group (P ⁇ 0.05).
  • the blood glucose of HFD+GC group was higher than that of SD+GC group (P ⁇ 0.05).
  • there was no increase in blood glucose in SD+GC compared with the SD group indicating that the basal diet combined with glucocorticoid modeling was unsuccessful at 6 weeks, but the high-fat combined with glucocorticoid model was successful.
  • the blood glucose of the rosiglitazone group was statistically different (P ⁇ 0.05).
  • the blood glucose of each of the administration groups of the composition of the present invention was also significantly decreased, and the difference was statistically significant, while the J group was more obvious than the other groups, and the effect of reducing the body weight by combining the original group J, suggesting that the composition of the present invention Weight inhibition may be the cause of hypoglycemia.
  • triglyceride level of the model group is not increased compared with the normal group from the influence of triglyceride TG. It is surprising that the high fat feeding group has the lowest triglyceride level in the HFD group. This is basically consistent with the change of 6 weeks prior to the previous administration.
  • the hepatic lobule was seen in the normal SD group, and the hepatic plate structure of the central artery and surrounding hepatic cells was seen in the lobule. No obvious pathological changes were observed.
  • the SD+GC group a small amount of hepatocytes were slightly steatosis in the hepatic lobules, and tiny lipid droplets were observed in the hepatocytes.
  • the HFD and HFD+GC groups hepatic cells in the hepatic lobules showed obvious fatty degeneration, and the hepatocytes were filled with vesicle-like lipid droplets.
  • Example 8 Comparative experiment of the lipid-lowering and liver-protecting composition of the present invention and Lipitor
  • This experiment includes two parts: prophylactic and therapeutic administration of lipid-lowering and liver-protecting composition (HQS). Healthy male Sprague-Dawley rats were randomly divided into normal group (SD) and model group. The normal group was fed with basal diet. The model was fed with high-fat diet, freely drinking water and eating. At the same time, 10 rats were selected. The modular rats were model group (HFD).
  • Prophylactic administration of lipid-lowering and liver-protecting composition Rat model rats were randomly divided into HQS-L-low-fat and liver-protecting composition low-dose group (1.2g/kg.d), HQS-H-lipid-protecting liver combination The high-dose group (2.4 g/kg.d), 8 rats in each group, was simultaneously administered at the same time.
  • lipid-lowering and liver-protecting composition After 7 weeks of continuous high-fat feeding of the model rats, the body weight of the model group was significantly higher than that of the normal group (SD rats fed with basic diet), and the module FPG, TC, The levels of TG and LDL-C were also significantly higher than those of the normal group, indicating that the model was successful at this time, with statistical differences, as shown in Figure 24.
  • Rats with a body weight exceeding 20% of normal rats were selected as obese rat models, and were randomly divided into HQS-L-low-fat and liver-protecting composition low-dose group (1.2g/kg.d), HQS-H-lipid-protected liver
  • the high-dose group (2.4 g/kg.d) and the Lipitor-positive group (Lipitor, 2 mg/kg), 8 rats in each group were administered after 7 weeks of high-fat feeding.
  • Rats in the normal group and the model group were given the same amount of normal saline, and each administration group was given the corresponding test drug once a day.
  • the high- and low-dose groups of the lipid-lowering and liver-protecting composition are the lipid-lowering and liver-protecting compositions of the present invention, wherein the mass ratio of Astragalus: Pueraria: Mulberry is 1:2:1.
  • preventive administration prevention group is administered while feeding high fat diet
  • the dosage of low-dose (HQS-L) and lipid-lowering liver-protecting composition high dose (HQS-H) of lipid-lowering and liver-protecting composition is set according to the effective dose set by the previous multi-drug test, and the dose is 1.2g/kg respectively. .d and 2.4g/kg.d.
  • the dosage of low-dose (HQS-L) and lipid-lowering liver-protecting composition high dose (HQS-H) of lipid-lowering and liver-protecting composition is set according to the effective dose set by the previous multi-drug test, and the dose is 1.2g/kg respectively. .d and 2.4g/kg.d.
  • the dose of Lipitor is set according to the clinical equivalent dose of human, and the dose is 2g/kg.d.
  • Body weight changes, eating, fur, activity, etc. were observed every day during the experiment, and body weight changes were recorded weekly.
  • Prophylactic administration At 7 weeks of administration and 12 weeks of administration, the rats were fasted for 12 hours, anesthetized with ether, blood was taken from the fundus venous plexus, heparin was anticoagulated, and plasma was collected at 4 ° C (3000 r/min, 15 min). Determination of fasting blood glucose (FPG), cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL-C);
  • FPG fasting blood glucose
  • TC cholesterol
  • TG triglyceride
  • LDL-C low density lipoprotein
  • Therapeutic administration After 9 weeks of administration and 14 weeks of administration, the rats were fasted for 12 hours, anesthetized with ether, blood was taken from the fundus venous plexus, heparin was anticoagulated, and plasma was collected by centrifugation (3000 r/min, 15 min) at 4 °C. FPG, TC, TG, LDL-C were measured.
  • the rats in each group were weighed and intraperitoneally injected with 10% chloral hydrate (0.35 ml/100 g body weight). The rats were anesthetized. The rats were anesthetized and fixed on the anatomical plate, and the abdominal aorta was sacrificed. The blood sample obtained was allowed to stand for 2 hours, centrifuged at 3000 r/min for 30 minutes to obtain serum, and part of it was whole blood, and the device was placed in a refrigerator at -20 ° C for use.
  • the rats in the prophylactic administration group were subjected to a living micro-CT experiment one week before dissection.
  • a micro-CT system for multifunctional 3D scanning of animals such as rats, mice and rabbits.
  • each group of rats was firstly anesthetized with 10% chloral hydrate for 1-2 hours, and placed in the trough for abdominal ablation scan.
  • the parameters of bone parameters, subcutaneous fat, visceral fat and fatty liver CT were measured.
  • the experiment was conducted at the Medical Experimental Research Center of the Third affiliated Hospital of Southern Medical University.
  • the body weight changes of the rats in each group at different times during the period of prophylactic administration and therapeutic administration are shown in Tables 4 to 5.
  • the weight of the drug-administered group was significantly lower than that of the model group, which was close to the normal group weight, and was significantly better than the Lipitor group (Lipitor group).
  • the weight of the Lipitor group was significantly increased. , close to the model group.
  • the microCT experiment was performed one week before the administration, and it was obvious that the weight growth of the rats was slow, especially in the group of the lipid-lowering and liver-protecting group.
  • the levels of FPG, TC, TG, and LDL-C in each group of rats were 7 weeks after the prophylactic administration and 9 weeks after the treatment, as shown in Figs.
  • the model group compared with the normal group (SD group), FPG, TC, TG, LDL- C water levels were significantly elevated and were statistically different.
  • the model group compared with the normal group (SD group), FPG, TC, TG, LDL-C
  • SD group normal group
  • FPG lipid-lowering and liver-protecting group
  • HQS-H group high-dose group of lipid-lowering and liver-protecting group
  • HQS-L group low-dose group of lipid-lowering and liver-protecting group
  • the lipid-lowering and liver-protecting composition is superior to the Lipitor group.
  • Homocysteine is a sulfur-containing amino acid in the human body and an important intermediate in the metabolism of methionine and cysteine. Hypercysteinia is a risk factor for cardiovascular and cerebrovascular diseases, osteoporosis, and neurological diseases.
  • HFD group the level of the model group
  • HQS-H group the high- and low-dose group of the lipid-lowering liver-protecting composition
  • HQS-H group the high- and low-dose group of the lipid-lowering liver-protecting composition
  • Glutamine transpeptidase is already one of the liver function tests. As shown in Fig. 31, by detecting the level of GTT1 in the liver, it is known that the level of the model group is significantly increased, while the high- and low-dose group of the lipid-lowering liver-protecting composition (HQS-H group, HQS-L group), Lipitor group (Lipitor Group) can be significantly reduced.
  • Lipid ectopic deposition refers to the accumulation of lipids in non-adipose tissue in the body, mostly in the liver, muscles and pancreas, and the most prone to occur in the liver. As shown in Fig. 32, by detecting lipids (TC, TG) in the liver, it was found that the liver lipids in the model group (HFD group) were significantly increased, while the administration group significantly improved liver lipid deposition, and the prevention group was compared. The treatment group was more significant.
  • the lipid-lowering and liver-protecting composition prevents the hepatic steatosis of the high- and low-dose group (prevention of HQS-H group and HQS-L group) from being significantly relieved, and is superior to the lipid-lowering and liver-protecting composition in the treatment of high- and low-dose groups (treatment of HQS-H)
  • the group and the HQS-L group were all more effective in the high dose group.
  • the hepatic steatosis of the lipid-lowering and liver-protecting group was significantly improved compared with the model group, and it was superior to the Lipitor group; among them, A represents the normal group, I represents the model group, and K represents the Lipu. Proper group.
  • White fat usually includes subcutaneous white fat and visceral white fat. It is currently believed that subcutaneous white fat and brown fat are involved in maintaining the homeostasis and metabolic balance of the body's energy, while visceral white fat is involved in the development of metabolic diseases.
  • Subcutaneous white fat mainly includes groin, underarm and parathyroid fat; visceral white fat mainly includes epididymal fat, perirenal fat and mesenteric fat.
  • white adipose tissue may exhibit brown fat phenotype after being subjected to certain physiological stimuli (cold exposure), hormone stimuli (such as Irisin), and the body receiving medical treatment (such as PPAR gamma agonist or ⁇ -adrenergic stimulation). If the lipid droplets become smaller, the number of strands is too large. Since browning of white fat shows that energy balance is converted from energy storage to energy expenditure, it has become a new target for the treatment of metabolic diseases such as obesity.
  • the high- and low-dose groups of the lipid-lowering and liver-protecting group can all improve brown and white.
  • HQS-H group, HQS-L group can all improve brown and white.
  • Lipitor Lipitor group
  • the epididymal fat coefficient and perirenal fat coefficient of the obese rat model established by pure high-fat feeding model were significantly higher than those of the normal group.
  • the lipid-lowering and liver-protecting composition administration group can significantly reduce the epididymal fat coefficient and the perirenal fat coefficient (reduced white fat, increase the brown-white ratio) of the model animals, and the reduction is obvious, and the white fat is browned.
  • the genetic effect is obvious; the consumption of a large amount of fatty acids while reducing white fat and increasing brown fat plays a role in lowering blood fat (one of the mechanisms of lowering blood fat).
  • Fig. 38 shows the white fat content after 14 weeks of prophylactic administration. It can be seen from the figure that the fat percentage of the low-dose group of the lipid-lowering and liver-protecting composition is remarkably lowered.
