WO2016145983A1 - 异喹啉生物碱的医药用途 - Google Patents

异喹啉生物碱的医药用途 Download PDF

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WO2016145983A1
WO2016145983A1 PCT/CN2016/074781 CN2016074781W WO2016145983A1 WO 2016145983 A1 WO2016145983 A1 WO 2016145983A1 CN 2016074781 W CN2016074781 W CN 2016074781W WO 2016145983 A1 WO2016145983 A1 WO 2016145983A1
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phellodendron
cork base
group
cork
diabetes
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PCT/CN2016/074781
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English (en)
French (fr)
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李萍
徐晓军
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中国药科大学
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Priority claimed from CN201510118942.4A external-priority patent/CN106138046B/zh
Priority claimed from CN201510118943.9A external-priority patent/CN106138047A/zh
Priority claimed from CN201510118945.8A external-priority patent/CN106138049B/zh
Priority claimed from CN201510118944.3A external-priority patent/CN106138048B/zh
Priority claimed from CN201610064099.0A external-priority patent/CN107007599A/zh
Application filed by 中国药科大学 filed Critical 中国药科大学
Publication of WO2016145983A1 publication Critical patent/WO2016145983A1/zh

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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine

Definitions

  • the invention relates to the field of natural medicine, relates to the medical use of isoquinoline alkaloids, in particular to the medical use of cork base or a salt thereof or a crystal form thereof.
  • Diabetes is a metabolic disease characterized by hyperglycemia caused by defects in insulin secretion and/or its biological dysfunction. It is a genetic susceptibility disease with environmental factors involved in genetics. It is a chronic systemic disease. Metabolic diseases, which seriously endanger human health, and actively carry out prevention and treatment of diabetes have become major social public health problems.
  • Type II diabetes is a pathological process from insulin resistance with insulin deficiency to insulin secretion deficiency with insulin resistance.
  • Type II diabetes is currently recognized as a complex genetic disease caused by multiple factors or interactions caused by environmental factors and lifestyle changes, but the etiology of type 2 diabetes has not yet been fully elucidated.
  • Western medicine currently uses diet, exercise therapy sulfonylureas, biguanides, thiazolidinediones, ⁇ -glucosidase inhibitors, repaglinide, insulin, etc. to treat type 2 diabetes, only thiazolidinediones Improve insulin resistance. While the patients are treated with western medicine, complications such as arteriosclerosis, coronary heart disease and hypertension are constantly appearing. The complex mechanism of type II diabetes and the systemic diseases caused by it are the weak links of western medicine treatment. Whether it is a sulphur-promoting agent or a thiazolidinedione sensitizer, it has no obvious weight loss effect in experimental and clinical research, and obesity is an important pathological basis of type 2 diabetes and even metabolic syndrome.
  • Type II diabetes is not only a disorder of glucose metabolism, but also a disorder of fat metabolism.
  • People with type 2 diabetes caused by obesity usually have a high-sugar and high-fat diet and are often accompanied by hyperlipidemia.
  • most obese people do not develop diabetes, indicating that the body's own condition plays an important role. Under the same toxic effects, susceptible people can easily develop into type II diabetes, and insensitive people may not develop disease for a lifetime or delay the onset of disease.
  • Modern medicine can't change the susceptibility of the body, but it can reduce the early incentives as much as possible by changing lifestyles, losing weight, etc., and prolonging the compensation period as much as possible, thus delaying the occurrence of type 2 diabetes and even avoiding the onset.
  • the classification standard formulated by the Ministry of Health formulated the "Guidelines for the Clinical Research of New Drugs for Treating Diabetes Mellitus (Diabetes)" is the most widely used classification method, namely, Yin Deficiency Syndrome, Qi and Yin Deficiency Syndrome, Yin and Yang. Two types of deficiency syndrome and blood stasis syndrome.
  • Nonalcoholic fatty liver disease is a clinical pathological syndrome characterized by no history of excessive drinking, accumulation of fat in hepatocytes caused by various causes, and hepatic steatosis and lipid accumulation. It is also a common clinical condition.
  • Diabetic nephropathy is one of the most important complications of diabetes.
  • the incidence rate in China is also on the rise. It has become the second leading end stage renal disease, second only to glomerulonephritis, due to the complexity.
  • Metabolic disorders, kidney damage in diabetes, especially once entering the clinical proteinuria the condition is generally irreversible, often progressive development until end-stage renal disease, often more difficult than other kidney diseases, is the main cause of death in diabetic patients One of them, the pathogenesis of DN and the development of therapeutic drugs have received great attention from the medical community.
  • Modern medicine begins with diet control, blood sugar control, blood pressure reduction, lipid metabolism adjustment, or the use of dialysis, kidney transplantation and other treatments, there is no effective Western medicine can prevent the process of DN kidney damage.
  • the main clinical diagnosis is characterized by albumin excretion rate and albumin creatinine ratio.
  • Cork is a traditional Chinese medicine commonly used in China. It was originally published in "Shen Nong's Herbal Classic", formerly known as “Shumu”, which is listed as the top grade. Its taste is bitter and cold. It belongs to the kidney and bladder. It has heat and dampness, and the bay fire is steamed. The effect of detoxification treatment is used for damp heat and filth, jaundice and urinary redness, tingling itch, hot sedative pain, athlete's foot sputum, bone steaming heat, night sweats, nocturnal emission, swollen poison, eczema.
  • Phellodendron preparations have been widely used in clinical practice, such as treatment of sinusitis, acute and chronic pus, acute and chronic osteomyelitis, chronic colitis, skin erosion, purulent skin lesions, wet dermatitis, catastrophic stomatitis, external swelling Pain, anal pain, and external washing after surgery, treatment of soft tissue contusion, swelling and other diseases.
  • Phellodendrine is an isoquinoline alkaloid isolated from the stem bark of Phylhdendron chinensis Schneid. and P. velutipes. It has hypotensive blood pressure, anti-nephritis, and inhibition of cellular immune response. Central nervous system inhibition and other effects.
  • Phellodendron The pharmacological effects of Phellodendron are mainly: antihypertensive effect, mainly manifested by intravenous injection of cats, rats and dogs can cause hypotension, and can enhance the adrenergic and norepinephrine pressor response, inhibit artificial asphyxia and stimulate vagus nerve
  • the pressurization reaction to the middle end inhibits the contraction of the cat's nictitating membrane from the stimulation of the preganglionic fibers.
  • autonomic nerve blockade which shows that corkaline inhibits the central nervous system, inhibits spontaneous activity and various reflexes in mice; muscle relaxation;
  • the induction period of the cellular immune response period has an inhibitory effect.
  • Isoquinoline alkaloids are useful in the preparation of compositions for the treatment or prevention of diabetes.
  • the isoquinoline alkaloid is used to prepare a composition for protecting islet cells, or for repairing damaged islet cells, or for preparing a composition against insulin resistance.
  • Isoquinoline alkaloids are useful in the preparation of compositions for the treatment or prevention of hyperlipidemia.
  • the isoquinoline alkaloid is used to prepare a composition for treating or preventing nonalcoholic fatty liver disease.
  • Isoquinoline alkaloids are useful in the preparation of compositions for the treatment or prevention of diabetic nephropathy.
  • the isoquinoline alkaloid is phellodendron.
  • the isoquinoline alkaloid is a cork base or a pharmaceutically acceptable salt, hydrate or anhydrate thereof, or a crystalline form thereof, which is commercially available or prepared by a known method.
  • the pharmaceutically acceptable salts of cork base include: inorganic acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate and phosphate; organic acid salts such as acetate, benzoate, maleic acid Salt, fumarate, malate, citrate, oxalate, lactate, succinate, tartrate, alkyl sulfonate or aryl sulfonate, cysteine or other amino acid a salt; also includes a basic salt such as a sodium salt, a potassium salt, and a calcium salt;
  • the pharmaceutically acceptable salt of cork base includes its crystalline form.
  • composition of the present invention is characterized in that the medicine, health care product, or functional food, excipient or carrier is an excipient or carrier commonly used in the pharmaceutical or food field, such as a diluent, a disintegrating agent, a lubricant, etc. .
  • composition of the present invention is characterized by preventing or treating type I or type II diabetes.
  • FBG fasting blood glucose test
  • corkaline has a significant effect on reducing blood lipids, and unexpectedly found that corkaline can lower the levels of cholesterol, triglyceride and low density lipoprotein, and unexpectedly found that cork base can be improved.
  • the level of high density lipoprotein can be used to prevent and/or treat diseases or symptoms associated with hyperlipidemia.
  • corkal has a significant therapeutic or preventive effect on nonalcoholic fatty liver, and unexpectedly found that corkaline effectively alleviates nonalcoholic fatty liver degeneration caused by high fat diet, improving the cause Liver function damage caused by steatosis.
  • the cortisol pharmaceutically acceptable salt is prepared according to conventional techniques in the art, or
  • the phlegm medicinal salt is prepared as follows, the cork base monomer is taken, dissolved in chloroform, added with a chloroform acid solution, the precipitate is precipitated, and dried by filtration to obtain a pharmaceutically acceptable salt monomer of phellodendron, dissolved in ethanol, placed in a refrigerator, Crystallization, filtration and drying, to obtain a pharmaceutically acceptable salt of cork base, the purity of which is above 98%;
  • the Phellodendrine according to the present invention has a CAS number of 6873-13-18, and its molecular formula is C 20 H 24 NO 4 , and has a relative molecular mass of 342, and has the following structure:
  • the structural formula of the citric acid citrate salt of the present invention is as follows:
  • the combined extracts are adsorbed by HPD100 macroporous adsorption resin column chromatography, and dried cork medicinal materials
  • the mass ratio of the macroporous adsorption resin is 1:5, the loading speed is controlled at 2.0 ml/min, and the adsorption is statically adsorbed for 2.0 h after the loading; after the adsorption is completed, the impurity is eluted with 3.0 BV of water; 30% is used again.
