WO2006054370A1 - Gluconeogenesis inhibiting agent - Google Patents

Gluconeogenesis inhibiting agent Download PDF

Info

Publication number
WO2006054370A1
WO2006054370A1 PCT/JP2005/008569 JP2005008569W WO2006054370A1 WO 2006054370 A1 WO2006054370 A1 WO 2006054370A1 JP 2005008569 W JP2005008569 W JP 2005008569W WO 2006054370 A1 WO2006054370 A1 WO 2006054370A1
Authority
WO
WIPO (PCT)
Prior art keywords
acid
corosolic acid
gluconeogenesis
corosolic
inhibiting agent
Prior art date
Application number
PCT/JP2005/008569
Other languages
French (fr)
Inventor
Futoshi Matsuyama
Yutaka Seino
Yuichiro Yamada
Masaya Hosokawa
Shinpei Fujimoto
Mitsuo Fukushima
Naoya Ueda
Kotaro Yamada
Original Assignee
Use-Techno Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Use-Techno Corporation filed Critical Use-Techno Corporation
Publication of WO2006054370A1 publication Critical patent/WO2006054370A1/en

Links

Classifications

    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present invention relates to a gluconeogenesis inhibiting agent.
  • Banaba leaf provides a banana extract which contains a certain amount of corosolic acid and has a hyperglicemia inhibiting action or a blood glucose depressant action (Japanese Patent Application Laid-Open No. 2000-169384).
  • Terpenes such as corosolic acid are widely contained in plants. However, it costs a great deal to identify terpenes that can work as active ingredients and to put them to good use. This is because of too many kinds of terpenes, and because, with respect to each kind of terpene, isolation, purification, molecular level experiments, non-clinical tests and clinical tests must be followed. On the other hand, if the sites, functional groups and conf ⁇ gurational structures relating to activities of terpenes are specified, it will facilitate terpenes to be put to good use.
  • corosolic acid and analogous compounds thereof can inhibit gluconeogenesis, and thus completed the present invention.
  • the present invention provides a gluconeogenesis inhibiting agent which contains as an active ingredient corosolic acid, an analogous compound of corosolic acid, or a pharmaceutically acceptable salt thereof.
  • the above-mentioned glucogenesis inhibiting agent has the following characteristics:
  • liver hepatopathy
  • Such a glucogenesis inhibiting agent can inhibit gluconeogenesis to suppress blood glucose elevation.
  • the gluconeogenesis inhibiting agent of the present invention exhibits a remarkable reduction in side effects such as hypoglycemia and hepatopathy (the administration of corosolic acid at 1,000 mg/kg is confirmed to give no side effect). Furthermore, the gluconeogenesis inhibiting agent of the present invention can suppress blood glucose elevation to prevent hypertension, obesity, hyperlipidemia
  • maslinic acid As preferred examples of the analogous compound of corosolic acid, there may be mentioned maslinic acid, tormentic acid, ursolic acid, asiatic acid, oleanolic acid, and 2 ⁇ ,19 ⁇ -dihydroxy-3-oxo-urs-12-en-28-oic acid. These compounds have the same backbone (basic skeleton) as corosolic acid, and show a remarkable gluconeogenesis inhibiting action and a hyperglicemia inhibiting action similarly to corosolic acid.
  • the gluconeogenesis inhibiting agent of the present invention is preferably administered to a subject that has a blood glucose level beyond the normal value, that is, satisfies the following condition (i) or (ii):
  • a fasting blood glucose level of 110 mg/dL or more (i) a fasting blood glucose level of 110 mg/dL or more; (ii) an OGTT (oral glucose tolerance test) value at 2 hours of 140 mg/dL or more.
  • OGTT oral glucose tolerance test
  • Fig. 1 is graphs showing the changes in glucose production in rat liver in relation to time.
  • Fig. 2 is a mass spectrum obtained by analyzing a corosolic acid standard solution.
  • Fig. 3 is a calibration curve derived from chromatograms of corosolic acid standard solutions.
  • Fig. 4 is a calibration curve derived from chromatograms of blood samples supplemented with corosolic acid.
  • Fig. 5 is chromatograms of a corosolic acid standard solution, and of blood samples taken from a dog administered orally with corosolic acid.
  • Fig. 6 is graphs showing the changes in any time blood glucose levels of GK rats in relation to time.
  • Fig. 7 is graphs showing the changes in blood glucose levels of GK rats in an OGTT in relation to time.
  • Fig. 8 is graphs showing the changes in body weight of Zucker fatty rats (fa/fa) in relation to time.
  • Fig. 9 is graphs showing the changes in blood glucose levels of subjects in relation to time.
  • Fig. 10 is graphs showing the changes in blood insulin levels of subjects in relation to time.
  • Fig. 11 is graphs showing the changes in blood insulin levels of subjects in relation to time.
  • Fig. 12 is graphs showing the changes in blood insulin levels of subjects in relation to time.
  • the gluconeogenesis inhibiting agent of the present invention contains as active ingredients corosolic acid, analogous compounds of corosolic acid, or pharmaceutically acceptable salts thereof.
  • Analogous compounds of corosolic acid mean triterpenes which have a structure-activity correlation similar to that of corosolic acid.
  • Corosolic acid is a triterpene (C 30 H 48 O 4 ; MW 472) represented by the following formula.
  • Preferred examples of the analogous compound of corosolic acid include maslinic acid, tormentic acid, ursolic acid, asiatic acid, oleanolic acid and 2 ⁇ ,19 ⁇ -dihydroxy-3-oxo-urs-12-en-28-oic acid.
  • Triterpenes preferably have at least one hydroxyl at the 2- and 3- positions thereof, and more preferably, have further at least one hydroxyl at the 29- and 30- positions and carboxyl at the 28- position thereof.
  • Such triterpenes include ursane type pentacyclic triterpenes and oleanane type pentacyclic triterpenes.
  • the following compounds 1) - 40) are examples of the ursane type pentacyclic triterpene: 1) Desfontainic acid
  • the following compounds 41) - 100) are examples of the oleanane type pentacyclic triterpene:
  • corosolic acid can be converted to the ether product by reacting the hydroxyl group of corosolic acid with a halogenated alkyl (such as CH 3 Br and CH 3 (CH 2 ) n Br).
  • a halogenated alkyl such as CH 3 Br and CH 3 (CH 2 ) n Br.
  • Corosolic acid also can be converted to the ketone product by the oxidation of one or both of hydroxyl groups at the 2- and 3- positions.
  • Examples of the pharmaceutically acceptable salt of corosolic acid or the analogous compound thereof include an alkali metal salt, an alkali earth metal salt, and an ammonium salt.
  • a salt formed between corosolic acid or an analogous compound thereof and sodium, potassium, calcium, magnesium, ammonia, dimethylamine, diethylamine, trimethylamine, tetramethylammonium, monoethanol amine, diethanol amine, or triethanol amine is preferred. (Extraction and purification of triterpene)
  • Triterpene an active ingredient of the gluconeogenesis inhibiting agent, can be obtained by extracting from banaba (Lagerstroemia Speciosa, Linn, or Pers.), loquat, soapberry (sapindus mukurossi), perilla or guava, and it is preferably by extracting from banaba leaf.
  • banaba Lagerstroemia Speciosa, Linn, or Pers.
  • loquat soapberry
  • soapberry sapindus mukurossi
  • perilla or guava preferably by extracting from banaba leaf.
  • the banaba leaf may be subjected to extraction with water or alcohol to get a banaba extract.
  • the banaba extract may be further concentrated if necessary.
  • the banaba leaf may be optionally cut into pieces for extraction, but is preferably cut to improve extraction efficiency.
  • the piece has a size which is adjusted depending on equipment used for the succeeding extraction process, but has preferably a size of 1 mm square or larger. A fine size that does not lead to clogging is most preferable.
  • the banaba leaf may be cut into a similar size of strips.
  • banaba leaf When banaba leaf is immersed in water, 1 kg of banaba leaf is preferably immersed in 2 L or more of water at room temperature or 10-60 0 C for 24 hours or more. The leaf is three times or more immersed in water. while the water is refreshed every immersion. Then, the leaf is three to five times subjected to hot water extraction with hot water, by boiling, and with steam, while the water is also refreshed every time. Furthermore, the leaf is pressed, extracted with hot water, and finally extracted with a solvent of water 50%/alcohol 50%, preferably at 0-100 0 C.
  • the leaf is pressed under a pressure of 1-3 arm, at an inner temperature of 100 0 C or more, and most preferably for an extraction time of about 3-10 min. If an inner pressure is beyond 1.5 atm, an extraction time is shortened to 1-5 min, wherein ideally the leaf is at first extracted at 1.1-1.3 atm for 2 min, then twice at 1.3-1.6 atm. A long time extraction should be avoided to prevent the triterpene from blowing out. Such a condition is also necessary because what kinds of tannins and chlorophylls will blow out varies depending on pressure, solvent, time and temperature. For the same reason, a mixed solvent of water and alcohol is finally used for extraction. The banaba leaf thus extracted is filtered while removing water, and dried in the sun or by a drier.
  • Water is preferably removed by centrifugation, followed by drying.
  • the leaf is preferably dried with warm air at 30-60 0 C or cold air, more preferably with air having as low humidity as possible.
  • the dried banaba leaf is extracted with hexane or a mixed solvent of hexane and water.
  • a resin or an active charcoal is preferably used to remove tannins or chlorophylls.
  • ethanol or an aqueous ethanol solution (ethanol content: 90% or more) is used for extraction.
  • the leaf is preferably subjected to hot water extraction with steam, pressure in a pressure vessel, and dewatering by a centrifugal separator. If an inner pressure is beyond 1.5 atm, a pressure time is preferably kept within 10 min. If hot air has a temperature of beyond 100 0 C, a drying time is kept within 10 min.
  • Concentration after extraction is preferably done for a relatively short time under a reduced pressure, because the concentrated product may be kept at a high temperature for a long time to deteriorate an active ingredient.
  • the extract liquid obtained by the aforementioned manner is filtered, concentrated at a temperature of 60 0 C or less under a reduced pressure to give a solid, which is then dried at a temperature of 50-70 0 C under a reduced pressure (higher than that at the concentration). A solid thus obtained can be pulverized to give a powdery concentrate.
  • ingredients contained in the banaba extract (white extract) or the concentrate thereof thus obtained there may be mentioned corosolic acid, maslinic acid, tormentic acid, ursolic acid, oleanolic acid, ⁇ -amirinic acid, ⁇ -amirinic acid, asiatic acid, 18 ⁇ -glycyrrhetic acid, tannins, chlorophylls and hemicellulose.
  • Corosolic acid is contained by 3-50% or more.
  • the banaba extract or the concentrate thereof may be in a liquid or solid state, or in paste form.
  • the banaba extract or the concentrate thereof is preferably stored at room temperature or in a refrigerator to be shielded from light and desiccated.
  • Triterpenes may be extracted and concentrated in the following manner.
  • the dried banaba leaf is firstly heated under reflux to extract in n-hexane for 1 hour, in purified water for 1 hour. Then, decocted banaba leaf is heated under reflux in ethanol for 1 hour, followed by filtering the extract, which is then concentrated under a reduced pressure to get a solid.
  • the banaba extract or the concentrate thereof thus obtained may be used as it is as the gluconeogenesis inhibiting agent, but is preferably purified as far as possible from tannins, chlorophylls and fibers to isolate the active ingredients (triterpenes).
  • the isolation and purification may be done according to a known method.
  • corosolic acid may be isolated and purified by the following procedure.
  • the banaba extract is suspended in water, and at first partitioned in ether, hexane or the like to remove a low polar component.
  • the aqueous layer is then successively eluted with water, methanol and acetone using Diaion HP-20 column chromatography or the like.
  • the methanol eluate fraction containing corosolic acid is then subjected to separation and purification by silica gel column chromatography and high performance liquid chromatography (HPLC) to isolate the corosolic acid.
  • the extract may be directly separated by silica gel column chromatography, and then purified by HPLC.
  • Corosolic acid may be isolated and purified in the following manner.
  • the extract (the banaba extract or the banaba extract concentrate) is suspended in water, and solvent-fractionated with hexane or ether. After filtration by suction, among water soluble fractions, organic solvent soluble fractions and insoluble fractions, the insoluble fractions are selected to use.
  • the insoluble fractions are applied to silica gel column chromatography using a mixed solvent of dichloromethane and methanol.
  • Corosolic acid-containing fractions obtained by the silica gel column chromatography are applied to an ion-exchange resin, and HPLC is used to separate corosolic acid.
  • the HPLC condition is as follows.
  • the triterpenes isolated and purified from the banaba extract or the banaba extract concentrate may be used as they are, and may also be acylated (for example, acetylated) to use, or consecutively deacylated (for example, deacetylated) to use.
  • corosolic acid is preferably acylated (for example, acetylated), and then deacylated to use. Acylation (such as acetylation) followed by deacylation can give corosolic acid having a very high purity (approximately 100%).
  • acetylation of corosolic acid for example, corosolic acid isolated and purified from the banaba extract is dissolved in absolute pyridine, acetic anhydride is added thereto, and the mixture was left to stand at room temperature for about 12 hours.
  • the reaction solution is supplemented with ice water, and extracted a few times (about three times) with chloroform.
  • the chloroform layer is dewatered with sodium sulfate, filtered to remove sodium sulfate, and distilled under a reduced pressure to evaporate chloroform.