  • Fig. 39 shows the fat content in the liver of each group of rats after 14 weeks of prophylactic administration. It can be seen from the figure that the liver fat percentage of the low-dose group of the lipid-lowering and liver-protecting composition is significantly reduced. .
  • Fig. 40 shows cortical bone density, cancellous bone density, and total bone density of each group after 14 weeks of prophylactic administration (cortical bone density of each group from left to right in Fig. 40). , cancellous bone density, total bone density). It can be seen from the figure that the high and low dose groups of the lipid-lowering and liver-protecting composition also have an increase in the total bone density of the femur.
  • Example 9 Protective effect and mechanism of the lipid-lowering and liver-protecting composition of the invention on myocardial and skeletal muscle of diabetic rats
  • the SD rats were first raised in the quarantine room to observe whether they were healthy or not. After 3 days of adaptive feeding, the rats were fasted for 12-14 hours, an appropriate amount of ether was anesthetized, and blood was taken from the fundus venous plexus. Heparin sodium injection was used as the injection. The anticoagulant was aspirated at 4 ° C (3000 rpm, 10 min) to extract the fasting blood glucose (FBG), triglyceride (TG), total cholesterol (TC) as the batch. Basic biochemical indicators of blood glucose and blood lipids in rats.
  • FBG fasting blood glucose
  • TG triglyceride
  • TC total cholesterol
  • the drug-administered group namely, normal group (16w), model group (16w), lipid-lowering and liver-protecting composition group (16w), normal group (23w), model group (23w), lipid-lowering and liver-protecting composition group (23w)
  • the total duration of drug intervention was the same for both courses. Except the normal group of rats fed the basal diet, the other groups of rats were fed high-fat diet until the end of the experiment.
  • test groups are detailed as follows:
  • D The lipid-lowering and liver-protecting composition group (the mass ratio of Astragalus, Puerariae and Mulberry skin is 1:2:1), therapeutic administration (23w);
  • the dose of lipid-lowering and liver-protecting composition was set to 2.4g ⁇ kg-1, and the prepared powder was dissolved in the gum arabic solution before use, and it was fully ground until it was completely dissolved. For distribution.
  • the dose of lipid-lowering and liver-protecting composition was set to 2.4g ⁇ kg-1, and the prepared powder was dissolved in the gum arabic solution before use, and it was fully ground until it was completely dissolved.
  • the human body weight (60kg) converted to the clinical equivalent dose of the rat after the oral administration, losartan potassium tablets
  • the dosage is set to 10mg/kg, the concentration of the drug is 1mg/mL, the amount of bromide is 0.4mg/kg, and the concentration is 1.2mg/mL.
  • the ground gum solution was prepared into a solution of 1 piece/100 mL and 1 piece/50 mL, respectively, and then poured into a centrifuge tube and stored in a refrigerator at 4 ° C, and it is now ready for use.
  • the rats were fasted for 14 hours, and the abdominal aorta was taken for 4-5mL. The supernatant was aspirated at 4°C (3000rpm, 10min), and 6 parts were dispensed. Each 50-100 ⁇ L was placed in the -80°C refrigerator. In the preservation, the content of the basic biochemical indicators to be tested.
  • the rats were quickly removed, placed in a petri dish preserved in a refrigerator at 4 ° C in a refrigerator, and then placed on an ice pack. The capsule and large blood vessels were removed along the junction of the chamber, and the apex was cut.
  • the glucose assay kit is detected by glucose oxidase-peroxidase assay, the total cholesterol (TC) assay kit is detected by cholesterol oxidase-peroxidase assay, and the triglyceride (TG) assay kit uses glycerol phosphate oxidase assay.
  • TC total cholesterol
  • TG triglyceride
  • - Peroxidase assay, low-density lipoprotein cholesterol (LDL-C) assay kits are detected by enzyme direct method, the specific operation and calculation method refer to the kit, homocysteine (Hcy), glutamyl
  • the transpeptidase (GTT1) was operated by enzyme-linked immunosorbent assay (ELISA) according to the kit instructions.
  • pancreatic tissue, myocardial tissue and skeletal muscle of each group were observed under a light microscope using a conventional HE staining procedure, and professional laboratory workers were asked to take pictures.
  • pancreatic acinar cells in the normal pancreas group are intact, the islet group is round or oval, the nucleus is clear, the boundary is clear, and the number of cells in each islet group is large and the cytoplasm is rich.
  • the cell size is uniform and the center of the cell is located (see Figures A1 and A2).
  • the amount of insulin in the model group was significantly reduced, the islet volume was significantly atrophied, and the morphology was irregular (see Figures B1 and B2); the islet endocrine cells were significantly reduced, some cells were cytoplasmic vacuolar degeneration, and some nuclei were pyknotic or Dissolved, disappeared, some areas of the cell gap increased, there is inflammatory cell infiltration around the island.
  • the islets of the lipid-lowering and liver-protecting composition for the prevention and administration of the rats were greatly improved, the number of islets was significantly increased, and the number of cells in the islets was increased.
  • the losartan and bromide-treated groups also showed varying degrees of improvement.
  • the myocardial tissues of the normal group of rats in the prevention and treatment were arranged neatly and uniformly, and the muscle cell size was regular (see Figures A1 and A2).
  • the model group of the prevention and treatment showed disorder of myocardial tissue, muscle.
  • the cell size is not uniform, obvious fat vacuole deposition is observed between the muscle fibers, the muscle fiber spacing is obviously widened, the fibers are obviously thinned, some muscle fibers have been dissolved and other pathological changes, and the lesions of the treatment model group rats are more serious (see Figure B1).
  • the muscle tissue of the normal group of rats in prevention and treatment was arranged in a complete, regular, clear, uniform muscle cell size, no cell atrophy, edema, muscle cell necrosis, fracture, and no fibrous tissue proliferation (see Figure A1, A2); prevention and treatment of the model group muscle tissue arrangement disorder, muscle cell size is uneven, atrophy is obvious, muscle cells large area edema, inflammatory cell infiltration is more obvious, large area of focal muscle tissue fracture (see Figure B1) B2); the muscle tissue arrangement of the rats in the prevention group and the treatment group of the lipid-lowering and liver-protecting group were uniform, the size of the muscle cells was uniform, the atrophy of the muscle cells was significantly reduced, the area of myocyte edema was small, the range was small, and the degree was light, and the prevention group was significantly better.
  • the treatment group (see Figures C and D); the bromide-fixed group had regular muscle tissue arrangement, mild inflammatory cell infiltration, and myocyte necrosis was rare (see Figure F).
  • the cardiomyocytes of the normal group were prevented from being red, the collagen fibers were blue, and the myocardial collagen fibers were evenly distributed, but the A2 myocardial collagen fibers were higher than A1 (see Figures A1 and A2).
  • myocardial interstitial and perivascular collagen components were accumulated, unevenly distributed, disordered, and connected to each other in a network.
  • B2 myocardial collagen fibers were higher than B1 (see Figures B1 and B2).
  • the myocardial collagen fibers were evenly distributed in each drug group, and a small amount of collagen was found around the myocardial cell interstitial and blood vessels (see Figures C, D, and E).
  • the skeletal muscle cells of the normal group were prevented from being red, the collagen fibers were blue, and the skeletal muscle collagen fibers were evenly distributed (see Figures A1 and A2).
  • the B2 skeletal muscle collagen fiber content was higher than B1 (see Figures B1 and B2).
  • the collagen fibers in the skeletal muscle of the lipid-lowering and liver-protecting group and the bromide group were more evenly distributed, and the content was less.
  • some collagen components were still found in the skeletal muscle cell interstitial and perivascular groups in the lipid-lowering and liver-protecting group. See Figure D).
  • the lipid-lowering and liver-protecting compositions can significantly reduce the content of GLU, TG and TC in diabetic rats.
  • Losartan can significantly reduce the content of GLU and TG, and the decrease of TC is not obvious; the bromide has obvious Hypoglycemic effect, but has a significant increase in TG, which may be the yang Caused by side effects of drugs.
  • the lipid-lowering and liver-protecting compositions can significantly reduce the content of Hcy and GGT1 in the liver of diabetic rats. Both losartan and bromide can reduce the Hcy content in the liver, but have a rise in GGT1. High effect (p ⁇ 0.01).
  • Example 10 The lipid-lowering and liver-protecting composition of the present invention improves the gene of obesity
  • the test is divided into 8 groups, each of which is as follows:
  • SPF-grade SD rats were randomly divided into normal group, glucocorticoid group, high-fat diet group, high-fat plus glucocorticoid group, and high-grade after two weeks of adaptive feeding in normal animal rooms (about 200-220 g).
  • Lipid plus glucocorticoid + lipid-lowering liver-protecting composition group glucocorticoid plus lipid-lowering liver-protecting composition group.
  • the normal group, the glucocorticoid group and the glucocorticoid + lipid-lowering and liver-protecting composition group were fed with the basic diet, and the other groups were fed with the high-fat diet.
  • the other groups began to administer prednisone acetate 3.5 mg/kg once a day, while high-fat plus glucocorticoid + lipid-lowering liver-protecting composition group and glucocortico
  • the lipid-lowering and liver-protecting composition (2.96g/kg) was started by intragastric administration, and the mass ratio of Astragalus: Pueraria: Mulberry was 1:2:1, continuous 12 week. Thereafter, the test operation was carried out as follows:
  • the precipitate was added with 30 ⁇ l of RNase-free water, and the precipitate was fully dissolved by a water bath at 58 ° C for 10 min.
  • RNA samples were assayed for OD-A260 and A280 (A260/A280 ratio of samples with high RNA purity > 1.7) at wavelengths of 260 nm and 280 nm using a DU800 nucleic acid/protein analyzer, and the RNA concentration was measured. Take a small amount of nucleic acid protein assay A260, calculate the RNA content. The A260/A280 was measured to be 1.8 to 2.0, and the purity of the RNA was confirmed. The rest was stored at -80 ° C for storage.
  • the RT reaction was carried out in accordance with the RT reagent instructions.
  • the RT reaction system is shown in the table below.
  • RNA volume 3.0 ⁇ g of total RNA based on the RNA concentration, and adjust the amount of RNase-free ultrapure water so that the total reaction volume is 20 ⁇ l.
  • the obtained cDNA product was stored at -20 °C.
  • Primers were designed according to the real time PCR primer design principles. The NCBI GenBank database was searched, and the designed primers were for different exons, and ⁇ -actin was used as the calibration gene for the internal control. PCR primers for MCP-1, UCP-1, PRDM16, Cidea and ⁇ -actin were designed by Primer Premier 5.0 software. The sequences of different primers are shown in Table 15. All PCR primers were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. .
