Method for the quantitative determination of kerelactone ester compounds

Abstract

To provide a method for accurately determining the quantity of a khellactone ester compound in a plant-derived extract.SOLUTION: The method for accurately determining the quantity of a khellactone ester compound in a plant-derived extract includes the steps of: calculating the purity of a khellactone ester compound and a quantitative standard material from an NMR spectrum of an NMR sample solution containing one of the khellactone ester compound and the quantitive standard material and a purity calculation standard material; acquiring a relative mole sensitivity of the khellactone ester compound to the quantitive standard material; and calculating the content of the khellactone esters compound in an extract from a quantitative chromatogram of the quantitative sample solution containing a plant-derived extract and a standard chromatogram of the quantitative standard solution containing a quantitative standard material. The quantitative standard material is a material which shows the absorption maximum in the wavelength range of 305-335nm in a UV spectrum.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for quantifying kerelactone ester compounds contained in plant extracts. [Background technology]

[0002] Long-life grass (also known as Peucedanum japonicum Thunb.), a plant belonging to the genus Peucedanum in the family Apiaceae, is an ingredient that has been used in cooking and as a folk medicine in Okinawa Prefecture and other areas since ancient times. It is known to be effective in preventing and treating diabetes and resolving obesity (Patent Document 1), has cell-activating effects, antioxidant effects, and melanin production inhibitory effects (Patent Document 2), inhibits heparanase activity and is effective in preventing and improving wrinkles (Patent Document 3), promotes ceramide synthesis (Patent Document 4), and has a Sirtuin-1 gene activating effect (Patent Document 5). [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2003-26694 [Patent Document 2] Japanese Patent Publication No. 2004-26697 [Patent Document 3] International Publication No. 2009 / 123215 [Patent Document 4] Japanese Patent Publication No. 2005-194239 [Patent Document 5] Patent No. 3521155 [Overview of the project] [Problems that the invention aims to solve]

[0004] Foods with functional claims can be provided to consumers with a display of the effects of the ingredients contained in the food (for example, the "anti-obesity, blood glucose lowering, and urinary function improvement" effects of the above-mentioned Chomeiso extract). When providing food or beverages containing the above-mentioned Chomeiso extract to consumers as foods with functional claims, it is necessary to ensure the content of keryllactone ester compounds, which are the functional ingredients with the above-mentioned effects, and to appropriately control quality. However, a highly accurate method for quantifying keryllactone ester compounds contained in plant extracts such as Chomeiso extract and other Apiaceae plant extracts has not yet been established, and proposals for such quantification methods are desired.

[0005] Generally, a known method for quantifying a target component involves subjecting a sample containing the target component to liquid chromatography, determining the peak area from the resulting chromatogram, and creating a calibration curve for the target component. Creating this calibration curve requires a highly pure and reliable standard of the target component. However, such standards for kerelactone ester compounds found in plant extracts, such as those from the Apiaceae family (e.g., Chomeiso extract), are not readily available, making accurate quantification using a calibration curve difficult.

[0006] In view of the above issues, the present invention aims to provide a method for accurately quantifying kerelactone ester compounds contained in plant extracts. [Means for solving the problem]

[0007] To solve the above problems, the present invention provides a method for quantifying at least one kerelactone ester compound contained in an extract from a plant, comprising: a purity calculation step of calculating the purity of the kerelactone ester compound and a quantitative standard substance using quantitative nuclear magnetic resonance spectroscopy (qNMR); a relative molar sensitivity acquisition step of obtaining the relative molar sensitivity of the kerelactone ester compound with respect to the quantitative standard substance with which the purity has been calculated; and a quantification step of calculating the content of the kerelactone ester compound in the extract from a quantitative chromatogram obtained by treating a quantitative sample solution containing the extract with liquid chromatography, a standard chromatogram obtained by treating a quantitative standard solution containing the quantitative standard substance with which the purity has been calculated with liquid chromatography, or a standard mixed quantitative chromatogram obtained by treating a standard mixed quantitative sample solution containing the extract and the quantitative standard substance with which the purity has been calculated with liquid chromatography, wherein the relative molar sensitivity acquisition step is performed on the kerelactone ester compound. The quantitative step includes the steps of: preparing an LC sample solution containing each of the kerelactone ester compounds and the quantitative standard substance at at least one molar concentration; obtaining an LC chromatogram by treating the various LC sample solutions by liquid chromatography; calculating the slope of each calibration curve showing the relationship between the molar concentration of each of the kerelactone ester compounds and the quantitative standard substance in the various LC sample solutions based on their respective purity and the respective peak areas of the kerelactone ester compounds and the quantitative standard substance in the LC chromatogram; and calculating the ratio of the slope of the calibration curve of the kerelactone esters to the slope of the calibration curve of the quantitative standard substance as the relative molar sensitivity of the kerelactone ester compounds, wherein the quantitative step includes the peak area of ​​the at least one quantitative standard substance whose purity has been calculated, obtained from the at least one standard chromatogram obtained by treating the at least one quantitative standard solution containing the quantitative standard substance whose purity has been calculated at at least one molar concentration by liquid chromatography,Alternatively, the present invention provides a method for quantifying kerelactone ester compounds, comprising the steps of: creating a standard calibration curve showing the relationship between the peak area of ​​at least one of the quantitative standard substances whose purity has been calculated, obtained from a standard mixed quantitative chromatogram obtained by treating the standard mixed quantitative sample solution containing the extract and the quantitative standard substance whose purity has been calculated at a predetermined molar concentration, by liquid chromatography; and calculating the content of each of the kerelactone ester compounds in the extract from the peak area of ​​the kerelactone ester compound obtained from a quantitative chromatogram obtained by treating the quantitative sample solution containing the extract by liquid chromatography, or the peak area of ​​the kerelactone ester compound obtained from a standard mixed quantitative chromatogram obtained by treating the standard mixed quantitative sample solution containing the extract and the quantitative standard substance by liquid chromatography, the slope of the standard calibration curve, and the relative molar sensitivity, wherein the quantitative standard substance is a substance that exhibits an absorption maximum at a wavelength of 305 to 335 nm in the UV spectrum. ,

