WO2006064583A1 - Terpene, method for determining its blood concentration, and method for analyzing its pharmacokinetics - Google Patents

Terpene, method for determining its blood concentration, and method for analyzing its pharmacokinetics Download PDF

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Publication number
WO2006064583A1
WO2006064583A1 PCT/JP2005/006517 JP2005006517W WO2006064583A1 WO 2006064583 A1 WO2006064583 A1 WO 2006064583A1 JP 2005006517 W JP2005006517 W JP 2005006517W WO 2006064583 A1 WO2006064583 A1 WO 2006064583A1
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Prior art keywords
terpene
acid
blood concentration
blood
pharmacokinetics
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PCT/JP2005/006517
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French (fr)
Inventor
Futoshi Matsuyama
Yutaka Seino
Mitsuo Fukushima
Shinpei Fujimoto
Kotaro Yamada
Toshihiro Miura
Koichi Okamoto
Naoya Ueda
Hiroyuki Morikawa
Tetsuo Kaneko
Masaya Hosokawa
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Use-Techno Corporation
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Publication of WO2006064583A1 publication Critical patent/WO2006064583A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2407/00—Assays, e.g. immunoassays or enzyme assays, involving terpenes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph

Definitions

  • the present invention relates to a terpene, a method for determining its blood concentration, and a method for analyzing its pharmacokinetics.
  • terpenes such as triterpene found in plants are known to have various physiological activities.
  • corosolic acid and triterpenes having the structurally-relative activity which are found in Banaba extracts obtained through hot- water extraction or alcohol extraction of dried Banaba leaf, have been studied focusing on the blood sugar depressant action in vivo and the GLUT4 translocation enhancing activity in skeletal muscle cells in vitro in rat, mouse, dog, and human, thus suggesting that they are likely usable for a blood sugar depressant and an anti- obestity agent:(Patent document 1). [0003]
  • an object of the present invention is to provide a method for determining precisely blood concentration of a terpene present in blood at a low concentration, a method for analyzing pharmacokinetics of a terpene using the method, and a terpene which has a pharmacokinetics analyzed using the method.
  • the present invention provides a method for determining blood concentration of a terpene, comprising the terpene in blood being detected and determined by a mass spectrometer.
  • the method for determining blood concentration of a terpene according to the present invention adopts a mass spectrometer, thereby the blood concentration of the terpene present in blood at a very low concentration can be precisely determined.
  • the mass spectrometer is preferably a quadruple mass spectrometer because the device significantly increases the accuracy of determination of blood concentration.
  • the blood is preferably fractionated by liquid chromatography to provide a fraction component, the terpene contained in which is detected by the mass spectrometer.
  • the procedure minimizes the effect of inclusions in blood, and increases the detection sensitivity.
  • the blood is more preferably centrifuged to provide a serum, an organic solvent extract from which is fractionated by the liquid chromatography.
  • corosolic acid or an analogous compound thereof is specifically preferred.
  • the preferred analogous compound is at least one triterpene selected from the group consisting of maslinic acid, ursolic acid, asiatic acid, oleanolic acid, tormentic acid, and 2 ⁇ , 19 ⁇ -dihydroxy-3-oxo-urs-12- en-28-oic acid.
  • the terpene is preferably a triterpene derived from Banaba, perilla, loquat, or guava. Those kinds of plants are rich in pharmaceutically useful triterpenes.
  • the present invention also provides a method for analyzing pharmacokinetics of a terpene, comprising the blood concentration of the terpene being determined with respect to time by the method for determining blood concentration of a terpene according to the present invention, wherein the terpene is present in blood of a living body with a terpene administrated.
  • the method for analyzing pharmacokinetics adopts the method for determining blood concentration of a terpene according to the present invention, thereby pharmacokinetics of the terpene can be analyzed at a high precision analysis even if the terpene is present in blood at a very low concentration.
  • the present invention further provides a terpene, comprising being administrated to a living body to have a pharmacokinetics which is analyzed by the method for analyzing pharmacokinetics of a terpene according to the present invention.
  • the terpene according to the present invention allows easy defining the optimum dose, and assures high safety after oral administration, because the terpene has a pharmacokinetics analyzed at a high precision by the method for analyzing pharmacokinetics according to the present invention.
  • FIG. 1 is a graph showing MS spectrum of corosolic acid. [Fig. 2]
  • Figure 2 is a graph showing the relation between peak area and concentration of corosolic acid in LC/MS/MS analysis.
  • Figure 3 is a graph showing the relation between peak area and concentration of corosolic acid in LC/MS/MS analysis.
  • Figure 4 is a graph showing the relation between peak area and concentration of corosolic acid in LC/MS/MS analysis.
  • Figure 4 is a graph showing the changes in peak intensity of corosolic acid with time in Example 1. Best Modes for Carrying Out the Invention
  • a method for determining precisely the concentration of a terpene present in blood at a very low concentration a method for analyzing pharmacokinetics of a terpene using the method, and a terpene which has a pharmacokinetics analyzed using the method.
  • the blood concentration of a terpene present in blood is determined by a mass spectrometer.
  • a preferable mass spectrometer is a quadruple mass spectrometer.
  • the quadruple mass spectrometer four rod-shaped electrodes are arranged apart from each other at an equal distance. Sample ions are charged in an ionization chamber and put into a space surrounded by these electrodes. A signal with a high frequency superimposed on a direct current voltage is applied to each of the four electrodes. The input ions fly among these electrodes while oscillating in an oscillation mode in accordance with Marsh's equation. Depending on the direct current voltage and the amplitude of the high frequency, only the ions having a specific mass are allowed to pass through the interelectrode space. By detecting the intensity of ions passed through the space using an ion detector, the quantity of ingredient having a specific mass in the sample can be determined. [0020]
  • the blood is preferably fractionated by liquid chromatography to provide a fraction, which is analyzed as an analytical sample by the mass spectrometer. That is, preferably, while the blood is fractionated by liquid chromatography, fractions thus prepared are successively analyzed by a mass spectrometer.
  • Such analysis may be suitably conducted using a known analyzer with liquid chromatography combined with mass spectrometer, such as LC/MS and LC/MS/MS.