  • the UCP-1 gene is abundantly expressed in brown adipose tissue and rarely expressed in white adipose tissue, and is a brown adipocyte-specific gene. As shown in Fig. 50, except for the high-fat diet group, the expression of UCP-1 gene was significantly decreased in the other groups, and the expression of UCP-1 gene in group I (HFD+GC+Example 2 composition group) was significantly higher. In group D (HFD+GC group), Huangqisan can promote the expression of brown adipose cell-specific gene UCP-1, indicating that Huangqisan has positive significance in resisting obesity.
  • group I HFD + GC + Example 2 composition group
  • group I significantly increased in Cidea gene expression, higher than other groups, indicating that Huangqisan has positive significance in resisting obesity.
  • the other groups showed a significant increase in MCP-1 gene expression compared to the normal group.
  • group D high-fat diet plus glucocorticoid group
  • the lipid-lowering and liver-protecting group I and J groups
  • Figure 53 shows that the PRDM16 transcription factor plays an important role in regulating brown fat and myoblast transformation, is essential for maintaining the brown fat phenotype, and also promotes brown fat formation and energy expenditure.
  • the lipid-lowering and liver-protecting composition has a significant effect on the gene of white fat browning.

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Abstract

一种降脂保肝组合物及其制备方法以及含有该组合物的药物，该组合物包含黄芪、葛根和桑白皮，且黄芪、葛根和桑白皮的质量比为黄芪∶葛根∶桑白皮＝(1～2)∶(1～2)∶(0～2)。该组合物及含有该组合物的药物能够用于肥胖型高脂血症、脂肪肝、肥胖症、高同型半胱氨酸血症的治疗以及心肌和骨骼肌的保护。

Description

一种降脂保肝组合物及其用途 技术领域
本发明涉及一种组合物及其用途，尤其是一种具有降低肥胖性高脂血症和改善脂肪肝的组合物及其用途。
背景技术
黄芪，又名绵芪，其主要活性成分是黄芪甲苷，具有抑制代谢综合征、延缓心肌缺血中心肌病变和改善心功能等作用，现代医学研究表明，黄芪有增强机体免疫功能、保肝、利尿、抗衰老、抗应激、降压和较广泛的抗菌作用。能消除实验性肾炎蛋白尿，增强心肌收缩力，调节血糖含量。黄芪不仅能扩张冠状动脉，改善心肌供血，提高免疫功能，而且能够延缓细胞衰老的进程。
葛根，为豆科植物野葛的干燥根，习称野葛。葛根中主要活性部位为总黄酮，其中葛根素是其主要活性成分之一。葛根素具有降糖、降脂、改善胰岛素抵抗作用，以及降压、扩张冠状动脉、改善心肌缺血、抗缺氧再灌注损伤、保护内皮功能以改善血液动力学、抑制血小板聚集等多种心血管保护作用，因此葛根素被广泛的应用于临床治疗糖尿病型冠心病和多种心血管疾病。
桑白皮为桑科植物桑的干燥根皮。其含黄酮类成分：桑素、桑皮色烯素、环桑素等。桑白皮中分离并证实了降血糖成分脱二氧亚胺基葡萄醇和MoranA降糖物质，经过试验，已经证实MoranA对四氧嘧啶诱发的高血糖小鼠有剂量依赖的降糖效果。
高脂血症，指血浆中脂质浓度超过正常范围。血脂是人体血浆内所含脂质的总称，其中包括胆固醇、甘油三脂、胆固醇脂、β-脂蛋白、磷脂、未脂化的脂酸等。当血清胆固醇超过正常值230毫克/100毫升，甘油三脂超过140毫克/100毫升，β-脂蛋白超过390毫克/100毫升以上时，即可称之为高血脂症。诱发高脂血症的原因多种多样。第一，基因决定，尤其是原发性的高脂血症。第二，其他疾病继发的，如肥胖、糖尿病、甲状腺功能减低、肾病综合征以及使用特定药物等，都可能诱发高脂血症。第三，吸烟、过多食用高糖高脂食物，都可能导致胆固醇或甘油三酯升高。肥胖型高脂血症是属于上述所述的第二种，由肥胖诱发的高脂血症。
脂肪肝，是指由于各种原因引起的肝细胞内脂肪堆积过多的病变。脂肪肝根据病因的不同，可以分为：肥胖性脂肪肝、酒精性脂肪肝、快速减肥性脂肪肝、营养不良性脂肪肝、糖尿病脂肪肝、药物性脂肪肝等。脂肪性肝病正严重威胁国人的健康，成为仅次于病毒性肝炎的第二大肝病，已被公认为隐蔽性肝硬化的常见原因。
已有将黄芪、葛根和桑白皮通过一定的比例组合用于治疗糖尿病、高脂蛋白血症的的报道，但将黄芪、葛根和桑白皮用于治疗肥胖型高脂血症和脂肪肝，目前还未有任何研究及报道。
发明内容
本发明的目的在于克服上述现有技术的不足之处而提供一种能够有效用于治疗肥胖型高脂血症和改善脂肪肝的降脂保肝组合物。同时，本发明还提供了所述降脂保肝组合物的制备方法及用途。
为实现上述目的，本发明采取的技术方案为：一种降脂保肝组合物，包含黄芪、葛根和桑白皮，且所 述黄芪、葛根和桑白皮的质量比为黄芪∶葛根∶桑白皮＝(1～2)∶(1～2)∶(0～2)。
本申请发明人经过大量研究及试验发现，如上所述特定比例关系的黄芪、葛根和桑白皮组成的组合物，在降肥胖性高脂血症和改善脂肪肝方面有着尤其显著的效果。
作为本发明所述降脂保肝组合物的优选实施方式，所述黄芪、葛根和桑白皮的质量比为黄芪∶葛根∶桑白皮＝2∶1∶1。
作为本发明所述降脂保肝组合物的优选实施方式，所述黄芪、葛根和桑白皮的质量比为黄芪∶葛根∶桑白皮＝1∶2∶1。
作为本发明所述降脂保肝组合物的优选实施方式，所述黄芪、葛根和桑白皮的质量比为黄芪∶葛根∶桑白皮＝1∶1∶2。
作为本发明所述降脂保肝组合物的优选实施方式，所述黄芪、葛根和桑白皮的质量比为黄芪∶葛根∶桑白皮＝1∶1∶1。
作为本发明所述降脂保肝组合物的优选实施方式，所述黄芪、葛根和桑白皮的质量比为黄芪∶葛根∶桑白皮＝2∶1∶0。
另外，本发明的另一目的在于提供一种如上所述的降脂保肝组合物的制备方法，为实现此目的，本发明采取的技术方案为：一种降脂保肝组合物的制备方法，包括以下步骤：将黄芪、葛根和桑白皮混合后以溶剂回流提取，提取后回收溶剂，并用大孔树脂对提取液精提，即得降脂保肝组合物。
另外，本发明还提供了一种用于治疗肥胖型高脂血症和脂肪肝的药物，所述药物包含如上所述的降脂保肝组合物。
另外，本发明还提供了如上所述降脂保肝组合物在用于制备预防或治疗肥胖性高脂血症的食品、药物和保健品中的用途。
另外，本发明还提供了如上所述降脂保肝组合物在用于制备预防或治疗脂肪肝的食品、药物和保健品中的用途。
另外，本发明还提供了如上所述的降脂保肝组合物在用于制备预防或治疗肥胖症的食品、药物和保健品中的用途。
另外，本发明还提供了如上所述的降脂保肝组合物在用于制备预防或治疗高半胱氨酸血症的食品、药物和保健品中的用途。
最后，所述的降脂保肝组合物在用于制备心肌和骨骼肌保护药物中的应用。
本发明所述降脂保肝组合物，能够有效用于肥胖性高脂血症和脂肪肝的治疗，产生显著的效果。本发明含有所述降脂保肝组合物的药物，能够有效治疗肥胖型高脂血症和脂肪肝，为肥胖性高脂血症和脂肪肝的治疗提供了新的选择。
附图说明
图1为对链脲佐菌素(STZ)-高脂饲料(HFD)联合诱导的2型糖尿病大鼠给药7周后，各组空腹血糖的对比图。
图2为对链脲佐菌素(STZ)-高脂饲料(HFD)联合诱导的2型糖尿病大鼠给药7周后，各组胆固醇的对比图。
图3为对链脲佐菌素(STZ)-高脂饲料(HFD)联合诱导的2型糖尿病大鼠给药7周后，各组甘油三酯的对比图。
图4为对链脲佐菌素(STZ)-高脂饲料(HFD)联合诱导的2型糖尿病大鼠给药7周后，各组低密度脂蛋白的对比图。
图5为对链脲佐菌素(STZ)-高脂饲料(HFD)联合诱导的2型糖尿病大鼠给药12周后，各组空腹血糖的对比图。
图6为对链脲佐菌素(STZ)-高脂饲料(HFD)联合诱导的2型糖尿病大鼠给药12周后，各组胆固醇的对比图。
图7为对链脲佐菌素(STZ)-高脂饲料(HFD)联合诱导的2型糖尿病大鼠给药12周后，各组甘油三酯的对比图。
图8为对链脲佐菌素(STZ)-高脂饲料(HFD)联合诱导的2型糖尿病大鼠给药12周后，各组谷丙转氨酶的对比图。