[0008] As the aforementioned quantitative standard substance, the compound shown in the following chemical structural formula (I) can be used.

[0009] [ka] In the above chemical structural formula (I), R1 represents hydrogen, an alkyl group having 1 to 5 carbon atoms, or an acetyl group; R2 represents hydrogen or a hydroxyl group; R3 represents hydrogen or an alkyl group having 1 to 5 carbon atoms; and R4 represents hydrogen or a 3-methyl-2-butenyl group.

[0010] As the aforementioned quantitative standard substance, 7-ethoxy-4-methylcoumarin, represented by the following chemical formula (II), can be used.

[0011] [ka]

[0012] In the quantitative analysis step, the content of the kerelactone ester compound can be calculated based on the following formulas (1) and (2).

[0013]

number

[0014]

number

[0015] In the above formulas (1) and (2), Ct K This represents "the content of the kerelactone ester compounds in the extract," and C K This represents "the molar concentration of the kerelactone ester compound in the quantitative sample solution or standard mixed quantitative sample solution," and M K represents "the molecular weight of the kerelactone ester compound," and C E represents "the concentration of the extract in the quantitative sample solution or standard mixed quantitative sample solution," S represents "the slope of the standard calibration curve when the standard calibration curve is prepared using the quantitative standard substance," and a K This represents "the peak area of ​​the kerelactone ester compound in the quantitative chromatogram or standard mixture quantitative chromatogram," and RMS K This represents the "relative molar sensitivity of the kerelactone ester compounds."

[0016] In the purity calculation step, the purity of the kerelactone ester compound and the quantitative standard substance can be measured by quantitative nuclear magnetic resonance spectroscopy (qNMR), and by the internal standard method. 1 H-qNMR measurement can be used.

[0017] As the plant, a plant belonging to the Apiaceae, Rubiaceae or Rosaceae family can be used, a plant belonging to the genus Glehnia of the Apiaceae family can be used, and Houttuynia cordata can be used.

[0018] The present invention can be used when the khellactone ester compounds are at least one selected from hyuganin D, cis-3'-acetyl-4'-tigloylkhellactone, peucedanocoumarin III, isosamidin, trans-3'-acetyl-4'-senecioylkhellactone, and pteryxin.

Advantages of the Invention

[0019] According to the present invention, a method for accurately quantifying khellactone ester compounds contained in an extract from a plant can be provided.

Modes for Carrying Out the Invention

[0020] Embodiments of the present invention will be described in detail. The method for quantifying khellactone ester compounds according to the present embodiment is a method for quantifying at least one khellactone ester compound contained in an extract from a plant. The quantification method according to the present embodiment includes a purity acquisition step, a relative molar sensitivity acquisition step, and a quantification step.

[0021] The khellactone ester compounds that can be quantified in the quantification method according to the present embodiment include, for example, hyuganin D having the following chemical structure (the first khellactone ester compound, chemical structural formula (A), C 20 H 22O7, Molecular weight: 374.38), cis-3'-acetyl-4'-tigloylkhellactone (a secondary kerrelactone ester compound, chemical structure (B), C 21 H 22 O7, Molecular weight: 386.40), peucedanocoumarin III (tertiary kerelactone ester compound, chemical structural formula (C), C 21 H 22 O7, Molecular weight: 386.40), isosamidin (a quaternary keratone ester compound, chemical structure (D), C 21 H 22 O7, Molecular weight: 386.40), trans-3'-acetyl-4'-senecioylkhellactone (a 5th kerrelactone ester compound, chemical structural formula (E), C 21 H 22 O7 (molecular weight: 386.40) and pterixin (a 6th kerelactone ester compound, chemical structure (F), C) 21 H 22 These are six types of kerelactone ester compounds (O7, molecular weight: 386.40).

[0022] [ka]

[0023] [ka]

[0024] [ka]

[0025] [ka]

[0026] [ka]

[0027] [ka]

[0028] The above plant extract may contain at least one of the above-mentioned first to sixth kerelactone ester compounds. The plant extract may also contain a different type of kerelactone ester compound along with the first to sixth kerelactone ester compounds, provided that the peaks of the different type of kerelactone ester compound and other compounds do not overlap with the peaks of the first to sixth kerelactone ester compounds in the quantitative chromatogram obtained in the quantitative process described later.