  • the resultant product can be recrystallized from hexane to give acetyl corosolic acid.
  • a method of deacylation a method wherein hydrolysis is performed with an alkali such as potassium hydroxide or sodium hydroxide may be mentioned. (Analysis of pharmacokinetics for triterpene)
  • the blood level and the urine level of a triterpene can be determined by LC/MS/MS (liquid chromatography/mass spectrometry/mass spectrometry).
  • a HPLC apparatus is used for LC, and a quadruple mass spectrometer is used for MS.
  • An ESI (electrospray ionization) method is preferable for ionization in LC/MS/MS.
  • the microanalysis of corosolic acid in blood by LC/MS/MS is conducted in the following manner.
  • the standard solution of corosolic acid is analyzed by LC/MS/MS to get the mass spectrum, and thereby to determine whether corosolic acid can be analyzed by LC/MS/MS or not.
  • a calibration curve is depicted from chromatograms for Standard solutions of corosolic acid to determine whether concentration of corosolic acid can be measured or not.
  • a blood sample supplemented with corosolic acid is pretreated (for deproteination, desalting and extraction), and an HPLC condition is so established that only the corosolic acid in the extract may be eluted at a certain time point.
  • Pretreated blood samples are analyzed under the HPLC condition to get chromatograms, from which a calibration curve is then depicted to determine whether the blood level of corosolic acid can be measured or not.
  • an analyte sample blood
  • LC/MS/MS LC/MS/MS under the established HPLC condition to determine the corosolic acid level in the sample.
  • the gluconeogenesis inhibiting agent containing a triterpene as an active ingredient can be used as a drug, an additive for food and drink, or a smoking material (such as tobacco).
  • the agent may be used in a solid, liquid or gas state, or in paste form, and may be made into a mist when used.
  • a triterpene powder isolated and purified can be pulverized by a nanoparticle pulverizer to get the finer powder.
  • the corosolic acid powder isolated and purified by HPLC has a particle size of about 10 ⁇ m, but the fine powder having a size of 10 nm - 100 nm can be obtained if pulverized by a nanoparticle pulverizer.
  • the particle pulverized to a nano-size can provide a higher effect even if used in a less amount.
  • a solvent is adequately selected depending on the fat solubility or water solubility of the triterpene.
  • an oil and fat (docosahexaenic acid) or an alcohol is adequate as a solvent for corosolic acid.
  • a triterpene may be glycosidated to increase water solubility, if it has a low water solubility.
  • the triterpene When used as a drug, the triterpene may be used in the form of powder, granule, tablet, capsule, injection or the like.
  • the granule or the tablet can be prepared by mixing powdery corosolic acid or the banaba extract concentrate with excipients such as lactose and starch.
  • the triterpene may be used together with digestion resistant dextrin or a sucrase inhibitor (such as L-arabinose or 1-deoxynojirimycine). Because the digestion resistant dextrin or a sucrase inhibitor inhibits absorption and digestion of glucose, a combination of the digestion resistant dextrin or a sucrase inhibitor with the gluconeogenesis inhibiting agent and sucrose provides a sweetener having a blood glucose depressant action.
  • the gluconeogenesis inhibiting agent can be used alone or in the form of a sweetener to add to drinks such as water, cooling drink, fruit juice, milk drink and alcoholic drink, as well as foods such as breads, noodles, rices, soybean curd, dairy product, soy sauce, miso (soybean paste) and cakes, as an additive for drink and food.
  • the gluconeogenesis inhibiting agent is preferably administered orally and intravenously, and particularly preferably orally.
  • the gluconeogenesis inhibiting agent As a preferred subject to whom the gluconeogenesis inhibiting agent is administered, there may be mentioned a person having a blood glucose level beyond the normal value (fasting blood glucose level: less than 110 mg/dL; OGTT value at 2 hours: less than 140 mg/dL).
  • the gluconeogenesis inhibiting agent When administered to animals other than humans, the gluconeogenesis inhibiting agent may be mixed to a feed to be ingested with it.
  • DMSO dimethyl sulfoxide
  • Fig. 1 is graphs showing the changes in glucose production in rat liver in relation to time. As shown in Fig. 1, there was no glucose production in liver at the time point of previous perfusion, but the addition of 2 mM lactic acid in the perfusate swiftly caused gluconeogenesis, resulting in a glucose production of 8.3 ⁇ mol/g/h after 30 min. Afterward, the addition of 200 ⁇ M corosolic acid inhibited gluconeogenesis by 30%. On the other hand, in the case of no corosolic acid added, the glucose production did not change.
  • Corosolic acid level in blood was determined by LC/MS/MS using an ESI method and a quadruple mass spectrometer. At first, corosolic acid standard solutions were analyzed by
  • Fig. 2 is a mass spectrum obtained by analyzing a corosolic acid standard solution. As shown in Fig. 2, an ion peak appeared at m/z value of 472, and hence it was revealed that corosolic acid (MW: 472) could be analyzed by LC/MS/MS .
  • Fig. 3 is a calibration curve depicted from chromatograms of corosolic acid standard solutions. Fig. 3 revealed that corosolic acid concentration could be determined at least within 1 ng/mL - 100 ng/mL.
  • a blood sample supplemented with corosolic acid was centrifuged by a centrifugal separator (3,000 rpm, 20 min), and 100 ⁇ L of the blood serum thus obtained was supplemented with 400 ⁇ L of acetonitrile. Then the resultant product was stirred by vortex, and deproteinated by centrifugation (15,000 rpm, 10 min). The acetonitrile layer was concentrated by a nitrogen gas, and supplemented with 300 ⁇ L of ethyl acetate, and then liquid/liquid extraction was carried out.
  • Fig. 4 is a calibration curve depicted from chromatograms of the blood samples supplemented with corosolic acid. Fig. 4 revealed that corosolic acid concentration in blood could be determined at least within 10 ng/mL - 100 ng/mL. Finally, blood samples taken from a dog administered orally with corosolic acid were analyzed by LC/MS/MS.
  • the dog that had fasted from the preceding day was administered orally with corosolic acid (20 mg/kg) and glucose (2 g/kg), and blood samples were collected just before administration, 30 min, 60 min, 90 min, 120 min, 180 min and 240 min after administration. These blood samples were pretreated in the same manner as described before to get samples for LC/MS/MS, which were then analyzed by LC/MS/MS in the same HPLC condition as described before.
  • Fig. 5 is chromatograms of a corosolic acid standard solution, and of the blood samples taken from a dog administered orally with corosolic acid.
  • (a) is a chromatogram of a corosolic acid standard solution, and
  • (b)-(h) are chromatograms of the blood samples collected just before administration, 30 min, 60 min, 90 min, 120 min, 180 min and 240 min after administration, respectively.
  • Fig. 5 since 90 min after administration, corosolic acid was detected in blood, and corosolic acid level in blood reached its maximum value (0.4 ⁇ g/mL) at the time point of 180 min after administration.
  • CRA represents corosolic acid.
  • GK rats a model animal for Type II diabetes, and Zucker fatty rats (fa/fa), a model animal for insulin resistance, were employed as test animals.
  • Wistar rats and Zucker fatty rats (+/+) were employed as control.
  • the animals were given feed with 0.0125% corosolic acid or without corosolic acid, and allowed to take it freely for about 2 weeks.
  • Feed intake, water intake, body weight, and blood glucose level were determined.
  • Glucose load in an OGTT was controlled to be 2 g/kg. Blood glucose levels were determined on the blood samples collected from animals.
  • GK rats are a model animal for insulin secretion failure specific to Asians. The animal suffers from diabetes due to insufficiency of insulin secretion.
  • Zucker fatty rats are a model animal for insulin resistance, and oversecretes insulin.
  • An individual having homologous pathogenic genes (fa genes) is expressed by Zucker fatty rat (fa/fa). Only this individual suffers from diabetes or falls in obesity. The wild type is expressed by Zucker fatty rat (+/+). This individual suffers from no diabetes and is free from obesity.
  • Fig. 6 is graphs showing the changes in any time blood glucose levels of GK rats in relation to time. As shown in Fig. 6, the any time blood glucose level in the corosolic acid administration group was significantly inhibited since 8th day after administration of corosolic acid in comparison with the non-administration group.
  • Fig. 7 is graphs showing the changes in blood glucose levels of GK rats in an OGTT in relation to time. As shown in Fig. 7, in the OGTT on GK rats, the rise in blood glucose level of the corosolic acid administration group was significantly inhibited at the time point of 90 min after glucose load in comparison with the non-administration group.
  • Fig. 8 is graphs showing the changes in body weight of Zucker fatty rats (fa/fa) in relation to time.
  • the body weight of the corosolic acid administration group was inhibited since 6th day after administration of corosolic acid in comparison with the non-administration group. This suggests that corosolic acid is also effective for a Westerner type of diabetes accompanied by obesity caused by insulin resistance.
  • the plot shown by "*" indicates data that had a significant difference found in comparison with the non-administration group.
  • CRA represents corosolic acid.
  • KK-Ay mice (aged 8 weeks, blood glucose level: 300 mg/100 mL), a model animal for Type II diabetes, were administered with various kinds of triterpenes to determine blood glucose levels in relation to time. The mice were given feed and water, and allowed to take it freely during the test.
  • PCB and CRA represent placebo and corosolic acid, respectively.
  • G and I represent blood glucose level and blood insulin level, respectively.
  • Time (min) shows time after administration.
  • Fig. 9 is graphs showing the changes in blood glucose levels of the subjects in relation to time.
  • Fig. 10 is graphs showing the changes in blood insulin levels of the subjects in relation to time.
  • CRA represents corosolic acid, and A, B and C in parentheses express names of the subjects.
  • a 75 g OGTT on eleven subjects was conducted to determine blood insulin level in relation to time. Afterward, the banaba extract containing corosolic acid was administered to the same eleven subjects continuously for
  • Fig. 11 is graphs showing the changes in blood insulin levels (average) of the subjects in relation to time.
  • Fig. 12 is graphs showing the changes in blood insulin levels (average) of the subjects in relation to time.
  • corosolic acid has water-insoluble property.
  • corosolic acid is a colorless transparent needle crystal, and soluble in an organic solvent such as ethanol and acetonitrile, but hardly dissolves in water.
  • Very low solubility in water is a large blockage against the analysis of biological reactions or pharmacokinetics of terpenes.
  • the solubility of corosolic acid in various kinds of solvents was studied by a conventionally used analytical HPLC (temperature: 25 0 C).
  • a buffer ionic strength: 0.1
  • an organic solvent ionic strength: 0.1
  • a mixed solution of surfactant/organic solvent ionic strength: 0.1
  • the pHs and solutes of the buffers used were as follows. pH 1 : HCl; pH 2: HCl; pH 3: citric acid; pH 4: citric acid; pH 5: citric acid; pH 6. citric acid; pH 7: phosphoric acid; pH 8: Tris; pH 9: boric acid; pH 10: glycine.
  • the organic solvents used were PG (propylene glycol), PEG400 (polyethylene glycol
  • the surfactants used were Tween 80 and BL9-EX. If the solubilities in the pH 1-10 buffers are determined, the pKa of corosolic acid can be obtained.
  • MeOH/0.05% TFA was used to give the shortest retention time. A high peak of absorption appeared around a wavelength of 210 nm. Thus, the solubility of corosolic acid was determined under the condition of eluent solution: 85% MeOH/0.05% TFA; and wavelength: 210 nm.
  • Corosolic acid was suspended in water and centrifuged to get a supernatant, which was then studied to determine whether corosolic acid could be detected by analytical HPLC (85% MeOH/0.05% TFA) or not. No corosolic acid was detected.
  • various concentrations of corosolic acid solutions were prepared, and they were analyzed by analytical HPLC.
  • the corosolic acid concentrations in the solutions were as follows: 0.1 mg/mL, 0.033 mg/mL, 0.01 mg/mL, 0.0033 mg/mL, 0.001 mg/mL, 0.00033 mg/mL and 0.0001 mg/mL.
  • Corosolic acid could be detected in four kinds of solutions having the higher concentrations.
  • the solubility of corosolic acid in the pH 5-8 buffers was studied.
  • the pH 5-8 buffers were prepared, 0.5 mg of corosolic acid was suspended in 10 mL of each of the buffers, and the suspension was shaken for about half a day. Afterward, the suspension was centrifuged by a centrifugal separator (5,000 RPM, 5 min, 25°C) to get a supernatant, which was then extracted with 10 mL of ethyl acetate. The organic layer was separated and concentrated. The concentrate was dissolved in 800 ⁇ L of the eluent (85% MeOH/0.05% TEA) to determine the corosolic acid concentration by analytical HPLC.
  • the organic solvent (PG 1 PEG) was administered from rat's femoral vein to confirm a reaction (dose: PG 250 ⁇ L; PEG 500 ⁇ L, administration time: about 2 min). The animal did not still fall in respiratory pause 30 min after administration. This suggested that corosolic acid could become an active ingredient in an injection or infusion.
  • the solubility of corosolic acid in the pH 4-8 buffers was studied.
  • the pH 4-8 buffers were prepared, 0.5 mg of corosolic acid was suspended in 10 mL of each of the buffers, and the suspension was shaken for about half a day. Afterward, the suspension was centrifiiged by a centrifugal separator