图9为对链脲佐菌素(STZ)-高脂饲料(HFD)联合诱导的2型糖尿病大鼠给药12周后，各组谷草转氨酶的对比图。
图10为对糖皮质激素(GC)+高脂饲料联合诱导的2型糖尿病大鼠给药6周后，各组空腹血糖的对比图。
图11为对糖皮质激素(GC)+高脂饲料联合诱导的2型糖尿病大鼠给药6周后，各组胆固醇的对比图。
图12为对糖皮质激素(GC)+高脂饲料联合诱导的2型糖尿病大鼠给药6周后，各组甘油三酯的对比图。
图13为对糖皮质激素(GC)+高脂饲料联合诱导的2型糖尿病大鼠给药10周后，各组空腹血糖的对比图。
图14为对糖皮质激素(GC)+高脂饲料联合诱导的2型糖尿病大鼠给药10周后，各组胆固醇的对比图。
图15为对糖皮质激素(GC)+高脂饲料联合诱导的2型糖尿病大鼠给药10周后，各组甘油三酯的对比图。
图16为对糖皮质激素(GC)+高脂饲料联合诱导的2型糖尿病大鼠给药12周后，SD组肝细胞形态图。
图17为对糖皮质激素(GC)+高脂饲料联合诱导的2型糖尿病大鼠给药12周后，SD+GC组肝细胞形态图。
图18为对糖皮质激素(GC)+高脂饲料联合诱导的2型糖尿病大鼠给药12周后，HFD组肝细胞形态图。
图19为对糖皮质激素(GC)+高脂饲料联合诱导的2型糖尿病大鼠给药12周后，HFD+GC组肝细胞形态图。
图20为对糖皮质激素(GC)+高脂饲料联合诱导的2型糖尿病大鼠给药12周后，罗格列酮组肝细胞形态图。
图21为对糖皮质激素(GC)+高脂饲料联合诱导的2型糖尿病大鼠给药12周后，黄芪∶葛根∶桑白 皮＝2∶1∶1组肝细胞形态图。
图22为对糖皮质激素(GC)+高脂饲料联合诱导的2型糖尿病大鼠给药12周后，黄芪∶葛根∶桑白皮＝2∶1∶0组肝细胞形态图。
图23为对糖皮质激素(GC)+高脂饲料联合诱导的2型糖尿病大鼠给药12周后，黄芪∶葛根∶桑白皮＝1∶2∶1组肝细胞形态图。
图24为经连续7周的高脂饲料喂养后得到的模型组大鼠(HFD组)与正常组大鼠(SD组)的体重以及FPG、TC、TG、LDL-C水平的对比图。
图25为预防性给药以及治疗性给药期间，各组大鼠的饮食量对比图。
图26为预防性给药7周后，各组大鼠FPG、TC、TG、LDL-C水平变化对比图。
图27为治疗性给药9周后，各组大鼠FPG、TC、TG、LDL-C水平变化对比图。
图28为预防性给药12周后，各组大鼠FPG、TC、TG、LDL-C水平变化对比图。
图29为治疗性给药14周后，各组大鼠FPG、TC、TG、LDL-C水平变化对比图。
图30为预防性给药15周以及治疗性给药15周后，匀浆肝脏，得到的各组大鼠肝脏中同型半胱氨酸水平对比图。
图31为预防性给药15周以及治疗性给药15周后，匀浆肝脏，得到的各组大鼠肝脏中谷氨酰转肽酶水平对比图。
图32为预防性给药15周以及治疗性给药15周后，匀浆肝脏，得到的各组大鼠肝脏中脂质(TC、TG)水平对比图。
图33为预防性给药15周以及治疗性给药15周后，各组大鼠肝细胞进行油红O染色后的脂肪变性对比图。
图34为预防性给药15周以及治疗性给药15周后，各组大鼠的棕色脂肪与附睾脂肪之比对比图。
图35为预防性给药15周以及治疗性给药15周后，各组大鼠的棕色脂肪与肾周脂肪之比对比图。
图36为治疗性给药15周后，各组大鼠的骨重量系数比对比图。
图37为利用Latheta LCT200对活体大鼠进行全身扫描得到的micro-CT扫描图。
图38为预防性预防给药14周后，各组大鼠活体中的白色脂肪含量对比图；
图39为预防性预防给药14周后，各组大鼠肝脏中的脂肪含量对比图；
图40为预防性给药14周后，各组大鼠股骨皮质骨密度、松质骨密度、总骨密度对比图；
图41为预防性给药16周以及治疗性给药16周后，各组大鼠胰腺组织病理结果图。
图42为预防性给药16周以及治疗性给药16周后，各组大鼠心肌组织病理结果图。
图43为预防性给药16周以及治疗性给药16周后，各组大鼠骨骼肌组织病理结果图。
图44为预防性给药16周以及治疗性给药16周后，各组Masson染色后心肌胶原纤维形态结果图。
图45为预防性给药16周以及治疗性给药16周后，各组心肌组织胶原相对含量对比图。
图46为预防性给药16周以及治疗性给药16周后，各组Masson染色后骨骼肌胶原纤维的形态结果图。
图47为预防性给药16周以及治疗性给药16周后，各组骨骼肌组织胶原相对含量对比图。
图48为治疗性给药16周后，各组糖尿病大鼠GLU、TG、TC含量对比图。
图49为治疗性给药16周后，匀浆肝脏，各组糖尿病大鼠Hcy、GGT1含量对比图。
图50为治疗性给药12周后，各组糖尿病大鼠UCP-1表达量对比图。
图51为治疗性给药12周后，各组糖尿病大鼠Cidea表达量对比图。
图52为治疗性给药12周后，各组糖尿病大鼠MCP-1表达量对比图。
图53为治疗性给药12周后，各组糖尿病大鼠PRDM16转录因子mRNA表达量对比图。
具体实施方式
为更好的说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。
实施例1
本发明降脂保肝组合物的一种实施例，本实施例降脂保肝组合物包含黄芪、葛根和桑白皮，且所述黄芪、葛根和桑白皮的质量比为黄芪∶葛根∶桑白皮＝2∶1∶1。
实施例2
本发明降脂保肝组合物的一种实施例，本实施例降脂保肝组合物包含黄芪、葛根和桑白皮，且所述黄芪、葛根和桑白皮的质量比为黄芪∶葛根∶桑白皮＝1∶2∶1。
实施例3
本发明降脂保肝组合物的一种实施例，本实施例降脂保肝组合物包含黄芪、葛根和桑白皮，且所述黄芪、葛根和桑白皮的质量比为黄芪∶葛根∶桑白皮＝1∶1∶2。
实施例4
本发明降脂保肝组合物的一种实施例，本实施例降脂保肝组合物包含黄芪、葛根和桑白皮，且所述黄芪、葛根和桑白皮的质量比为黄芪∶葛根∶桑白皮＝1∶1∶1。
实施例5
本发明降脂保肝组合物的一种实施例，本实施例降脂保肝组合物包含黄芪、葛根和桑白皮，且所述黄芪、葛根和桑白皮的质量比为黄芪∶葛根∶桑白皮＝2∶1∶0。
实施例6本发明降脂保肝组合物降脂保肝效果试验
1、本发明降脂保肝组合物对链脲佐菌素(STZ)-高脂饲料(HFD)联合诱导的2型糖尿病大鼠空腹血糖、总胆固醇、甘油三酯、低密度脂蛋白的影响
1.1试验分组
取雄性SD大鼠若干只，体重180～220g，在实验室条件下适应性饲养一周后，禁食16h，乙醚麻醉，于眼底静脉丛取血，肝素抗凝，4℃离心(3000rpm，10min)分取血浆，测定大鼠空腹血糖(Glu)、甘油三酯(TC)、总胆固醇(TG)作为该批次SD大鼠的基础生化指标。喂以基础饲料1周后，取10只作为正常组，其余作为模型组。大鼠禁食16h后，模型组腹腔注射链脲佐菌素(STZ)溶液35mg/Kg，正常组腹腔注射等体积柠檬酸盐缓冲液(PH＝4.4)，注射体积为5ml/Kg，注射10天后，禁食16h，眼底静脉丛取血，测定空腹血糖FBG，选取FBG＞7.2mmo/L的大鼠为2型糖尿病模型大鼠并给予高脂饲料。
将正常组大鼠取出10只，为A-正常组，然后将上述2型糖尿病模型大鼠随机分为6组，每组8只，6组分别为：B-模型组；G-试验组1；H-试验组2；I-试验组3；J-试验组4；K-试验组5。
1.2剂量设置
本发明组合物剂量为60g生药/日，按照BioS氏法(动物公斤体重剂量系数折算法)计算动物给药的临床等效剂量为：本发明组合物7g生药/kg。本发明组合物(黄芪∶葛根∶桑白皮)试验组1(2∶1∶1)；试验组2(1∶2∶1)；试验组3(1∶1∶2)；试验4(1∶1∶1)；试验组5(2∶1∶0)。
G：60g÷60kg×7×20.7875％×2＝2.91025g/Kg，按10ml/kg给药；精密称取2.91025g于10ml 0.5％的阿拉伯树胶中；
H：60g÷60kg×7×21.1695％×2＝2.96373g/Kg。按10ml/kg给药；精密称取2.96373g于10ml 0.5％的阿拉伯树胶中；
I：60g÷60kg×7×17.8993％×2＝2.50593g/Kg。按10ml/kg给药；精密称取2.50593g于10ml 0.5％的阿拉伯树胶中；
J：60g÷60kg×7×19.2917％×2＝2.70081g/Kg；按10ml/kg给药；精密称取2.70081g于10ml 0.5％的阿拉伯树胶中；
K：60g÷60kg×7×27.455％×2＝3.8479g/Kg。按10ml/kg给药；精密称取3.8479g于10ml 0.5％的阿拉伯树胶中。
1.3试验方法
正常组和模型组分别灌服10％阿拉伯树胶(溶媒)，G-试验组1、H-试验组2、I-试验组3、J-试验组4、K-试验组5分别灌胃给药实施例1～5的药物组合物(将实施例1～5的药物组合物中，黄芪、根和桑白皮混合后以溶剂回流提取，提取后回收溶剂，并用大孔树脂对提取液精提后灌胃给药)，各组动物灌胃给药，每天一次，按照10ml/Kg/d进行灌胃给药，分别测试给药7周后和给药12周后各组动物的空腹血糖(Glu)、甘油三酯(TC)、胆固醇(TG)、低密度脂蛋白(LDL-C)。
1.4试验结果
1.4.1给药7周后，各组动物的空腹血糖(Glu)、甘油三酯(TC)、胆固醇(TG)、低密度脂蛋白(LDL-C)见表1和附图1～4所示。
表1给药7周后的Glu、TC、TG、LDL-C结果(
Figure PCTCN2017079796-appb-000001
n＝8)
Figure PCTCN2017079796-appb-000002
与正常组相比，*P＜0.05，**P＜0.01，***P＜0.001；与模型组相比，ΔP＜0.05，ΔΔP＜0.01，ΔΔΔP＜0.001
由表1及附图1～4可知，与正常组相比，模型组血糖显著升高(P＜0.05)，表明STZ糖尿病小鼠造模成功。