  • a calibration curve is preliminarily prepared on the relation between intensity of peak corresponding to the molecular weight of a terpene to analyze and concentration of the terpene in a sample, and then blood concentration of the terpene is determined on the basis of the calibration curve.
  • Known concentrations of samples prepared to contain specific quantities of the terpene are analyzed by mass spectrometer.
  • the blood concentration of the terpene can be precisely determined.
  • the blood concentration can be determined in a concentration range of about 1 ng/mL or more. Particularly in a range of a very low concentration of about 1 - 60 ng/mL, conventional liquid chromatography analysis alone was difficult to detect corosolic acid or an analogous compound thereof.
  • a mass spectrometer has allowed high precision determination of blood concentration even at so low a concentration.
  • the above liquid chromatography preferably uses a reversed phase column of ODS, polymer base, and the like.
  • the mobile phase may be adequately selected from solvents, such as methanol/water liquid mixture, which are normally used in liquid chromatography depending on the kind of target terpene, column, or other variables.
  • solvents such as methanol/water liquid mixture
  • the blood is preferably centrimged to provide serum, an organic solvent extract of which is injected into the liquid chromatograph. The procedure removes proteins from the blood, which allows the liquid chromatography to extract stably with a high separability.
  • acetonitrile can be suitably used.
  • the present invention is useful for determining the blood concentration of corosolic acid or an analogous compound thereof as a terpene.
  • the particularly preferred analogous compound of corosolic acid includes at least one kind of triterpene selected from the group consisting of maslinic acid, ursolic acid, asiatic acid, oleanolic acid, tormentic acid, and 2 ⁇ ,19 ⁇ -dihydroxy-3-oxo-urs-12- en-28-oic acid.
  • the present invention is also favorably applied for determining a triterpene which has an active site structurally in common with aforementioned triterpenes, as an early insulin- secretion enhancer and a sugar-dependent early insulin-secretion enhancer.
  • the triterpene having a structurally common active site have at least one hydroxyl at the positions 2 and 3 thereof. More preferably, it has further at least one hydroxyl at the position 29 and
  • terpene includes an ursane type pentacyclic triterpene and an oleanane type pentacyclic triterpene.
  • ursane type pentacyclic triterpene are desfontainic acid, 2 ⁇ ,19 ⁇ -dihydroxy-3-oxo-l,12-ursadien-28-oic acid, 2 ⁇ ,20 ⁇ -dihydroxy-3-oxo-12-ursen-28-oic acid, 2 ⁇ ,3 ⁇ - dihydroxy-12,20(30)-usradien-28-oic acid, 2 ⁇ ,3 ⁇ -dihydroxy- 12,20(30)-ursadien-28-oic acid, 2 ⁇ ,3 ⁇ -dihydroxy-12-ursen-23-oic acid, 2 ⁇ ,3 ⁇ -dihydroxy-12-ursen-23-oic acid, l ⁇ ,2 ⁇ ,3 ⁇ ,19 ⁇ ,23- pntahydroxy-12-
  • oleanane type pentacyclic triterpene examples include 2 ⁇ ,3 ⁇ -dihidorxy ⁇ 12,18-oleanadiene-24,28-dioic acid, 2 ⁇ ,3 ⁇ - dihydroxy-12-oleanene-23,28-dioic acid, 2 ⁇ ,3 ⁇ -dihydroxy-12- oleanene-23,28-dioic acid, 2 ⁇ ,3 ⁇ -dihydroxy-12-oleanene-28,30- dioic acid, 2 ⁇ ,3 ⁇ -dihydroxy-12-oleanene-23-oic acid, 2 ⁇ ,3 ⁇ - dihydroxy-12-oleanene-28-oic acid, 2 ⁇ ,3 ⁇ -dihydroxy-12-oleanene- 28-oic acid, 2 ⁇ ,3 ⁇ -dihydroxy-12-oleanene-28-oic acid, 2 ⁇ ,3 ⁇ - dihydroxy-13(18)-oleanene-28-oic acid, 12 ⁇ ,13 ⁇ -
  • the method for determining blood concentration according to the present invention is applicable to the derivatives of the above triterpenes, including: ethers obtained from a reaction between the hydroxyl group of above triterpenes with a halogenated hydrocarbon such as CH 3 Br and CH 3 (CH 2 ) n Br; ketones or diketones obtained from selective oxidation of hydroxyl group at position 2 and/or 3 in the triterpenes.
  • ethers obtained from a reaction between the hydroxyl group of above triterpenes with a halogenated hydrocarbon such as CH 3 Br and CH 3 (CH 2 ) n Br
  • ketones or diketones obtained from selective oxidation of hydroxyl group at position 2 and/or 3 in the triterpenes.
  • the present invention is also useful for determining blood concentration of a triterpene which is derived from Banaba, perilla, loquat, or guava, and is further applicable to triterpenes derived from these plants using plant culture, gene manipulation, semi- synthesis, and the like.
  • the terpenes used for determining blood concentration and for analyzing pharmacokinetics may be prepared by the extraction of above plants or the like. A preferred method for pretreating and extracting Banaba leaf to provide triterpenes is described below.
  • the raw material Banaba Before extracting triterpenes from Banaba leaf, the raw material Banaba is subjected to pretreatment including hot water extraction. Although the collected raw material Banaba leaf can be directly subjected to extraction, it is preferably cut to fine chips before the pretreatment in order to extract efficiently. Although the chip may have a size which is adequately varied depending on equipment for the succeeding extraction process, the Banaba leaf is preferably cut to the chips having a size such as a 1 mm square or larger size. If the chips have a size of less than 1 mm square, the equipment is likely to get clogged with them in the extraction process.
  • the cut Banaba leaf chips are immersed in water at a ratio of
  • the chips are thrice or more immersed in water having a temperature of 10 - 60 0 C for 24 hours or more, while water is exchanged every immersion.
  • the chips are three to five times immersed in hot water, boiled, and contacted with steam to extract, while hot water is exchanged every time.
  • the hot water extraction is preferably done under 1 to 3 atm at 100 0 C or higher using a pressure vessel or the like.
  • the extraction is preferably conducted for about 3 - 10 minutes. A longer time for the extraction may lose corosolic acid during the pretreatment stage.
  • the chips are further treated by extraction using a solvent of water 50% and alcohol 50% having a preferable temperatures of 0 - 100 0 C.