[0029] The standard substance used for quantification can be any substance that has an absorption maximum at substantially the same wavelength as the 1st to 6th keralactone ester compounds to be quantified. For example, any substance that shows an absorption maximum at a wavelength of 305 to 335 nm in the UV spectrum is acceptable. Preferably, the standard substance is one whose peak appears before the peaks of the 1st to 6th keralactone ester compounds in the quantitative chromatogram obtained in the quantitative process described later (i.e., a substance with a shorter retention time than the 1st to 6th keralactone esters). Specifically, the substance shown in the following chemical structural formula (I) can be used.

[0030] [ka]

[0031] In the above chemical structural formula (I), R1 represents hydrogen, an alkyl group having 1 to 5 carbon atoms, or an acetyl group; R2 represents hydrogen or a hydroxyl group; R3 represents hydrogen or an alkyl group having 1 to 5 carbon atoms; and R4 represents hydrogen or a 3-methyl-2-butenyl group.

[0032] Examples of substances represented by the above chemical structural formula (I) include 7-ethoxy-4-methylcoumarin,C shown in the following chemical structural formula (II). 12 H 12 O3, molecular weight: 204.22), 7-methoxy-4-methylcoumarin (7-methoxy-4-methylcoumarin, C) shown in the chemical structural formula (III) below. 11 H 10 O3, molecular weight: 190.20), 7-methoxy-8-(3-methyl-2-butenyl)coumarin (also known as osthol, C3, shown in the chemical structural formula (IV) below) 15 H 16 O3, molecular weight: 244.29), 7-hydroxycoumarin (also known as umbrellaiferone, C9H6O3, molecular weight: 162.14), shown in the following chemical structure (V), and 7-ethoxycoumarin (C9H6O3, C9H6O3), shown in the following chemical structure (VI). 11 H 10 O3, molecular weight: 190.20), 7-acetoxy-4-methylcoumarin (7-acetoxy-4-methylcoumarin, C) shown in the following chemical structure formula (VII). 12 H 10 O4, molecular weight: 218.21), 5,7-dihydroxy-4-methylcoumarin (C4, molecular weight: 218.21), shown in the following chemical structural formula (VIII). 10 Examples include H8O4 (molecular weight: 192.17). Among these, 7-ethoxy-4-methylcoumarin (7-ethoxy-4-methylcoumarin, C) shown in the following chemical structural formula (II) is an example. 12 H 12 It is preferable to use O3 (molecular weight: 204.22) as a standard substance for quantification.

[0033] [ka]

[0034] [ka]

[0035] [ka]

[0036] [ka]

[0037] [ka]

[0038] [ka]

[0039] [ka]

[0040] Plants belonging to the genus Peucedanum in the family Apiaceae can be used as extraction materials. Examples of such plants include Chomeiso (also known as Botanbofu, scientific name: Peucedanum japonicum Thunb.). .The plants used as raw materials for extraction are not limited to the long-life grass mentioned above, but any plant containing at least one of the 1st to 6th kerulactone ester compounds mentioned above is acceptable. The plants include, for example, Angelica cartilaginomarginata, Angelica cartilaginomarginata var. lehmanniana, Ligusticum brachylobum, Ligusticum calophlebicum, Ligusticum elatum, Ligusticum involucratum, Musineon divaricatum, Musineon hookeri, Mutellina purpurea, Peucedanum decursivum, Peucedanum formosanum, Peucedanum harry-smithii, Peucedanum harrysmithii var. subglabrum, Peucedanum praeruptorum, Peucedanum terebinthaceum, Peucedanum verticillare, Peucedanum zhongdianensis, Phlojodicarpus sibiricus, Pleurospermum govanianum, Prionosciadium thapsoides, Pteryxia terebinthina, Pteryxia terebinthina var. terebinthina, Pteryxia terebinthina var.This could include plants of the Apiaceae family such as *California californica*, *Seseli condensatum*, *Seseli cuneifolium*, *Seseli eriocephalum*, *Seseli libanotis*, *Seseli mairei*, *Seseli mucronatum*, *Seseli seravschanicum*, *Xyloselinum leonidii*, and *Zizia aptera*; plants of the Rubiaceae family such as *Morinda citrifolia*; or plants of the Smilaxaceae family such as *Smilax riparia*.

[0041] There are no particular restrictions on the part of the plant used for extraction, and it can be selected as appropriate depending on the purpose. For example, when using Chomeiso (a type of plant in the Apiaceae family) as the extraction material, the fruit, leaves, stems, flowers, roots, etc. can be used, but among these, the roots are preferred.

[0042] Plant extracts can be prepared by drying the raw material, then either using it as is or by grinding it using a crushing machine, and finally subjecting it to extraction with an extraction solvent. The drying of the raw material may be done in the sun or using a commonly used drying machine.

[0043] As the extraction solvent, it is preferable to use a polar solvent, such as water or a hydrophilic organic solvent. In the extraction process, it is preferable to use these extraction solvents individually or in combination of two or more, at room temperature or below the boiling point of the solvent.

[0044] Water that can be used as an extraction solvent may include pure water, tap water, well water, mineral water, mineral water, hot spring water, spring water, fresh water, etc., as well as water that has undergone various treatments. Treatments applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, etc. Therefore, in this embodiment, water that can be used as an extraction solvent also includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate-buffered physiological saline, etc.