关于对空腹血糖的影响：从表1和附图1可看出，与模型组血糖相比，不同比例降脂保肝组合物给药 组具有显著性差异(P＜0.05)，有较明显的降低血糖作用；但各给药组之间并无差异。
关于对胆固醇的影响：从表1和附图2可看出，与正常组相比，模型组胆固醇显著升高(P＜0.05)；与模型组胆固醇相比，不同比例黄芪散给药组具有显著性差异(P＜0.05)；K组(2∶1∶0)与其他给药组相比具有显著性差异(P＜0.05)；各个给药组对胆固醇均有降低的作用，但K组(2∶1∶0)较其他组相比降低胆固醇的效果更明显。
关于对甘油三酯的影响：从表2和附图3可见，与正常组相比，模型组甘油三酯显著升高(P＜0.05)；与模型组相比G(2∶1∶1)、H(1∶2∶1)、J(1∶1∶1)有显著性差异(P＜0.05)，可以明显的降低甘油三酯水平。I组与模型组相比没有显著性差异(P＞0.05)，没有降低甘油三酯的作用。
关于低密度脂蛋白的影响：从表2和附图4可见，与正常组相比，模型组低密度脂蛋白显著升高(P＜0.05)；与模型组相比，各个给药组均具有显著性差异(P＜0.05)；K组与其他给药组相比具有显著性差异(P＜0.05)；不同比例的黄芪散给药组均可以降低低密度脂蛋白水平，但K组较其他组降低低密度脂蛋白水平的效果更明显。
以上结果表明，不同比例的配方对血糖血脂的影响不同。G(2∶1∶1)；H(1∶2∶1)；J(1∶1∶1)对血糖、胆固醇、甘油三酯、低密度脂蛋白均有明显的降低作用。I(1∶1∶2)，K(2∶1∶0)可以降低胆固醇、低密度脂蛋白、血糖的水平，但没有降低甘油三酯水平的作用。
1.4.2给药12周后，各组动物的空腹血糖(Glu)、胆固醇(TG)和甘油三酯(TC)见表2和附图5～7所示。
表2给药12周后各组空腹血糖、胆固醇和甘油三酯结果(
Figure PCTCN2017079796-appb-000003
n＝8)
Figure PCTCN2017079796-appb-000004
与正常组相比*P＜0.05，**P＜0.01，***P＜0.001；与模型组相比，ΔP＜0.05，ΔΔP＜0.01，ΔΔΔP＜0.001
结果分析：由表2和附图5～7可以看出，随着给药时间的加长，不同比例组合物在疗效上的作用差异更加明显。给药12周后，H组对血糖、胆固醇、甘油三酯的影响较其他组要好(P＜0.01)；G组对血糖血脂的影响次之。K组降低胆固醇的优势较为明显。J组可以降低血糖、胆固醇、及甘油三酯的水平，但其降低甘油三酯的作用较其他方面要好。I组可以降低胆固醇的水平，但对血糖血脂的影响不明显。
2、本发明降脂保肝组合物对2型糖尿病大鼠肝脏的影响(对谷草/谷丙转氨酶、胆红素的影响)
本试验分组同上，分别测试给药12周后各组动物的谷丙转氨酶(ALT)和谷草转氨酶(AST)，测试结果见表3和附图8～9所示。
表3给药12周后各组的谷丙转氨酶(ALT)、谷草转氨酶(AST)结果(
Figure PCTCN2017079796-appb-000005
n＝8)
Figure PCTCN2017079796-appb-000006
与正常组相比，*P＜0.05，**P＜0.01，***P＜0.001；与模型组相比，ΔP＜0.05，ΔΔP＜0.01，ΔΔΔP＜0.001
由表3和附图8～9可看出，与正常组相比，模型组谷草转氨酶(AST)有明显升高的趋势，但无显著性差异(P＞0.05)；但与模型组相比，各给药组的谷草转氨酶具有下降的趋势。与正常组相比，模型组谷丙转氨酶明显升高，有显著性差异(P＜0.05)，12周后2型糖尿病大鼠肝功能受损。与模型组相比，各给药组均显著降低谷草转氨酶水平(P＜0.05)，改善肝功能；与正常组相比，各给药组谷丙转氨酶没有显著性差异(P＞0.05)。由此可以得出，不同比例的本发明组合物均可预防2型糖尿病造成的肝功能异常的状况。
实施例7本发明降脂保肝组合物降脂保肝效果试验
1、本发明降脂保肝组合物对糖皮质激素(GC)+高脂饲料联合诱导的2型糖尿病大鼠空腹血糖(FPG)、胆固醇(TC)、甘油三酯(TG)的影响
1.1试验分组
本试验共分为9组，每组分别如下：
A——SD组，N＝8
B——SD+GC组，N＝10
C——HFD组，N＝8
D——HFD+GC组，N＝12
E——HFD+GC+罗格列酮组，N＝7
F——HFD+GC+实施例1组合物组，N＝9
G——HFD+GC+实施例5组合物组，N＝9
I——HFD+GC+实施例2组合物组，N＝10
J——SD+GC+实施例2组合物组，N＝10
1.2试验方法
SPF级别SD大鼠在普通级动物房适应性饲养两周后(约200-220g)，空腹取血测定基础水平。3天后，除正常基础饲料组外，其余各组开始灌胃醋酸泼尼松3.5mg/kg，一天一次，同时1小时后开始灌胃给药，高脂饲料喂养连续12周。在给药6周和给药10周，分别禁食12h，采血浆测各组空腹血糖、胆固醇、甘油三酯的指标，观察给药6周和给药10周后各组空腹血糖、胆固醇和甘油三酯的变化。
1.3试验结果
给药6周后各组空腹血糖、胆固醇和甘油三酯的变化结果如附图10～12所示。
由附图10可看出，从对空腹血糖FPG的影响来看，模型HFD+GC组与正常基础组相比，血糖升高，具有统计学差异(P＜0.05)。HFD+GC组大鼠血糖高于SD+GC组，也具有统计学差异(P＜0.05)。但SD+GC与SD组相比血糖无升高，表明基础饲料联合糖皮质激素造模在6周时未成功，但高脂联合糖皮质激素造模成功。与模型HFD+GC组相比，罗格列酮组大鼠血糖降低，具有统计学差异(P＜0.05)。此时，仅基础喂养的J组与模型组相比，血糖降低，具有降血糖作用，同时结合体重的影响，发现喂养基础饲料的J组血糖低于喂养高脂饲料的F、G、I组，体重也相应低于，提示本发明组合物对体重抑制作用可能是降糖的原因。
由附图11可看出，从对胆固醇TC的影响来看，模型HFD+GC组与正常基础组相比，胆固醇升高，具有统计学差异(P＜0.01)。各给药组均可以降低胆固醇，并且基础喂养的J组降胆固醇的幅度更大。
由附图12可看出，从对甘油三酯TG的影响来看，模型组的甘油三酯水平与正常组相比均未升高，奇怪的是高脂喂养组HFD组甘油三酯水平最低，表明糖尿病模型血浆中甘油三酯水平是降低的，这有待于进一步观察和思考。
给药10周后各组空腹血糖、胆固醇和甘油三酯的变化结果如附图13～15所示。
由附图13可看出，从对空腹血糖FPG的影响来看：与正常组相比，SD+GC、HFD、HFD+GC模型组血糖均升高，且HFD+GC模型组血糖最高，具有统计学差异。其原因在于糖皮质激素属于胰岛素拮抗激素，可以增强肝脏中的糖原异生，促进肝糖原分解，抑制外周组织对葡萄糖的摄取和利用，从而导致血糖升高。与HFD+GC模型组相比，罗格列酮组大鼠血糖降低，具有统计学差异(P＜0.001)。本发明组合物各给药组血糖也明显降低，具有统计学差异，而J组相比其他组而言，降糖幅度更明显，同时结合J组原方降低体重的影响，提示本发明组合物对体重抑制作用可能是降糖的原因。
由附图14可看出，从对胆固醇TC的影响来看，模型HFD、HFD+GC组与正常基础组相比，胆固醇升高，具有统计学差异(P＜0.01)。各给药组均可以降低胆固醇，并且基础喂养的J组、高脂喂养的F、G、I组降胆固醇的幅度更大。
由附图15可看出，从对甘油三酯TG的影响来看，模型组的甘油三酯水平与正常组相比均未升高，奇怪的是高脂喂养组HFD组甘油三酯水平最低，这与前期给药6周的变化情况基本一致。
2、本发明降脂保肝组合物对糖皮质激素(GC)+高脂饲料联合诱导的2型糖尿病大鼠肝细胞形态变化的影响
对上述1中给药12周后的A、B、C、D、E、F、G、I组，采用H-E染色法，在同一视野下观察各组大鼠肝脏细胞的形态变化。观察结果如附图16～23所示。
由附图16～23可看出，正常SD组大鼠可见肝小叶，小叶中可见中央动脉及周围肝细胞围城的肝板结构，未见明显病理改变。SD+GC组大鼠可见肝小叶中有少量肝细胞轻微脂肪变性，肝细胞中可见微小脂滴。HFD和HFD+GC组大鼠可见肝小叶中肝细胞中脂肪变性明显，肝细胞中充满了小泡样脂滴。在小叶中可见有点状坏死区域，周围可见炎性细胞浸润，且HFD+GC组大鼠脂肪变性更加明显。与HFD+GC组大鼠相比，罗格列酮组、F组、I组大鼠可见肝细胞脂肪变性极少，肝细胞中少见小泡样脂滴。而G组大鼠可见肝细胞脂肪变性明显，肝细胞中可见大的脂肪空泡。
实施例8本发明降脂保肝组合物与立普妥效果对比试验
1.试验分组
本实验包括降脂保肝组合物(HQS)预防给药和治疗给药两部分。健康雄性SD大鼠应性饲养一周后，随机分为正常组(SD)和造模组，正常组采用基础饲料喂养，造模组采用高脂饲料喂养，自由饮水和进食，同时选取10只造模组大鼠为模型组(HFD)。降脂保肝组合物预防给药：将造模组大鼠随机分为HQS-L-降脂保肝组合物低剂量组(1.2g/kg.d)、HQS-H-降脂保肝组合物高剂量组(2.4g/kg.d)，每组8只，同时造模同时给药。降脂保肝组合物治疗给药：将造模组大鼠连续高脂喂养7周后，造模组体重明显高于正常组(喂食基础饲料的SD大鼠)，造模组FPG、TC、TG、LDL-C水平也明显高于正常组，表明此时模型成功，具有统计学差异，具体如附图24所示。选择超过正常大鼠体重20％的大鼠为肥胖大鼠模型，随机分为HQS-L-降脂保肝组合物低剂量组(1.2g/kg.d)、HQS-H-降脂保肝组合物高剂量组(2.4g/kg.d)、立普妥阳性药组(Lipitor，2mg/kg)，每组8只，高脂喂养7周后再开始给药。正常组和模型组大鼠分别给予等量的生理盐水，各给药组给予相应试药，每日一次。其中，降脂保肝组合物高、低剂量组的给药为本发明的降脂保肝组合物，其中黄芪∶葛根∶桑白皮的质量比为1∶2∶1。