  • the Banaba leaf chips are dried in the drying process by solar drying or using a drier. Free water is preferably removed from the Banaba leaf chips by a centrifugal separator before drying. Warm air having 30 - 60 0 C or cool air with low humidity as far as possible is blown for drying.
  • the dried Banaba leaf chips are heated to extract under reflux in n-hexane for 1 hour, in purified water for 1 hour, and then in ethanol for 1 hour. A liquid extract is filtered, concentrated under a reduced pressure, and further dried up.
  • An ethanol extract provides mainly a white extract.
  • the white extract contains corosolic acid, maslinic acid, tormentic acid, ursolic acid, oleanolic acid, ⁇ -amirinic acid, ⁇ -amirinic acid, asiatic acid, 18 ⁇ -glycyrrhetic acid, tannins, chrolophylls, hemicellulose, and other compounds.
  • the content of corosolic acid in the extract is about 3 - 50% by weight.
  • the extract is either a paste liquid or a powder solid.
  • the extract is preferably stored to be desiccated and blocked from light at room temperature or in a refrigerator. [0038]
  • the fraction thus obtained is then passed through an ion exchange resin column, and is further fractionated by reversed phase HPLC using an ODS column, (mobile phase: 85% methanol/0.05% trifluoroacetic acid; flow rate: 6mL/min; detection wavelength: 210 nm) to obtain corosolic acid.
  • blood concentration of the terpene administrated to a living body is determined with respect to time by the above method for determining blood concentration of a terpene, allowing analyzing pharmacokinetics of the terpen.
  • Blood concentrations of a terpene in blood of a living body with the terpene administrated are determined at an interval of about 30 - 60 minutes after administrating, allowing analyzing absorption of the terpene into blood in detail.
  • the method for analyzing pharmacokinetics according to the present invention is particularly suitable for pharmacokinetics of a terpene orally administrated to a living body.
  • the method analyzing pharmacokinetics can be favorably applied for analyzing pharmacokinetics of a terpene administrated to mammalian such as human, dog, and mouse.
  • corosolic acid C 30 H 48 O 4 , molecular weight of 472
  • a triterpene a triterpene
  • the terpene according to the present invention comprises its pharmacokinetics after administrated being analyzed by the above method for analyzing pharmacokinetics of a terpene.
  • the terpene according to the present invention can have a readily optimized dose because the pharmaokinetics is accurately analyzed by the method for analyzing of the present invention, thus assuring high safety, for example, in oral administration. Consequently, the terpene according to the present invention can be favorably employed as an early insulin-secretion enhancer and a sugar- dependent early insulin-secretion enhancer for an anti-diabetes agent, an anti-obesity agent, a triglyceride reducer, and a cholesterol concentration reducer. [0043]
  • terpene may be added to nutrient supplements and general foods and drinks such as drink, noodle, dry food, cake, bread, liquor, fat, and seasoning, or may be formed to use in powder, tablet, liquid, and paste as a drug, or vaporized to use by a spraying device, tobacco, or the like.
  • the terpene is required to be soluble in water, it may be modified to a water-soluble derivative such as a glucoside, or may be used in a mixture with tannin, cellulose, chlorophyll, and the like which are extracted from plants to coexist.
  • a solution of the terpene in a solvent such as water and alcohol may be mixed with a starch, an indigestible dextrin, or the like to form a mixture, from which the solvent is then removed to get a solid to use.
  • a solution of corosolic acid in acetonitrile/formic acid (100 ng/mL) was prepared as a sample.
  • the sample was injected into the LC/MC/MC apparatus to draw the MS spectra of corosolic acid (Fig. 1).
  • the ionization was done by the electro-spray ionization method (ESI).
  • ESI electro-spray ionization method
  • Samples having their respective corosolic acid contents of 1 ng/mL and 10 ng/mL were similarly analyzed.
  • Figure 2 shows the plots and the calibration curve. The result confirmed that intensity of a parent peak can be measured to determine concentration of corosolic acid.
  • Example 1 To a dog (beagle) which was fasted for a day, corosolic acid and glucose were orally administrated by 20 mg/kg and 2 g/kg, respectively. Blood samples were collected from the dog before administration, 30, 60, 90, 120, 180, and 240 minutes after administration. The collected blood samples were treated with the same procedure as in the preparation of the calibration curve for blood concentration of corosolic acid, and analyzed by LC/MS/MS to determine the changes in blood concentration with time. [0052] Figure 4 shows the result.
  • Blood samples collected (a) before the administration, (b) 30 minutes after the administration, (c) 60 minutes after, (d) 90 minutes after, (e) 120 minutes after, (f) 180 minutes after, and (g) 240 minutes after, provide their respective graphs in Figure 4.
  • the corosolic acid peak near 12 minutes of retention time was detected in the blood sample collected (c) 60 minutes after the administration, reached the maximum in the blood sample collected (f) 180 minutes after the administration, and afterward decreased.
  • the maximum blood concentration of corosolic acid was 20 ⁇ g/mL.
  • LC/MS/MS could be employed to confirm, for the first time, the pharmacokinetics of corosolic acid.
  • Comparative Example 1 By the same procedure as in Example 1, the blood samples collected from a dog were centrifuged (3,000 rpm for 20 minutes) to obtain serum, 2 mL of which was divided into 200 ⁇ L aliquots to put in niicrotubes. A fourfold volume (800 ⁇ L) of acetonitrile was added to the each serum to remove proteins. After centrifuging (15,000 rpm for 5 minutes, 4 0 C):, the resulted supernatants were collected in a sample bottle. All the acetonitrile was vaporized to prepare a concentrate. The concentrate was then dissolved in 500 ⁇ L of aqueous solution of 95% methanol by volume (similar to the developing solvent for HPLC) to prepare a sample. The sample was analyzed by HPLC. The HPLC condition was as follows:
  • a method for determining precisely the concentration of a terpene present in blood at a very low concentration a method for analyzing pharmacokinetics of a terpene using the method, and a terpene which has a pharmacokinetics analyzed using the method.

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Abstract

[Problem] To provide a method for determining precisely blood concentration of a terpene present in blood at a low concentration, a method for analyzing pharmacokinetics of a terpene using the method for determining blood concentration of a terpene, and a terpene having pharmacokinetics which is analyzed using the method for analyzing pharmacokinetics of a terpene. [Solution] The method for determining blood concentration of a terpene comprises detecting the terpene in blood by a mass spectrometer to determine blood concentration of the terpene.