  • the ethanol was eluted at 3.0 BV, and the eluate was collected and concentrated under reduced pressure at 50 ° C to obtain an extract;
  • the 200-300 mesh silica gel column chromatography of the extract is basified by 10% ammonia water: the weight ratio of the extract to the alkalized silica gel of the filler is 1:30, first eluted with chloroform-methanol 30:1, 5.0 BV, and then The mixture was eluted with 5.0 BV methanol, and the combined methanol fractions were collected; the methanol eluate was concentrated and dried under reduced pressure at 45 ° C to obtain crude crude phloem;
  • the crude crude cork base was first dissolved in a small amount of ethanol, and then the concentrated HC1 solution was added to pH 3.0, placed in a refrigerator at 4 ° C for 24 hours, crystallized, filtered and dried, and the above method was repeatedly recrystallized to obtain a purity of 98% of the hydrochloride. .
  • the crude cork base was first dissolved in a small amount of ethanol, and then the citric acid solution was added to a pH of 3.0, placed in a refrigerator at 4 ° C for 24 hours, crystallized, filtered and dried, and the above method was repeatedly recrystallized to obtain a citric acid cork having a purity of 98%. Alkali salt.
  • the peak positions are 3404.71, 3058.97, 2835.29, 2737.91, 1612.26, 1529.37, 1456.07, 1336.58, 1451.80, 1355.27, 1262.70, 1356.27, 1262.70, 1226.76, 1210.56, 1118.94, 1114.01, 1122.24, 1102.05, 1009.90. , 878.82, 848.68, 837.53, 802.99, 733.58, 698.08 cm-1.
  • the peak present at 2500 cm-1 to 2300 cm-1 is likely to be the N-H stretching vibration of the tertiary amine salt R3NH+.
  • the remaining peaks may correspond to an alkyl C-H bending vibration, a C-N stretching vibration, and an out-of-plane bending vibration of the benzene ring C-H.
  • Figure 1 shows the effect of corkaline on pancreatic tissue of type I diabetic mice through the upper, middle and lower parts
  • Figure 1 shows the normal group of mouse pancreatic tissue
  • Figure 1 shows the pancreatic tissue of the STZ group of mice
  • FIG. 1 shows pancreatic tissue of the STZ + cork base group of mice.
  • Figure 2 shows the effect of corkaline on oral glucose tolerance in type 2 diabetic mice
  • control was in the normal diet group
  • HFD was in the high-fat diet group
  • HFD+Phellodendrine was in the high-fat+Phellodendrine group.
  • FIG. 3 shows the effect of corkaline on insulin tolerance in type 2 diabetic mice
  • control was in the normal diet group
  • HFD was in the high-fat diet group
  • HFD+Phellodendrine was in the high-fat+Phellodendrine group.
  • Figure 4 shows the effects of corkaline on liver tissue of mice with nonalcoholic fatty liver disease through the upper left, upper right, lower left, and lower right parts;
  • the upper left part of Figure 4 shows the liver tissue of the normal group of mice
  • the upper right part of Figure 4 shows the liver tissue of the high fat group mice
  • FIG. 4 The lower left part of Figure 4 shows the liver tissue of the lovastatin group mice
  • the lower right portion of Figure 4 shows the liver tissue of the phellodendron mouse.
  • Figure 5 shows renal HE staining through the upper, middle and lower parts
  • Figure 5 shows the kidney tissue of the normal group of mice
  • the middle part of Figure 5 shows the kidney tissue of the STZ group of mice
  • Figure 5 shows the kidney tissue of the STZ + cork base group mice.
  • FIG. 6 shows renal PAS staining through the upper, middle and lower parts
  • Figure 6 shows the kidney tissue of the normal group of mice
  • the middle part of Figure 6 shows the kidney tissue of the STZ group of mice
  • FIG. 6 shows the kidney tissue of the STZ + cork base group mice.
  • the sample is: phellodendrine, dosage 2.0440mg
  • the unit of abscissa is temperature (°C);
  • the sample is: phellodendrine, the dosage is 6.3330mg
  • the unit of abscissa is temperature (°C);
  • the invention relates to the following table
  • C57BL mice were randomly divided into 3 groups, normal control group (10) and STZ group (20).
  • STZ group received continuous intraperitoneal injection of STZ (50mg/Kg) for 5 days. After two weeks, fasting blood glucose was measured. The blood glucose was greater than or equal to 13.8mmol/L.
  • the STZ group was randomly divided into STZ group and phellodendron treatment group, 10 in each group. The phellodendron treatment group was treated with oral gavage (15 mg/kg) for 10 weeks, and the fasting blood glucose was measured after fasting for 4 hours. Shown, there is a significant statistical difference.
  • the scale is 20 ⁇ m and the magnification is 400 times.
  • this study used a classic type II diabetes model, which was randomly divided into 3 groups, normal control group (10 rats), model group (10 rats), and cork base treatment group. (10 rats), the model group and the phellodendron treatment group were treated with a high-fat diet (basic feed supplemented with 20% lard) for 8 weeks, and the phellodendron-treated group was treated with oral gavage (50 mg/kg) on a high-fat diet.
  • Administration after the end of the administration, the fasting overnight measurement, fasting blood glucose, glucose tolerance, and insulin tolerance were measured.
  • the experimental data were analyzed for variance and the results were expressed as x ⁇ s. The results showed that the treatment effect of phellodendron was obvious and there was a statistically significant difference.
  • the glucose tolerance test is an oral glucose load test to understand the body's ability to regulate blood glucose after eating glucose. Through the glucose tolerance test, sugar metabolism abnormalities can be found early, which is currently recognized as the gold standard for diagnosis of diabetes. In the case of elevated blood glucose but not yet reached the diagnostic criteria for diabetes, OGTT can be used for differential diagnosis in order to determine whether or not diabetes is present. Under normal circumstances, the body has a mechanism to maintain blood sugar, oral glucose, rapid rise in blood glucose after a brief rise, that is, normal glucose tolerance, the corresponding area under the oral glucose tolerance curve is small, the use of sugar in diabetic patients, oral glucose After the blood sugar rises rapidly, the blood sugar declines slowly, that is, the corresponding under-line area of glucose tolerance decreases. In this experiment, the glucose tolerance decreases.
  • mice were fasted overnight. After oral administration of blood glucose 2g/kg, the mice were tested for blood glucose at 0, 15, 30, 60, 90, and 120 minutes, and the corresponding line area was calculated using GraphPad Prism software. AUC)
  • Insulin tolerance test method mice were fasted overnight, mice were injected with insulin 0.75 IU/kg intraperitoneally at 0, 30, 60, 90, 120, 150 min, blood glucose was measured at the tip of the tail, and the corresponding line area was calculated using GraphPad Prism software. (AUC)
  • the raw data of glucose tolerance is as follows:
  • control is a normal diet group
  • HFD is a high-fat diet group
  • HFD+Phellodendrine group is a high-fat diet.
  • the insulin tolerance test is an experiment that reflects the body's sensitivity to insulin.
  • the main feature of type II diabetes is insulin resistance, which is less sensitive to insulin. That is, the same time insulin is injected with the same insulin in the same time, as shown in Figure 3, high fat. Reduced sensitivity to insulin after modeling, injecting the same amount of insulin.
  • the blood glucose level at each point was higher than that of the normal group, and the corresponding underline area was also higher than that of the normal group.
  • the blood glucose level at each time point after treatment with phellodendron was lower than that of the model group, and the corresponding line area was lower than that of the high fat group. Model group. The results indicate that phellodendron can effectively increase insulin sensitivity and improve diabetes.
  • mice C57BL mice, SPF grade, male, body weight (20 ⁇ 2) g, were randomly divided into two groups, the first group of 12, the normal group, given normal feed, the remaining mice (44) were divided into the second group, Give high-fat diet (basic feed with 20% lard, 1.25% cholesterol, 0.5% sodium cholate) for free feeding and drinking. After 8 weeks of continuous feeding, 4 mice were sacrificed from the normal control group and the fatty liver model group. Serum and liver biochemical parameters and liver histopathological sections were compared between the two groups to determine whether the nonalcoholic fatty liver model mice were Established successfully.
  • mice Thirty-two successful mice were randomly divided into 4 groups: normal control group (8 rats), model group (8 rats), corkaline treatment group (8 rats), and positive control group (8 lovastatin group). ), continuous intragastric administration for 8 weeks. During the administration period, except for the standard feed of the normal control group, the other groups continued to be given a high-fat diet until the end of the experiment.
  • the phellodendron treatment group was administered by oral gavage (50 mg/kg) on a high-fat diet
  • the positive control group was administered by oral lovastatin (60 mg/kg) on a high-fat diet.
  • the experimental animals were kept in the SPF animal room, 12h light 12h night, free diet and drinking water, the animal state was normal during the experiment.
  • liver function related indicators serum ALT, AST,
  • Phellodendron can significantly reduce total cholesterol (TC) and triglyceride (TG) in plasma, increase high density lipoprotein (HDL), lower low density lipoprotein (LDL), and reduce aspartate in plasma.
  • Acid transaminase (AST), alanine aminotransferase (ALT), pathological section showed that it effectively relieves non-alcoholic fatty liver degeneration caused by high fat diet and improves liver function damage caused by steatosis.