2.给药方法
2.1预防性给药(预防组是边高脂饲料喂养边给药)；
2.1.1剂量设置
降脂保肝组合物低剂量(HQS-L)和降脂保肝组合物高剂量(HQS-H)的剂量设置根据前期多次药效试验所设的有效剂量，剂量分别为1.2g/kg.d和2.4g/kg.d。
2.1.2预防性给药方法
每日一次，连续给药15周。
2.2治疗性给药(治疗组是高脂喂养7周后再开始给药)。
2.2.1剂量设置
降脂保肝组合物低剂量(HQS-L)和降脂保肝组合物高剂量(HQS-H)的剂量设置根据前期多次药效试验所设的有效剂量，剂量分别为1.2g/kg.d和2.4g/kg.d。立普妥剂量是按照人临床等效剂量所设，剂量为2g/kg.d。
2.2.2治疗性给药方法
每日一次，连续给药15周。
3试验操作方法
3.1体重
实验过程中每天观察各组大鼠体重变化、进食、皮毛、活动等情况，每周记录体重变化。
3.2糖脂生化指标
预防给药：于给药7周末及给药12周末，大鼠禁食不禁水12h，乙醚麻醉，眼底静脉丛取血，肝素抗凝，4℃离心(3000r/min，15min)分取血浆，测定空腹血糖(FPG)、胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL-C)；
治疗给药：于给药9周末及给药14周末，大鼠禁食不禁水12h，乙醚麻醉，眼底静脉丛取血，肝素抗凝，4℃离心(3000r/min，15min)分取血浆，测定FPG、TC、TG、LDL-C。
3.3实验取材及脏器系数
实验结束时，各组大鼠称重，并用10％水合氯醛腹腔注射(0.35ml/100g体重)，将大鼠麻醉，大鼠麻醉后仰卧固定于解剖板上，进行腹主动脉取血处死，所获血标本一部分静置2h后3000r/min离心30min得血清，一部分为全血，并分装置于-20℃冰箱备用。迅速取出心、肝、脾、肺、肾、附睾脂肪组织、肾周脂肪组织、棕色脂肪组织、股骨等脏器组织，称重，分装，放置冻存管-80℃冷冻保存。同时计算各脏器系数，脏器系数＝脏器质量/体重×100％。同时取一部分肝脏组织用于HE染色，一部分肝脏组织用于油红O染色。
3.4肝脏脂质水平测定
从-80℃冷藏冰箱里取一管肝脏组织。检测时取组织约100mg，加入异丙醇，体积1ml/50mg。匀浆后放置过夜。第二天离心后取上清，测定其中TC和TG含量，测得的结果换算为每千克脏器中含有的脂质含量以g计，结果换算成g/kg。分子量：甘油三酯639g/mol；总胆固醇386.65g/mol。
3.5肝脏同型半胱氨酸、谷氨酰转肽酶测定
从-80℃冷藏冰箱里取一管肝脏组织。检测时取组织约100mg，用PBS洗去血污。剪成小块放入组织研磨器中，加入1ml PBS，制成匀浆，然后置于-20℃过夜。经过反复冻融2次处理破坏细胞膜后，将组织匀浆于4℃5000g离心5分钟取上清，取适量上清液立即实验。测定方法按照ELISA法，按照试剂盒说明书操作。
3.6大鼠活体micro-CT实验
预防给药组大鼠在解剖前的一周，进行活体micro-CT实验。利用Latheta LCT200小动物CT，一款针对大鼠、小鼠及兔子等动物进行多功能3D扫描的micro-CT系统。实验前，各组大鼠先用10％水合氯醛腹腔注射麻醉1-2h，放入槽中行腹平扫描，对大鼠骨参数、皮下脂肪、内脏脂肪、脂肪肝CT值等参数进行测定。该实验在南方医科大学第三附属医院医学实验研究中心进行。
4.试验结果
4.1体重曲线变化比较
在预防给药和治疗给药期间，各组大鼠不同时期的体重变化如表4～5所示。
表4预防给药期间，各组大鼠不同时期的体重变化结果(
Figure PCTCN2017079796-appb-000007
n＝8)
Figure PCTCN2017079796-appb-000008
表5治疗给药期间，各组大鼠不同时期的体重变化结果(
Figure PCTCN2017079796-appb-000009
n＝8)
Figure PCTCN2017079796-appb-000010
Figure PCTCN2017079796-appb-000011
结果分析：如表4～5所示，正常组(SD组)与模型组(HFD组)大鼠体重相比可知：模型组的体重持续增加，且具有统计学差异，表明大鼠肥胖模型成功。从降脂保肝组合物预防给药和治疗给药后大鼠的体重结果分析可知：降脂保肝组合物高剂量组(HQS-H组)、降脂保肝组合物低剂量组(HQS-L组)均对肥胖具有较好的预防作用，给药组体重明显低于模型组，接近于正常组体重，且明显优于立普妥组(Lipitor组)，立普妥组体重显著增加，接近模型组。预防组大鼠在给药前一周做了microCT实验，明显感觉大鼠体重增长缓慢，尤其降脂保肝组合物给药组大鼠体重有所下降。
如附图25所示，通过统计各组大鼠平均每天每只的饮食情况，可知喂高脂饲料的各组大鼠饮食情况之间无差异。表明各组大鼠体重的变化与饮食量无关。
4.2糖脂生化指标比较
4.2.1预防给药7周以及治疗给药9周后糖脂生化指标比较
预防给药7周以及治疗给药9周后，各组大鼠FPG、TC、TG、LDL-C水平变化如图26～27所示。
从附图26～27所示的预防给药7周和治疗给药9周糖脂结果来看，模型组(HFD组)与正常组(SD组)相比，FPG、TC、TG、LDL-C水平均显著升高，且具有统计学差异。与模型组相比，降脂保肝组合物高剂量组(HQS-H组)、降脂保肝组合物低剂量组(HQS-L组)均有较好的降糖、降脂作用，且有一定的剂量依赖性。
4.2.2预防给药12周以及治疗给药14周后糖脂生化指标比较
预防性给药12周以及治疗给药14周后，各组大鼠FPG、TC、TG、LDL-C水平变化如附图28～29所示。
从图28～29所示的预防给药12周和治疗给药14周糖脂结果来看，模型组(HFD组)与正常组(SD组)相比，FPG、TC、TG、LDL-C水平均显著升高，且具有统计学差异。与模型组相比，降脂保肝组合物高剂量组(HQS-H组)、降脂保肝组合物低剂量组(HQS-L组)均能较好的降糖、降脂，且具有一定剂量依赖性。并且在降糖降脂方面，降脂保肝组合物优于立普妥组。
4.3同型半胱氨酸、谷氨酰转肽酶比较
预防性给药15周以及治疗性给药15周后，各组大鼠肝脏中同型半胱氨酸水平和谷氨酰转肽酶水平如图30～31所示。
同型半胱氨酸(Hcy)是人体内的一种含硫氨基酸，为蛋氨酸和半胱氨酸代谢过程中的重要中间产物。Hcy过高是心脑血管疾病、骨质疏松、神经系统疾病的危险因素。如附图30所示，通过检测各组大鼠肝脏中Hcy水平，发现模型组(HFD组)水平显著升高，而降脂保肝组合物高、低剂量组(HQS-H组、HQS-L组)均显著降低，表明给药组的大鼠脂肪肝明显得到改善。
谷氨酰转肽酶(GTT1)已经是肝功能检查项目之一。如附图31所示，通过检测肝脏中GTT1水平，可知模型组水平显著升高，而降脂保肝组合物高、低剂量组(HQS-H组、HQS-L组)、立普妥组(Lipitor 组)均可显著降低。
4.4肝脏脂质比较
预防性给药15周以及治疗性给药15周后，各组大鼠肝脏中脂质(TC、TG)水平如图32所示。
脂质异位沉积是指体内脂质堆积在非脂肪组织，多发生在肝脏、肌肉和胰腺，最易发生的部位是肝脏。如附图32所示，通过检测肝脏中脂质(TC、TG)，发现模型组(HFD组)肝脏脂质显著升高，而给药组均可显著改善肝脏脂质沉积，且预防组较治疗组更加显著。
4.5肝脏油红O染色比较
预防性给药15周以及治疗性给药15周后，各组大鼠肝细胞进行油红O染色后的脂肪变性对比如图33所示。
如附图33所示，正常组肝细胞内几乎未见脂滴。模型组染色后大量大泡样的脂滴，相互融合，且高脂喂养时间越久，脂滴越明显。降脂保肝组合物预防高、低剂量组(预防HQS-H组、预防HQS-L组)的肝细胞脂肪变性明显减轻，优于降脂保肝组合物治疗高低剂量组(治疗HQS-H组、治疗HQS-L组)，均以高剂量组减轻程度更明显。降脂保肝组合物高低治疗剂量组的肝细胞脂肪变性与模型组相比，也明显得到改善，且优于立普妥组；其中，A代表正常组，I代表模型组，K代表立普妥组。
4.6棕色、白色脂肪系数比较
4.6.1白色脂肪系数比较
哺乳动物主要含有白色脂肪(WAT)棕色脂肪组织(BAT)两种。而白色脂肪通常又包括皮下白色脂肪和内脏白色脂肪。目前认为皮下白色脂肪和棕色脂肪参与维持机体能量的稳态和代谢的平衡，而内脏白色脂肪则参与代谢性疾病的发生。皮下白色脂肪主要包括腹股沟、腋下、甲状腺旁脂肪；内脏白色脂肪主要包括附睾脂肪、肾周脂肪、肠系膜脂肪。人体的相关研究认为，内脏白色脂肪与胰岛素抵抗、糖尿病等心血管疾病危险因素的相关性比皮下白色脂肪更高。探讨动物不同部位脂肪组织形态和功能差异，可以更好地为代谢综合征的发病机制提供依据。
研究发现，白色脂肪组织在受到某些生理刺激(冷暴露)，激素刺激(如Irisin)以及机体接受药物治疗(如PPARγ激动剂或β-肾上腺素能刺激)后，可出现棕色脂肪表型特征，如脂滴变小，线粒过数目过多。由于白色脂肪棕色化显示了能量平衡由能量储存转为能量支出，因而成为治疗肥胖等代谢性疾病的新靶点。
预防性给药15周以及治疗性给药15周后，各组大鼠的附睾脂肪和肾周脂肪系数如表6～7所示。
表6预防性给药15周以及治疗性给药15周后，各组大鼠的附睾脂肪和肾周脂肪系数
Figure PCTCN2017079796-appb-000012
表7治疗性给药15周以及治疗性给药15周后，各组大鼠的附睾脂肪和肾周脂肪系数
Figure PCTCN2017079796-appb-000013
结果分析：如表6～7所示，附睾脂肪和肾周脂肪作为白色脂肪，降脂保肝组合物高、低剂量组(HQS-H组、HQS-L组)均显著降低白色脂肪系数，且优于立普妥组。