[0045] Examples of hydrophilic organic solvents that can be used as extraction solvents include lower aliphatic alcohols having 1 to 5 carbon atoms, such as methanol, ethanol, propyl alcohol, and isopropyl alcohol; lower aliphatic ketones, such as acetone and methyl ethyl ketone; and polyhydric alcohols having 2 to 5 carbon atoms, such as 1,3-butylene glycol, propylene glycol, and glycerin.

[0046] When using a mixture of two or more polar solvents as an extraction solvent, the mixing ratio can be adjusted as appropriate. For example, when using a mixture of water and a lower aliphatic alcohol, it is preferable to mix 1 to 90 parts by volume of the lower aliphatic alcohol with 10 parts by volume of water; when using a mixture of water and a lower aliphatic ketone, it is preferable to mix 1 to 40 parts by volume of the lower aliphatic ketone with 10 parts by volume of water; and when using a mixture of water and a polyhydric alcohol, it is preferable to mix 10 to 90 parts by volume of the polyhydric alcohol with 10 parts by volume of water.

[0047] The extraction process is not particularly limited as long as it allows the first to sixth keralactone ester compounds contained in the raw material to be eluted into the extraction solvent, and can be carried out according to conventional methods. For example, the raw material can be immersed in an extraction solvent in an amount (by mass ratio) of 5 to 15 times the amount of the raw material, and the soluble components can be extracted at room temperature or under reflux heating. After extraction, the extract can be obtained by filtering to remove the extraction residue. A paste-like concentrate can be obtained by distilling off the solvent from the obtained extract, and a dried product can be obtained by further drying this concentrate.

[0048] The method for isolating and purifying the first to sixth kerelactone ester compounds from the extract, concentrate, or dried extract obtained as described above is not particularly limited and can be carried out by conventional methods. For example, the extract can be dissolved in a mobile phase and subjected to column chromatography using a porous material such as silica gel or alumina, or a porous resin such as styrene-divinylbenzene copolymer or polymethacrylate, to recover fractions containing each of the first to sixth kerelactone ester compounds. In this case, the mobile phase can be appropriately selected depending on the stationary phase used, but for example, when separating the extract by column chromatography using a porous synthetic adsorbent such as Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation), which is a porous synthetic adsorbent resin, the mobile phase can be water, methanol, or a mixture thereof. Furthermore, fractions containing each of the first to sixth kerelactone ester compounds obtained by column chromatography may be purified using any organic compound purification method, such as preparative column chromatography using octadecylsilylated silica gel, organic silica hybrid support, phenylsilylated silica gel, adamantylsilylated silica gel, triacontylsilylated silica gel, polyvinyl alcohol (PVA)-based polymer support, etc. as packing materials, recrystallization, sublimation purification, or liquid-liquid counterflow extraction.

[0049] [Purity acquisition process] In the process of obtaining the purity of the first to sixth kerlactone ester compounds and quantitative standard substances obtained as described above by quantitative nuclear magnetic resonance spectroscopy (qNMR), the measurement method for quantitative nuclear magnetic resonance spectroscopy (qNMR) is not particularly limited and can be carried out by a conventional method. The measurement method for quantitative nuclear magnetic resonance spectroscopy (qNMR) is as follows: 1 In addition to the internal standard method, H-qNMR measurement methods include external standard methods such as ERETIC, PULCON, QUANTAS, and methods using double sample tubes, among others. 13 Examples include 13C-qNMR measurements. Purity can be obtained using any quantitative nuclear magnetic resonance (qNMR) spectroscopy (QNMR) measurement method capable of obtaining accurate purity.

[0050] In this embodiment, DSS-d6 was used as the standard substance for purity calculation, and the internal standard method was used. 1 The following will be given as an example of a method for measuring purity using individual signals derived from the first to sixth kerlactone ester compounds and the quantitative standard substance by H-qNMR measurement, but the method is not limited to this embodiment. For example, the first to sixth kerlactone ester compounds and the quantitative standard substance 1 Purity may also be measured by 1H-qNMR, quantitatively calculating any multiple signals obtained from each substance individually, and obtaining the average value. In this case, the signals used for quantitative calculation should be those where the signals of impurities do not overlap with the signals of the first to sixth kerelactone esters and the quantitative standard substance. Preferably, when comparing the purity values ​​calculated using each signal, any signals that deviate are considered to appear due to the influence of impurities, measurement, data processing, etc., so the average value should be calculated after excluding such deviating signals. In particular, if the purity of the first to sixth kerelactone ester compounds and the quantitative standard substance to be measured is expected to be less than 98%, care should be taken to check for outliers, and outliers should be appropriately excluded.

[0051] Each of the first to sixth kerelactone ester compounds described above, along with a purity standard substance of known purity (e.g., DSS-d6), is dissolved in a solvent (e.g., DMSO-d6) to prepare the first to sixth NMR sample solutions. Similarly, a quantitative standard substance and a purity standard substance of known purity (e.g., DSS-d6), along with a solvent (e.g., DMSO-d6), is dissolved in a solvent (e.g., DMSO-d6) to prepare the seventh NMR sample solution. The kerelactone ester compounds used to prepare the first to sixth NMR sample solutions may be commercially available pure products or purified products obtained by isolation and purification from plant extracts. In this embodiment, the first to seventh NMR sample solutions containing the first to sixth kerelactone esters and quantitative standard substances, along with the purity standard substance, are prepared, and the purity is calculated using the so-called internal standard method. However, the method is not limited to this. For example, in addition to the first to seventh NMR sample solutions containing the first to sixth kerelactone esters and the quantitative standard substance, respectively, which are the target of purity calculation, an eighth NMR sample solution containing the purity calculation standard substance may be prepared, and the purity may be calculated using the so-called external standard method.