Description

DESCRIPTION
TERPENE, METHOD FOR DETERMINING ITS BLOOD CONCENTRATION, AND METHOD FOR ANALYZING ITS PHARMACOKINETICS Technical Field
[0001]
The present invention relates to a terpene, a method for determining its blood concentration, and a method for analyzing its pharmacokinetics. Background Art
[0002]
Many of terpenes such as triterpene found in plants are known to have various physiological activities. For example, corosolic acid and triterpenes having the structurally-relative activity, which are found in Banaba extracts obtained through hot- water extraction or alcohol extraction of dried Banaba leaf, have been studied focusing on the blood sugar depressant action in vivo and the GLUT4 translocation enhancing activity in skeletal muscle cells in vitro in rat, mouse, dog, and human, thus suggesting that they are likely usable for a blood sugar depressant and an anti- obestity agent:(Patent document 1). [0003]
For such physiologically active drugs, it is important to analyze in detail their pharmacokinetics which they are administrated to living bodies to follow. The analysis of pharmacokinetics examines a process by which a drug is taken into a living body to subject itself to absorption, distribution, metabolism, and excretion. For the analysis, the administrated drug must be determined with respect to a change in blood concentration, a half life of blood concentration, an excretion rate, and the others. The analysis of pharmacokinetics reveals the structural changes of the drug, for example, caused by oxidation, reduction, hydrolysis, and conjugation represented by glucuronic acid bond through the process from absorption to excretion of the drug. [0004]
Once the analysis of pharmacokinetics identifies the sites, functional groups, configurations, and other characteristics associated with the physiological activities, it is expected that useful terpenes can be easily found out among many natural terpenes, and that natural terpenes can be easily converted to their derivatives such as alkoxides, glycosides, halides, ketonates and diketonates, and further subjected to a technique such as plant culture, liquid culture and gene manipulation to develop more pharmaceutically effective drugs. [Patent document 1] Japanese Patent Laid-Open No. 2000-169384
Disclosure of the Invention [Problems to be Solved by the Invention] [0005]
The pharmacokinetics for aforementioned terpenes was, however, not fully analyzed. It is likely because of a following fact. Precise analysis of pharmacokinetics for terpenes requires that their blood concentrations can be determined at a very low concentration of pg/mL - ng/mL. A conventional technique such as high performance liquid chromatography (HPLC) alone, however, is difficult to detect the terpenes at so low concentrations, and there has not yet been established a method for determining blood concentrations of terpenes at a high precision. [0006]
In this regard, an object of the present invention is to provide a method for determining precisely blood concentration of a terpene present in blood at a low concentration, a method for analyzing pharmacokinetics of a terpene using the method, and a terpene which has a pharmacokinetics analyzed using the method. [Means to Solve the Problems] [0007] The inventors of the present invention conducted intensive studies to solve the above problems, and found that the terpene present in blood at a low concentration, which cannot be detected by HPLC or the like alone, can be detected by a mass spectrometer, and thus completed the present invention. [0008]
The present invention provides a method for determining blood concentration of a terpene, comprising the terpene in blood being detected and determined by a mass spectrometer. [0009] The method for determining blood concentration of a terpene according to the present invention adopts a mass spectrometer, thereby the blood concentration of the terpene present in blood at a very low concentration can be precisely determined.
[0010]
The mass spectrometer is preferably a quadruple mass spectrometer because the device significantly increases the accuracy of determination of blood concentration.
[0011]
In the method for determining the blood concentration, the blood is preferably fractionated by liquid chromatography to provide a fraction component, the terpene contained in which is detected by the mass spectrometer. The procedure minimizes the effect of inclusions in blood, and increases the detection sensitivity.
Furthermore, the blood is more preferably centrifuged to provide a serum, an organic solvent extract from which is fractionated by the liquid chromatography.
[0012]
Among the above terpenes, corosolic acid or an analogous compound thereof is specifically preferred. The preferred analogous compound is at least one triterpene selected from the group consisting of maslinic acid, ursolic acid, asiatic acid, oleanolic acid, tormentic acid, and 2α, 19α-dihydroxy-3-oxo-urs-12- en-28-oic acid.
[0013]
The terpene is preferably a triterpene derived from Banaba, perilla, loquat, or guava. Those kinds of plants are rich in pharmaceutically useful triterpenes. [0014]
The present invention also provides a method for analyzing pharmacokinetics of a terpene, comprising the blood concentration of the terpene being determined with respect to time by the method for determining blood concentration of a terpene according to the present invention, wherein the terpene is present in blood of a living body with a terpene administrated. [0015]
The method for analyzing pharmacokinetics adopts the method for determining blood concentration of a terpene according to the present invention, thereby pharmacokinetics of the terpene can be analyzed at a high precision analysis even if the terpene is present in blood at a very low concentration. [0016] The present invention further provides a terpene, comprising being administrated to a living body to have a pharmacokinetics which is analyzed by the method for analyzing pharmacokinetics of a terpene according to the present invention. The terpene according to the present invention allows easy defining the optimum dose, and assures high safety after oral administration, because the terpene has a pharmacokinetics analyzed at a high precision by the method for analyzing pharmacokinetics according to the present invention. Brief Description of the Drawings [Fig. 1]
Figure 1 is a graph showing MS spectrum of corosolic acid. [Fig. 2]
Figure 2 is a graph showing the relation between peak area and concentration of corosolic acid in LC/MS/MS analysis. [Fig. 3] Figure 3 is a graph showing the relation between peak area and concentration of corosolic acid in LC/MS/MS analysis. [Fig- 4]
Figure 4 is a graph showing the changes in peak intensity of corosolic acid with time in Example 1. Best Modes for Carrying Out the Invention
[Effect of the Invention] [0017]
According to the present invention, there are provided a method for determining precisely the concentration of a terpene present in blood at a very low concentration, a method for analyzing pharmacokinetics of a terpene using the method, and a terpene which has a pharmacokinetics analyzed using the method. [0018]
According to the present invention, the blood concentration of a terpene present in blood is determined by a mass spectrometer.