  • mice and blood lipid levels were measured after the end of the administration.
  • the experimental data were analyzed for variance and the results were expressed as x ⁇ s.
  • the results showed that corkaline had significant effects on reducing high fat and had statistically significant differences.
  • C57BL mice were randomly divided into 3 groups, normal control group (10 rats) and STZ group (20 rats).
  • STZ group received continuous intraperitoneal injection of STZ (50mg/Kg) for 5 days. After two weeks, fasting blood glucose was measured. The blood glucose was greater than or equal to 13.8mM.
  • the STZ group was then randomly divided into the STZ group and the phellodendron treatment group. The STZ group was only normally fed. The phellodendron treatment group was administered orally (15 mg/kg). After 12 weeks, the mice were placed in the metabolic cage of the mouse.
  • the urine Free diet and drinking water, the urine is used for analysis, the measured indicators are the classic indicators of clinical detection of diabetic nephropathy, as shown in Table 14-16, the STZ group compared with the blank control group, the significant increase is more than 10 times, This indicates that the diabetic mice have severe renal damage. As shown in Figure 1 and 2, the STZ group has obvious organ damage compared with the normal group. Based on the above table, the successful model of diabetic nephropathy can be obtained.
  • the present invention performs PAS staining on the kidney. And HE staining.
  • Figures 5 and 6 show that corkaline can effectively repair the organ damage of diabetic nephropathy, as shown in the STZ group, the glomerular mesangial area is widened, the matrix is increased, the glomerular basement membrane is thickened, and the renal tubular basement membrane is Thickening and division, showing the typical characteristics of diabetic nephropathy, showing good modeling, and the repair of cork base to a certain extent, the mesangial area is relatively reduced, improving the stromal hyperplasia and thickening of the renal tubule basement, indicating that the treatment effect is obvious The effect is remarkable.
  • Preparation process take cork base through 100 mesh sieve, add starch, magnesium stearate, mix well, make granules, dry, tablet, and get.
  • Preparation process take cork base through 100 mesh sieve, add starch, magnesium stearate and mix evenly, make granules, dry, capsule, and get.
  • Preparation process take cork base through 100 mesh sieve, add 1.9mL water for injection to dissolve, add sodium chloride for injection to isotonic, adjust the pH value to 7 ⁇ 7.1, filter, refrigerate for 24 hours, add water for injection to the specified amount, Filtered, potted, sterilized, that is.
  • Dry yeast 5g warm water 90ml, water a little, flour 150g, corkaline 5mg, vegetable oil 10g, low sodium salt a little
  • Biscuits Sprinkle the yeast in warm water, stir and dissolve, add the phellodendron. Add flour, stir, add vegetable oil, knead into smooth dough; dough into 0.2cm thick slices; press out the shape, puncture the hole, sprinkle water on the surface, sprinkle with a little low sodium salt, ferment at room temperature for 10 minutes; oven preheat At 120 degrees, it is placed on the upper layer and baked for about 10 minutes to obtain a food containing corkine.
  • Phellodendron monomer (purity of 91% ⁇ 92%) l00mg, first dissolved in 10ml chloroform, added 10mL 0.1M HCL chloroform solution, precipitated, filtered and dried to obtain a container of phellodendron hydrochloride, dissolved in 1ml of ethanol, set the refrigerator 4 After standing at °C for 24h, crystallization, filtration and drying, to obtain a container of phellodendron hydrochloride, the purity reached 98% or more.
  • Phellodendron monomer (purity: 91% to 92%) 100 mg, first dissolved in 10 ml of chloroform, added with 10 mL of 0.1 M H 2 SO 4 chloroform solution, precipitated, and dried by filtration to obtain a tablet of phlebosulfate, dissolved in 1 ml of ethanol, filtered The filtrate was placed in a refrigerator at 4 ° C for 24 hours, crystallization, and dried by filtration to obtain a tablet of phleboxine sulfate having a purity of 98% or more.
  • Phellodendron monomer (purity of 91% to 92%) l00mg, first dissolved in 10ml chloroform, add 10mL 0.1M chloroform solution, precipitate precipitation, filter and dry, to obtain the celloside phosphate monomer, dissolved in 1ml ethanol, filtered, filtrate Placed in a refrigerator at 4 ° C for 24 h, crystallization, filtration and drying, to obtain a phosphate cellosite monomer, the purity reached 98% or more.
  • Phellodendron monomer (purity of 91% ⁇ 92%) l00mg, first dissolved in 10ml chloroform, add 10mL 0.1M chloroform solution of fruit acid, precipitated, filtered and dried to obtain pectin monomer, dissolved in 1ml of ethanol, filtered The filtrate was placed in a refrigerator at 4 ° C for 24 hours, crystallization, and dried by filtration to obtain a corrugated base of pectin, and the purity was over 98%.
  • Phellodendron monomer (purity 91% ⁇ 92%) l00mg, first dissolved in 10ml chloroform, add 10mL 0.1M oxalic acid chloroform solution, precipitate precipitation, filtered and dried to obtain oxalic acid corkine monomer, dissolved with 1ml ethanol, filtered, filtrate Placed in the refrigerator at 4 ° C for 24 h, crystallization, filtration and drying, to obtain oxalic acid cork base monomer, the purity reached 98% or more.
  • Phellodendron monomer (purity of 91% ⁇ 92%) l00mg, first dissolved in 10ml chloroform, add 10mL 0.1M succinic acid chloroform solution, precipitated, filtered and dried to obtain succinic acid berberine monomer, dissolved in 1ml of ethanol, filtered The filtrate was placed in a refrigerator at 4 ° C for 24 hours, crystallization, and dried by filtration to obtain a succinic acid phellodendrin monomer having a purity of 98% or more.
  • Phellodendron monomer (purity 91% ⁇ 92%) l00mg, first dissolved in 10ml chloroform, adding 10mL 0.1M acetic acid chloroform solution, precipitation, filtration and drying, to obtain cellulose acetate base, dissolved in 1ml ethanol, filtered, filtrate Placed in a refrigerator at 4 ° C for 24 h, crystallization, filtration and drying, to obtain a cellulose acetate base, the purity of 98% or more.
  • Phellodendron monomer (purity 91% ⁇ 92%) l00mg, first dissolved in 10ml chloroform, add 10mL 0.1M propionate chloroform solution, precipitate precipitation, filtered and dried to obtain propionic acid cork base monomer, dissolved with 1ml ethanol, filtered The filtrate was placed in a refrigerator at 4 ° C for 24 hours, crystallization, and dried by filtration to obtain a propionate propionate monomer having a purity of 98% or more.
  • the combined extracts were adsorbed by HPD100 macroporous adsorption resin column chromatography.
  • the mass ratio of dried Phellodendron chinense to macroporous adsorption resin was 1:5, the loading speed was controlled at 2.0 ml/min, and the static adsorption was 2.0 after loading. h; after the adsorption is completed, the impurity is eluted with 3.0BV of water; then 3.0BV is eluted with 30% ethanol, and the eluate is collected and concentrated under reduced pressure at 50 ° C to obtain an extract;
  • the 200-300 mesh silica gel column chromatography of the extract is basified by 10% ammonia water: the weight ratio of the extract to the alkalized silica gel of the filler is 1:30, first eluted with chloroform-methanol 30:1, 5.0 BV, and then The mixture was eluted with 5.0 BV methanol, and the combined methanol fractions were collected; the methanol eluate was concentrated and dried under reduced pressure at 45 ° C to obtain crude crude phloem;
  • the crude crude cork base was first dissolved in a small amount of ethanol, and then the concentrated HC1 solution was added to pH 3.0, placed in a refrigerator at 4 ° C for 24 hours, crystallized, filtered and dried, and the above method was repeatedly recrystallized to obtain a purity of 98% of the hydrochloride. .
  • Test equipment and conditions Nuclear magnetic resonance spectrum test using Bruker Avance 400 nuclear magnetic resonance instrument (Switzerland), according to the superconducting pulse Fourier transform NMR spectroscopy method.
  • the solvent was deuterated dimethyl sulfoxide (DMSO-d 6 ) and TMS was used as an internal standard.
  • DMSO-d 6 deuterated dimethyl sulfoxide
  • TMS was used as an internal standard.
  • the observation frequency is 400.1 MHz
  • the observation frequency is 100.6 MHz.
  • Mass spectrometry was performed using a Brucker Amazon SL ion trap mass spectrometer (Switzerland).
  • the sample was dissolved in water and analyzed by direct injection.
  • the combined extracts were adsorbed by HPD100 macroporous adsorption resin column chromatography.
  • the mass ratio of dried Phellodendron chinense to macroporous adsorption resin was 1:5, the loading speed was controlled at 2.0 ml/min, and the static adsorption was 2.0 after loading. h; after the adsorption is completed, the impurity is eluted with 3.0BV of water; then 3.0BV is eluted with 30% ethanol, and the eluate is collected and concentrated under reduced pressure at 50 ° C to obtain an extract;
  • the 200-300 mesh silica gel column chromatography of the extract is basified by 10% ammonia water: the weight ratio of the extract to the alkalized silica gel of the filler is 1:30, first eluted with dichloromethane-methanol 30:1, 5.0 BV, and then The mixture was eluted with 5.0 BV methanol, and the combined methanol fractions were collected; the methanol eluate was concentrated and dried under reduced pressure at 45 ° C to obtain crude crude phloem;
  • the crude cork base was first dissolved in a small amount of ethanol, and then the citric acid solution was added to a pH of 3.0, placed in a refrigerator at 4 ° C for 24 hours, crystallized, filtered and dried, and the above method was repeatedly recrystallized to obtain a citric acid cork having a purity of 98%. Alkali salt.