[0052] Next, each of the NMR sample solutions from NMR 1 to 7 is 1 The 1st to 7th NMR spectra are obtained by quantitative NMR treatment using H-qNMR measurement, and the purities P1 to P6 of the 1st to 6th kerelactone ester compounds are calculated from each NMR spectrum using the following formulas (3-1) to (3-6), and the purity P of the standard substance for quantification is determined. S This is calculated using the following formula (4).

[0053]

number

[0054]

number

[0055] In the above formulas (3-1) to (3-6) and formula (4), P1 to P6 represent the "purity of the first to sixth kerelactone ester compounds," and P S This represents the "purity of the standard substance for quantitative analysis," and P IS A represents the "purity of the standard substance used for purity calculation (e.g., DSS-d6)", A1 to A6 represent the "signal area of ​​the 1st to 6th kerelactone ester compounds in the 1st to 6th NMR spectra", and A S represents the "signal area of ​​the quantitative standard in the 7th NMR spectrum," and A IS H represents the signal area of ​​the standard substance used for purity calculation (e.g., DSS-d6) in each NMR spectrum, H1~H6 represents the number of protons derived from the signal areas A1~A6 of the 1st~6th kerelactone ester compounds in the 1st~6th NMR spectra, H S This is "Signal area A of the quantitative standard in the 7th NMR spectrum S It represents the number of protons derived from H IS This refers to the signal area A of the standard substance used for purity calculation (e.g., DSS-d6) in each NMR spectrum. IS M represents the number of protons derived from, and M1 to M6 represent the molecular weight of the first to sixth kerelactone ester compounds. S represents the "molecular weight of the standard substance for quantitative analysis," and M IS W represents the molecular weight of the standard substance used for purity calculation (e.g., DSS-d6), W1 to W6 represent the weighed values ​​(mg) of the first to sixth keralactone ester compounds during the preparation of the first to sixth NMR sample solutions, and W S This represents the "weighing value (mg) of the quantitative standard substance during the preparation of the 7th NMR sample solution," and W IS This represents the "weighed value (mg) of the standard substance used for purity calculation (e.g., DSS-d6) during the preparation of the 1st to 7th NMR sample solutions."

[0056] Generalizing the above formulas (3-1) to (3-6), they can be expressed by the following formula (3).

[0057]

number

[0058] In the above formula (3), P K This represents the "purity of kerelactone ester compounds," and P IS This represents the "purity of the standard substance used for calculating purity," and A K This represents the "signal area in the NMR spectrum of kerelactone ester compounds," and A IS H represents the signal area in the NMR spectrum of the standard substance used for purity calculation. K "Signal area A in the NMR spectrum of kerelactone ester compounds" K It represents the number of protons derived from H IS This refers to "Signal area A in the NMR spectrum of a standard substance used for purity calculation." IS It represents the number of protons derived from M K This represents the "molecular weight of kerelactone ester compounds," and M IS This represents the "molecular weight of the standard substance used for purity calculation," and W K This represents the "weighed value of kerelactone ester compounds in NMR sample solution," and W IS This represents the "weighed value of the standard substance used to calculate purity in the NMR sample solution."

[0059] Signal areas A1-A6,A of each kerlactone ester compound, quantitative standard, and purity standard (e.g., DSS-d6) in each NMR spectrum. S ,A IS and the signal areas A1~A6,A S ,A IS Proton numbers H1~H6,H S ,H IS This can be calculated, for example, using NMR analysis software (VnmrJ version 3.2, manufactured by Varian).

[0060] [Relative Molar Sensitivity Acquisition Process] Each of the first to sixth kerelactone ester compounds whose purity has been obtained as described above is mixed with the quantitative standard substance whose purity has been obtained as described above and dissolved in a predetermined solvent (for example, a mixed solvent of water and methanol) to prepare the first to sixth LC sample solutions containing each kerelactone ester compound. Each of the first to sixth LC sample solutions only needs to be prepared at least one (for example, six) molar concentration. In this embodiment, the method for preparing the first to sixth LC sample solutions containing each of the first to sixth kerelactone ester compounds and the quantitative standard substance is described as an example, but the embodiment is not limited to this. For example, three types of LC sample solutions (first-second to third-second LC sample solutions) containing two compounds selected from the first to sixth kerelactone ester compounds may be prepared. In this case, the two kerelactone ester compounds contained in each LC sample solution only need to be those whose peaks do not overlap in the LC chromatogram obtained by subjecting the LC sample solution to liquid chromatography. Preferably, the LC sample solution can be prepared by combining two kerelactone ester compounds whose peaks are not adjacent to each other in the LC chromatogram obtained by subjecting a sample solution containing all of the first to sixth kerelactone ester compounds to liquid chromatography. For example, a first-second LC sample solution containing the first kerelactone ester compound and the fourth kerelactone ester compound, a second-second LC sample solution containing the second kerelactone ester compound and the fifth kerelactone ester compound, and a third-second LC sample solution containing the third kerelactone ester compound and the sixth kerelactone ester compound may be prepared. Alternatively, a seventh-third LC sample solution may be prepared by dissolving a pure quantitative standard substance in a predetermined solvent, along with the first-third to sixth-third LC sample solutions obtained by dissolving each of the purified first to sixth kerelactone ester compounds in a predetermined solvent.