[0019]
A preferable mass spectrometer is a quadruple mass spectrometer. In the quadruple mass spectrometer, four rod-shaped electrodes are arranged apart from each other at an equal distance. Sample ions are charged in an ionization chamber and put into a space surrounded by these electrodes. A signal with a high frequency superimposed on a direct current voltage is applied to each of the four electrodes. The input ions fly among these electrodes while oscillating in an oscillation mode in accordance with Marsh's equation. Depending on the direct current voltage and the amplitude of the high frequency, only the ions having a specific mass are allowed to pass through the interelectrode space. By detecting the intensity of ions passed through the space using an ion detector, the quantity of ingredient having a specific mass in the sample can be determined. [0020]
The blood is preferably fractionated by liquid chromatography to provide a fraction, which is analyzed as an analytical sample by the mass spectrometer. That is, preferably, while the blood is fractionated by liquid chromatography, fractions thus prepared are successively analyzed by a mass spectrometer.
Such analysis may be suitably conducted using a known analyzer with liquid chromatography combined with mass spectrometer, such as LC/MS and LC/MS/MS. [0021] A calibration curve is preliminarily prepared on the relation between intensity of peak corresponding to the molecular weight of a terpene to analyze and concentration of the terpene in a sample, and then blood concentration of the terpene is determined on the basis of the calibration curve. Known concentrations of samples prepared to contain specific quantities of the terpene are analyzed by mass spectrometer. The intensities of peak corresponding to the molecular weight of the terpene and the terpene concentrations in the samples are plotted to find out a relationship between them, and then a linear recurrence is applied to the plots to prepare a calibration curve. [0022]
Within a concentration range where the above plots gives a linear relation to allow drawing the calibration curve, the blood concentration of the terpene can be precisely determined. For an example case of corosolic acid or an analogous compound thereof, the blood concentration can be determined in a concentration range of about 1 ng/mL or more. Particularly in a range of a very low concentration of about 1 - 60 ng/mL, conventional liquid chromatography analysis alone was difficult to detect corosolic acid or an analogous compound thereof. A mass spectrometer, however, has allowed high precision determination of blood concentration even at so low a concentration. [0023]
The above liquid chromatography preferably uses a reversed phase column of ODS, polymer base, and the like. When a reversed phase column is applied, the mobile phase may be adequately selected from solvents, such as methanol/water liquid mixture, which are normally used in liquid chromatography depending on the kind of target terpene, column, or other variables. [0024] For fractionating a blood by liquid chromatography, the blood is preferably centrimged to provide serum, an organic solvent extract of which is injected into the liquid chromatograph. The procedure removes proteins from the blood, which allows the liquid chromatography to extract stably with a high separability. For the above organic solvent, acetonitrile can be suitably used. [0025]
The present invention is useful for determining the blood concentration of corosolic acid or an analogous compound thereof as a terpene. The particularly preferred analogous compound of corosolic acid includes at least one kind of triterpene selected from the group consisting of maslinic acid, ursolic acid, asiatic acid, oleanolic acid, tormentic acid, and 2α,19α-dihydroxy-3-oxo-urs-12- en-28-oic acid. [0026]
The present invention is also favorably applied for determining a triterpene which has an active site structurally in common with aforementioned triterpenes, as an early insulin- secretion enhancer and a sugar-dependent early insulin-secretion enhancer. The triterpene having a structurally common active site have at least one hydroxyl at the positions 2 and 3 thereof. More preferably, it has further at least one hydroxyl at the position 29 and
30, and has carboxyl at the position 28 thereof. An example of the terpene includes an ursane type pentacyclic triterpene and an oleanane type pentacyclic triterpene. [0027] Examples of the ursane type pentacyclic triterpene are desfontainic acid, 2α,19α-dihydroxy-3-oxo-l,12-ursadien-28-oic acid, 2ξ,20β-dihydroxy-3-oxo-12-ursen-28-oic acid, 2α,3α- dihydroxy-12,20(30)-usradien-28-oic acid, 2α,3β-dihydroxy- 12,20(30)-ursadien-28-oic acid, 2β,3β-dihydroxy-12-ursen-23-oic acid, 2α,3α-dihydroxy-12-ursen-23-oic acid, lα,2α,3β,19α,23- pntahydroxy-12-ursen-28-oic acid, 2α,3β,7α,19α,23-pentahydroxy-
12-ursen-28-oic acid, lβ,2α,3α,19α-tetrahydroxy-12-ursen-28-oic acid, lβ,2α,3β,19α-tetrahydroxy-12-ursen-28-oic acid, lβ,2β,3β,19α-tetrahydroxy-12-ursen-28-oic acid, 2α,3β,6β,19α- tetrahydroxy-12-ursen-28-oic acid, 2α,3β,6β,23-tetrahydroxy-12- ursen-28-oic acid, 2α,3β,7α,19α-tetrahydroxy-12-ursen-28-oic acid,