  • Corosolic acid was detected in the concentrates derived from the pH 4, 7 and 8 suspensions, but it was revealed that suspensions of pH 6 or more acidity needs improvement of HPLC detection sensitivity by increasing the addition amount of a buffer or ethyl acetate, or using a halogenated solvent (such as dichloromethane) in place of ethyl acetate.
  • a halogenated solvent such as dichloromethane
  • the PG solution of corosolic acid (4 mg/mL) and the PEG solution of corosolic acid (5 mg/mL) were prepared. These solutions (the PG solution 200 ⁇ L, the PEG solution 400 ⁇ L) were administered from the femoral veins of four rats, and carotid cannulation was conducted to collect blood samples, which were studied to determine whether corosolic acid in blood could be detected by analytical HPLC or not. The samples collected just before administration, 1 min, 5 min, 10 min, 30 min and 60 min after administration were centrifuged (10,000 RPM, 5 min, 4°C) to get each 150 ⁇ L of blood plasma, to which 4-fold amount of acetonitrile was added to obtain the suspension.
  • the suspension was deproteinated by centrifugation (10,000 RPM, 5 min, 4 0 C), and the supernatant thus obtained was analyzed by analytical HPLC.
  • Both the PG solution and the PEG solution provided a peak of corosolic acid that made an analysis with the calibration curve (methanol solution) possible. They also provided the approximately same change in blood level during the 60 min.
  • the group administered with the PEG solution would theoretically provide a blood level two times as much as the group administered with the PG solution, but the result was out of the expectation. This was considered to be due to an unsaturated suspension administered or an individual difference existing among rats.
  • the solubility of corosolic acid in the pH 6 and 5 buffers was studied.
  • the pH 6 and 5 buffers were prepared, 1.0 mg of corosolic acid was suspended in 20 mL of each of the buffers, and the suspension was shaken for about half a day. Afterward, the suspension was centrifuged by a centrifugal separator (5,000 RPM, 5 min, 25°C) to get a supernatant, which was then extracted with 20 mL of ethyl acetate. The organic phase was separated and concentrated. The concentrate was dissolved in 800 ⁇ L of the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC.
  • the suspension before the centrifugation was also studied to determine the corosolic acid concentration by analytical HPLC. No corosolic acid was detected in the suspension before the centrifugation. But the concentrates from the pH 6 and 5 suspensions provided a peak of corosolic acid to that made an analysis with the calibration curve possible. Thus, corosolic acid was suspended in about 30-50 mL of each of the pH 4 and 3 buffers to conduct analytical HPLC (as described later). The DMSO solution of corosolic acid (8 mg/mL) was prepared.
  • This solution (100 ⁇ L and 200 ⁇ L) was administered to four rats via the femoral veins, and carotid cannulation was conducted to collect blood samples, which were studied to determine whether corosolic acid in blood could be detected by analytical HPLC or not.
  • the samples collected just before administration, 1 min, 5 min, 10 min, 30 min and 60 min after administration were centrifuged (15,000 RPM, 5 min, 4°C) to get each 150 ⁇ L of blood plasma, to which 4-fold amount of acetonitrile was added to obtain the suspension.
  • the suspension was deproteinated by centrifugation (15,000 RPM, 5 min, 4°C), and the supernatant thus obtained was analyzed by analytical HPLC.
  • the suspension was deproteinated by centrifugation (15,000 RPM, 5 min, 4°C).
  • the corosolic acid concentration was studied by analytical HPLC. A peak of corosolic acid that made an analysis with the calibration curve possible was provided, and was relatively stable.
  • the corosolic acid concentration was 0.035 mg/mL, and it was demonstrated that corosolic acid could dissolve in blood (pH 7.4) several times as much as in the pH 7 buffer. This suggested that corosolic acid formed a protein binding in blood.
  • a sufficient amount of corosolic acid is considered to dissolve in the blood (2,400 mL) of a human (60 kg, adult).
  • the solubility of corosolic acid in ethanol is high, but the toxicity of ethanol is not negligible.
  • PG/PEG ratio volume ratio
  • solubility parameter affinity to water
  • the solubility of corosolic acid in the pH 4 buffer was studied.
  • the pH 4 buffer was prepared, 1.0 mg of corosolic acid was suspended in 30 mL of the buffer, and the suspension was shaken for about half a day.
  • the suspension was centrifuged by a centrifugal separator (5,000 RPM, 5 min, 25 0 C) to get a supernatant, which was then extracted with 30 rriL of ethyl acetate.
  • the organic phase was separated and concentrated.
  • the concentrate was dissolved in 800 ⁇ L of the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC.
  • the suspension before the centrifugation was also studied to determine the corosolic acid concentration by analytical HPLC.
  • the solubility of corosolic acid in the pH 4 and 3 buffers was studied.
  • the pH 4 and 3 buffers were prepared, 1.0 mg of corosolic acid was suspended in 30 mL of each of the buffers, and the suspension was shaken for about half a day. Afterward, the suspension was centrifuged by a centrifugal separator (5,000 RPM, 5 min, 25 0 C) to separate a supernatant, which was then extracted with 30 mL of ethyl acetate. The organic phase was separated and concentrated. The concentrate was dissolved in 800 ⁇ L of the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC. Corosolic acid could be detected in the concentrate derived from the pH 4 suspension, but not detected in the concentrate derived from the pH 3 suspension. It was demonstrated that improvement of HPLC detection sensitivity needed increasing amount of the buffer or ethyl acetate.
  • 5% aqueous ⁇ -cyclodextrin solution and 5% aqueous ⁇ -cyclodextrin solution were prepared, 1 mg of corosolic acid was suspended in 1 mL of each of these solutions, and the suspension was shaken for about half a day by a stirrer. The suspension was centrifuged (15,000 RPM, 5 min, 25°C) by a centrifugal separator to separate a supernatant, which was studied to determine the corosolic acid concentration by analytical HPLC. The aqueous ⁇ -cyclodextrin solution provided no corosolic acid to detect, but the aqueous ⁇ -cyclodextrin solution provided a peak of corosolic acid that made an analysis with the calibration curve possible.
  • the solubility of corosolic acid in the pH 4 buffer was studied.
  • the pH 4 buffer was prepared, 1.0 mg of corosolic acid was suspended in 50 mL of the buffer, and the suspension was shaken for about half a day.
  • the suspension was centrifuged by a centrifugal separator (5,000 RPM, 5 min, 25°C) to separate a supernatant, which was then extracted with 50 mL of ethyl acetate.
  • the organic phase was separated and concentrated.
  • the concentrate was dissolved in 800 ⁇ L of the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC. 1 mg of corosolic acid was added to 100 ⁇ L of a PG 80%/MCG 20% mixed solvent. The mixture obtained was shaken, and then left to stand for about half a day.
  • the resultant product was centrifuged (15,000 RPM, 5 min, 25 0 C) by a centrifugal separator to separate a supernatant, which was then diluted by 1, 000-fold with the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC.
  • the solubility of corosolic acid in this mixed solvent was not found to be improved in comparison with that in PQ PEG or MCG alone, as found in the PG /PEG mixed solvent. It was revealed that a PG/MCG mixed solvent system could not be expected to improve the solubility to such an extent that it might be applied to goods (a drug).
  • 1% aqueous ⁇ -cyclodextrin solution was prepared, 1 mg of corosolic acid was suspended in 1 mL of the solution, and the suspension was shaken for about half a day by a stirrer. The suspension was centrifuged (15,000 RPM, 5 min, 25 0 C) by a centrifugal separator to separate a supernatant, which was studied to determine the corosolic acid concentration by analytical
  • the suspensions were centrifuged by a centrifugal separator (5,000 RPM, 5 min, 25 0 C) to separate supernatants, which were then extracted with 30 mL and 50 mL of ethyl acetate respectively.
  • the organic phases were separated and concentrated.
  • Each of the concentrates was dissolved in 800 ⁇ L of the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC.
  • Corosolic acid could be detected in any of the concentrates, but they provided no peak of corosolic acid that made an analysis with the calibration curve possible. But a plot graph (ordinate: logC tota i; abscissa: pH) depicted from data of the solubility in the pH 10-6 buffers gave stable data. 1 mg of corosolic acid was added to 100 ⁇ L of a PG 40%/PEG
  • STZ streptozotocin
  • a 0.5% CMC (carboxymethylcellulose) solution control
  • a suspension of corosolic acid 5 mg in a 0.5% CMC solution 1 mL was administered to another rat in the same way.
  • Carotid cannulation was conducted just before administration, 30 min, 60 min, 90 min, 120 min and 180 min after administration to collect blood samples.
  • the blood samples were centrifuged (15,000 RPM, 5 min, 4°C) by a centrifugal separator to separate 20 uL of blood plasma.
  • CMC solution (control) was administered to one rat forcibly once by a disposable injection syringe and a rat oral probe (2 mL/kg), and the suspensions obtained by suspending each 5 mg of corosolic acid, ursolic acid and oleanolic acid in a 0.5% CMC solution 1 mL were administered to the other three rats in the same way.
  • Carotid cannulation was conducted just before administration, 30 min, 60 min, 90 min, 120 min and 180 min after administration to collect blood samples.
  • the blood samples were centrifuged (15,000 RPM, 5 min, 4°C) by a centrifugal separator to separate 20 ⁇ L of blood plasma.
  • Glucose CII-test Wako (Wako Pure Chemical Industries, Ltd.) was used to determine blood glucose level by the Mutarose GOD (glucose oxidase) method, while the absorbance was determined by a spectrophotometer (505 nm). Concentration was calculated using the calibration curve obtained by analyzing standard solutions, resulting in a pre-value of 300-500 mg/dL with a remarkable dispersion.
  • the control, the corosolic acid suspension, and the oleanolic acid suspension provided a continuous rise in blood glucose level. This was considered to be due to a continuously given stimulation because of the operation.
  • the rat administered with the ursolic acid suspension exhibited a fall in blood glucose level since 90 min after administration. This was considered to be because of the fact that the rat could maintain the respiratory tract by a tube to suffer from less stimulation although it had fallen in dyspnea until then.
  • pH 10 and 9 buffers glycine buffer (pH 9.98, ionic strength 0.1) and borate buffer (pH 9.02, ionic strength 0.1)
  • pH 10 and 9 buffers glycine buffer (pH 9.98, ionic strength 0.1) and borate buffer (pH 9.02, ionic strength 0.1)
  • 0.5 g of every extract powder was added to 20 mL of every buffer. Then the resultant product was shaken for 1 min by voltex, and left to stand for 30 min. The suspension image was recorded by a digital camera.
  • PBA was confirmed to give clearly a deposit in either of the pH 10 and 9 buffers.
  • the solubility of corosolic acid was observed to be higher in an alkaline side (pH 10).
  • M and PBW were observed to have a higher solubility than PBA, but were confirmed to give a deposit by careful observation in both pH 10 and 9 buffers. It was revealed that the solubility in a more acidic side was hard to study unless the
  • the solubility of corosolic acid in the pH 3 buffer was studied.
  • the pH 3 buffer was prepared, 15 mg of corosolic acid was suspended in 300 mL of the buffer, and the suspension was shaken for about half a day.
  • the suspension was centrifuged by a centrifugal separator (5,000 RPM, 5 min, 25°C) to get a supernatant, which was then extracted with 300 mL of ethyl acetate.
  • the organic phase was separated and concentrated.
  • the concentrate was dissolved in 800 ⁇ L of the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC.
  • the concentrate provided a peak of corosolic acid that made an analysis with a calibration curve possible. It was judged that the data thus obtained was reliable data, because it was coincident with the plot graph (ordinate: logQ ota i; abscissa: pH).
  • Corosolic acid was administered to a beagle dog raised for an experiment, and the corosolic acid concentration in serum was determined by analytical HPLC. Blood samples were collected just before administration (blank), 30 min, 60 min, 90 min, 120 min and 180 min after administration. Every 200 ⁇ L from 2 mL of the sample (serum) at every time point was transferred into a microtube. Every sample thus separated was supplemented with 4-fold amount of acetonitrile (800 ⁇ L) and deproteinated.
  • the resultant product was centrifuged (15,000 RPM, 5 min, 4°C) by a centrifugal separator to separate a supernatant, which was then gathered in a sample bottle for every time point and concentrated completely.
  • the concentrate was dissolved in 500 ⁇ L of the eluent (85% MeOH/0.05% TFA) to confirm by analytical HPLC whether the concentrate could provide a peak of the corosolic acid under the present HPLC condition (standard solution was a corosolic acid methanol solution) or not (whether the corosolic acid concentration could be determined or not).
  • standard solution was a corosolic acid methanol solution
  • the present invention provides a novel antidiabetic drug which has both properties of a biguanide-based drug that has a gluconeogenesis inhibiting action and a hyperglycemia inhibiting action, and a nateglinide-based drug that has an early insulin secretion stimulating action, and which has lowered side effects in comparison with these drugs.
  • the present invention allows use of corosolic acid (an example of a triterpene), analogous compounds of corosolic acid, and pharmaceutically acceptable salts thereof as drugs or food and drink additives. Furthermore, the present invention specifies the sites, functional groups, and structures of triterpenes relating to their activity to facilitate discovery or synthesis of novel useful compounds and development of more effective and safer medicaments.