[0061] Next, LC chromatograms of each of the 1st to 6th LC sample solutions at various molar concentrations are obtained by subjecting them to liquid chromatography, and the peak areas of the 1st to 6th keralactone ester compounds and the quantitative standard substances in each LC chromatogram are determined. The liquid chromatography conditions (e.g., liquid chromatograph apparatus, stationary phase, mobile phase, etc.) are the same for each LC sample solution.

[0062] Then, regression analysis was performed with the peak areas of the 1st to 6th keralactone ester compounds and the quantitative standard substance as dependent variables, and the molar concentrations of the 1st to 6th keralactone ester compounds based on the above purity and the molar concentrations of the quantitative standard substance based on the above purity in each of the 1st to 6th LC sample solutions as independent variables, with the constant term set to 0. Regression lines (1st to 6th calibration curves) for each keralactone ester compound and a regression line (standard calibration curve) for the quantitative standard substance were created, and the slopes S1 to S6 of each regression line (1st to 6th calibration curve) and the slope S of the regression line (standard calibration curve) were determined. S The relative molar sensitivities RMS1 to RMS6 for each of the 1st to 6th keralactone ester compounds are calculated as the slope S of the standard calibration curve, which is the regression line of the quantitative standard substance. S The ratios of the slopes S1 to S6 of the 1st to 6th calibration curves to the given value are calculated using the following formulas (a) to (f). RMS1 = S1 / S S …(a) RMS2 = S2 / S S …(b) RMS3 = S3 / S S …(c) RMS4 = S4 / S S …(d) RMS5 = S5 / S S …(e) RMS6 = S6 / S S …(f)

[0063] [Quantitative process] A sample solution for quantification is prepared by dissolving a plant extract in a predetermined solvent (e.g., a mixed solvent of water and methanol). Simultaneously, a standard solution for quantification containing the standard substance at a predetermined molar concentration is prepared by dissolving a standard substance for quantification, whose purity has been determined, in a predetermined solvent (e.g., a mixed solvent of water and methanol).

[0064] The plant extract used in preparing the quantitative sample solution may be obtained by extraction from the raw material in the same manner as the plant extract that can be used in the purity acquisition step described above, or it may be obtained by purification using a conventionally known method. Alternatively, the quantitative sample solution may be prepared using food or beverages containing the plant extract. When preparing the quantitative sample solution using such food or beverages, for example, the food or beverage may be dissolved or dispersed in the solvent (e.g., a mixed solvent of water and methanol), and the filtrate obtained by filtering with a filter or the like may be used to prepare the quantitative sample solution.

[0065] Furthermore, if it is difficult to prepare the quantitative sample solution by dissolving or dispersing plant extracts or food and beverages containing plant extracts in the above solvent, the sample pretreatment method is not particularly limited and can be carried out by conventional methods. Examples include extraction by solvent (e.g., water, alcohols, acetonitrile, ethyl acetate, n-hexane, chloroform, etc., either alone or in mixtures), supercritical extraction (e.g., supercritical carbon dioxide), subcritical extraction (e.g., subcritical water), Soxhlet extraction, steam distillation, precipitation using solvents of different polarities (e.g., alcohol precipitation, acetonitrile precipitation), liquid-liquid partitioning, enzymatic treatment, treatment with acid or alkali, solid-phase extraction (SPE), holding liquid extraction (SLE), resin treatment, ultrafiltration, salting out, dialysis, etc. Using any of these analytical pretreatment methods, a fraction containing kerelactone ester compounds can be prepared, dissolved or dispersed in the above solvent (e.g., a mixed solvent of water and methanol), and the filtrate filtered through a filter or the like can be prepared as the quantitative sample solution.

[0066] When preparing the quantitative sample solution by dissolving or dispersing a plant extract or food or beverage containing a plant extract in the solvent, or when preparing the quantitative sample solution using any of the above-mentioned pre-analytical treatments, a quantitative standard substance whose purity has been determined may be added at a predetermined molar concentration to prepare a standard mixed quantitative sample solution.

[0067] A quantitative chromatogram is obtained by subjecting the quantitative sample solution to liquid chromatography, and the peak areas a1 to a6 of the 1st to 6th kerelactone ester compounds in the quantitative chromatogram are determined. At the same time, a standard chromatogram is obtained by subjecting the quantitative standard solution to liquid chromatography, and the peak area a of the quantitative standard substance in the standard chromatogram is determined. S The following can also be determined. The liquid chromatography treatment conditions (e.g., liquid chromatograph apparatus, stationary phase, mobile phase, etc.) for the quantitative sample solution and the quantitative standard solution should be the same.

[0068] A chromatogram for the standard mixture quantification is obtained by subjecting the sample solution for standard mixture quantification to liquid chromatography, and the peak areas a1 to a6 of the 1st to 6th keralactone ester compounds and the peak area a of the quantification standard substance in the chromatogram are determined. S You may also request this.