  • Test equipment and conditions Nuclear magnetic resonance spectrum test using Bruker Avance 400 nuclear magnetic resonance instrument (Switzerland), according to the superconducting pulse Fourier transform NMR spectroscopy method.
  • the solvent was deuterated dimethyl sulfoxide (DMSO-d 6 ) and TMS was used as an internal standard.
  • DMSO-d 6 deuterated dimethyl sulfoxide
  • TMS was used as an internal standard.
  • the observation frequency is 400.1 MHz
  • the observation frequency is 100.6 MHz.
  • Mass spectrometry was performed using a Bruker Amazon SL ion trap mass spectrometer (Switzerland).
  • the sample was dissolved in water and analyzed by direct injection.

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Abstract

异喹啉生物碱在制备用于治疗或预防糖尿病、糖尿病肾病、高脂血症、非酒精性脂肪肝的组合物中的用途。所述异喹啉生物碱为黄柏碱或其药用盐、水合物、无水物、晶型。

Description

异喹啉生物碱的医药用途 技术领域
本发明涉及天然药物领域，涉及异喹啉生物碱的医药用途，具体涉及黄柏碱或其盐或其晶型的医药用途。
背景技术
糖尿病是一种由于胰岛素分泌缺陷及(或)其生物学作用障碍引起的以高血糖为特征的代谢疾病，是在遗传基础上，有环境因素参与的遗传易感性疾病，是一种慢性全身性代谢疾病，严重危害人类健康，积极开展糖尿病的防治工作已成为主要的社会公共卫生问题。
I型糖尿病的发病原因主要是由于胰岛素分泌绝对缺少，II型糖尿病是从胰岛素抵抗为主伴胰岛素相对不足到胰岛素分泌不足为主伴胰岛素抵抗的病理过程。目前公认II型糖尿病是一种在环境因素、生活方式的改变的作用下由多个基因分别或相互作用所导致的复杂遗传病，但是II型糖尿病的病因尚未完全阐明。
西医目前常采用饮食、运动疗法磺酰脲类、双胍类、噻唑烷二酮类、α-葡萄糖苷酶抑制剂、瑞格列奈、胰岛素等来治疗II型糖尿病，仅噻唑烷二酮类可改善胰岛素抵抗。病人在用西药治疗的同时，又不断地出现动脉硬化、冠心病和高血压等并发症，II型糖尿病的复杂机制及其导致的全身病变是西药治疗的薄弱环节。不论是磺脉类的促泌剂还是噻唑烷二酮类的增敏剂在实验和临床研究中都不具备明显的减肥功效，而肥胖是2型糖尿病乃至代谢综合征的重要病理基础。
II型糖尿病不仅是糖代谢紊乱疾病，还是脂肪代谢紊乱疾病。肥胖引起的II型糖尿病患者通常都有高糖和高脂饮食，并常伴有高脂血症。有研究表明提示血糖“正常”的肥胖患者己有β细胞分泌功能的异常。但是，大部分的肥胖者并不发展为糖尿病，说明机体的自身状况起重要作用。在同样的毒性作用下,易感人群很容易发展成II型糖尿病，而不敏感人群可能终身不发病，或者延迟发病。现代医学并不能够改变机体的易感性，但可以通过改变生活方式，减肥等手段来尽可能地减少早期的诱因，尽量的延长代偿期，从而延迟2型糖尿病的发生，甚至避免发病。
现代II型糖尿病的治疗观点已从已往的单纯控制血糖转为降糖、降脂、降压、改善胰岛素抵抗等多环节治疗。但是，对于II型糖尿病患者，药量需逐渐增加，投药种类也常由单一用药逐渐变为联合用药，这不可避免地要考虑药物代谢对肝肾的副作用。故从传统中药中寻找低毒、疗效肯定的天然药物来治疗是目前研糖尿病究热点之一。
卫生部制定颁布的《中药新药治疗消渴病(糖尿病)的临床研究指导原则》中所制定的分类标准是目前采用最广泛的分类方法,即阴虚热盛证、气阴两虚证、阴阳两虚证和血疲气滞证四型。
非酒精性脂肪肝是一种无过量饮酒史，由各种原因引起的肝细胞内脂肪堆积，以肝细胞脂肪变性和脂质蓄积为主要特征的临床病理综合征，也是一种临床常见病症。
糖尿病肾病(diabetic nephropathy，DN)是糖尿病最重要的并发症之一，我国的发病率亦呈上升趋势，目前已成为终末期肾病的第二位，仅次于肾小球肾炎，由于存在复杂的代谢紊乱，糖尿病发生肾脏损害，特别是一旦进入临床蛋白尿期，病情一般不可逆转，往往呈进行性发展直至终末期肾病，往往比其他肾脏疾病的治疗更加困难，是引起糖尿病患者死亡的主要原因之一，DN的发病机制和治疗药物的研制受到了医学界极大的重视。现代医学上多从饮食控制、血糖控制、降压、调整脂代谢等方面着手，或是采用透析、肾移植等治疗手段，尚无疗效确切的西药能阻止DN肾功能损害的进程。临床其主要诊断的特征为白蛋白排泄率，和白蛋白肌酐比值。
黄柏为我国常用的传统中药，始载于《神农本草经》，原名“檗木”，列为上品，其性味苦、寒，主归肾、膀胱经，具有清热燥湿，湾火除蒸，解毒疗疫之功效，用于湿热汚痢，黄疸尿赤，带下阴痒，热淋淫痛，脚气痿蹵，骨蒸劳热，盗汗，遗精，肿毒，湿疹。现代药理研究表明，黄柏具有抗菌、抗真菌、抗炎、抗肝炎、抗肾炎、抗溃病、抗氧化、抗痛风、降血压、降血糖、抗肿瘤、抗心力衰竭、抑制细胞免疫等作用。目前黄柏制剂在临床上已经广泛运用，如治疗鼻窦炎、急慢性脓耳、急慢性骨髓炎、慢性结肠炎、皮肤糜烂、脓拖性皮损、湿疫皮炎、溃窃性口腔炎、外痔肿痛、肛痛以及术后外洗、治疗软组织挫伤、消肿等病症。(《黄柏中黄柏碱的提取纯化工艺研究》，《西南交 通大学硕士研究生学位论文》，罗鸿，2012年)。
黄柏碱(Phellodendrine)是从芸香科植物黄柏(Phelhdendron chinensis Schneid.)、威氏黄柏的茎皮中提取分离出来的一种异喹啉生物碱，其具有降血压、抗肾炎、抑制细胞免疫反应、中枢神经抑制等作用。
黄柏碱的药理作用主要有：降压作用，主要表现为静注于猫和鼠和犬均可引起降压，并能增强肾上腺素和去甲肾上腺素的升压反应，抑制人工窒息及刺激迷走神经向中端的升压反应，抑制刺激节前纤维而起的猫瞬膜收缩。随剂量增大，降压作用强度及持续时间也增加；植物神经阻断作用，表现为黄柏碱对中枢神经有抑制作用，能抑制小鼠的自发活动和各种反射；肌肉松驰作用；对细胞免疫应答期的诱导期有抑制作用。
《黄柏中几种生物碱的分离、鉴定及促胰岛素分泌活性筛选》(《中国医药指南》，2011年3月，第9卷，第7期，第54-55页，周明伟、范明松、季宇斌、唐意红)记载了：从黄柏的生物碱部分分离得到3个化合物，分别为小檗碱、药根碱、黄柏碱。研究发现，在葡萄糖浓度为5.6mmol/L时，黄柏总碱中的3种生物碱对胰岛素的分泌均无明显的促进作用；而当葡萄糖浓度为16.7mmol/L时，小檗碱能够明显的促进细胞的胰岛素分泌。阳性药格列美脲在低糖浓度和高糖浓度时，都能够促进胰岛素分泌。
未见有关黄柏碱、晶型及其盐对于糖尿病有预防或治疗作用的报道。
未见有关黄柏碱、晶型及其盐对于高脂血症有预防或治疗作用的报道。
未见有关黄柏碱、晶型及其盐对于非酒精性脂肪肝有预防或治疗作用的报道。
未见有关黄柏碱、晶型及其盐对于糖尿病肾病有预防或治疗作用的报道。
未见有关黄柏碱晶型的报道。
未见有关黄柏碱晶型及其盐的制备工艺的报道。
发明内容
异喹啉生物碱用于制备治疗或预防糖尿病的组合物。
异喹啉生物碱用于制备保护胰岛细胞、或、修复受损胰岛细胞的组合物，或，用于制备对抗胰岛素抵抗的组合物。
异喹啉生物碱用于制备治疗或预防高脂血症的组合物。
异喹啉生物碱用于制备治疗或预防非酒精性脂肪肝的组合物。
异喹啉生物碱用于制备治疗或预防糖尿病肾病的组合物。
异喹啉生物碱为黄柏碱。
异喹啉生物碱为市售或按已知方法制备的黄柏碱或其药用盐、水合物或无水物、或晶型。
黄柏碱药用盐包括：无机酸盐例如盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硝酸盐和磷酸盐；有机酸盐例如乙酸盐，苯甲酸盐，马来酸盐，富马酸盐，苹果酸盐，柠檬酸盐，草酸盐，乳酸盐，琥珀酸盐，酒石酸盐，烷基磺酸盐或芳基磺酸盐，半胱氨酸盐或其它氨基酸盐；还包括碱式盐如钠盐、钾盐和钙盐；
黄柏碱药用盐包括，其晶型。
本发明的组合物,其特征为：药物、保健品、或功能性食品,赋形剂或载体为制药或食品领域中常用的赋形剂或载体，如稀释剂，崩解剂，润滑剂等。
本发明的组合物,其特征为：为通过口服或注射形式使用
本发明的组合物,其特征为：预防或治疗I、或II型糖尿病。