Landscapes

  • Health & Medical Sciences (AREA)
  • Diabetes (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Emergency Medicine (AREA)
  • Epidemiology (AREA)
  • Endocrinology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Steroid Compounds (AREA)

Abstract

The present invention provides a gluconeogenesis inhibiting agent containing as an active ingredient corosolic acid, an analogous compound of corosolic acid, or a pharmaceutically acceptable salt thereof.

Description

DESCRIPTION
GLUCONEOGENESIS INHIBITING AGENT Technical Field
The present invention relates to a gluconeogenesis inhibiting agent. Background Art
It is known that hot-water extraction or alcohol extraction of dried
Banaba leaf provides a banana extract which contains a certain amount of corosolic acid and has a hyperglicemia inhibiting action or a blood glucose depressant action (Japanese Patent Application Laid-Open No. 2000-169384).
Disclosure of the Invention
Terpenes such as corosolic acid are widely contained in plants. However, it costs a great deal to identify terpenes that can work as active ingredients and to put them to good use. This is because of too many kinds of terpenes, and because, with respect to each kind of terpene, isolation, purification, molecular level experiments, non-clinical tests and clinical tests must be followed. On the other hand, if the sites, functional groups and confϊgurational structures relating to activities of terpenes are specified, it will facilitate terpenes to be put to good use. The present inventors found that corosolic acid and analogous compounds thereof (triterpenes similar to corosolic acid in structure-activity correlation) can inhibit gluconeogenesis, and thus completed the present invention.
The present invention provides a gluconeogenesis inhibiting agent which contains as an active ingredient corosolic acid, an analogous compound of corosolic acid, or a pharmaceutically acceptable salt thereof. The above-mentioned glucogenesis inhibiting agent has the following characteristics:
(1) it remarkably inhibits gluconeogenesis during fasting;
(2) it remarkably inhibits gluconeogenesis 90-180 minutes after meal; (3) it increases insurin sensitivity;
(4) it has a remarkably reduced incidence of side effects in liver (hepatopathy); and
(5) it is particularly suitable for oral administration.
Such a glucogenesis inhibiting agent can inhibit gluconeogenesis to suppress blood glucose elevation. The gluconeogenesis inhibiting agent of the present invention exhibits a remarkable reduction in side effects such as hypoglycemia and hepatopathy (the administration of corosolic acid at 1,000 mg/kg is confirmed to give no side effect). Furthermore, the gluconeogenesis inhibiting agent of the present invention can suppress blood glucose elevation to prevent hypertension, obesity, hyperlipidemia
(hypercholesteremia and hypertriglycemia), arteriosclerosis and the like.
As preferred examples of the analogous compound of corosolic acid, there may be mentioned maslinic acid, tormentic acid, ursolic acid, asiatic acid, oleanolic acid, and 2α,19α-dihydroxy-3-oxo-urs-12-en-28-oic acid. These compounds have the same backbone (basic skeleton) as corosolic acid, and show a remarkable gluconeogenesis inhibiting action and a hyperglicemia inhibiting action similarly to corosolic acid.
The gluconeogenesis inhibiting agent of the present invention is preferably administered to a subject that has a blood glucose level beyond the normal value, that is, satisfies the following condition (i) or (ii):
(i) a fasting blood glucose level of 110 mg/dL or more; (ii) an OGTT (oral glucose tolerance test) value at 2 hours of 140 mg/dL or more.
Brief Description of the Drawings
Fig. 1 is graphs showing the changes in glucose production in rat liver in relation to time.
Fig. 2 is a mass spectrum obtained by analyzing a corosolic acid standard solution.
Fig. 3 is a calibration curve derived from chromatograms of corosolic acid standard solutions. Fig. 4 is a calibration curve derived from chromatograms of blood samples supplemented with corosolic acid.
Fig. 5 is chromatograms of a corosolic acid standard solution, and of blood samples taken from a dog administered orally with corosolic acid.
Fig. 6 is graphs showing the changes in any time blood glucose levels of GK rats in relation to time.
Fig. 7 is graphs showing the changes in blood glucose levels of GK rats in an OGTT in relation to time.
Fig. 8 is graphs showing the changes in body weight of Zucker fatty rats (fa/fa) in relation to time. Fig. 9 is graphs showing the changes in blood glucose levels of subjects in relation to time.
Fig. 10 is graphs showing the changes in blood insulin levels of subjects in relation to time.
Fig. 11 is graphs showing the changes in blood insulin levels of subjects in relation to time.
Fig. 12 is graphs showing the changes in blood insulin levels of subjects in relation to time.
Best Modes for Carrying Out the Invention
The gluconeogenesis inhibiting agent of the present invention contains as active ingredients corosolic acid, analogous compounds of corosolic acid, or pharmaceutically acceptable salts thereof. Analogous compounds of corosolic acid mean triterpenes which have a structure-activity correlation similar to that of corosolic acid.
Corosolic acid is a triterpene (C30H48O4; MW 472) represented by the following formula.
Figure imgf000005_0001
Preferred examples of the analogous compound of corosolic acid include maslinic acid, tormentic acid, ursolic acid, asiatic acid, oleanolic acid and 2α,19α-dihydroxy-3-oxo-urs-12-en-28-oic acid. Triterpenes preferably have at least one hydroxyl at the 2- and 3- positions thereof, and more preferably, have further at least one hydroxyl at the 29- and 30- positions and carboxyl at the 28- position thereof. Such triterpenes include ursane type pentacyclic triterpenes and oleanane type pentacyclic triterpenes.
The following compounds 1) - 40) are examples of the ursane type pentacyclic triterpene: 1) Desfontainic acid
2) 2,19α-Dihydroxy-3-oxo-l,12-ursadien-28-oic acid
3) 2x,20β-Dihydroxy-3-oxo-12-ursen-28-oic acid 4) 2α,3α-Dihydroxy-12,20(30)-ursadien-28-oic acid
5) 2α,3β-Dihydroxy-12,20(30)-ursadien-28-oic acid
6) 2β,3β-Dihydroxy-12-ιιrsen-23-oic acid
7) 2α,3α-Dihydroxy-12-ursen-28-oic acid 8) lα,2α,3β,19α,23-Pentahydroxy-12-ursen-28-oic acid
9) 2α,3β,7α,19α,23-Pentahydroxy-12-ursen-28-oic acid
10) lβ,2α,3α,19α-Tetrahydroxy-12-ursen-28-oic acid
11) 1 β,2α,3β,19α-Tetrahydroxy-12-ursen-28-oic acid
12) lβ,2β,3β,19α-Tetrahydroxy-12-ursen-28-oic acid 13) 2α,3β,6β,19α-Tetrahydroxy-12-ursen-28-oic acid
14) 2α,3β,6β,23-Tetτahydroxy-12-ursen-28-oic acid
15) 2α,3β,7α,19α-Tetrahydroxy-12-ursen-28-oic acid
16) 2α,3α,7β,19α-Tetrahydroxy-12-ursen-28-oic acid
17) 2α,3β,13β,23-Tetrahydroxy-ll-ursen-28-oic acid 18) 2α,3α,19α,23-Tetrahydroxy-12-ursen-28-oic acid
19) 2α,3β,19α,23-Tetrahydroxy-12-ursen-28-oic acid
20) 2α,3α,19α,24-Tetrahydroxy-12-ursen-28-oic acid
21) 2α,3β,19α,24-Tetrahydroxy-12-ursen-28-oic acid
22) 2α,3β,23-Trihydroxy-ll-oxo-12-ursen-28-oic acid 23) 2α,3β,24-Trihydroxy-12,20(30)-ursadien-28-oic acid
24) 2α,3β,27-Trihydroxy-28-ursanoic acid
25) 2α,3β,19α-Trihydroxy-12-ursene-23,28-dioic acid
26) 2α,3β,19α-Trihydroxy-12-ursene-24,28-dioic acid
27) lβ,2β,3β-Trihydroxy-12-ursen-23-oic acid 28) 2α,3β,6β-Trihydroxy-12-ursen-28-oic acid
29) 2α,3α,19α-Trihydroxy-12-ursen-28-oic acid 30) 2α,3β,19α-Triliydroxy-12-ursen-28-oic acid
31) 2α,3α,23-Trihydroxy-12-ursen-28-oic acid
32) 2α,3β,23-Trihydroxy-12-ursen-28-oic acid
33) 2α,3α,24-Trihydroxy-12-ursen-28-oic acid 34) 2α,3β,24-Trihydroxy-12-ursen-28-oic acid
35) 2α,3β,27-Ursanetriol
36) 12-Ursene- 1 β,2α,3β, 11 α,20β-pentol
37) 12-Ursene-lβ,2α,3β,Hα-tetrol
38) 12-Ursene-2α,3β,l lα,20β-tetrol 39) 12-Ursene-2α,3β,llα-triol
40) 12-Ursene-2α,3β,28-triol
The following compounds 41) - 100) are examples of the oleanane type pentacyclic triterpene:
41) 2α,3β-Dihydroxy-12,18-oleanadiene-24,28-dioic acid 42) 2α,3β-Dihydroxy-12-oleanene-23,28-dioic acid
43) 2β,3β-Dihydroxy-12-oleanene-23,28-dioic acid
44) 2β,3β-Dihydroxy-12-oleanene-28,30-dioic acid
45) 2β,3β-Dihydroxy-12-oleanen-23-oic acid
46) 2β,3β-Dihydroxy-12-oleanen-28-oic acid 47) 2α,3α-Dihydroxy-12-oleanen-28-oic acid
48) 2α,3β-Dihydroxy-12-oleanen-28-oic acid
49) 2α,3β-Dihydroxy-13(18)-oleanen-28-oic acid
50) 12β,13β-Epoxy-2α,3β,21β,22β-tetrahydroxy-30-oleananoic acid
51) 13,28-Epoxy-2α,3β,16α,22β-tetrahydroxy-30-oleananoic acid 52) 13β,28-Eρoxy-2α,3β,16α,22β-tetrahydroxy-30-oleananoic acid
53) 12-Oleanene-2α,3α-diol 54) 12-Oleanene-2α,3β-diol
55) 13(18)-Oleanene-2α,3α-diol
56) 13(18)-Oleanene-2β,3β-diol
57) 18-Oleanene-2α,3β-diol 58) 18-Oleanene-2α,3α-diol
59) 12-Oleanene-2α,3 β, 16β,21 β,22α,28-hexol
60) 12-Oleanene- 1 β,2α,3 β, 11 α-tetrol
61) 12-Oleanene-2β,3β,23,28-tetrol
62) 12-Oleanene-2α,3β,llα-triol 63) 12-Oleanene-2β,3β,28-triol
64) 12- Oleanene-2α,3β,23-triol
65) 13(18)-Oleanene-2α,3β,llα-triol
66) 2β,3β,6β,16α,23-Pentahydroxy-12-oleanen-28-oic acid
67) 2β,3β,16β,21β,23-Pentahydroxy-12-oleanen-28-oic acid 68) 2β,3β,16α,23,24-Pentahydroxy-12-oleanen-28-oic acid
69) 2β,3β,13β,16α-Tetrahydroxy-23,28-oleananedioic acid
70) 2β,3β,16β,21β-Tetrahydroxy-12-oleanen-24,28-dioic acid
71) 2β,3β,16α,23-Tetrahydroxy-12-oleanen-24,28-dioic acid
72) 2β,3β,22β,27-Tetrahydroxy-12-oleanen-23,28-dioic acid 73) 2α,3β,6β,23-Tetrahydroxy-12-oleanen-28-oic acid
74) 2β,3β,6α,23-Tetrahydroxy-12-oleanen-28-oic acid
75) 2β,3β,6β,23-Tetrahydroxy-12-oleanen-28-oic acid
76) 2β,3β,16β,21β-Tetrahydroxy-12-oleanen-28-oic acid
77) 2β,3β,16α,23-Tetrahydroxy-12-oleanen-28-oic acid 78) 2α,3β,19α,23-Tetrahydroxy-12-oleanen-28-oic acid
79) 2α,3β,19β,23-Tetrahydroxy-12-oleanen-28-oic acid 80) 2α,3β,19α,24-Tetrahydroxy-12-oleanen-28-oic acid
81) 2α,3β,21β,23-Tetrahydroxy-12-oleanen-28-oic acid
82) 2α,3β,23,24-Tetrahydroxy-12-oleanen-28-oic acid
83) 2β,3β,23-Trihydroxy-5,12-oleanadien-28-oic acid 84) 2α,3α,24-Triliydroxy-ll,13(18)-oleanadien-28-oic acid
85) 2α,3β,13β-Trihydroxy-28-oleananoic acid
86) 2β,3β,16α-Trihydroxy-12-oleanene-23,28-dioic acid
87) 2α,3β,18β-Trihydroxy-12-oleanene-23,28-dioic acid
88) 2α,3β,19α-Trihydroxy-12-oleanene-23,28-dioic acid 89) 2α,3β,19β-Trihydroxy-12-oleanene-23,28-dioic acid
90) 2α,3β,19α-Trihydroxy-12-oleanene-24,28-dioic acid
91) 2α,3β,19β-Trihydroxy-12-oleanene-24,28-dioic acid
92) 2β,3β,23-Trihydroxy-12-oleanene-28,30-dioic acid
93) 2β,3β,27-Trihydroxy-12-oleanene-23,28-dioic acid 94) 2α,3β,18β-Trihydroxy-12-oleanen-28-oic acid
95) 2α,3β,19α-Trihydroxy-12-oleanen-28-oic acid
96) 2α,3β,19α-Trihydroxy-12-oleanen-29-oic acid
97) 2α,3β,21β-Trihydroxy-12-oleanen-28-oic acid
98) 2α,3α,23-Trihydroxy-12-oleanen-28-oic acid 99) 2α,3α,24-Trihydroxy-12-oleanen-28-oic acid
100) 2α,3β,30-Trihydroxy-12-oleanen-28-oic acid
Furthermore, as the analogous compound of corosolic acid, the ether products (including glycosides) or the ketone products of corosolic acid, maslinic acid and the like may be mentioned. For example, corosolic acid can be converted to the ether product by reacting the hydroxyl group of corosolic acid with a halogenated alkyl (such as CH3Br and CH3(CH2)nBr). Corosolic acid also can be converted to the ketone product by the oxidation of one or both of hydroxyl groups at the 2- and 3- positions.
Examples of the pharmaceutically acceptable salt of corosolic acid or the analogous compound thereof include an alkali metal salt, an alkali earth metal salt, and an ammonium salt. Specifically, a salt formed between corosolic acid or an analogous compound thereof and sodium, potassium, calcium, magnesium, ammonia, dimethylamine, diethylamine, trimethylamine, tetramethylammonium, monoethanol amine, diethanol amine, or triethanol amine is preferred. (Extraction and purification of triterpene)
Triterpene, an active ingredient of the gluconeogenesis inhibiting agent, can be obtained by extracting from banaba (Lagerstroemia Speciosa, Linn, or Pers.), loquat, soapberry (sapindus mukurossi), perilla or guava, and it is preferably by extracting from banaba leaf. In order to obtain a triterpene from banaba leaf, the banaba leaf may be subjected to extraction with water or alcohol to get a banaba extract. The banaba extract may be further concentrated if necessary.
The banaba leaf may be optionally cut into pieces for extraction, but is preferably cut to improve extraction efficiency. The piece has a size which is adjusted depending on equipment used for the succeeding extraction process, but has preferably a size of 1 mm square or larger. A fine size that does not lead to clogging is most preferable. The banaba leaf may be cut into a similar size of strips.
When banaba leaf is immersed in water, 1 kg of banaba leaf is preferably immersed in 2 L or more of water at room temperature or 10-600C for 24 hours or more. The leaf is three times or more immersed in water. while the water is refreshed every immersion. Then, the leaf is three to five times subjected to hot water extraction with hot water, by boiling, and with steam, while the water is also refreshed every time. Furthermore, the leaf is pressed, extracted with hot water, and finally extracted with a solvent of water 50%/alcohol 50%, preferably at 0-1000C.
The leaf is pressed under a pressure of 1-3 arm, at an inner temperature of 1000C or more, and most preferably for an extraction time of about 3-10 min. If an inner pressure is beyond 1.5 atm, an extraction time is shortened to 1-5 min, wherein ideally the leaf is at first extracted at 1.1-1.3 atm for 2 min, then twice at 1.3-1.6 atm. A long time extraction should be avoided to prevent the triterpene from blowing out. Such a condition is also necessary because what kinds of tannins and chlorophylls will blow out varies depending on pressure, solvent, time and temperature. For the same reason, a mixed solvent of water and alcohol is finally used for extraction. The banaba leaf thus extracted is filtered while removing water, and dried in the sun or by a drier. Water is preferably removed by centrifugation, followed by drying. The leaf is preferably dried with warm air at 30-600C or cold air, more preferably with air having as low humidity as possible. The dried banaba leaf is extracted with hexane or a mixed solvent of hexane and water. For pretreatment, a resin or an active charcoal is preferably used to remove tannins or chlorophylls. Finally, ethanol or an aqueous ethanol solution (ethanol content: 90% or more) is used for extraction.
In the consecutive processes, the leaf is preferably subjected to hot water extraction with steam, pressure in a pressure vessel, and dewatering by a centrifugal separator. If an inner pressure is beyond 1.5 atm, a pressure time is preferably kept within 10 min. If hot air has a temperature of beyond 1000C, a drying time is kept within 10 min.
Concentration after extraction is preferably done for a relatively short time under a reduced pressure, because the concentrated product may be kept at a high temperature for a long time to deteriorate an active ingredient. The extract liquid obtained by the aforementioned manner is filtered, concentrated at a temperature of 600C or less under a reduced pressure to give a solid, which is then dried at a temperature of 50-700C under a reduced pressure (higher than that at the concentration). A solid thus obtained can be pulverized to give a powdery concentrate. As ingredients contained in the banaba extract (white extract) or the concentrate thereof thus obtained, there may be mentioned corosolic acid, maslinic acid, tormentic acid, ursolic acid, oleanolic acid, α-amirinic acid, β-amirinic acid, asiatic acid, 18β-glycyrrhetic acid, tannins, chlorophylls and hemicellulose. Corosolic acid is contained by 3-50% or more. The banaba extract or the concentrate thereof may be in a liquid or solid state, or in paste form. The banaba extract or the concentrate thereof is preferably stored at room temperature or in a refrigerator to be shielded from light and desiccated.
Triterpenes may be extracted and concentrated in the following manner. The dried banaba leaf is firstly heated under reflux to extract in n-hexane for 1 hour, in purified water for 1 hour. Then, decocted banaba leaf is heated under reflux in ethanol for 1 hour, followed by filtering the extract, which is then concentrated under a reduced pressure to get a solid.
The banaba extract or the concentrate thereof thus obtained may be used as it is as the gluconeogenesis inhibiting agent, but is preferably purified as far as possible from tannins, chlorophylls and fibers to isolate the active ingredients (triterpenes). The isolation and purification may be done according to a known method. For example, corosolic acid may be isolated and purified by the following procedure.