[0069] Peak area a in a standard chromatogram or standard chromatogram for quantitative analysis of mixtures S A regression analysis was performed with the dependent variable being the molar concentration of the quantitative standard substance based on its purity in the quantitative standard solution as the independent variable, and the constant term set to 0. A regression line (quantitative standard calibration curve) for the quantitative standard substance was created, and the slope S of the regression line (quantitative standard calibration curve) was calculated. S The following calculation is performed. Assuming that a regression line (quantitative calibration curve for the first to sixth kerelactone ester compounds) similar to that for the quantitative standard substance is created for each of the first to sixth kerelactone ester compounds, the slopes S1' to S6' of the said regression line (quantitative calibration curve for the first to sixth kerelactone esters) are expressed by the following equations (5-1) to (5-6).

[0070]

number

[0071] The processing conditions for liquid chromatography (e.g., liquid chromatograph apparatus, stationary phase type, mobile phase type, etc.) used when determining relative molar sensitivities RMS1 to RMS6 are the same for all LC sample solutions from the 1st to the 6th. The slope S of the standard calibration curve related to the quantitative standard substance is also determined. S The relative molar sensitivities RMS1 to RMS6, expressed as the ratio of the slopes S1 to S6 of the 1st to 6th calibration curves related to the 1st to 6th keralactone ester compounds, are considered to remain constant even if the processing conditions of the liquid chromatography treatment change. Therefore, the slope S of the quantitative calibration curve S By multiplying ' by the respective relative molar sensitivities RMS1 to RMS6, it is possible to accurately calculate the slopes S1' to S6' of the regression lines (quantitative calibration curves for the first to sixth quantitative substances) for each of the first to sixth keralactone ester compounds, assuming that regression lines (quantitative calibration curves) similar to those for the quantitative standard substances have been created for each of the first to sixth keralactone ester compounds.

[0072] Also, the slope S S ', the peak areas a1 to a6 of the 1st to 6th kerlactone ester compounds in the quantitative chromatogram or standard mixed quantitative chromatogram, and the peak area a of the quantitative standard substance in the standard chromatogram or standard mixed quantitative chromatogram. S Therefore, the respective content Ct1 to Ct6 of the 1st to 6th keralactone ester compounds in the plant extract is calculated using the following formulas (1-1) to (1-6) and formulas (2-1) to (2-6).

[0073]

number

[0074]

number

[0075] In the above formulas (1-1) to (1-6) and formulas (2-1) to (2-6), Ct1 to Ct6 represent the "content of each of the first to sixth keralactone ester compounds per 1 g of plant extract (g / g)", C1 to C6 represent the "molar concentration of each of the first to sixth keralactone ester compounds in the quantitative sample solution (mol / L)", M1 to M6 represent the "molecular weight of each of the first to sixth keralactone ester compounds", and C E represents the "concentration of the plant extract in the quantitative sample solution (mg / mL)," and a1 to a6 represent the "peak areas of the first to sixth kerelactone ester compounds in the quantitative chromatogram."

[0076] As described above, the content of each of the first to sixth kerelactone ester compounds can be calculated. By calculating the sum of the calculated content of each of the first to sixth kerelactone ester compounds, the kerelactone ester compounds in the plant extract can be quantified with high accuracy.

[0077] Generalizing the above formulas (1-1) to (1-6) and (2-1) to (2-6), they can be expressed as follows: formulas (1) and (2).

[0078]

number

[0079]

number

[0080] In the above formulas (1) and (2), Ct K This represents the "content of keryllactone ester compounds in plant extracts," and C K This represents the "molar concentration of kerelactone ester compounds in the quantitative sample solution," and M K This represents the "molecular weight of kerelactone ester compounds," and C Erepresents "the concentration of the extract of the Umbelliferae plant in the sample solution for quantification", S represents "the slope of the standard calibration curve when a standard calibration curve is created using the standard substance for quantification", and a K represents "the peak area of the kelulactone ester compounds in the spectrum for quantification", and RMS K represents "the relative molar sensitivity of the kelulactone ester compounds".

[0081] As described above, in the present embodiment, a standard substance for quantification containing a chemical structure common to the kelulactone ester compounds contained in the plant extract is used, and the standard substance for quantification is quantified using a calibration curve. The first to sixth kelulactone ester compounds are quantified by calculation using the relative molar sensitivity RMS expressed as the relationship with the standard substance for quantification. Therefore, according to the present embodiment, the content of at least one kind of kelulactone ester compound contained in the plant extract can be easily and accurately determined.

[0082] The embodiments described above are described to facilitate the understanding of the present invention and are not described to limit the present invention. Therefore, each element disclosed in the above embodiments is intended to include all design changes and equivalents belonging to the technical scope of the present invention.

[0083] In the above embodiment, when the first to sixth kelulactone ester compounds are commercially available pure products and their purity is known, the purity acquisition step of calculating the purity of each kelulactone ester compound may not be performed, and only the relative molar sensitivity needs to be acquired. Needless to say, the purity of each kelulactone ester compound may also be calculated using a commercially available pure product with a known purity.

Example

[0084] Hereinafter, the present invention will be described in more detail by giving test examples and the like, but the present invention is not limited to the following test examples and the like.

[0085] 〔Test Example 1〕Liquid chromatography analysis of the standard substance for quantification Various quantitative standard substances shown by the above chemical structural formulas (II) to (VIII) were dissolved in methanol and filtered through a membrane filter to prepare various test solutions.