在黄柏碱的药理活性研究中，本发明意外地发现黄柏碱具有显著的减低血糖作用。本发明通过(1)STZ造模的糖尿病小鼠，通过(2)高脂诱导的2型糖尿病动物模型(DIO小鼠)，进行了禁食血糖检测(FBG)。
在黄柏碱的药理活性研究中，本发明意外地发现黄柏碱具有显著的减低血脂作用，意外地发现黄柏碱能降低胆固醇，甘油三酯和低密度脂蛋白的水平,出人意料地发现黄柏碱能提升高密度脂蛋白的水平，从而可用于预防和、或治疗与高血脂有关的疾病或症状。
在黄柏碱的药理活性研究中，本发明意外地发现黄柏碱具有显著的治疗或预防非酒精性脂肪肝作用，意外地发现黄柏碱有效缓解高脂饲料引起的非酒精性脂肪肝变性，改善因脂肪变性而导致的肝功能损害。
黄柏碱药用盐按照本领域的常规技术制备、或、
黄柏碱药用盐按照如下方式制备，取黄柏碱单体，用氯仿溶解，加入氯仿的酸溶液，析出沉淀，过滤干燥，得到黄柏碱的药用盐单体，用乙醇溶解，置冰箱放置，析晶，过滤干燥，得到黄柏碱的药用盐，纯度达到98％以上；
本发明所述的黄柏碱(Phellodendrine)，CAS号为6873-13-18，其分子式 是C20H24NO4，相对分子质量342，具有式如下的结构：
Figure PCTCN2016074781-appb-000001
本发明所述的盐酸黄柏碱的结构式如下：
Figure PCTCN2016074781-appb-000002
本发明所述的柠檬酸黄柏碱盐结构式如下：
Figure PCTCN2016074781-appb-000003
黄柏碱的盐酸盐、柠檬酸盐的优选的制备方法
(1)总生物碱的提取
配制质量分数为3.0％的NaCl溶液，调其PH为3.0；干燥黄柏药材用该溶液以料液比1:8浸泡提取2次，每次48h，合并提取液；
(2)黄柏碱的粗分
将合并提取液通过HPD100型大孔吸附树脂柱层析吸附，干燥的黄柏药材 与大孔吸附树脂的质量比为1:5，上样速度控制在2.0ml/min，上样完毕后静止吸附2.0h；吸附完毕后，先用3.0BV的水洗脱除杂；再用30％乙醇洗脱3.0BV，收集洗脱液，于50℃减压浓缩得浸膏；
(3)黄柏碱的纯化精制
将浸膏通过10％氨水碱化的200-300目硅胶柱层析：浸膏与填料碱化硅胶的重量比为1:30，先采用三氯甲烷-甲醇30:1洗脱5.0BV，再用5.0BV甲醇洗脱，收集合并甲醇洗脱部分；将甲醇洗脱液于45℃条件下减压浓缩干燥，得黄柏碱粗品；
(4)盐酸黄柏碱结晶的制备
将黄柏碱粗品先用少量乙醇溶解，再滴加浓HC1调溶液PH至3.0，置冰箱4℃条件下放置24h，析晶，过滤干燥，上述方法反复重结晶得到纯度达到98％的盐酸黄柏碱。
(5)柠檬酸黄柏碱盐结晶的制备
将黄柏碱粗品先用少量乙醇溶解，再滴加柠檬酸调溶液PH至3.0，置冰箱4℃条件下放置24h，析晶，过滤干燥，上述方法反复重结晶得到纯度达到98％的柠檬酸黄柏碱盐。
黄柏碱晶型鉴定报告
本品外观为白色粉末，为了鉴定晶型对其进行了如下表征：
由黄柏碱的拉曼谱图可知：黄柏碱该晶型的出峰位置在3027.98,2934.40,1612.19,1454.43,1346.48,803.70,757.01,710.76,670.10,594.22,447.83,418.96,400.67,283.32,221.56,176.72,103.27,83.85cm-1。
由黄柏碱的DSC图谱可知：黄柏碱该晶型在升温到250℃左右开始熔化。
由黄柏碱的TGA图谱可知：黄柏碱该晶型的分解温度是270.13℃。
黄柏碱的XRD图谱及数据
黄柏碱X射线衍射数据
Anode Cu，Wavelength 1 1.5406A°，Wavelength 2 1.54443A°
Generator 40KV，Generator 40mA
Step 0.02°，StepTime 0.3s，Start Angle 3.00°
End Angle 40.00°，Slit1.0/1.0/Ni/0.2，Date Mar-26-2015
NO.Angle d value Intensity Intensity％
Figure PCTCN2016074781-appb-000004
黄柏碱的红外图谱
由该谱图可知，其出峰位置为3404.71,3058.97,2835.29,2739.91,1612.26,1529.37,1456.07,1336.58,1451.80,1355.27,1262.70,1356.27,1262.70,1226.76,1210.56,1180.94,1114.01,1122.24,1027.05,1009.90,878.82,848.68,837.53,802.99,733.58,698.08cm-1。
在3404.71cm-1位置出现一个强而且较宽的峰，此峰应该为黄柏碱酚羟基与结构中形成氢键的羟基伸缩振动，
3058.97cm-1出现的则是其结构中苯环C-H的伸缩振动，
2835.29cm-1和2739.91cm-1对应的则是其结构中烷基C-H的伸缩振动。
在2500cm-1到2300cm-1存在的峰有可能是叔胺盐R3NH+的N-H伸缩振动。
峰1612.26cm-1和1529.37cm-1极有可能是结构中苯环的C＝C伸缩振动。
其余的峰对应的可能是烷基C-H弯曲振动、C-N的伸缩振动以及苯环C-H的面外弯曲振动。
基于上述发现，本发明现已完成。为了便于理解，下面通过附图和具体实施例对本发明的黄柏碱在治疗糖尿病药物中的用途进行详细的描述。需要特别指出的是，具体实施例和附图仅是为了说明，显然本领域的技术人员可以根据本文说明，对本发明进行各种修正或改变，这些修正和改变也将纳入本发明范围之内。
附图说明
图1.黄柏碱对I型糖尿病小鼠胰腺组织的影响
图2.黄柏碱对II型糖尿病小鼠口服糖耐量的影响
图3.黄柏碱对II型糖尿病小鼠胰岛素耐量的影响
图4.黄柏碱对非酒精性脂肪肝小鼠肝脏组织的影响
图5：肾脏HE染色
图6：肾脏PAS染色
图7.黄柏碱的拉曼图谱
图8.黄柏碱的DSC图谱
图9.黄柏碱的TGA图谱
图10.黄柏碱的XRD图谱及数据
图11.黄柏碱的红外图谱
图12.黄柏碱的盐酸盐的CNMR图
图13.黄柏碱的盐酸盐的HNMR图
图14.黄柏碱的盐酸盐的MS图
图15.黄柏碱的柠檬酸盐的CNMR图
图16黄柏碱的柠檬酸盐的HNMR图
图17黄柏碱的柠檬酸盐的MS图
图1的详细说明
图1通过上中下三个部分展示黄柏碱对I型糖尿病小鼠胰腺组织的影响；
图1的上部展示正常组小鼠胰腺组织；
图1的中部展示STZ组小鼠胰腺组织；
图1的下部展示STZ+黄柏碱组小鼠胰腺组织。
图2的详细说明
图2展示黄柏碱对II型糖尿病小鼠口服糖耐量的影响；
图2中，control为正常饮食组，HFD为高脂饮食组，HFD+Phellodendrine为高脂+Phellodendrine组。
图3的详细说明
图3展示黄柏碱对II型糖尿病小鼠胰岛素耐量的影响
图3中，control为正常饮食组，HFD为高脂饮食组，HFD+Phellodendrine为高脂+Phellodendrine组。
图4的详细说明
图4通过左上、右上、左下、右下四个部分展示黄柏碱对非酒精性脂肪肝小鼠肝脏组织的影响；
图4的左上部展示正常组小鼠肝脏组织；
图4的右上部展示高脂组小鼠肝脏组织；
图4的左下部展示洛伐他汀组小鼠肝脏组织；
图4的右下部展示黄柏碱小鼠肝脏组织。
图5的详细说明
图5通过上中下三个部分展示肾脏HE染色；
图5的上部展示正常组小鼠肾脏组织；
图5的中部展示STZ组小鼠肾脏组织；
图5的下部展示STZ+黄柏碱组小鼠肾脏组织。
图6的详细说明
图6通过上中下三个部分展示肾脏PAS染色；
图6的上部展示正常组小鼠肾脏组织；
图6的中部展示STZ组小鼠肾脏组织；
图6的下部展示STZ+黄柏碱组小鼠肾脏组织。
图8的详细说明
样品是：phellodendrine,用量2.0440mg
横坐标单位是温度(℃)；
图9的详细说明
样品是：phellodendrine,用量6.3330mg
横坐标单位是温度(℃)；
图10的详细说明
横轴：2THETA SCALE；
本发明涉及如下表格
表1.黄柏碱对STZ造模的I型糖尿病小鼠的禁食血糖的影响。
表2.黄柏碱对STZ造模的小鼠胰岛数目的影响
表3.黄柏碱对高脂造模的II型糖尿病禁食血糖的影响
表4.正常组小鼠糖耐量原始数据及相应线下面积
表5.高脂组小鼠糖耐量原始数据及相应线下面积
表6.黄柏碱治疗高脂饮食小鼠糖耐量原始数据及线下面积
表7.黄柏碱对口服糖耐量线下面积数据分析
表8.正常组小鼠糖胰岛素耐量原始数据及线下面积
表9.高脂组小鼠糖胰岛素耐量原始数据及线下面积
表10.黄柏碱治疗高脂饮食小鼠糖胰岛素耐量原始数据
表11.黄柏碱对高脂饮食小鼠糖胰岛素耐量线下统计分析
表12.各组小鼠血脂指标的变化
表13.各组小鼠肝功能相关指标的变化
表14.黄柏碱对STZ造模的糖尿病肾病的血糖的影响
表15.黄柏碱对STZ造模的糖尿病肾病的尿量的影响
表16.黄柏碱对STZ造模的糖尿病肾病的UAE(24h尿白蛋白排泄率)的影响.