4.6.2棕色/白色脂肪系数比较
预防性给药15周以及治疗性给药15周后，各组大鼠的棕色脂肪与白色脂肪之比如图34～35所示。
如附图34～35所示，通过将棕色脂肪与白色脂肪之比也可以看出来，降脂保肝组合物高、低剂量组(HQS-H组、HQS-L组)均可提高棕白比，优于立普妥(Lipitor组)。
综上，采用纯高脂喂养造模建立的肥胖大鼠模型的附睾脂肪系数、肾周脂肪系数都明显高于正常组大鼠。从给药上来看，降脂保肝组合物给药组能明显降低模型动物的附睾脂肪系数、肾周脂肪系数(降低白色脂肪、提高棕白比)，且降低明显，并且对白色脂肪棕色化的基因影响明显；在降低白色脂肪、提高棕色脂肪的同时消耗了大量的脂肪酸起到了降血脂的作用(降血脂的机理之一)。
4.7骨重量系数比比较
如附图36所示，通过股骨与体重的比，发现正常组(SD组)与模型组(HFD组)之间无差异，而降脂保肝组合物高、低剂量组(HQS-H组、HQS-L组)与模型组相比，骨重量系数均显著升高，且具有统计学差异，表明降脂保肝组合物具有提高骨质量的效果，且优于立普妥(Lipitor组)。
4.8活体成像观察药物的影响
通过活体成像显示药物对骨胳、肝、脂肪的影响如图37～40所示。利用Latheta LCT200对活体大鼠进行全身扫描，通过计算脂肪率，我们发现与正常组相比，模型组大鼠脂肪率和肝脂肪率明显升高，且具有统计学差异。与模型组相比，降脂保肝组合物高低剂量组的脂肪率和肝脏脂肪率明显降低，且具有剂量依赖性。同时对股骨的总骨密度也有所提升。
如附图38所示，其显示了预防给药14周后的白色脂肪含量，从图中可以看出降脂保肝组合物高低剂量组的脂肪率明显降低。
如附图39所示，其显示了预防性给药14周后，各组大鼠肝脏中的脂肪含量对比，从图中可以看出降脂保肝组合物高低剂量组的肝脏脂肪率明显降低。
如附图40所示，其显示了预防性给药14周后，各组大鼠皮质骨密度、松质骨密度、总骨密度(图40中从左至右依次为各组的皮质骨密度、松质骨密度、总骨密度)。从图中可以看出降脂保肝组合物高低剂量组对股骨的总骨密度也有所提升。
实施例9本发明降脂保肝组合物对糖尿病大鼠心肌和骨骼肌的保护作用及其机制研究
1.试验分组
SD大鼠先在检疫室中饲养，观察其是否健康有无病毒携带，适应性喂养3天后，禁食不禁水12-14h，适量乙醚麻醉，于眼底静脉丛取血，采用肝素钠注射液作为抗凝剂，4℃离心(3000rpm，10min)吸取上清液，测定大鼠空腹血糖(fasting blood glucose，FBG)、甘油三酯(triglyceride，TG)、总胆固醇(total cholesterol，TC)作为该批次大鼠的血糖、血脂的基础生化指标。取血后适应5天，以37mg/kg的剂量一次性腹腔注射链脲佐菌素(STZ)柠檬酸缓冲液(留取12只大鼠作为正常组并腹腔注射等体积的缓冲盐溶液)，确保在30min内注射完毕且尽量避免光照。3周后待大鼠血糖稳定，眼底静脉丛取血测空腹血糖(FBG)和糖耐量，选择糖耐量异常(2hBG≥7.8mmol·L-1)的大鼠归入成模大鼠，禁食不禁水12-14h，取血测空腹血糖(FBG)、总胆固醇(TC)、甘油三酯(TG)，根据FBG、TG、TC水平以及体重随机分组，本实验研究周期分为STZ成模后16w(简称16w模型组)和23w(STZ造模后给予高脂饲料7w后再开始给药，简称23w模型组)两个病程，因此相应设置两个病程的正常组和降脂保肝组合物给药组，即正常组(16w)、模型组(16w)、降脂保肝组合物组(16w)、正常组(23w)、模型组(23w)、降脂保肝组合物组(23w)、氯沙坦组(23w)和溴吡斯的明组(23w)，两个病程的药物干预总时间长相同。除正常组大鼠喂养基础饲料之外，其他组大鼠均喂养高脂饲料直至实验结束。
明晰起见，将8个试验分组详列如下：
A1——正常组，预防给药(16w)；
B1——模型组，预防给药(16w)；
A2——正常组，治疗给药(23w)；
B2——模型组，治疗给药(23w)；
C——降脂保肝组合物组(黄芪、葛根和桑白皮的质量比为1∶2∶1)，预防给药(16w)；
D——降脂保肝组合物组(黄芪、葛根和桑白皮的质量比为1∶2∶1)，治疗给药(23w)；
E——氯沙坦组，治疗给药(23w)；
F——溴吡斯的明组，治疗给药(23w)。
2.试验方法
2.1预防性给药(预防组是STZ成模后开始给药)
2.1.1剂量设置
本实验在本课题组前期研究的基础上，降脂保肝组合物给药剂量设为2.4g·kg-1，临用前将制备粉末溶于阿拉伯胶溶液中充分研磨直至其完全溶解，现配现用。
2.1.2给药方法
一日一次，给药16周。
2.2治疗性给药(治疗组是STZ成模后给予高脂饲料7w后再开始给药)。
2.2.1剂量设置
本实验在本课题组前期研究的基础上，降脂保肝组合物给药剂量设为2.4g·kg-1，临用前将制备粉末溶于阿拉伯胶溶液中充分研磨直至其完全溶解，现配现用。根据2015版《中华人民共和国药典》说明和阳性药说明书，按临床生药量计算，以人体重(60kg)折算大鼠的临床等效剂量换算后灌胃给药，氯沙坦钾片 剂量设定为10mg/kg，配药浓度为1mg/mL，溴吡斯的明片剂量设定为12mg/kg，配药浓度为1.2mg/mL，临用前放入小型研钵中用研棒尽量研碎用阿拉伯树胶溶液分别配制成1片/100mL和1片/50mL的溶液，随后倒入离心管中4℃冰箱保存，现配现用。
2.2.2给药方法
一日一次，给药16周。
2.3试验操作方法
2.3.1实验取材
实验末次给药后禁食不禁水14h，腹主动脉取血4-5mL，4℃离心(3000rpm，10min)吸取上清液，分装6份，每份50-100μL，放置在-80℃冰箱中保存，待测基础生化指标含量。随后立即脱颈处死大鼠迅速取出心脏，放在4℃冰箱保存过的冰生理盐水的培养皿中冲洗后于冰袋上操作，沿房室交界处剪除包膜、大血管，剪取心尖部约1.5mm3的心肌组织和骨骼肌组织，装于加有4％多聚甲醛固定液的EP管中保存，分别用于HE染色、Masson染色和TUNEL实验检测来观察模型组大鼠的心肌形态学改变以及药物对其的保护作用；各剪取约50mg的胰腺组织放置于4％多聚甲醛固定液中保存，用于观察模型组大鼠胰腺病变情况和降脂保肝组合物的干预作用。
2.3.2基础生化指标检测方法
葡萄糖测定试剂盒采用葡萄糖氧化酶-过氧化物酶法检测，总胆固醇(TC)测定试剂盒采用胆固醇氧化酶-过氧化物酶法检测，甘油三酯(TG)测定试剂盒采用甘油磷酸氧化酶-过氧化物酶法检测，低密度脂蛋白胆固醇(LDL-C)测定试剂盒均采用酶直接法检测，具体操作及计算方法参照试剂盒进行、同型半胱氨酸(Hcy)、谷氨酰转肽酶(GTT1)采用酶联免疫法(ELISA)法，按照试剂盒说明书进行操作。
2.3.3HE染色
采用常规的HE染色步骤，在光学显微镜下观察各组大鼠胰腺组织、心肌组织和骨骼肌组织的病变改变情况，并且请专业实验员拍照。
2.3.4Masson三色染色
采用常规的Masson三色染色步骤，于光学显微镜下观察各组大鼠心肌和骨骼肌组织胶原纤维的病变情况，请专业实验员拍照并统计各组大鼠心肌胶原含量。读片时每张标本随机选取6个区域拍照，采用Image ProPlus图像分析系统进行心肌组织胶原相对含量。
3.试验结果
3.1胰腺组织病理结果
如附图41所示，胰腺组正常组胰腺腺泡细胞均包膜完整，胰岛团为圆形或卵圆形，胞核清晰，边界清晰，每个胰岛团内细胞数量较多，胞质丰富；细胞大小均匀，核居细胞中央(见图A1、A2)。与正常组比较，模型组大鼠胰岛素量显著减少，胰岛体积明显萎缩，形态不规则(见图B1、B2)；胰岛内分泌细胞明显减少、部分细胞胞浆空泡变性，某些细胞核固缩或溶解、消失，有些区域细胞间隙增大，小岛周围有炎性细胞浸润。和模型组比较，降脂保肝组合物预防给药和治疗给药大鼠(见图C、D)的胰岛有很大改善，胰岛数量明显增加，胰岛内细胞有所增多。氯沙坦和溴吡斯的明给药组也有不同程度的改善。
3.2心肌组织病理结果
如附图42所示，预防和治疗的正常组大鼠心肌组织排列整齐、均匀，肌细胞大小规则(见图A1、A2)，预防和治疗的模型组大鼠表现出心肌组织排列紊乱，肌细胞大小不均匀，肌纤维间可见明显的脂肪空泡沉积，肌纤维间隔明显增宽，纤维明显变细，部分肌纤维已发生溶解等病理改变，并且治疗模型组大鼠的病变更为严重(见图B1、B2)；降脂保肝组合物预防组和治疗组大鼠均表现出肌纤维排列规则，纤维间未见脂肪沉积，也未见纤维溶解等，但治疗组大鼠肌纤维间偶尔可见极少的脂肪空泡(见图C、D)；氯沙坦治疗组大鼠心肌组织排列基本整齐但仍能看到较多的脂肪空泡(见图E)。
3.3骨骼肌组织病理结果
如附图43所示，预防和治疗的正常组大鼠肌组织排列完整、规则、清晰，肌细胞大小均匀，未见细胞萎缩、水肿及肌细胞坏死、断裂，无纤维组织增生现象(见图A1、A2)；预防和治疗的模型组肌组织排列紊乱，肌细胞大小不均匀，萎缩明显，肌细胞大面积水肿，炎性细胞浸润较明显，大面积灶状肌组织断裂(见图B1、B2)；降脂保肝组合物预防组和治疗组大鼠肌组织排列规则，肌细胞大小均匀，肌细胞萎缩明显减少，肌细胞水肿区域少、范围小、程度轻，并且预防组明显优于治疗组(见图C、D)；溴吡斯的明给药组肌组织排列规则，轻度炎细胞浸润，肌细胞坏死断裂少见(见图F)。
3.4Masson染色观察心肌胶原纤维改变
如附图44所示，预防和治疗的正常组大鼠心肌细胞染成红色，胶原纤维蓝色，心肌胶原纤维分布均匀，但A2心肌胶原纤维含量高于A1(见图A1、A2)。模型组心肌细胞间质、血管周围可见胶原成分堆积，分布不均匀，排列紊乱，相互连接成网状，B2心肌胶原纤维含量高于B1(见图B1、B2)。各药物组心肌胶原纤维分布较均匀，心肌细胞间质、血管周围见少量胶原成分(见图C、D、E)。