2α,3α,7β,19α-tetrahydroxy-12-ursen-28-oic acid, 2α,3β,13β,23- tetrahydroxy-12-ursen-28-oic acid, 2α,3α,19α,23-tetrahydroxy-12- ursen-28-oic acid, 2α,3β,19α,23-tetrahydroxy-12-ursen-28-oic acid, 2α,3α,19α,24-tetrahydroxy-12-ursen-28-oic acid, 2α,3β,19α,24- tetrahydroxy-12-ιιrsen-28-oic acid, 2α,3β,23-trihydroxy-ll-oxo-12- ursen-28-oic acid, 2α,3β,24-trihydroxy-12,20(30)-ιιrsadien-28-oic acid,
2α,3β,27-trihydroxy-28-ursanoic acid, 2α,3β,19α-trihydroxy-12- ursene-23,28-dioic acid, 2α,3β,19α-trihydroxy-12-ursene-24,28- dioic acid, lβ,2β,3β-trihydroxy-12-ursene-23-oic acid, 2α,3β,6β- trihydroxy-12-ursene-28-oic acid, 2α,3α,19α-trihydroxy-12-ursene- 28-oic acid, 2α,3β,19α-trihydroxy-12-ιirsene-28-oic acid, 2α,3α,23- trihydroxy-12-ursene-28-oic acid, 2α,3β,23-trihydroxy-12-ursene- 28-oic acid, 2α,3α,24-trihydroxy-12-ursene-28-oic acid, 2α,3β,24- trihydroxy-12-ursene-28-oic acid, 2α,3β,27-ursanetriol, 12-ursene- lβ,2α,3β,llα,20β-pentol, 12-ursene-lβ,2α,3β,llα-tetrol, 12-ursene- 2α,3β,llα,20β-tetrol, and 12-ursene-2α,3β,l lα-triol. [0028]
Examples of the oleanane type pentacyclic triterpene are 2α,3β-dihidorxy~12,18-oleanadiene-24,28-dioic acid, 2α,3β- dihydroxy-12-oleanene-23,28-dioic acid, 2β,3β-dihydroxy-12- oleanene-23,28-dioic acid, 2β,3β-dihydroxy-12-oleanene-28,30- dioic acid, 2β,3β-dihydroxy-12-oleanene-23-oic acid, 2β,3β- dihydroxy-12-oleanene-28-oic acid, 2α,3α-dihydroxy-12-oleanene- 28-oic acid, 2α,3β-dihydroxy-12-oleanene-28-oic acid, 2α,3β- dihydroxy-13(18)-oleanene-28-oic acid, 12β,13β-epoxy-
2α,3β,21β,22β-tetrahydroxy-30-oleanoic acid, 13,28-epoxy-
2α,3β,16α,22β-tetrahydroxy-30-oleanoic acid, 13β,28-epoxy-
2α,3β,16α,22β-tetrahydroxy-30-oleanoic acid, 12-oleanene-2α,3α- diol, 12-oleanene-2α,3β-diol, 13(18)-oleanene-2α,3α-diol, 13(18)- oleanene-2β,3β-diol, 18-oleanene-2α,3β-diol, 18-oleanene-2α,3α- diol, 12-oleanene-2α,3β,16β,21β,22α,28-hexol, 12-oleanene- lβ,2α,3β,llα-tetrol, 12-oleanene-2β,3β,23,28-tetrol 12-oleanene- 2α,3β,llα-triol, 12-oleanene-2β,3β,28-triol, 12-oleanene-2α,3β,23- triol, 13(18)-oleanene-2α,3β,llα-triol, 2β,3β,6β,16α,23- pentahydroxy-12-oleanene-28-oic acid, 2β,3β,16β,21β,23- pentahydroxy-12-oleanene-28-oic acid, 2β,3β,16α,23,24- pentahydoxy-12-oleanene-28-oic acid, 2β,3β,13β,16α-tetrahydroxy- 23,28-oleananedoic acid, 2β,3β,16β,21β-tetrahydroxy-12-oleanene- 24,28-dioic acid, 2β,3β,16α,23-tetrahydroxy-12-oleanene-24,28- dioic acid, 2β,3β,22β,27-tetrahydroxy-12-oleanene-23,28-dioic acid, 2α,3β,6β,23-tetrahydroxy-12-oleanene-28-oic acid, 2β,3β,6α,23-tetrahydroxy-12-oleanene-28-oic acid, 2β,3β,6β,23- tetrahydroxy-12-oleanene-28-oic acid, 2β,3β,16β,21β-tetrahydroxy- 12-oleanene-28-oic acid, 2β,3β,16α,23-tetrahydroxy-12-oleanene- 28-oic acid, 2α,3β,19α,23-tetrahydropxy-12-oleanene-28-oic acid, 2α,3β,19β,23-tetrahydroxy-12-oleanene-28-oic acid, 2α,3β,19α,24- tetrahydroxy-12-oleanene-28-oic acid, 2α,3β,21β,23-tetrahydroxy- 12-oleanene-28-oic acid, 2α,3β,23,24-tetrahydroxy-12-oleanene-28- oic acid, 2β,3β,23-trihydroxy-5,12-oleanadien-28-oic acid, 2α,3α,24-trihy doxy- 11,13(18)-oleanadien-28-oic acid, 2α,3β,13β- trihydroxy-28-oleanoic acid, 2β,3β,16α-trihydroxy-12-oleanene-
23,28-dioic acid, 2α,3β,18β-trihydroxy-12-oleanene-23,28-dioic acid, 2α,3β,19α-trihydroxy-12-oleanene-23,28-dioic acid,
2α,3β,19β-trihydroxy-12-oleanene-23,28-dioic acid, 2α,3β,19α- trihydroxy-12-oleanene-24,28-dioic acid, 2α,3β,19β-trihydroxy-12- oleanene-24,28-dioic acid, 2α,3β,23-trihydroxy-12-oleanene-28,30- dioic acid, 2α,3β,27-trihydroxy-12-oleanene-23,28-dioic acid, 2α,3β,18β-trihydoxy-12-oleanene-28-oic acid, 2α,3β,19α- trihydroxy-12-oleanene-28-oic acid, 2α,3β,19α-trihydroxy-12- oleanene-29-oic acid, 2α,3β,21β-trihydroxy-12-oleanene-28-oic acid, 2α,3α,23-trihydroxy-12-oleanene-28-oic acid, 2α,3α,24- trihydroxy-12-oleanene-28-oic acid, and 2α,3α,30-trihydroxy-12- oleanene-28-oic acid. [0029]
Furthermore, the method for determining blood concentration according to the present invention is applicable to the derivatives of the above triterpenes, including: ethers obtained from a reaction between the hydroxyl group of above triterpenes with a halogenated hydrocarbon such as CH3Br and CH3(CH2)nBr; ketones or diketones obtained from selective oxidation of hydroxyl group at position 2 and/or 3 in the triterpenes. [0030]
The present invention is also useful for determining blood concentration of a triterpene which is derived from Banaba, perilla, loquat, or guava, and is further applicable to triterpenes derived from these plants using plant culture, gene manipulation, semi- synthesis, and the like.
[0031]
The terpenes used for determining blood concentration and for analyzing pharmacokinetics may be prepared by the extraction of above plants or the like. A preferred method for pretreating and extracting Banaba leaf to provide triterpenes is described below.
[0032]
Before extracting triterpenes from Banaba leaf, the raw material Banaba is subjected to pretreatment including hot water extraction. Although the collected raw material Banaba leaf can be directly subjected to extraction, it is preferably cut to fine chips before the pretreatment in order to extract efficiently. Although the chip may have a size which is adequately varied depending on equipment for the succeeding extraction process, the Banaba leaf is preferably cut to the chips having a size such as a 1 mm square or larger size. If the chips have a size of less than 1 mm square, the equipment is likely to get clogged with them in the extraction process.