表17.黄柏碱对STZ造模的糖尿病肾病的UACR(ug尿白蛋白/mg肌酐比值)的影响.
具体实施方式
实施例1
为了研究黄柏碱对I型糖尿病血糖影响，本实验采用经典的I型糖尿病模型，使用C57BL小鼠随机分为3组，正常对照组(10只)，STZ组(20只)。STZ组连续腹腔注射STZ(50mg/Kg)5天，两周后测定禁食血糖，血糖大于等于13.8mmol/L为造模成功。然后将STZ组随机分为STZ组和黄柏碱治疗组，每组10只，黄柏碱治疗组采用口服灌胃(15mg/kg)10周后，禁食4h后测定禁食血糖结果如图1所示，具有显著的统计学差异。
动物的一般情况:
胰岛组织病理学检查：石蜡切片、HE染色，观察病理变化
表1.黄柏碱对STZ造模的I型糖尿病血糖的影响.
Figure PCTCN2016074781-appb-000005
注：*P<0.05，**P<0.01，***P<0.001，与STZ组相比。
结果表明黄柏碱能够有效的降低STZ造成的I型糖尿病的血糖
表2.黄柏碱对STZ造模的小鼠胰岛数目的影响
Figure PCTCN2016074781-appb-000006
注：*P<0.05，**P<0.01，***P<0.001，与STZ组相比。
随机选取胰腺病理切片9个视野进行统计。附图1的结果显示STZ造模后胰腺萎缩，组织结构坏死，通过黄柏碱治疗后胰岛组织形态结构恢复明显。
注：标尺为20μm，放大倍数400倍。
实施例2
为了研究黄柏碱对II型糖尿病的影响，本实验采用经典的II型糖尿病模型，使用C57BL小鼠随机分为3组，正常对照组(10只)，模型组(10只)，黄柏碱治疗组(10只)，其中模型组和黄柏碱治疗组采用高脂饮食(基础饲料加入20％猪油)造模8周，黄柏碱治疗组在高脂饮食同时采用口服灌胃(50mg/kg)方式给药，给药结束后测定禁食过夜测定、空腹血糖、葡萄糖耐量和胰岛素耐量。实验数据进行方差分析，结果以x±s表示。结果显示，黄柏碱治疗效果明显，具有显著统计学差异。
实验结果：
表3.黄柏碱对高脂造模的II型糖尿病禁食血糖的影响
Figure PCTCN2016074781-appb-000007
注：***P<0.001，与正常组相比；*P<0.05，***P<0.001，与高脂组相比
糖耐量试验是一种口服葡萄糖负荷试验，用以了解机体对进食葡萄糖后的血糖调节能力。通过糖耐量试验，可以早期发现糖代谢异常，是目前公认的诊断糖尿病的金标准，在血糖增高但尚未达到糖尿病诊断标准时，为明确是否患糖尿病，可以采用OGTT进行鉴别诊断。正常情况下，机体有一套维持血糖的机制，口服葡萄糖，血糖短暂升高后迅速恢复正常，即糖耐量正常，相应的口服糖耐量曲线线下面积较小，糖尿病患者糖的利用障碍，口服葡萄糖后血糖迅速升高，血糖下降速度较慢，即糖耐量减退相应的线下面积较大，该实验中糖耐量减退，
如图2所示黄柏碱治疗组和正常组的糖耐量曲线均在模型组曲线下，相应的线下面积也均低于模型组，且具有统计意义。
糖耐量实验方法：小鼠禁食过夜，小鼠口服血糖2g/kg后分别在0,15,30,60,90,120min时，尾尖采血测定相应血糖，使用GraphPad Prism软件计算相应线下面积(AUC)
胰岛素耐受实验方法：小鼠禁食过夜，小鼠腹腔注射胰岛素0.75IU/kg后分别在0,30,60,90,120,150min时，尾尖采血测定相应血糖，使用GraphPad Prism软件计算相应线下面积(AUC)
糖耐量原始数据如下：
表4.正常组小鼠糖耐量原始数据
Figure PCTCN2016074781-appb-000008
表5.高脂组小鼠糖耐量原始数及相应线下面积
Figure PCTCN2016074781-appb-000009
表6.黄柏碱治疗高脂饮食小鼠糖耐量原始数及线下面积
Figure PCTCN2016074781-appb-000010
图2.黄柏碱对II型糖尿病小鼠口服糖耐量的影响
注：control为正常饮食组，HFD为高脂饮食组，HFD+Phellodendrine组为高脂
表7.黄柏碱对口服糖耐量线下面积数据分析
Figure PCTCN2016074781-appb-000011
胰岛素耐量试验是反映机体对胰岛素敏感性的实验，II型糖尿病的主要特征为胰岛素抵抗，对胰岛素敏感性降低，即注射相同胰岛素相同时间内血糖高于正常机体，如图3所示，高脂造模后对胰岛素敏感性降低，注射相同量的胰岛素 后各个点的血糖值均高于正常组，其相应的线下面积也高于正常组，给予黄柏碱治疗后各个时间点的血糖数值低于模型组，相应线下面积低于高脂诱导的模型组。结果表明黄柏碱能够有效的增加胰岛素敏感性改善糖尿病。
表8.正常组小鼠糖胰岛素耐量原始数据
Figure PCTCN2016074781-appb-000012
表9.高脂组小鼠糖胰岛素耐量原始数据
Figure PCTCN2016074781-appb-000013
表10.黄柏碱治疗组小鼠糖胰岛素耐量原始数据
Figure PCTCN2016074781-appb-000014
图3.黄柏碱对II型糖尿病小鼠胰岛素耐量的影响
表11.黄柏碱对II型糖尿病小鼠糖胰岛素耐量线下统计分析
Figure PCTCN2016074781-appb-000015
注：*P<0.05，**P<0.01，***P<0.001，与高脂组比较
可见：与正常组相比，模型组小鼠空腹血糖值显著升高，说明II型糖尿病模型造模成功。同时，与模型组相比，黄柏碱有显著的降低空腹血糖的作用和增加葡萄糖耐受、增强胰岛素敏感性。
实施例3
黄柏碱对高脂血症和非酒精性脂肪肝的治疗作用，
1、实验动物及方法：
C57BL小鼠，SPF级，雄性，体重(20±2)g，随机分成两组，第一组12只，为正常组，给与正常饲料，其余小鼠(44只)分为第二组，给与高脂饲料(基础饲料加入20％猪油，1.25％胆固醇，0.5％的胆酸钠)自由摄食、饮水。连续喂养8周后，从正常对照组和脂肪肝模型组分别处死4只小鼠，比较两组小鼠的血清和肝脏生化指标，以及肝脏组织病理切片，判断非酒精性脂肪肝模型小鼠是否建立成功。
将造模成功小鼠32只随机分为4组，分别为正常对照组(8只)，模型组(8只)，黄柏碱治疗组(8只)，阳性对照组(洛伐他汀组8只)，连续灌胃给药8周。给药期间，除正常对照组标准饲料饲养外，其余各组继续给予高脂饮食，直到实验结束。黄柏碱治疗组在高脂饮食同时采用口服灌胃(50mg/kg)方式给药，阳性对照组在高脂饮食同时采用口服灌胃洛伐他汀(60mg/kg)方式给药)
2、观察指标：
a动物的一般情况
实验动物在SPF动物房中饲养，12h光照12h黑夜，自由饮食饮水，实验期间动物状态正常。
b肝功能相关指标：血清ALT、AST，
c血脂相关指标：TC、TG、HDL、LDL
d病理学检查：HE染色
3、实验结果：
结果表明：黄柏碱均可明显降低血浆中的总胆固醇(TC)和甘油三酯(TG)，升高高密度脂蛋白(HDL)，降低低密度脂蛋白(LDL)，降低血浆中天门冬氨酸转氨酶(AST),丙氨酸转氨酶(ALT)，病理切片结果显示有效缓解高脂饲料引起的非酒精性脂肪肝变性，改善因脂肪变性而导致的肝功能损害。
给药结束后测定小鼠、血脂水平。实验数据进行方差分析，结果以x±s表示。结果显示，黄柏碱对降低高脂效果明显，具有显著统计学差异。
表12.各组小鼠血脂指标的变化
Figure PCTCN2016074781-appb-000016
Figure PCTCN2016074781-appb-000017
表13各组小鼠肝功能相关指标的变化
Figure PCTCN2016074781-appb-000018
Figure PCTCN2016074781-appb-000019
实施例4
为了研究黄柏碱对糖尿病肾病血糖影响，使用C57BL小鼠随机分为3组，正常对照组(10只)，STZ组(20只)。STZ组连续腹腔注射STZ(50mg/Kg)5天，两周后测定禁食血糖，血糖大于等于13.8mM为糖尿病造模成功。然后将STZ组随机分为STZ组和黄柏碱治疗组，STZ组仅正常饲养，黄柏碱治疗组采用口服灌胃(15mg/kg)，12周后，将小鼠放入小鼠代谢笼中，自由饮食饮水，将尿液用于分析，所测指标均为临床上糖尿病肾病的检测的经典指标，如表14-16所示STZ组相比空白对照组，显著升高均在10倍以上，说明糖尿病小鼠肾功能出现严重损伤，如图1、2所示STZ组相比正常组器官损伤明显。综合以上表图可以得出糖尿病肾病造模成功。
表14.结果表明黄柏碱能够有效的降低STZ造成的血糖升高，逆转由糖尿病肾病引起的尿量增加(表15)，有效的降低糖尿病肾病引起的24h蛋白排泄率升高(表16)。临床上常用尿液中白蛋白和肌酐的比值评估肾功能，黄柏碱的有效的降低糖尿肾病引起的白蛋白和肌酐的比值升高(表17)。尿量、24h蛋白排泄率白蛋白和肌酐的比值的结果表明黄柏碱治疗很好的改善了肾功能。
为了更进一步的评估肾脏器官性损伤的改变，本发明对肾脏进行PAS染色 和HE染色。图5，6显示：黄柏碱能够有效的修复糖尿病肾病的器官性损伤逆转，如图所示STZ组肾小球系膜区增宽，基质增加，肾小球基膜增厚，肾小管基膜增厚及分裂，呈现出极为典型的糖尿病肾病特征，显示造模良好，而黄柏碱在一定程度上修复，系膜区相对缩小，改善了基质增生以及肾小管基膜增厚，说明治疗作用明显效果显著。
表14.黄柏碱对STZ造模的糖尿病肾病的血糖的影响(单位：mM).