The banaba extract is suspended in water, and at first partitioned in ether, hexane or the like to remove a low polar component. The aqueous layer is then successively eluted with water, methanol and acetone using Diaion HP-20 column chromatography or the like. The methanol eluate fraction containing corosolic acid is then subjected to separation and purification by silica gel column chromatography and high performance liquid chromatography (HPLC) to isolate the corosolic acid. Alternatively, the extract may be directly separated by silica gel column chromatography, and then purified by HPLC.
Corosolic acid may be isolated and purified in the following manner. The extract (the banaba extract or the banaba extract concentrate) is suspended in water, and solvent-fractionated with hexane or ether. After filtration by suction, among water soluble fractions, organic solvent soluble fractions and insoluble fractions, the insoluble fractions are selected to use. The insoluble fractions are applied to silica gel column chromatography using a mixed solvent of dichloromethane and methanol. Corosolic acid-containing fractions obtained by the silica gel column chromatography are applied to an ion-exchange resin, and HPLC is used to separate corosolic acid. The HPLC condition is as follows. Column: reversed phase ODS; eluent: 85% MeOH (methanol)/0.05% TFA (trifluoroacetic acid); flow rate: 6 mL/min; wavelength: 210 nm. The triterpenes isolated and purified from the banaba extract or the banaba extract concentrate may be used as they are, and may also be acylated (for example, acetylated) to use, or consecutively deacylated (for example, deacetylated) to use. For example, corosolic acid is preferably acylated (for example, acetylated), and then deacylated to use. Acylation (such as acetylation) followed by deacylation can give corosolic acid having a very high purity (approximately 100%).
For acetylation of corosolic acid, for example, corosolic acid isolated and purified from the banaba extract is dissolved in absolute pyridine, acetic anhydride is added thereto, and the mixture was left to stand at room temperature for about 12 hours. The reaction solution is supplemented with ice water, and extracted a few times (about three times) with chloroform. Next, the chloroform layer is dewatered with sodium sulfate, filtered to remove sodium sulfate, and distilled under a reduced pressure to evaporate chloroform. The resultant product can be recrystallized from hexane to give acetyl corosolic acid. As an example of a method of deacylation, a method wherein hydrolysis is performed with an alkali such as potassium hydroxide or sodium hydroxide may be mentioned. (Analysis of pharmacokinetics for triterpene)
The understanding of pharmacokinetics, that is, the absorption, distribution, metabolism and excretion of a drug in body needs the determination of blood level and urine level of the drug in relation to time.
The blood level and the urine level of a triterpene can be determined by LC/MS/MS (liquid chromatography/mass spectrometry/mass spectrometry). A HPLC apparatus is used for LC, and a quadruple mass spectrometer is used for MS. An ESI (electrospray ionization) method is preferable for ionization in LC/MS/MS.
For example, the microanalysis of corosolic acid in blood by LC/MS/MS is conducted in the following manner. At first, the standard solution of corosolic acid is analyzed by LC/MS/MS to get the mass spectrum, and thereby to determine whether corosolic acid can be analyzed by LC/MS/MS or not. Then, a calibration curve is depicted from chromatograms for Standard solutions of corosolic acid to determine whether concentration of corosolic acid can be measured or not. Then, a blood sample supplemented with corosolic acid is pretreated (for deproteination, desalting and extraction), and an HPLC condition is so established that only the corosolic acid in the extract may be eluted at a certain time point. Pretreated blood samples are analyzed under the HPLC condition to get chromatograms, from which a calibration curve is then depicted to determine whether the blood level of corosolic acid can be measured or not. Finally, an analyte sample (blood) is pretreated as described above, and applied to LC/MS/MS under the established HPLC condition to determine the corosolic acid level in the sample.
(A method for using the gluconeogenesis inhibiting agent)
The gluconeogenesis inhibiting agent containing a triterpene as an active ingredient can be used as a drug, an additive for food and drink, or a smoking material (such as tobacco). The agent may be used in a solid, liquid or gas state, or in paste form, and may be made into a mist when used.
A triterpene powder isolated and purified can be pulverized by a nanoparticle pulverizer to get the finer powder. For example, the corosolic acid powder isolated and purified by HPLC has a particle size of about 10 μm, but the fine powder having a size of 10 nm - 100 nm can be obtained if pulverized by a nanoparticle pulverizer. The particle pulverized to a nano-size can provide a higher effect even if used in a less amount. In order to prepare a triterpene solution, a solvent is adequately selected depending on the fat solubility or water solubility of the triterpene.
For example, an oil and fat (docosahexaenic acid) or an alcohol is adequate as a solvent for corosolic acid. A triterpene may be glycosidated to increase water solubility, if it has a low water solubility.
When used as a drug, the triterpene may be used in the form of powder, granule, tablet, capsule, injection or the like. The granule or the tablet can be prepared by mixing powdery corosolic acid or the banaba extract concentrate with excipients such as lactose and starch. The triterpene may be used together with digestion resistant dextrin or a sucrase inhibitor (such as L-arabinose or 1-deoxynojirimycine). Because the digestion resistant dextrin or a sucrase inhibitor inhibits absorption and digestion of glucose, a combination of the digestion resistant dextrin or a sucrase inhibitor with the gluconeogenesis inhibiting agent and sucrose provides a sweetener having a blood glucose depressant action. The gluconeogenesis inhibiting agent can be used alone or in the form of a sweetener to add to drinks such as water, cooling drink, fruit juice, milk drink and alcoholic drink, as well as foods such as breads, noodles, rices, soybean curd, dairy product, soy sauce, miso (soybean paste) and cakes, as an additive for drink and food.
The gluconeogenesis inhibiting agent is preferably administered orally and intravenously, and particularly preferably orally.
As a preferred subject to whom the gluconeogenesis inhibiting agent is administered, there may be mentioned a person having a blood glucose level beyond the normal value (fasting blood glucose level: less than 110 mg/dL; OGTT value at 2 hours: less than 140 mg/dL). When administered to animals other than humans, the gluconeogenesis inhibiting agent may be mixed to a feed to be ingested with it.
(Examples)
The present invention will now be explained in greater detail through examples below, but not limited by these examples.
(Example 1 : Confirmation of gluconeogenesis inhibiting action of corosolic acid)
The effect of corosolic acid on the gluconeogenesis in liver was confirmed by the following experiment. Wistar male rats (body weight about 200 g) after fasting for 24 hours were anesthetized with pentbarbital to carry out liver perfusion experiment. A 370C perfusate supplemented with oxygen was perfused from portal vein at a rate of 10 mL/min and recovered at inferior vena cava. At first, the perfusate was previously perfused for 20 min, and then supplemented with 2 mM lactic acid. After 30 min, a solution (200 μM) of corosolic acid in
DMSO (dimethyl sulfoxide) was added into the perfusate for 10 min. On the other hand, DMSO without dissolved corosolic acid was perfused as the control.
Fig. 1 is graphs showing the changes in glucose production in rat liver in relation to time. As shown in Fig. 1, there was no glucose production in liver at the time point of previous perfusion, but the addition of 2 mM lactic acid in the perfusate swiftly caused gluconeogenesis, resulting in a glucose production of 8.3 μmol/g/h after 30 min. Afterward, the addition of 200 μM corosolic acid inhibited gluconeogenesis by 30%. On the other hand, in the case of no corosolic acid added, the glucose production did not change.
These results revealed that corosolic acid inhibited gluconeogenesis in liver. In Fig. 1, CRA represents corosolic acid.
(Example 2: Determination of corosolic acid level in blood)
Corosolic acid level in blood was determined by LC/MS/MS using an ESI method and a quadruple mass spectrometer. At first, corosolic acid standard solutions were analyzed by
LC/MS/MS.
Fig. 2 is a mass spectrum obtained by analyzing a corosolic acid standard solution. As shown in Fig. 2, an ion peak appeared at m/z value of 472, and hence it was revealed that corosolic acid (MW: 472) could be analyzed by LC/MS/MS .
Fig. 3 is a calibration curve depicted from chromatograms of corosolic acid standard solutions. Fig. 3 revealed that corosolic acid concentration could be determined at least within 1 ng/mL - 100 ng/mL.
Next, blood samples supplemented with corosolic acid were analyzed by LC/MS/MS.
For pretreatment, 500 μL of a blood sample supplemented with corosolic acid was centrifuged by a centrifugal separator (3,000 rpm, 20 min), and 100 μL of the blood serum thus obtained was supplemented with 400 μL of acetonitrile. Then the resultant product was stirred by vortex, and deproteinated by centrifugation (15,000 rpm, 10 min). The acetonitrile layer was concentrated by a nitrogen gas, and supplemented with 300 μL of ethyl acetate, and then liquid/liquid extraction was carried out. The ethyl acetate layer was concentrated and dried, which was then dissolved in 100 μL of a elution solvent (95% aqueous methanol solution) to provide a sample for LC/MS/MS. The HPLC condition was so established that only corosolic acid in the extract might be eluted independently at a certain time. Fig. 4 is a calibration curve depicted from chromatograms of the blood samples supplemented with corosolic acid. Fig. 4 revealed that corosolic acid concentration in blood could be determined at least within 10 ng/mL - 100 ng/mL. Finally, blood samples taken from a dog administered orally with corosolic acid were analyzed by LC/MS/MS. The dog that had fasted from the preceding day was administered orally with corosolic acid (20 mg/kg) and glucose (2 g/kg), and blood samples were collected just before administration, 30 min, 60 min, 90 min, 120 min, 180 min and 240 min after administration. These blood samples were pretreated in the same manner as described before to get samples for LC/MS/MS, which were then analyzed by LC/MS/MS in the same HPLC condition as described before.
Fig. 5 is chromatograms of a corosolic acid standard solution, and of the blood samples taken from a dog administered orally with corosolic acid. In Fig. 5, (a) is a chromatogram of a corosolic acid standard solution, and
(b)-(h) are chromatograms of the blood samples collected just before administration, 30 min, 60 min, 90 min, 120 min, 180 min and 240 min after administration, respectively. As shown in Fig. 5, since 90 min after administration, corosolic acid was detected in blood, and corosolic acid level in blood reached its maximum value (0.4 μg/mL) at the time point of 180 min after administration. In Fig. 5, CRA represents corosolic acid.
These results revealed that blood levels of terpenes including corosolic acid could be determined by LC/MS/MS. Further, it was demonstrated that corosolic acid could be absorbed as it is in body without being decomposed nor modified, and that corosolic acid could be administered orally. (Example 3 : Non-clinical test)
In order to analyze the biological reaction and drug effect of terpenes including corosolic acid, the following animal test was conducted.
GK rats, a model animal for Type II diabetes, and Zucker fatty rats (fa/fa), a model animal for insulin resistance, were employed as test animals.
Wistar rats and Zucker fatty rats (+/+) (aged 7 weeks) were employed as control. The animals were given feed with 0.0125% corosolic acid or without corosolic acid, and allowed to take it freely for about 2 weeks. Feed intake, water intake, body weight, and blood glucose level were determined. Glucose load in an OGTT was controlled to be 2 g/kg. Blood glucose levels were determined on the blood samples collected from animals.
GK rats are a model animal for insulin secretion failure specific to Asians. The animal suffers from diabetes due to insufficiency of insulin secretion. Zucker fatty rats are a model animal for insulin resistance, and oversecretes insulin. An individual having homologous pathogenic genes (fa genes) is expressed by Zucker fatty rat (fa/fa). Only this individual suffers from diabetes or falls in obesity. The wild type is expressed by Zucker fatty rat (+/+). This individual suffers from no diabetes and is free from obesity.
As an early symptom of diabetes, abnormal intake of water may be mentioned. Thus, feed intake and water intake per day were determined. The results are shown in Table 1 and Table 2. In Table 1 and Table 2, CRA represents corosolic acid. (Table 1)
Figure imgf000021_0001
(Table 2)
Figure imgf000021_0002
As shown in Table 1 and Table 2, in Wistar rats, GK rats and Zucker fatty rats (+/+), there was found no significant difference in feed intake and water intake between the corosolic acid administration group and the non-administration group. On the other hand, in Zucker fatty rats (fa/fa), the corosolic acid administration group showed a significantly larger water intake compared with the non-administration group. This suggests that corosolic acid and terpenes having the same structure-activity correlation have a diuretic action, a renal function improving action and an antihypertensive action.
Fig. 6 is graphs showing the changes in any time blood glucose levels of GK rats in relation to time. As shown in Fig. 6, the any time blood glucose level in the corosolic acid administration group was significantly inhibited since 8th day after administration of corosolic acid in comparison with the non-administration group.
Fig. 7 is graphs showing the changes in blood glucose levels of GK rats in an OGTT in relation to time. As shown in Fig. 7, in the OGTT on GK rats, the rise in blood glucose level of the corosolic acid administration group was significantly inhibited at the time point of 90 min after glucose load in comparison with the non-administration group.
Fig. 8 is graphs showing the changes in body weight of Zucker fatty rats (fa/fa) in relation to time. As shown in Fig. 8, in Zucker fatty rats (fa/fa), the body weight of the corosolic acid administration group was inhibited since 6th day after administration of corosolic acid in comparison with the non-administration group. This suggests that corosolic acid is also effective for a Westerner type of diabetes accompanied by obesity caused by insulin resistance. In Fig. 6, Fig. 7 and Fig. 8, the plot shown by "*" indicates data that had a significant difference found in comparison with the non-administration group. CRA represents corosolic acid.
No effect of corosolic acid was found on the any time blood glucose levels of Zucker fatty rats (fa/fa), but in the OGTT on Zucker fatty rats (fa/fa), the rise in blood glucose level of the corosolic acid administration group was significantly inhibited at the time point of 30 min after glucose load in comparison with the non-administration group.
(Example 4: Non-clinical test)
KK-Ay mice (aged 8 weeks, blood glucose level: 300 mg/100 mL), a model animal for Type II diabetes, were administered with various kinds of triterpenes to determine blood glucose levels in relation to time. The mice were given feed and water, and allowed to take it freely during the test.
Corosolic acid, asiatic acid,
2α,19α-dihydroxy-3-oxo-urs-12-en-28-oic acid and tormentic acid exhibited significant hypoglycemic effects. Maslinic acid did not exhibit a significant hypoglycemic effect, but exhibited a remarkable tendency toward reducing blood glucose level. These results revealed that corosolic acid and analogous compounds thereof had an inhibitory action against the rise in blood glucose level. (Example 5: Clinical test) Three patients (A, B and C) with borderline diabetes had fasted since
8 p.m. (no glucose loaded), and were administered with a placebo once or with a drug (corosolic acid) once at 9 a.m. of the following day to determine the succeeding change in blood glucose level and blood insulin level. Blood samples were collected just before administration (0 min), 30 min, 60 min, 90 min, 120 min and 180 min after administration. The subjects were provided with no information about whether a placebo or a drug was given (crossover, double blind). 10 mg of corosolic acid was administered once (single time), and tests were carried out twice for each of the placebo and the drug. The test was conducted after obtaining informed consent from each subject according to the Code of Ethics, Medical Jurisprudence.