3.5Masson染色观察心肌组织胶原相对含量
如附图45所示，与正常组比较模型组大鼠心肌组织胶原相对含量明显增加(P＜0.01)；与模型组比较，降脂保肝组合物预防组、治疗组和氯沙坦组能不同程度减少心肌组织胶原相对含量(P＜0.01)，提示降脂保肝组合物和氯沙坦可以明显抑制胶原纤维增生，其中黄芪散治疗组效果优于预防组。
3.6Masson染色观察骨骼肌胶原纤维改变
如附图46所示，预防和治疗的正常组大鼠骨骼肌细胞染成红色，胶原纤维蓝色，骨骼肌胶原纤维分布均匀(见图A1、A2)。模型组骨骼肌细胞间质、血管周围可见胶原纤维堆积，分布不均匀，排列紊乱，相互连接成网状，B2骨骼肌胶原纤维含量高于B1(见图B1、B2)。降脂保肝组合物预防组和溴吡斯的明组骨骼肌胶原纤维分布较均匀，含量较少；但降脂保肝组合物治疗组骨骼肌细胞间质、血管周围仍见部分胶原成分(见图D)。
3.7Masson染色观察骨骼肌组织胶原相对含量
如附图47所示，与正常组比较模型组大鼠骨骼肌组织胶原相对含量明显增加(P＜0.01)；与模型组比较，降脂保肝组合物预防组、治疗组和氯沙坦组能不同程度减少骨骼肌组织胶原相对含量(P＜0.01)，提示降脂保肝组合物和氯沙坦的明可以明显抑制胶原纤维增生，其中降脂保肝组合物预防组效果优于治疗组。
3.8糖脂生化指标比较
如附图48所示，降脂保肝组合物均能明显降低糖尿病大鼠GLU、TG、TC含量，氯沙坦可显著降低GLU、TG含量，对TC降低不明显；溴吡斯的明有降糖作用，但对TG有明显的升高作用，这可能是该阳 性药的副作用所致。
如附图49所示，降脂保肝组合物均能明显降低糖尿病大鼠肝脏中Hcy、GGT1含量，氯沙坦和溴吡斯的明均可降低肝脏中Hcy含量，但对GGT1都具有升高作用(p＜0.01)。
实施例10本发明降脂保肝组合物改善肥胖的基因
1、试验分组
本试验共分为8组，每组分别如下：
A——SD组，N＝8
B——SD+GC组，N＝10
C——HFD组，N＝8
D——HFD+GC组，N＝12
I——HFD+GC+实施例2组合物组，N＝10
J——SD+GC+实施例2组合物组，N＝10
2.试验方法
SPF级别SD大鼠在普通级动物房适应性饲养两周后(约200-220g)，将大鼠随机分为正常组、糖皮质激素组、高脂饲料组、高脂加糖皮质激素组、高脂加糖皮质激素+降脂保肝组合物组、糖皮质激素加降脂保肝组合物组。正常组、糖皮质激素组和糖皮质激素+降脂保肝组合物组喂以基础饲料，其他各组喂以高脂饲料。除正常组和高脂饲料组给予等量生理盐水外，其余各组开始灌胃醋酸泼尼松3.5mg/kg，一天一次，同时高脂加糖皮质激素+降脂保肝组合物组和糖皮质激素加降脂保肝组合物组1小时后开始灌胃给药降脂保肝组合物(2.96g/kg)，其中黄芪∶葛根∶桑白皮的质量比为1∶2∶1，连续12周。其后，按照以下方法进行试验操作：
(1)总RNA的提取
①收集动物的白色脂肪与棕色脂肪，用RNA保存液保存。存放于-80℃冰箱。
②解冻组织，先用镊子拿出样本，放于滤纸上吸干保存液，称重，后转移到另一干净的1.5ml Ep管中，加入1mltrizol，用小剪刀在trizol液中剪碎，于玻璃匀浆器中砧碎至肉眼看不到大颗粒。于10000rpm离心5min，仔细将上层油状透明层弃去(脂肪层)。
③每管加氯仿0.2ml，剧烈振摇混匀15s，置室温5min使之分层，4℃10000×g离心5min，仔细将上层水相移至另一1.5ml离心管中。
④每管加入0.5ml异丙醇，室温放置10min，4℃10000×g离心10min。小心将上层清液弃去，用70％乙醇1ml洗涤沉淀，4℃5000×g离心5min，弃上清，室温放置使乙醇完全挥发，管底可见白色小点即RNA。
⑤沉淀中加入30μl无RNase水，58℃水浴10min使沉淀充分溶解。
(2)RNA浓度、纯度的检测
用DU800核酸/蛋白分析仪在波长260nm和280nm处分别测定RNA样本的OD-A260和A280(RNA纯度高的样本的A260/A280比值＞1.7)，并计量RNA浓度。取少量用核酸蛋白测定仪测定 A260，计算RNA含量。并测定A260/A280为1.8～2.0，证实RNA的纯度。其余放-80℃冻存备用。
(3)RT合成cDNA
利用上步提取的RNA样品，参照RT试剂说明书进行RT反应。RT反应体系如下表所示。
Figure PCTCN2017079796-appb-000014
*根据RNA浓度计算所加总RNA量3.0μg的RNA体积，再调整无RNA酶超纯水用量，使反应总体积为20μl。
在RT-PCR热循环仪中设置如下热循环程序进行反应(下表)：
Figure PCTCN2017079796-appb-000015
反应结束后，所得cDNA产物置-20℃保存。
(4)Real time PCR
①PCR引物设计与合成
按照real time PCR引物设计原则设计引物。检索NCBI GenBank数据库，所设计引物分别针对不同的外显子，以β-actin作为内参对照的校准基因。应用Primer Premier5.0软件分别设计MCP-1、UCP-1、PRDM16、Cidea和β-actin的PCR引物，不同引物的序列见表15，所有PCR引物均由上海生工生物工程技术服务有限公司合成。
Figure PCTCN2017079796-appb-000016
②Real time PCR反应产物验证
按SYBR qPCR Mix试剂盒的操作说明书配制如下反应体系(下表)
Figure PCTCN2017079796-appb-000017
用ABI7500型real time PCR仪进行real time PCR，扩增条件如下表所示：
Figure PCTCN2017079796-appb-000018
③Real time PCR检测目的基因和内参β-actin基因在各组脂肪组织中的表达
在50μl反应体系内，含有模板cDNA200ng，其余的反应成分、扩增条件及融解曲线的设置均同产物验证实验。分别扩增MCP-1、UCP-1、PRDM16、Cidea和β-actin的DNA片段。采用2-ΔΔCT法对real time PCR结果进行数据分析，其中正常对照组的数值均为1。
3.试验结果
3.1棕色脂肪组织中UCP-1基因表达
UCP-1基因在棕色脂肪组织中表达丰富，在白色脂肪组织中极少表达，为棕色脂肪细胞特异性基因。如附图50所示，除高脂饲料组外，其余各组在UCP-1基因表达上都明显下降，其中I组(HFD+GC+实施例2组合物组)的UCP-1基因表达明显高于D组(HFD+GC组)，说明黄芪散能够促进棕色脂肪细胞特异性基因UCP-1的表达，说明黄芪散在抵抗肥胖上具有积极意义。
3.2棕色脂肪组织中Cidea基因表达
如附图51所示，I组(HFD+GC+实施例2组合物组)在Cidea基因表达上明显上升，高于其他各组，说明黄芪散在抵抗肥胖上具有积极意义。
3.3白色脂肪组织中MCP-1基因表达
如附图52所示，与正常组相比，其余各组在MCP-1基因表达上都明显上升。较D组(高脂饲料加糖皮质激素组)，降脂保肝组合物给药组(I、J组)在MCP-1基因表达上都有明显下降。
3.4棕色脂肪组织中PRDM16基因表达
附图53显示，PRDM16转录因子在调控棕色脂肪和成肌细胞转化中起重要作用，是保持棕色脂肪表型所必需的，也可促进棕色脂肪形成、能量消耗。
综上可知，降脂保肝组合物对白色脂肪棕色化的基因影响明显。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

Claims (13)

  1. 一种降脂保肝组合物，其特征在于，包含黄芪、葛根和桑白皮，且所述黄芪、葛根和桑白皮的质量比为黄芪∶葛根∶桑白皮＝(1～2)∶(1～2)∶(0～2)。
  2. 如权利要求1所述的降脂保肝组合物，其特征在于，所述黄芪、葛根和桑白皮的质量比为黄芪∶葛根∶桑白皮＝2∶1∶1。
  3. 如权利要求1所述的降脂保肝组合物，其特征在于，所述黄芪、葛根和桑白皮的质量比为黄芪∶葛根∶桑白皮＝1∶2∶1。
  4. 如权利要求1所述的降脂保肝组合物，其特征在于，所述黄芪、葛根和桑白皮的质量比为黄芪∶葛根∶桑白皮＝1∶1∶2。
  5. 如权利要求1所述的降脂保肝组合物，其特征在于，所述黄芪、葛根和桑白皮的质量比为黄芪∶葛根∶桑白皮＝1∶1∶1。
  6. 如权利要求1所述的降脂保肝组合物，其特征在于，所述黄芪、葛根和桑白皮的质量比为黄芪∶葛根∶桑白皮＝2∶1∶0。
  7. 如权利要求1～6任一项所述的降脂保肝组合物的制备方法，其特征在于，包括以下步骤：将黄芪、葛根和桑白皮混合后以溶剂回流提取，提取后回收溶剂，并用大孔树脂对提取液精提，即得降脂保肝组合物。
  8. 一种用于治疗肥胖性高脂血症和脂肪肝的药物，其特征在于，包含如权利要求1～6任一项所述的降脂保肝组合物。
  9. 如权利要求1～6任一项所述的降脂保肝组合物在用于制备预防或治疗肥胖性高脂血症的食品、药物和保健品中的用途。
  10. 如权利要求1～6任一项所述的降脂保肝组合物在用于制备预防或治疗脂肪肝的食品、药物和保健品中的用途。
  11. 如权利要求1～6任一项所述的降脂保肝组合物在用于制备预防或治疗肥胖症的食品、药物和保健品中的用途。
  12. 如权利要求1～6任一项所述的降脂保肝组合物在用于制备预防或治疗高同型半胱氨酸血症的食品、药物和保健品中的用途。
  13. 如权利要求1～6任一项所述的降脂保肝组合物在用于制备心肌和骨骼肌保护药物中的应用。
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