[0033]
The cut Banaba leaf chips are immersed in water at a ratio of
1 kg of Banaba leaf to 2 liter or more of water. The chips are thrice or more immersed in water having a temperature of 10 - 600C for 24 hours or more, while water is exchanged every immersion.
Then, the chips are three to five times immersed in hot water, boiled, and contacted with steam to extract, while hot water is exchanged every time. The hot water extraction is preferably done under 1 to 3 atm at 1000C or higher using a pressure vessel or the like. The extraction is preferably conducted for about 3 - 10 minutes. A longer time for the extraction may lose corosolic acid during the pretreatment stage.
[0034] After the hot water extraction, the chips are further treated by extraction using a solvent of water 50% and alcohol 50% having a preferable temperatures of 0 - 1000C.
[0035]
After completing the pretreatment, the Banaba leaf chips are dried in the drying process by solar drying or using a drier. Free water is preferably removed from the Banaba leaf chips by a centrifugal separator before drying. Warm air having 30 - 600C or cool air with low humidity as far as possible is blown for drying.
[0036] The dried Banaba leaf chips are heated to extract under reflux in n-hexane for 1 hour, in purified water for 1 hour, and then in ethanol for 1 hour. A liquid extract is filtered, concentrated under a reduced pressure, and further dried up.
[0037]
An ethanol extract provides mainly a white extract. The white extract contains corosolic acid, maslinic acid, tormentic acid, ursolic acid, oleanolic acid, α-amirinic acid, β-amirinic acid, asiatic acid, 18β-glycyrrhetic acid, tannins, chrolophylls, hemicellulose, and other compounds. The content of corosolic acid in the extract is about 3 - 50% by weight. The extract is either a paste liquid or a powder solid. The extract is preferably stored to be desiccated and blocked from light at room temperature or in a refrigerator. [0038]
Further purification of the extract allows corosolic acid and other ingredients to be isolated. For example, hexane or ether is added to an aqueous suspension of the extract to get a mixture, which is then filtered by suction to separate a water-soluble fraction, an organic solvent-soluble fraction, and an insoluble fraction. The insoluble fraction is treated by silica gel column chromatography (mobile phase: solvent mixture of dichloromethane and methanol) to obtain a corosolic acid -containing fraction. The fraction thus obtained is then passed through an ion exchange resin column, and is further fractionated by reversed phase HPLC using an ODS column, (mobile phase: 85% methanol/0.05% trifluoroacetic acid; flow rate: 6mL/min; detection wavelength: 210 nm) to obtain corosolic acid.
[0039] In the method for analyzing pharmacokinetics according to the present invention, blood concentration of the terpene administrated to a living body is determined with respect to time by the above method for determining blood concentration of a terpene, allowing analyzing pharmacokinetics of the terpen. For example,
Blood concentrations of a terpene in blood of a living body with the terpene administrated are determined at an interval of about 30 - 60 minutes after administrating, allowing analyzing absorption of the terpene into blood in detail. The method for analyzing pharmacokinetics according to the present invention is particularly suitable for pharmacokinetics of a terpene orally administrated to a living body. [0040]
Specifically, the method analyzing pharmacokinetics can be favorably applied for analyzing pharmacokinetics of a terpene administrated to mammalian such as human, dog, and mouse. [0041]
For example, corosolic acid (C30H48O4, molecular weight of 472), a triterpene, was administrated to a dog, and then the pharmacokinetics was analyzed by the above method, revealing for the first time that corosolic acid administrated can subject itself to no modification reaction to be taken into blood. [0042]
The terpene according to the present invention comprises its pharmacokinetics after administrated being analyzed by the above method for analyzing pharmacokinetics of a terpene. The terpene according to the present invention can have a readily optimized dose because the pharmaokinetics is accurately analyzed by the method for analyzing of the present invention, thus assuring high safety, for example, in oral administration. Consequently, the terpene according to the present invention can be favorably employed as an early insulin-secretion enhancer and a sugar- dependent early insulin-secretion enhancer for an anti-diabetes agent, an anti-obesity agent, a triglyceride reducer, and a cholesterol concentration reducer. [0043]
The uses of terpene are not specifically limited, and the terpene may be added to nutrient supplements and general foods and drinks such as drink, noodle, dry food, cake, bread, liquor, fat, and seasoning, or may be formed to use in powder, tablet, liquid, and paste as a drug, or vaporized to use by a spraying device, tobacco, or the like. [0044]
If the terpene is required to be soluble in water, it may be modified to a water-soluble derivative such as a glucoside, or may be used in a mixture with tannin, cellulose, chlorophyll, and the like which are extracted from plants to coexist. Alternatively, a solution of the terpene in a solvent such as water and alcohol may be mixed with a starch, an indigestible dextrin, or the like to form a mixture, from which the solvent is then removed to get a solid to use.