Figure PCTCN2016074781-appb-000020
注：*P<0.05，**P<0.01，***P<0.001，与STZ组相比。
表15.黄柏碱对STZ造模的糖尿病肾病的尿量的影响(单位：g).
Figure PCTCN2016074781-appb-000021
表16.黄柏碱对STZ造模的糖尿病肾病的UAE(24h尿白蛋白排泄率，单位：μg)的影响.
Figure PCTCN2016074781-appb-000022
表17.黄柏碱对STZ造模的糖尿病肾病的UACR(μg尿白蛋白/mg肌酐 比值)的影响.
Figure PCTCN2016074781-appb-000023
注：*P<0.05，**P<0.01，***P<0.001，与STZ组相比。
实施例5
黄柏碱的制剂
黄柏碱片剂
黄柏碱10mg,淀粉88g,硬脂酸镁3g
制备工艺：取黄柏碱过100目筛，加淀粉、硬脂酸镁混合均匀，制成颗粒，干燥，压片，即得。
黄柏碱胶囊
黄柏碱10mg,淀粉88g,硬脂酸镁3g
制备工艺：取黄柏碱过100目筛，加淀粉、硬脂酸镁混合均匀，制成颗粒，干燥，装胶囊，即得。
黄柏碱注射液
黄柏碱47mg，注射用氯化钠7mg
制备工艺：取黄柏碱过100目筛，加1.9mL注射用水溶解，加入注射用氯化钠至等渗，调节pH值至7～7.1，滤过，冷藏24小时，加注射用水至规定量，滤过，灌封，灭菌，即得。
实施例6
黄柏碱的食品制剂
干酵母5g、温水90ml、水少许、面粉150g、黄柏碱5mg，植物油10g、低钠盐少许
饼干做法：把酵母撒在温水里搅拌倒溶化，加入黄柏碱。加入面粉搅拌，再加入植物油，揉成光滑的面团；面团制成0.2cm厚的薄片；压出造型，刺洞，表面撒上水，撒上一点低钠盐，室温发酵10分钟；烤箱预热120度，放在上层，烤约10分钟，得到含黄柏碱的食品。
实施例7
黄柏碱单体(纯度在91％～92％)l00mg，先用10ml氯仿溶解，加入10mL 0.1M HCL氯仿溶液，析出沉淀，过滤干燥，得到盐酸黄柏碱单体，用1ml乙醇溶解，置冰箱4℃条件下放置24h，析晶，过滤干燥，得到盐酸黄柏碱单体，纯度达到98％以上。
黄柏碱单体(纯度在91％～92％)l00mg，先用10ml氯仿溶解，加入10mL 0.1M H2SO4氯仿溶液，析出沉淀，过滤干燥，得到硫酸黄柏碱单体，用1ml乙醇溶解，过滤，滤液置冰箱4℃条件下放置24h，析晶，过滤干燥，得到硫酸黄柏碱单体，纯度达到98％以上。
黄柏碱单体(纯度在91％～92％)l00mg，先用10ml氯仿溶解，加入10mL 0.1M磷酸氯仿溶液，析出沉淀，过滤干燥，得到磷酸黄柏碱单体，用1ml乙醇溶解，过滤，滤液置冰箱4℃条件下放置24h，析晶，过滤干燥，得到磷酸黄柏碱单体，纯度达到98％以上。
黄柏碱单体(纯度在91％～92％)l00mg，先用10ml氯仿溶解，加入10mL 0.1M果酸氯仿溶液，析出沉淀，过滤干燥，得到果酸黄柏碱单体，用1ml乙醇溶解，过滤，滤液置冰箱4℃条件下放置24h，析晶，过滤干燥，得到果酸黄柏碱单体，纯度达到98％以上。
黄柏碱单体(纯度在91％～92％)l00mg，先用10ml氯仿溶解，加入10mL 0.1M草酸氯仿溶液，析出沉淀，过滤干燥，得到草酸黄柏碱单体，用1ml乙醇溶解，过滤，滤液置冰箱4℃条件下放置24h，析晶，过滤干燥，得到草酸黄柏碱单体，纯度达到98％以上。
黄柏碱单体(纯度在91％～92％)l00mg，先用10ml氯仿溶解，加入10mL 0.1M琥珀酸氯仿溶液，析出沉淀，过滤干燥，得到琥珀酸黄柏碱单体，用1ml乙醇溶解，过滤，滤液置冰箱4℃条件下放置24h，析晶，过滤干燥，得到琥珀酸黄柏碱单体，纯度达到98％以上。
黄柏碱单体(纯度在91％～92％)l00mg，先用10ml氯仿溶解，加入10mL 0.1M醋酸氯仿溶液，析出沉淀，过滤干燥，得到醋酸黄柏碱单体，用1ml乙醇溶解，过滤，滤液置冰箱4℃条件下放置24h，析晶，过滤干燥，得到醋酸黄柏碱单体，纯度达到98％以上。
黄柏碱单体(纯度在91％～92％)l00mg，先用10ml氯仿溶解，加入10mL 0.1M丙酸氯仿溶液，析出沉淀，过滤干燥，得到丙酸黄柏碱单体，用1ml乙醇溶解，过滤，滤液置冰箱4℃条件下放置24h，析晶，过滤干燥，得到丙酸黄柏碱单体，纯度达到98％以上。
实施例8
盐酸黄柏碱的制备方法
(1)总生物碱的提取
配制质量分数为3.0％的NaCl溶液，调其PH为3.0；干燥黄柏药材用该溶液以料液比1:8浸泡提取2次，每次48h，合并提取液；
(2)黄柏碱的粗分
将合并提取液通过HPD100型大孔吸附树脂柱层析吸附，干燥的黄柏药材与大孔吸附树脂的质量比为1:5，上样速度控制在2.0ml/min，上样完毕后静止吸附2.0h；吸附完毕后，先用3.0BV的水洗脱除杂；再用30％乙醇洗脱3.0BV，收集洗脱液，于50℃减压浓缩得浸膏；
(3)黄柏碱的纯化精制
将浸膏通过10％氨水碱化的200-300目硅胶柱层析：浸膏与填料碱化硅胶的重量比为1:30，先采用三氯甲烷-甲醇30:1洗脱5.0BV，再用5.0BV甲醇洗脱，收集合并甲醇洗脱部分；将甲醇洗脱液于45℃条件下减压浓缩干燥，得黄柏碱粗品；
(6)盐酸黄柏碱结晶的制备
将黄柏碱粗品先用少量乙醇溶解，再滴加浓HC1调溶液PH至3.0，置冰箱4℃条件下放置24h，析晶，过滤干燥，上述方法反复重结晶得到纯度达到98％的盐酸黄柏碱。
(5)盐酸黄柏碱的结构鉴定
测试仪器及条件：核磁共振谱测试采用Bruker Avance 400型核磁共振仪(瑞士)，按超导脉冲傅里叶变换核磁共振波谱方法通则。溶剂采用氘代二甲基亚砜(DMSO-d6)，TMS为内标。对1H核,观察频率为400.1MHz，对13C核，观察频率为100.6MHz。质谱测试采用Brucker Amazon SL型离子阱质谱仪(瑞士)，
样品用水溶解，直接进样分析。
Positive ESI-MS：m/z 342.1[M+H]+,推断本品的分子式为C20H24NO4。1H-NMR(400MHz，DMSO-d6)δ：6.85，6.73，6.72，6.66(4H,s,C-2，3，10，11)，4.77(1H,d,J＝10.0Hz，H-8b)，4.70(1H,m,H-14)，4.58(1H,d,J＝15.2Hz，H-8a)，3.77(3H,s,OCH3)，3.75(3H,s,OCH3)，3.66(1H,m,H-6)，3.60(1H,m,H-6)，3.28(1H,dd,J＝5.6,18.4Hz,H-13a)，3.16(3H,N-CH3)，3.14(2H,m,H-5)，2.96(1H,dd,J＝10,18.6Hz,H-13b)。13C-NMR(100MHz，DMSO-d6)，δ：22.6(C-5)，33.0(C-13)，49.2(N-CH3)，51.6(C-6)，55.7(OCH3at C-3)，55.7(OCH3at C-10)，62.0(C-8)，64.3(C-14)，109.9(C-1)，112.5(C-9)，113.4(C- 4)，114.7(C-12)，116.4(C-13)，118.8(C-4a)，121.8(C-12a)，124.1(C-8a)，145.5(C-11)，146.8(C-2)，147.1(C-3)，147.9(C-10)。综合1H,13C-NMR及ESI-MS等波谱数据，与文献对照，确定本品为盐酸黄柏碱。
实施例9柠檬酸黄柏碱盐的制备方法
(1)总生物碱的提取
配制质量分数为3.0％的NaCl溶液，调其PH为3.0；干燥黄柏药材用该溶液以料液比1:8浸泡提取2次，每次48h，合并提取液；
(2)黄柏碱的粗分
将合并提取液通过HPD100型大孔吸附树脂柱层析吸附，干燥的黄柏药材与大孔吸附树脂的质量比为1:5，上样速度控制在2.0ml/min，上样完毕后静止吸附2.0h；吸附完毕后，先用3.0BV的水洗脱除杂；再用30％乙醇洗脱3.0BV，收集洗脱液，于50℃减压浓缩得浸膏；
(3)黄柏碱的纯化精制