The test results are shown in Table 3. In Table 3, PCB and CRA represent placebo and corosolic acid, respectively. G and I represent blood glucose level and blood insulin level, respectively. Time (min) shows time after administration. (Table 3)
Figure imgf000024_0001
Fig. 9 is graphs showing the changes in blood glucose levels of the subjects in relation to time. Fig. 10 is graphs showing the changes in blood insulin levels of the subjects in relation to time. In Fig. 9 and Fig. 10, CRA represents corosolic acid, and A, B and C in parentheses express names of the subjects.
As shown in Table 3, Fig. 9 and Fig. 10, if the subjects were loaded with no glucose, there were found no significant differences in blood glucose level and blood insulin level at any point of time between cases where they were administered with corosolic acid and with the placebo.
In an OGTT on the same three subjects as in the above test, when they were administered with corosolic acid, a rise in blood insulin level and inhibition of the rise in blood glucose level were found at the time point of 30 min after glucose load.
These results revealed that corosolic acid stimulated secretion of insulin depending on blood glucose. (Example 6: Clinical test)
A 75 g OGTT on eleven subjects was conducted to determine blood insulin level in relation to time. Afterward, the banaba extract containing corosolic acid was administered to the same eleven subjects continuously for
1 year, and another 75 g OGTT on them was conducted to determine blood insulin level in relation to time. Fig. 11 is graphs showing the changes in blood insulin levels (average) of the subjects in relation to time.
A 75 g OGTT on seven subjects was conducted having a fasting blood glucose level of 110 mg/dL or more to determine blood insulin level in relation to time. Afterward, the banaba extract containing corosolic acid was administered to the same seven subjects continuously for 1 year, and another 75 g OGTT on them was conducted to determine blood insulin level in relation to time. Fig. 12 is graphs showing the changes in blood insulin levels (average) of the subjects in relation to time.
As shown in Fig. 11 and Fig. 12, after ingesting the banaba extract continuously for 1 year, the blood insulin level significantly increased at the time point of 30 min after glucose load in comparison with the level before the ingesting. This result demonstrated that corosolic acid stimulated secretion of insulin depending on blood glucose.
(Example 7: Analysis of corosolic acid solution by analytical HPLC)
Many terpenes including corosolic acid have water-insoluble property. For example, corosolic acid is a colorless transparent needle crystal, and soluble in an organic solvent such as ethanol and acetonitrile, but hardly dissolves in water. Very low solubility in water is a large blockage against the analysis of biological reactions or pharmacokinetics of terpenes. Thus, the solubility of corosolic acid in various kinds of solvents was studied by a conventionally used analytical HPLC (temperature: 250C).
As a solvent, a buffer (ionic strength: 0.1), an organic solvent, and a mixed solution of surfactant/organic solvent were used. The pHs and solutes of the buffers used were as follows. pH 1 : HCl; pH 2: HCl; pH 3: citric acid; pH 4: citric acid; pH 5: citric acid; pH 6. citric acid; pH 7: phosphoric acid; pH 8: Tris; pH 9: boric acid; pH 10: glycine. The organic solvents used were PG (propylene glycol), PEG400 (polyethylene glycol
400), ethanol and soybean oil. The surfactants used were Tween 80 and BL9-EX. If the solubilities in the pH 1-10 buffers are determined, the pKa of corosolic acid can be obtained.
75%, 80%, and 85% MeOH/0.05% TFA were used as eluents (mobile phases) to confirm the retention time and the detecting wavelength of a solution of corosolic acid in methanol (1 mg/mL). Shodex Asahipak ODP-50 6D (50χ 10.0 mm K)) was used as a column. The flow rate was set to be 1.0 mL/min, and UV was used to detect corosolic acid. The retention times were about 30 min, about 20 min, and about 12 min, and 85%
MeOH/0.05% TFA was used to give the shortest retention time. A high peak of absorption appeared around a wavelength of 210 nm. Thus, the solubility of corosolic acid was determined under the condition of eluent solution: 85% MeOH/0.05% TFA; and wavelength: 210 nm.
Corosolic acid was suspended in water and centrifuged to get a supernatant, which was then studied to determine whether corosolic acid could be detected by analytical HPLC (85% MeOH/0.05% TFA) or not. No corosolic acid was detected.
In order to depict a calibration curve, various concentrations of corosolic acid solutions were prepared, and they were analyzed by analytical HPLC. The corosolic acid concentrations in the solutions were as follows: 0.1 mg/mL, 0.033 mg/mL, 0.01 mg/mL, 0.0033 mg/mL, 0.001 mg/mL, 0.00033 mg/mL and 0.0001 mg/mL. Corosolic acid could be detected in four kinds of solutions having the higher concentrations.
The solubility of corosolic acid in the pH 5-8 buffers was studied. The pH 5-8 buffers were prepared, 0.5 mg of corosolic acid was suspended in 10 mL of each of the buffers, and the suspension was shaken for about half a day. Afterward, the suspension was centrifuged by a centrifugal separator (5,000 RPM, 5 min, 25°C) to get a supernatant, which was then extracted with 10 mL of ethyl acetate. The organic layer was separated and concentrated. The concentrate was dissolved in 800 μL of the eluent (85% MeOH/0.05% TEA) to determine the corosolic acid concentration by analytical HPLC. The suspension before the centrifugation was studied to determine the corosolic acid concentration by analytical HPLC. No corosolic acid was detected in the suspension before the centrifugation. Corosolic acid was detected in the concentrates derived from the pH 6, 7 and 8 suspensions, but a peak area of corosolic acid varied, and a sample of apparently too high concentration was found. This was considered to be caused by contamination of the actual material (corosolic acid) when the supernatant was separated. It was revealed that the other suspensions than the pH 8 suspension brought no reliable data.
The organic solvent (PG1 PEG) was administered from rat's femoral vein to confirm a reaction (dose: PG 250 μL; PEG 500 μL, administration time: about 2 min). The animal did not still fall in respiratory pause 30 min after administration. This suggested that corosolic acid could become an active ingredient in an injection or infusion.
The solubility of corosolic acid in the pH 4-8 buffers was studied. The pH 4-8 buffers were prepared, 0.5 mg of corosolic acid was suspended in 10 mL of each of the buffers, and the suspension was shaken for about half a day. Afterward, the suspension was centrifiiged by a centrifugal separator
(5,000 RPM, 5 min, 25°C) to get a supernatant, which was then extracted with 10 mL of ethyl acetate. The organic phase was separated and concentrated. The concentrate was dissolved in 800 μL of the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC. The suspension before the centrifugation was also studied to determine the corosolic acid concentration by analytical HPLC. No corosolic acid was detected in the suspension before the centrifugation. Corosolic acid was detected in the concentrates derived from the pH 4, 7 and 8 suspensions, but it was revealed that suspensions of pH 6 or more acidity needs improvement of HPLC detection sensitivity by increasing the addition amount of a buffer or ethyl acetate, or using a halogenated solvent (such as dichloromethane) in place of ethyl acetate.
The PG solution of corosolic acid (4 mg/mL) and the PEG solution of corosolic acid (5 mg/mL) were prepared. These solutions (the PG solution 200 μL, the PEG solution 400 μL) were administered from the femoral veins of four rats, and carotid cannulation was conducted to collect blood samples, which were studied to determine whether corosolic acid in blood could be detected by analytical HPLC or not. The samples collected just before administration, 1 min, 5 min, 10 min, 30 min and 60 min after administration were centrifuged (10,000 RPM, 5 min, 4°C) to get each 150 μL of blood plasma, to which 4-fold amount of acetonitrile was added to obtain the suspension. The suspension was deproteinated by centrifugation (10,000 RPM, 5 min, 40C), and the supernatant thus obtained was analyzed by analytical HPLC. Both the PG solution and the PEG solution provided a peak of corosolic acid that made an analysis with the calibration curve (methanol solution) possible. They also provided the approximately same change in blood level during the 60 min. The group administered with the PEG solution would theoretically provide a blood level two times as much as the group administered with the PG solution, but the result was out of the expectation. This was considered to be due to an unsaturated suspension administered or an individual difference existing among rats.
The solubility of corosolic acid in the pH 6 and 5 buffers was studied. The pH 6 and 5 buffers were prepared, 1.0 mg of corosolic acid was suspended in 20 mL of each of the buffers, and the suspension was shaken for about half a day. Afterward, the suspension was centrifuged by a centrifugal separator (5,000 RPM, 5 min, 25°C) to get a supernatant, which was then extracted with 20 mL of ethyl acetate. The organic phase was separated and concentrated. The concentrate was dissolved in 800 μL of the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC. The suspension before the centrifugation was also studied to determine the corosolic acid concentration by analytical HPLC. No corosolic acid was detected in the suspension before the centrifugation. But the concentrates from the pH 6 and 5 suspensions provided a peak of corosolic acid to that made an analysis with the calibration curve possible. Thus, corosolic acid was suspended in about 30-50 mL of each of the pH 4 and 3 buffers to conduct analytical HPLC (as described later). The DMSO solution of corosolic acid (8 mg/mL) was prepared. This solution (100 μL and 200 μL) was administered to four rats via the femoral veins, and carotid cannulation was conducted to collect blood samples, which were studied to determine whether corosolic acid in blood could be detected by analytical HPLC or not. The samples collected just before administration, 1 min, 5 min, 10 min, 30 min and 60 min after administration were centrifuged (15,000 RPM, 5 min, 4°C) to get each 150 μL of blood plasma, to which 4-fold amount of acetonitrile was added to obtain the suspension. The suspension was deproteinated by centrifugation (15,000 RPM, 5 min, 4°C), and the supernatant thus obtained was analyzed by analytical HPLC. Any samples collected 1 min after administration provided a peak of corosolic acid that made an analysis with the calibration curve (methanol solution) possible. Animals administered with 200 μL of the solution fell in respiratory pause 50 min after administration. This was considered to be due to a trouble in cannulation or to clogging of a blood vessel by a deposit. It was revealed from the comparison of blood levels in samples collected 1 min and 5 min after administration that the group administered with 200 μL of the solution provided a blood level of about two times as much as the group administered with 100 μL of the solution, in accordance with the theory. The change in blood level during the 60 min was a little different from those obtained by the administration of the PG solution and the PEG solution. This was considered to be due to individual difference existing among rats, different kinds of solvents, or unsaturated PG or PEG solutions. The solubility of corosolic acid in rat's blood was studied. Carotid cannulation on rat (one head) was conducted to collect a blood sample, which was then centrifuged (15,000 RPM, 5 min, 40C) to separate a supernatant. 1 mg of corosolic acid was suspended in 1 mL of the blood plasma, and the suspension was shaken for about half a day. The resultant product was centrifuged (15,000 RPM, 5 min, 4°C) by a centrifugal separator to separate a supernatant, to which 4-fold amount of acetonitrile was added to obtain the suspension. The suspension was deproteinated by centrifugation (15,000 RPM, 5 min, 4°C). The corosolic acid concentration was studied by analytical HPLC. A peak of corosolic acid that made an analysis with the calibration curve possible was provided, and was relatively stable. The corosolic acid concentration was 0.035 mg/mL, and it was demonstrated that corosolic acid could dissolve in blood (pH 7.4) several times as much as in the pH 7 buffer. This suggested that corosolic acid formed a protein binding in blood. A sufficient amount of corosolic acid is considered to dissolve in the blood (2,400 mL) of a human (60 kg, adult). The solubility of corosolic acid in ethanol is high, but the toxicity of ethanol is not negligible. Thus, a relatively low toxic PG/PEG mixed solvent was studied to obtain a PG/PEG ratio (volume ratio) which gives a solubility parameter (affinity to water) coincident with that of ethanol. Because ethanol (EtOH), PG and PEG have their respective solubility parameters of 12.55, 14.00 and 11.61, if PG:PEG=X:(1-X), then an equation: 14.00X+11.61(l-X)=12.55, and hence X-0.39 is given. Therefore, a ratio of PG 40%/PEG 60% was established.
The solubility of corosolic acid in the pH 4 buffer was studied. The pH 4 buffer was prepared, 1.0 mg of corosolic acid was suspended in 30 mL of the buffer, and the suspension was shaken for about half a day.
Afterward, the suspension was centrifuged by a centrifugal separator (5,000 RPM, 5 min, 250C) to get a supernatant, which was then extracted with 30 rriL of ethyl acetate. The organic phase was separated and concentrated. The concentrate was dissolved in 800 μL of the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC. The suspension before the centrifugation was also studied to determine the corosolic acid concentration by analytical HPLC.
1 mg of corosolic acid was added to 100 μL of a PG 40%/PEG 60% mixed solvent and 100 μL of a PG 30%/PEG 70% mixed solvent. The mixture obtained was shaken, and then left to stand for about half a day. The resultant products were centrifuged (15,000 RPM, 5 min, 25°C) by a centrifugal separator to separate supernatants, which were then diluted by 1, 000-fold with the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC. The solubility of corosolic acid in these mixed solvents was not found to be improved in comparison with that in PG or PEG alone.
The solubility of corosolic acid in the pH 4 and 3 buffers was studied. The pH 4 and 3 buffers were prepared, 1.0 mg of corosolic acid was suspended in 30 mL of each of the buffers, and the suspension was shaken for about half a day. Afterward, the suspension was centrifuged by a centrifugal separator (5,000 RPM, 5 min, 250C) to separate a supernatant, which was then extracted with 30 mL of ethyl acetate. The organic phase was separated and concentrated. The concentrate was dissolved in 800 μL of the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC. Corosolic acid could be detected in the concentrate derived from the pH 4 suspension, but not detected in the concentrate derived from the pH 3 suspension. It was demonstrated that improvement of HPLC detection sensitivity needed increasing amount of the buffer or ethyl acetate.
1 mg of corosolic acid was added to 100 μL of a PG 50%/PEG 50% mixed solvent. The mixture obtained was shaken, and then left to stand for about half a day. The resultant product was centrifuged (15,000 RPM, 5 min, 250C) by a centrifugal separator to separate a supernatant, which was then diluted by 1, 000-fold with the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC. The solubility of corosolic acid in this mixed solvent was not found to be improved in comparison with that in PG or PEG alone, as found in the PG
40%/PEG 60% and PG 30%/PEG 70% mixed solvents. It was revealed that a PG/PEG mixed solvent system could not be expected to improve the solubility to such an extent that it might be applied to goods (a drug).
5% aqueous α-cyclodextrin solution and 5% aqueous γ-cyclodextrin solution were prepared, 1 mg of corosolic acid was suspended in 1 mL of each of these solutions, and the suspension was shaken for about half a day by a stirrer. The suspension was centrifuged (15,000 RPM, 5 min, 25°C) by a centrifugal separator to separate a supernatant, which was studied to determine the corosolic acid concentration by analytical HPLC. The aqueous α-cyclodextrin solution provided no corosolic acid to detect, but the aqueous γ-cyclodextrin solution provided a peak of corosolic acid that made an analysis with the calibration curve possible.