[Examples] [0045]
The present invention is described in more detail with respect to examples and comparative examples below. [0046] (Analysis of authentic product of corosolic acid by LC/MS/MS)
An authentic product of corosolic acid which is a triterpene was analyzed using "API3000" (product name of Applied Biosystems, Inc. U.S.A.) which is an apparatus of liquid chromatograph/tandem quadruple mass spectrometer (LC/MS/MS). [0047]
First, a solution of corosolic acid in acetonitrile/formic acid (100 ng/mL) was prepared as a sample. The sample was injected into the LC/MC/MC apparatus to draw the MS spectra of corosolic acid (Fig. 1). The ionization was done by the electro-spray ionization method (ESI). As shown in Fig. 1, a peak appeared at a position of m/z=472 which corresponds to the molecular weight of corosolic acid, confirming that the LC/MC/MC apparatus was able to detect corosolic acid. [0048] Samples having their respective corosolic acid contents of 1 ng/mL and 10 ng/mL were similarly analyzed. The peak intensities of peaks at m/z=472 (hereinafter referred to as the "parent peak") in the MS spectra were plotted against the concentration of corosolic acid to get plots, to which linear recurrence was applied to provide a calibration curve of corosolic acid. Figure 2 shows the plots and the calibration curve. The result confirmed that intensity of a parent peak can be measured to determine concentration of corosolic acid. [0049]
Preparation of calibration curve for corosolic acid in blood> Corosolic acid was added to a blood sampled from a dog
(beagle) to get a concentration of 10 ng/mL. A 500 μL aliquot of the blood was centrifuged (3,000 rpm for 20 minutes) to obtain a serum. A 400 μL of acetonitrile was added to 100 μL aliquot of the serum to mix them together in a vortex mixer, followed by centrifugal separation (15,000 rpm for 10 minutes) to remove proteins. The acetonitrile layer was enriched by blowing nitrogen gas, and subjected to liquid-liquid extraction with 300 μL of ethyl acetate. The ethyl acetate layer was then dried. The resulted solid was dissolved in 100 μL of eluent (95% aqueous methanol). Thus prepared solution was adopted as a sample for LC/MS/MS analysis. [0050]
The prepared sample and other samples prepared in the same procedure to be different only in concentration of corosolic acid, were analyzed by LC/MS/MS to draw a calibration curve shown in
Fig. 3. Based on the calibration curve, it was confirmed that blood concentration of corosolic acid can be determined within a concentration range of approximately 1 - 100 ng/mL. [0051] <Changes in blood concentration of corosolic acid with time>
(Example 1) To a dog (beagle) which was fasted for a day, corosolic acid and glucose were orally administrated by 20 mg/kg and 2 g/kg, respectively. Blood samples were collected from the dog before administration, 30, 60, 90, 120, 180, and 240 minutes after administration. The collected blood samples were treated with the same procedure as in the preparation of the calibration curve for blood concentration of corosolic acid, and analyzed by LC/MS/MS to determine the changes in blood concentration with time. [0052] Figure 4 shows the result. Blood samples collected (a) before the administration, (b) 30 minutes after the administration, (c) 60 minutes after, (d) 90 minutes after, (e) 120 minutes after, (f) 180 minutes after, and (g) 240 minutes after, provide their respective graphs in Figure 4. The horizontal axis of Fig. 4 shows retention time in the LC section, and the vertical axis shows intensity of peak at m/z=472. in MS. As seen in Fig. 4, the corosolic acid peak (near 12 minutes of retention time) was detected in the blood sample collected (c) 60 minutes after the administration, reached the maximum in the blood sample collected (f) 180 minutes after the administration, and afterward decreased.
The maximum blood concentration of corosolic acid was 20 μg/mL. [0053]
The above results showed that a triterpene such as corosolic acid administrated to a living body can subject itself to neither decomposition nor modification to be taken in the body. The phenomenon is contrast to a fact that most of sugar-dependent early insulin secretion enhancers made of protein including peptide is digested and decomposed. The molecular weight of corosolic acid is 472. For a compound having this magnitude of molecular weight, it is generally difficult to predict whether or not the compound is decomposed and then absorbed. Nevertheless,
LC/MS/MS could be employed to confirm, for the first time, the pharmacokinetics of corosolic acid. [0054] (Comparative Example 1) By the same procedure as in Example 1, the blood samples collected from a dog were centrifuged (3,000 rpm for 20 minutes) to obtain serum, 2 mL of which was divided into 200 μL aliquots to put in niicrotubes. A fourfold volume (800 μL) of acetonitrile was added to the each serum to remove proteins. After centrifuging (15,000 rpm for 5 minutes, 40C):, the resulted supernatants were collected in a sample bottle. All the acetonitrile was vaporized to prepare a concentrate. The concentrate was then dissolved in 500 μL of aqueous solution of 95% methanol by volume (similar to the developing solvent for HPLC) to prepare a sample. The sample was analyzed by HPLC. The HPLC condition was as follows:
• Column: Shodex Asahipak ODP-50 (polymer-base) 6D (250 x 10.0 mm LD.)
• Flow rate: 1.0 mL/min
• Detection: UV absorption (210 nm) [0055]
For all the samples prepared from the blood after administration of corosolic acid, no peak corresponding to corosolic acid (near 12 minutes of retention time) was observed.
[0056]
The above result showed that HPLC alone does not allow to determine blood concentration of corosolic acid, as seen in the case of Comparative Example 1, and thus can not analyze pharmacokinetics of corosolic acid at all, and that the analysis using the mass spectrometer, as seen in the case of Example 1, allows to determine the blood concentration of corosolic acid administrated to a living body at a high accuracy, and can analyze the pharmacokinetics of corosolic acid. Industrial Applicability
According to the present invention, there are provided a method for determining precisely the concentration of a terpene present in blood at a very low concentration, a method for analyzing pharmacokinetics of a terpene using the method, and a terpene which has a pharmacokinetics analyzed using the method.

Claims

1. A method for determining blood concentration of a terpene, comprising the terpene in blood being detected and determined by a mass spectrometer.
2. The method for determining blood concentration of a terpene as in claim 1, wherein the mass spectrometer is a quadruple mass spectrometer.
3. The method for determining blood concentration of a terpene as in claim 1 or 2, wherein the blood is fractionated by liquid chromatography to provide a fraction component, the terpene contained in which is detected by the mass spectrometer.
4. The method for determining blood concentration of a terpene as in claim 3, wherein the blood is centrifuged to provide a serum, an organic solvent extract from which is fractionated by the liquid chromatography.
5. The method for determining blood concentration of a terpene as in any of claims 1-4, wherein the terpene is corosolic acid or an analogous compound thereof.
6. The method for determining blood concentration of a terpene as in claim 5, wherein the analogous compound of corosolic acid is at least one triterpene selected from the group consisting of maslinic acid, ursolic acid, asiatic acid, oleanolic acid, tormentic acid, and 2α,19α-dihydroxy-3-oxo-urs-12-en-28-oic acid.
7. The method for determining blood concentration of a terpene as in any of claims 1-6, wherein the terpene is a triterpene obtained from Banaba, perilla, loquat, or guava.
8. A method for analyzing pharmacokinetics of a terpene, comprising the blood concentration of the terpene being determined with respect to time by the method for determining blood concentration of a terpene as in any of claims 1-7, wherein the terpene is present in blood of a living body with a terpene administrated.
9. A terpene, comprising being administrated to a living body to have a pharmacokinetics which is analyzed by the method for analyzing pharmacokinetics of a terpene as in claim 8.
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