将浸膏通过10％氨水碱化的200-300目硅胶柱层析：浸膏与填料碱化硅胶的重量比为1:30，先采用二氯甲烷-甲醇30:1洗脱5.0BV，再用5.0BV甲醇洗脱，收集合并甲醇洗脱部分；将甲醇洗脱液于45℃条件下减压浓缩干燥，得黄柏碱粗品；
(7)盐酸黄柏碱结晶的制备
将黄柏碱粗品先用少量乙醇溶解，再滴加柠檬酸调溶液PH至3.0，置冰箱4℃条件下放置24h，析晶，过滤干燥，上述方法反复重结晶得到纯度达到98％的柠檬酸黄柏碱盐。
(6)柠檬酸黄柏碱盐的的结构鉴定
测试仪器及条件：核磁共振谱测试采用Bruker Avance 400型核磁共振仪(瑞士)，按超导脉冲傅里叶变换核磁共振波谱方法通则。溶剂采用氘代二甲基亚砜(DMSO-d6)，TMS为内标。对1H核,观察频率为400.1MHz，对13C核，观察频率为100.6MHz。质谱测试采用Bruker Amazon SL型离子阱质谱仪(瑞士)，
样品用水溶解，直接进样分析。
Positive ESI-MS：m/z 342.1[M+H]+，Negative ESI-MS:190.9[M-H]-,推断 本品的分子式为C20H24NO4·C6H8O7；1H-NMR(400MHz，DMSO-d6)δ：6.85，6.73，6.71，6.65(4H,s,H-2,3,10,11)，4.74(1H,d,J＝10.0Hz，H-8b)，4.71(1H,m,H-14)，4.57(1H,d,J＝15.2Hz,H-8a)，3.77(3H,s,OCH3)，3.75(3H,s,OCH3)，3.66(1H,m,H-6)，3.60(1H,m,H-6)，3.29(1H,dd,J＝4.8,18.0Hz，H-13a)，3.14(3H,N-CH3，C-5)，2.95(1H,dd,J＝9.6,18.0Hz,H-13b),2.51(0.5H,s,H-2',4')；13C-NMR(100MHz,DMSO-d6),δ:22.7(C-5)，33.0(C-13)，49.3(N-CH3)，51.6(C-6)，55.7(OCH3at C-3)，55.7(OCH3at C-10)，62.0(C-8)，64.4(C-13a)，109.9(C-1)，112.5(C-9)，113.4(C-4)，114.7(C-12)，116.4(C-13)，118.9(C-4a)，121.8(C-12a)，124.1(C-8a)，145.5(C-11)，146.8(C-2)，147.1(C-3)，147.9(C-10),44.6(C-2',4')，71.1(C-3')，171.4(C-1',5')，177.2(C-6')。综合1H,13C-NMR及ESI-MS等波谱数据，与文献对照，确定本品为柠檬酸黄柏碱盐。

Claims (10)

  1. 异喹啉生物碱用于制备治疗或预防糖尿病的组合物。
  2. 异喹啉生物碱用于制备治疗或预防糖尿病肾病的组合物。
  3. 异喹啉生物碱用于制备治疗或预防高脂血症的组合物。
  4. 异喹啉生物碱用于制备治疗或预防非酒精性脂肪肝的组合物。
  5. 根据权利要求1-2中的组合物,其特征为：用于制备预防或治疗I型糖尿病、或、用于制备保护胰岛细胞、修复受损的胰岛细胞的组合物；或、用于制备预防或治疗II型糖尿病、或、制备对抗胰岛素抵抗的组合物。
  6. 根据权利要求1-4中的组合物,其特征为：异喹啉生物碱用于制备保护糖尿病肾病人的肾脏组织、或、修复糖尿病肾病人的受损的肾脏组织的组合物。
  7. 根据权利要求1-4中的组合物,其特征为：异喹啉生物碱为黄柏碱或其药用盐、水合物或无水物。
  8. 根据权利要求1-4中的组合物,其特征为：
    其中黄柏碱药用盐包括：无机酸盐例如盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硝酸盐和磷酸盐；有机酸盐例如乙酸盐，苯甲酸盐，马来酸盐，富马酸盐，苹果酸盐，柠檬酸盐，草酸盐，乳酸盐，琥珀酸盐，酒石酸盐，烷基磺酸盐或芳基磺酸盐，半胱氨酸盐或其它氨基酸盐。还包括碱式盐如钠盐、钾盐和钙盐。
  9. 根据权利要求1-4中的组合物,其特征为：
    黄柏碱晶型以下列的X射线衍射图表征，其以2θO角、晶面间距d(AO)、相对强度(Intensity％)来表示：
    Figure PCTCN2016074781-appb-100001
    Figure PCTCN2016074781-appb-100002
  10. 根据权利要求1-8中的组合物,其特征为：
    黄柏碱药用盐按照如下方式制备：
    取黄柏碱单体，用氯仿溶解，加入氯仿的酸溶液，析出沉淀，过滤干燥，得到黄柏碱的药用盐单体，用乙醇溶解，置冰箱放置，析晶，过滤干燥，得到黄柏碱的药用盐，纯度达到98％以上；
    或；
    黄柏碱的盐酸盐、柠檬酸盐的优选的制备方法
    (1)总生物碱的提取
    配制质量分数为3.0％的NaCl溶液，调其PH为3.0；干燥黄柏药材用该溶液以料液比1:8浸泡提取2次，每次48h，合并提取液；
    (2)黄柏碱的粗分
    将合并提取液通过HPD100型大孔吸附树脂柱层析吸附，干燥的黄柏药材与大孔吸附树脂的质量比为1:5，上样速度控制在2.0ml/min，上样完毕后静止吸附2.0h；吸附完毕后，先用3.0BV的水洗脱除杂；再用30％乙醇洗脱3.0BV，收集洗脱液，于50℃减压浓缩得浸膏；
    (3)黄柏碱的纯化精制
    将浸膏通过10％氨水碱化的200-300目硅胶柱层析：浸膏与填料碱化硅胶的重量比为1:30，先采用三氯甲烷-甲醇30:1洗脱5.0BV，再用5.0BV甲醇洗脱，收集合并甲醇洗脱部分；将甲醇洗脱液于45℃条件下减压浓缩干燥，得黄柏碱粗品；
    (4)盐酸黄柏碱结晶的制备
    将黄柏碱粗品先用少量乙醇溶解，再滴加浓HC1调溶液PH至3.0，置冰箱4℃条件下放置24h，析晶，过滤干燥，上述方法反复重结晶得到纯度达到98％的盐酸黄柏碱；
    (5)柠檬酸黄柏碱盐结晶的制备
    将黄柏碱粗品先用少量乙醇溶解，再滴加柠檬酸调溶液PH至3.0，置冰箱4℃条件下放置24h，析晶，过滤干燥，上述方法反复重结晶得到纯度达到98％的柠檬酸黄柏碱盐。
PCT/CN2016/074781 2015-03-17 2016-02-28 异喹啉生物碱的医药用途 WO2016145983A1 (zh)

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CN201510118943.9A CN106138047A (zh) 2015-03-17 2015-03-17 一种异喹啉生物碱的预防或治疗糖尿病的用途
CN201510118945.8A CN106138049B (zh) 2015-03-17 2015-03-17 异喹啉生物碱防治高血脂症和非酒精性脂肪肝的用途
CN201510118944.3A CN106138048B (zh) 2015-03-17 2015-03-17 一种异喹啉生物碱的盐的降糖降脂保肝的用途
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101153039A (zh) * 2006-09-30 2008-04-02 中国科学院上海药物研究所 13,13a-二氢小檗碱衍生物及其药物组合物和用途
CN102107006A (zh) * 2009-12-25 2011-06-29 奇复康药物研发(苏州)有限公司 用于治疗糖尿病的偶联药物及其医药用途

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101153039A (zh) * 2006-09-30 2008-04-02 中国科学院上海药物研究所 13,13a-二氢小檗碱衍生物及其药物组合物和用途
CN102107006A (zh) * 2009-12-25 2011-06-29 奇复康药物研发(苏州)有限公司 用于治疗糖尿病的偶联药物及其医药用途

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