The solubility of corosolic acid in the pH 4 buffer was studied. The pH 4 buffer was prepared, 1.0 mg of corosolic acid was suspended in 50 mL of the buffer, and the suspension was shaken for about half a day.
Afterward, the suspension was centrifuged by a centrifugal separator (5,000 RPM, 5 min, 25°C) to separate a supernatant, which was then extracted with 50 mL of ethyl acetate. The organic phase was separated and concentrated. The concentrate was dissolved in 800 μL of the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC. 1 mg of corosolic acid was added to 100 μL of a PG 80%/MCG 20% mixed solvent. The mixture obtained was shaken, and then left to stand for about half a day. The resultant product was centrifuged (15,000 RPM, 5 min, 250C) by a centrifugal separator to separate a supernatant, which was then diluted by 1, 000-fold with the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC. The solubility of corosolic acid in this mixed solvent was not found to be improved in comparison with that in PQ PEG or MCG alone, as found in the PG /PEG mixed solvent. It was revealed that a PG/MCG mixed solvent system could not be expected to improve the solubility to such an extent that it might be applied to goods (a drug).
1% aqueous β-cyclodextrin solution was prepared, 1 mg of corosolic acid was suspended in 1 mL of the solution, and the suspension was shaken for about half a day by a stirrer. The suspension was centrifuged (15,000 RPM, 5 min, 250C) by a centrifugal separator to separate a supernatant, which was studied to determine the corosolic acid concentration by analytical
HPLC. As a result, the aqueous solution provided a peak of corosolic acid that made an analysis with the calibration curve possible. The three samples were stable, and hence it was judged that they provided reliable data. But it was revealed that aqueous α-, β-, or γ-cyclodextrin solution could not be expected to improve the solubility to such an extent that it might be applied to goods (a drug). The solubility of corosolic acid in the pH 5 and 4 buffers was studied. The pH 5 and 4 buffers were prepared. 1.5 mg of corosolic acid was suspended in 30 mL of the pH 5 buffer, and 2.5 mg of corosolic acid was suspended in 50 mL of the pH 4 buffer. The suspensions were shaken for about half a day. Afterward, the suspensions were centrifuged by a centrifugal separator (5,000 RPM, 5 min, 250C) to separate supernatants, which were then extracted with 30 mL and 50 mL of ethyl acetate respectively. The organic phases were separated and concentrated. Each of the concentrates was dissolved in 800 μL of the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC.
Corosolic acid could be detected in any of the concentrates, but they provided no peak of corosolic acid that made an analysis with the calibration curve possible. But a plot graph (ordinate: logCtotai; abscissa: pH) depicted from data of the solubility in the pH 10-6 buffers gave stable data. 1 mg of corosolic acid was added to 100 μL of a PG 40%/PEG
40%/MCG 20% mixed solvent. The mixture obtained was shaken, and then left to stand for about half a day. The resultant product was centrifuged (15,000 RPM, 5 min, 25°C) by a centrifugal separator to separate a supernatant, which was then diluted by 1, 000-fold with the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC. The solubility of corosolic acid in this mixed solvent was not found to be improved in comparison with that in PG, PEG or MCG alone, as found in the PG /PEG mixed solvent and the PG /MCG mixed solvent. It was revealed that a PG/PEG/MCG mixed solvent system could not be expected to improve the solubility to such an extent that it might be applied to goods (a drug). STZ (streptozotocin) was intraperitoneally administered to two rats to prepare a model for diabetes. A 0.5% CMC (carboxymethylcellulose) solution (control) was administered to one rat forcibly once by a disposable injection syringe and a rat oral probe (2 mL/kg), and a suspension of corosolic acid 5 mg in a 0.5% CMC solution 1 mL was administered to another rat in the same way. Carotid cannulation was conducted just before administration, 30 min, 60 min, 90 min, 120 min and 180 min after administration to collect blood samples. The blood samples were centrifuged (15,000 RPM, 5 min, 4°C) by a centrifugal separator to separate 20 uL of blood plasma. Glucose CII-test Wako (Wako Pure Chemical
Industries, Ltd.) was used to determine blood glucose level by the Mutarose GOD (glucose oxidase) method, while the absorbance was determined by a spectrophotometer (505 nm). Concentration was calculated using the calibration curve obtained by analyzing standard solutions, resulting in a pre-value of about 500 mg/dL, which was a rather high value. Both the control and the corosolic acid suspension provided a continuous rise in blood glucose level. This was considered to be due to a continuously given stimulation because of the operation. But the corosolic acid suspension provided a more moderate rise. Among four rats intraperitoneally administered with STZ, a 0.5%
CMC solution (control) was administered to one rat forcibly once by a disposable injection syringe and a rat oral probe (2 mL/kg), and the suspensions obtained by suspending each 5 mg of corosolic acid, ursolic acid and oleanolic acid in a 0.5% CMC solution 1 mL were administered to the other three rats in the same way. Carotid cannulation was conducted just before administration, 30 min, 60 min, 90 min, 120 min and 180 min after administration to collect blood samples. The blood samples were centrifuged (15,000 RPM, 5 min, 4°C) by a centrifugal separator to separate 20 μL of blood plasma. Glucose CII-test Wako (Wako Pure Chemical Industries, Ltd.) was used to determine blood glucose level by the Mutarose GOD (glucose oxidase) method, while the absorbance was determined by a spectrophotometer (505 nm). Concentration was calculated using the calibration curve obtained by analyzing standard solutions, resulting in a pre-value of 300-500 mg/dL with a remarkable dispersion. The control, the corosolic acid suspension, and the oleanolic acid suspension provided a continuous rise in blood glucose level. This was considered to be due to a continuously given stimulation because of the operation. The rat administered with the ursolic acid suspension exhibited a fall in blood glucose level since 90 min after administration. This was considered to be because of the fact that the rat could maintain the respiratory tract by a tube to suffer from less stimulation although it had fallen in dyspnea until then.
1 mg of corosolic acid was added to 100 μL of a PG 40%/PEG 50%/EtOH 10% mixed solvent. The mixture obtained was shaken, and then left to stand for about half a day. The resultant product was centrifuged (15,000 RPM, 5 min, 25°C) by a centrifugal separator to separate a supernatant, which was then diluted by 1, 000-fold with the eluent (85%
MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC. The solubility of corosolic acid in this mixed solvent was not found to be improved in comparison with that in PG, PEG or EtOH alone, as found in the PG /PEG mixed solvent and the PG /MCG mixed solvent. It was revealed that a PG/PEG/EtOH mixed solvent system could not be expected to improve the solubility to such an extent that it might be applied to goods (a drug).
The solubility of Colosolia PBW (Lot No =020641) (banaba water extract), Colosolia PBA (Lot No.=40722010) (banaba hot water extract) and Colosolia M (Lot No. =40905005) (a mixture extract of banaba hot water extract and banaba alcohol extract) (all of them made by Use Techno
Corporation) in the pH 10 and 9 buffers (glycine buffer (pH 9.98, ionic strength 0.1) and borate buffer (pH 9.02, ionic strength 0.1)) were studied (room temperature). 0.5 g of every extract powder was added to 20 mL of every buffer. Then the resultant product was shaken for 1 min by voltex, and left to stand for 30 min. The suspension image was recorded by a digital camera. PBA was confirmed to give clearly a deposit in either of the pH 10 and 9 buffers. The solubility of corosolic acid was observed to be higher in an alkaline side (pH 10). M and PBW were observed to have a higher solubility than PBA, but were confirmed to give a deposit by careful observation in both pH 10 and 9 buffers. It was revealed that the solubility in a more acidic side was hard to study unless the extract powder was reduced to a content of less than 0.5 g/20 mL (=2.5%).
The solubility of corosolic acid in the pH 3 buffer was studied. The pH 3 buffer was prepared, 15 mg of corosolic acid was suspended in 300 mL of the buffer, and the suspension was shaken for about half a day.
Afterward, the suspension was centrifuged by a centrifugal separator (5,000 RPM, 5 min, 25°C) to get a supernatant, which was then extracted with 300 mL of ethyl acetate. The organic phase was separated and concentrated. The concentrate was dissolved in 800 μL of the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC.
The concentrate provided a peak of corosolic acid that made an analysis with a calibration curve possible. It was judged that the data thus obtained was reliable data, because it was coincident with the plot graph (ordinate: logQotai; abscissa: pH).
The solubility of Colosolia PBW, PBA and M in the pH 8-5 buffers (Tris buffer (pH 7.99, ionic strength 0.1), phosphate buffer (pH 7.04, ionic strength 0.1), citrate buffer (pH 5.96, ionic strength 0.1) and citrate buffer (pH 4.98, ionic strength 0.1)) was studied (room temperature). 0.1 g of every extract powder was added to 20 mL of every buffer. Then the resultant product was shaken for 1 min by voltex, and left to stand for 30 min. The suspension image was recorded by a digital camera. PBA was confirmed to give clearly a deposit in any of the pH 8-5 buffers. The solubility of corosolic acid was observed to be higher in an alkaline side. M and PBW were observed to have a higher solubility than PBA, but M was confirmed to give a deposit the more easily in the more acidic side, and PBW was confirmed to give a deposit in any buffer by careful observation. This suggested that the extract containing terpenes including corosolic acid likewise has a critical problem in its solubility to study the biological reaction and pharmacokinetics.
1 mg of corosolic acid was added to 100 μL of a 1% Tween-PG 40%/PEG 60% solution, and 2 mg of corosolic acid was added to 100 μL of a
1% Tween-EtOH solution. The mixtures obtained were shaken, and then left to stand for about half a day. The resultant products were centriftiged (15,000 RPM, 5 min, 25°C) by a centrifugal separator to separate supernatants, which were then diluted by 1, 000-fold with the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC. The solubility of corosolic acid in these solutions was not found to be improved in comparison with that in PG/PEG mixed solvent, PG/MCG mixed solvent, PG alone, PEG alone or EtOH alone. It was revealed that the solution could not be expected to improve the solubility to such an extent that it might be applied to goods (a drug). 5 mg of corosolic acid was added to 100 μL of 1% BL-9EX-PG
40%/PEG 60% solution and 100 μL of 1% BL-9EX-EtOH solution, and the mixtures obtained were shaken. Corosolic acid was completely dissolved in 1% BL-9EX-EtOH solution. The suspension of corosolic acid in 1% BL-9EX-PG 40%/PEG 60% solution was left to stand for about half a day, and centrifuged (15,000 RPM, 5 min, 250C) by a centrifugal separator to separate a supernatant, which was then diluted by 1, 000-fold with the eluent (85% MeOH/0.05% TFA) to determine the corosolic acid concentration by analytical HPLC. The solubility of corosolic acid in this solution was not found to be improved in comparison with that in PG/PEG mixed solvent. It was revealed that if a surfactant (such as Tween 80, BL-9EX) is added, the solubility is lowered instead. Further, it was revealed that this solution could not be expected to improve the solubility to such an extent that it might be applied to goods (a drug). On the other hand, 1% BL-9EX-EtOH solution allows theoretically a solubility of 50 mg/mL or more, if there is no mistake in experimental procedures (such as weighing). But oral administration of this solution could possibly bring toxicity. Ethanol can be orally administered as a pharmaceutical additive within the maximum dose of 1 mL/day. Thus, it is demonstrated that ethanol is preferably formulated as an ethanol solution which is not filled in a capsule, but mixed into a syrup. The solubility of Colosolia PBW, PBA and M in the pH 4-1 buffers
(citrate buffer (pH 3.98, ionic strength 0.1), citrate buffer (pH 3.04, ionic strength 0.1), hydrochloride buffer (pH 2.03, ionic strength 0.1) and hydrochloride buffer (pH 1.26, ionic strength 0.1)) were studied (room temperature). 0.1 g of every extract powder was added to 20 mL of every buffer. Then the resultant product was shaken for 1 min by voltex, and left to stand for 30 min. The suspension image was recorded by a digital camera. PBA was confirmed to give clearly a deposit in any buffer of pH 4-1. The solubility of corosolic acid was observed to be relatively higher in an alkaline side. M and PBW were confirmed to give a deposit, which was observed to be formed the more remarkably in the more acidic side. Corosolic acid was administered to a beagle dog raised for an experiment, and the corosolic acid concentration in serum was determined by analytical HPLC. Blood samples were collected just before administration (blank), 30 min, 60 min, 90 min, 120 min and 180 min after administration. Every 200 μL from 2 mL of the sample (serum) at every time point was transferred into a microtube. Every sample thus separated was supplemented with 4-fold amount of acetonitrile (800 μL) and deproteinated. The resultant product was centrifuged (15,000 RPM, 5 min, 4°C) by a centrifugal separator to separate a supernatant, which was then gathered in a sample bottle for every time point and concentrated completely. The concentrate was dissolved in 500 μL of the eluent (85% MeOH/0.05% TFA) to confirm by analytical HPLC whether the concentrate could provide a peak of the corosolic acid under the present HPLC condition (standard solution was a corosolic acid methanol solution) or not (whether the corosolic acid concentration could be determined or not). The sample collected before the administration of corosolic acid (negative control) allowed, under the present
HPLC condition, detection of other blood ingredients than corosolic acid, but no corosolic acid (retention time: about 12 min). The samples collected after the administration were analyzed by analytical HPLC, but provided no peak of corosolic acid that made an analysis with the calibration curve (methanol solution) possible. A banaba clear extract spray liquid was administered to rats, which had been administered intraperitoneally with STZ to prepare a model for diabetes, to study the influence of the extract on blood glucose level. Five of ten heads were administered with a physiological saline (Ohtsuka physiological saline injection) (control), and the remaining five heads were administered with a banaba clear extract (p-280: obtained by dissolving a stock solution of decolorized banaba extract in water to give a concentration of 10 ppm) once by inhalation respectively. Inhalation time was 5 min. Blood samples were collected just before administration, 30 min, 60 min, 90 min, 120 min, 180 min and 360 min after administration. Blood samples were collected from the tail vein incised by a knife, and blood glucose level was determined by a small-sized blood glucose measuring device (Glutest E) and an electrode (Glutest sensor). There was confirmed at any time point no significant difference in blood glucose level between the banaba extract inhalation group and the control inhalation group. Industrial Applicability
The present invention provides a novel antidiabetic drug which has both properties of a biguanide-based drug that has a gluconeogenesis inhibiting action and a hyperglycemia inhibiting action, and a nateglinide-based drug that has an early insulin secretion stimulating action, and which has lowered side effects in comparison with these drugs.
Terpenes, particularly triterpenes, have been noticed to be a treasure house for drugs. But they have scarcely been used for drugs or food and drink additives, because they have too large molecular weight, and are difficult to isolate and purify or synthesize. The present invention allows use of corosolic acid (an example of a triterpene), analogous compounds of corosolic acid, and pharmaceutically acceptable salts thereof as drugs or food and drink additives. Furthermore, the present invention specifies the sites, functional groups, and structures of triterpenes relating to their activity to facilitate discovery or synthesis of novel useful compounds and development of more effective and safer medicaments.

Claims

1. A gluconeogenesis inhibiting agent containing as an active ingredient corosolic acid, an analogous compound of corosolic acid, or a pharmaceutically acceptable salt thereof.
2. The gluconeogenesis inhibiting agent according to claim 1, wherein the analogous compound of corosolic acid is at least one selected from the group consisting of maslinic acid, tormentic acid, ursolic acid, asiatic acid, oleanolic acid and 2α,19α-dihydroxy-3-oxo-urs-12-en-28-oic acid.
3. The gluconeogenesis inhibiting agent according to claim 1 or
2, wherein the gluconeogenesis inhibiting agent inhibits gluconeogenesis during fasting.
4. The gluconeogenesis inhibiting agent according to any one of claims 1 to 3, wherein the gluconeogenesis inhibiting agent inhibits gluconeogenesis 90 to 180 minutes after meal.
5. The gluconeogenesis inhibiting agent according to any one of claims 1 to 4, wherein the gluconeogenesis inhibiting agent increases insulin sensitivity.
6. The gluconeogenesis inhibiting agent according to any one of claims 1 to 5, wherein the gluconeogenesis inhibiting agent has a reduced incidence of hepatopathy.
7. The gluconeogenesis inhibiting agent according to any one of claims 1 to 6, wherein the gluconeogenesis inhibiting agent is orally administered to inhibit gluconeogenesis.
8. The gluconeogenesis inhibiting agent according to any one of claims 1 to 7, wherein the gluconeogenesis inhibiting agent is administered to a subject that satisfies the following condition (i) or (ii):
(i) a fasting blood glucose level of 110 mg/dL or more;
(ii) an OGTT (oral glucose tolerance test) value at 2 hours of 140 mg/dL or more.
PCT/JP2005/008569 2004-11-16 2005-04-28 Gluconeogenesis inhibiting agent WO2006054370A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2004332083 2004-11-16
JP2004-332083 2004-11-16

Publications (1)

Publication Number Publication Date
WO2006054370A1 true WO2006054370A1 (en) 2006-05-26

Family

ID=36406917

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2005/008569 WO2006054370A1 (en) 2004-11-16 2005-04-28 Gluconeogenesis inhibiting agent

Country Status (1)

Country Link
WO (1) WO2006054370A1 (en)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2908310A1 (en) * 2006-11-14 2008-05-16 Phytodia Use of product from plants comprising extracts, fractions and/or molecules as TGR5 specific agonist, for human or animal the preparation of drug to prevent and/or treat metabolic disorder, obesity and type II diabetes
US7994179B2 (en) 2007-12-20 2011-08-09 Astrazeneca Ab Carbamoyl compounds as DGAT1 inhibitors 190
US8003676B2 (en) 2006-05-30 2011-08-23 Astrazeneca Ab 1,3,4-oxadiazole derivatives as DGAT1 inhibitors
EP2440220A1 (en) * 2009-06-12 2012-04-18 Generex Pharmaceuticals, Inc. Compositioins and preparation methods of compositions for prevention and treatment of hypertension
US8188092B2 (en) 2009-06-19 2012-05-29 Astrazeneca Ab Substituted pyrazines as DGAT-1 inhibitors
CN101619088B (en) * 2009-08-04 2012-07-04 上海朴颐化学科技有限公司 Maslinic acid derivative as well as preparation and application thereof
CN104739840A (en) * 2013-12-26 2015-07-01 韩嘉 Novel use of asiatic acid in preparation of drugs for preventing and treating hyperlipidemia and fatty liver

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000169384A (en) * 1998-12-09 2000-06-20 Yuusu Techno Corporation:Kk Blood glucose level increase inhibitor or hypoglycemic agent
JP2002212171A (en) * 2000-11-17 2002-07-31 Takeda Chem Ind Ltd Isoxazole derivative
JP2003081858A (en) * 2001-09-13 2003-03-19 Nippon Yakuyo Shokuhin Kenkyusho:Kk Prophylactic and therapeutic agent for diabetes mellitus and health food
JP2003081860A (en) * 2001-06-25 2003-03-19 Kanebo Ltd Agent for treatment of diabetes
JP2003119148A (en) * 2001-08-07 2003-04-23 Kanebo Ltd Agent for ameliorating side-effect of thiazolidine derivative
WO2004022083A1 (en) * 2002-09-04 2004-03-18 Dsm Ip Assets B.V. A nutritional and therapeutic composition of an insulin sensitizer and a peptide fraction
JP2004215562A (en) * 2003-01-14 2004-08-05 Yuusu Techno Corporation:Kk Additive for food and drink, medicinal composition, glut4 translocating agent and translocating method

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000169384A (en) * 1998-12-09 2000-06-20 Yuusu Techno Corporation:Kk Blood glucose level increase inhibitor or hypoglycemic agent
JP2002212171A (en) * 2000-11-17 2002-07-31 Takeda Chem Ind Ltd Isoxazole derivative
JP2003081860A (en) * 2001-06-25 2003-03-19 Kanebo Ltd Agent for treatment of diabetes
JP2003119148A (en) * 2001-08-07 2003-04-23 Kanebo Ltd Agent for ameliorating side-effect of thiazolidine derivative
JP2003081858A (en) * 2001-09-13 2003-03-19 Nippon Yakuyo Shokuhin Kenkyusho:Kk Prophylactic and therapeutic agent for diabetes mellitus and health food
WO2004022083A1 (en) * 2002-09-04 2004-03-18 Dsm Ip Assets B.V. A nutritional and therapeutic composition of an insulin sensitizer and a peptide fraction
JP2004215562A (en) * 2003-01-14 2004-08-05 Yuusu Techno Corporation:Kk Additive for food and drink, medicinal composition, glut4 translocating agent and translocating method

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8003676B2 (en) 2006-05-30 2011-08-23 Astrazeneca Ab 1,3,4-oxadiazole derivatives as DGAT1 inhibitors
WO2008074935A3 (en) * 2006-11-14 2008-09-12 Univ Pasteur Biologically active compositions, methods for producing the same and biological applications
FR2908310A1 (en) * 2006-11-14 2008-05-16 Phytodia Use of product from plants comprising extracts, fractions and/or molecules as TGR5 specific agonist, for human or animal the preparation of drug to prevent and/or treat metabolic disorder, obesity and type II diabetes
US7994179B2 (en) 2007-12-20 2011-08-09 Astrazeneca Ab Carbamoyl compounds as DGAT1 inhibitors 190
US9629884B2 (en) 2009-06-12 2017-04-25 Generex Pharmaceuticals, Inc. Compositions and methods for increasing lifespan and health span
EP2440220A1 (en) * 2009-06-12 2012-04-18 Generex Pharmaceuticals, Inc. Compositioins and preparation methods of compositions for prevention and treatment of hypertension
EP2440220A4 (en) * 2009-06-12 2012-12-12 Generex Pharm Inc Compositioins and preparation methods of compositions for prevention and treatment of hypertension
US9050277B2 (en) 2009-06-12 2015-06-09 Generex Pharmaceuticals, Inc. Combined Geum japonicum and Centella asiatica extracts for the therapeutic treatment of heart failure
US9950019B2 (en) 2009-06-12 2018-04-24 Generex Pharmaceuticals, Inc. Compositions and methods for the prevention and treatment of brain diseases and conditions
US9283255B2 (en) 2009-06-12 2016-03-15 Generex Pharmaceuticals, Inc. Compositions and methods for the prevention and treatment of red blood cell coagulation
US8188092B2 (en) 2009-06-19 2012-05-29 Astrazeneca Ab Substituted pyrazines as DGAT-1 inhibitors
CN101619088B (en) * 2009-08-04 2012-07-04 上海朴颐化学科技有限公司 Maslinic acid derivative as well as preparation and application thereof
CN104739840A (en) * 2013-12-26 2015-07-01 韩嘉 Novel use of asiatic acid in preparation of drugs for preventing and treating hyperlipidemia and fatty liver

Similar Documents

Publication Publication Date Title
US6541522B2 (en) Methods of using compositions containing hypotriglyceridemically active stilbenoids
WO2006054370A1 (en) Gluconeogenesis inhibiting agent
US10028970B2 (en) Composition comprising cashew apple extract
WO2007094497A1 (en) Ameliorating agent for metabolic syndrome
JP6346564B2 (en) Composition comprising chicory extract
EP1311257A2 (en) Compositions containing hypoglycemically active stilbenoids
AU2012266308A1 (en) Composition comprising cashew apple extract
JP4686173B2 (en) Processed acerola containing polyphenol and / or vitamin C
US20150196498A1 (en) Hydroxytrosol containing extract obtained from olives and solids containing residues of olive oil extraction
US20100015258A1 (en) Rice Bran Extracts and Methods of Use Thereof
US7767236B2 (en) Plant seed extract composition and process for producing the same
JP2006169236A (en) Glycogenesis inhibitor
Kawamura et al. Investigation of the safety and antihyperglycemic effect of Apios americana flower intake as a food material in normal and diabetic mice
KR20180000586A (en) A pharmaceutical composition comprising extract from germinated gemmule of bean for preventing or treating metabolic disease
JP2001278804A (en) Lipid metabolism-improving agent and food containing the same
JP2010270096A (en) Glucose absorption inhibitor
JP5186084B2 (en) Robofruit-containing saponin and its use
Jung et al. Effect of onion (Allium cepa) ultra-high pressure processing and hot water extracts on the serum cholesterol level in high cholesterol-fed rats
EP4169522A1 (en) Pharmaceutical composition containing saururus chinensis fraction, and preparation method therefor
KR100406184B1 (en) Anti-cholesterol compositions containing acetone extract of sea squirt shell
JP2001139945A (en) Antioxidant
WO2004000338A1 (en) Propolis extract and process for producing the same, and propolis-extract-containing antihypertension drug, food preparation and propolis composition
JP2002173437A (en) Medicinal composition including extract of loquat core for controlling amount of lipid in humor
WO2018168333A1 (en) Bile acid synthesis promoter, composition for promoting bile acid synthesis and food composition for promoting bile acid synthesis
KR100408088B1 (en) Anti-cholesterol compositions containing acetone extract of sea urchin shell

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS KE KG KM KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 05738865

Country of ref document: EP

Kind code of ref document: A1