[0086] Various test solutions were subjected to liquid chromatography under the following conditions to obtain LC chromatograms. <Liquid chromatography conditions> Liquid chromatograph system: 1250 Infinity II Prime LC (manufactured by Agilent Technologies) Detector: G7117C (diode array, detection wavelength: 320nm, manufactured by Agilent Technologies) Injection volume: 15μL Column: SunShell C30 (2.6μm, 3.0mm x 100mm, manufactured by Chromanic Technologies) Column temperature: 40℃ Mobile phase: (A) Water: Trifluoroacetic acid: Methanol: Tetrahydrofuran = 400:0.2:50:55 (volume ratio) (B) Acetonitrile 0-50 min: (B) 0% 50-55 min: (B) 95% 55-70 min: (B) 0% Flow rate: 1.0mL / min Analysis time (Run time):70min

[0087] From the UV spectra obtained by the diode array detector, it was confirmed that all of the various standard substances for quantification represented by the above chemical structural formulas (II) to (VIII) have absorption maxima at wavelengths of 305 to 335 nm. From the obtained LC chromatograms, it was confirmed that all of the various standard substances for quantification represented by the above chemical structural formulas (II) to (VIII) can be used as standard substances for quantification. Among the above standard substances for quantification, 7-ethoxy-4-methylcoumarin represented by the chemical structural formula (II) is moderately retained by the stationary phase in the liquid chromatography process in the quantification step, has a shorter retention time than the retention times of the first to sixth khellactone ester compounds, does not overlap with the peaks of the first to sixth khellactone ester compounds, and has an absorption maximum at wavelengths of 305 to 335 nm similar to that of the first to sixth khellactone ester compounds. Therefore, it was inferred that it is more suitable as a standard substance for quantification.

[0088] [Test Example 2] Measurement of the purity of khellactone ester compounds and standard substances for quantification (1) Preparation of khellactone ester compounds and standard substances for quantification Six types of khellactone ester compounds (hyuganin D (hyuganin D, the first khellactone ester compound, chemical structural formula (A), C 20 H 22 O7, molecular weight: 374.38), cis-3'-acetyl-4'-tigloylkhellactone (cis-3'-acetyl-4'-tigloylkhellactone, the second khellactone ester compound, chemical structural formula (B), C 21 H 22 O7, molecular weight: 386.40), peucedanocoumarin III (peucedanocoumarin III, the third khellactone ester compound, chemical structural formula (C), C 21 H 22 O7, molecular weight: 386.40), isosamidin (isosamidin, the fourth khellactone ester compound, chemical structural formula (D), C 21 H 22O7, Molecular weight: 386.40), trans-3'-acetyl-4'-senecioylkhellactone (a 5th kerrelactone ester compound, chemical structural formula (E), C 21 H 22 O7 (molecular weight: 386.40) and pterixin (a 6th kerelactone ester compound, chemical structure (F), C) 21 H 22 O7, molecular weight: 386.40)) and the quantitative standard substance shown in the above chemical structural formula (II) (7-ethoxy-4-methylcoumarin (C 12 H 12 We prepared each of the following: O3 (molecular weight: 204.22).

[0089] 7-ethoxy-4-methylcoumarin was prepared as a standard substance for quantification using a commercially available reagent (manufactured by Tokyo Chemical Industry Co., Ltd.). The first to sixth keralactone ester compounds were isolated and purified from the roots of *Chomeiso* (a type of grass) as follows.

[0090] 5 kg of the roots of Chomeiso were heated and extracted at 80°C with 10 times the volume of 50% by mass ethanol to obtain an extract (820 g). This extract (750 g) was subjected to a ion HP-20 (Mitsubishi Chemical Corporation) column, and eluted in the order of water, 30% by mass methanol, 60% by mass methanol, and methanol to obtain a methanol eluate (117 g). The methanol eluate (97.5 g) was fractionated by silica gel column, ODS column, and preparative HPLC (column: YMC-Triart C18 (YMC Corporation), methanol:water = 65:35) to obtain a fraction containing the first to third keratolactone ester compounds and a fraction containing the fourth to sixth keratolactone ester compounds.

[0091] The fractions containing the first to third kerelactone ester compounds were subjected to preparative HPLC (column: YMC-Triart C18 (YMC Corporation), acetonitrile:water = 40:60) to obtain crude purified products of the first to third kerelactone ester compounds. The crude purified products were subjected to preparative HPLC (column: Kinetex C18 (Phenomenex), methanol:water = 60:40) and recycled preparative HPLC (column: JAIGEL-GS310 (Nippon Analytical Industry Co., Ltd.) x 2, MeOH) to isolate 59 mg of the first kerelactone ester compound. Furthermore, the crude purified products were subjected to recycled preparative HPLC (column: JAIGEL-GS310 (Nippon Analytical Industry Co., Ltd.) x 2, MeOH) and preparative HPLC (column: Capcell Pak ADME-HR (Osaka Soda Co., Ltd.), acetonitrile:water = 50:50) to isolate 93 mg of the second kerelactone ester compound. Furthermore, the crude product was subjected to recycled preparative HPLC (column: JAIGEL-GS310 (manufactured by Nippon Analytical Industry Co., Ltd.) x 2, MeOH) and preparative HPLC (column: Capcell Pak ADME-HR (manufactured by Osaka Soda Co., Ltd.), acetonitrile:water = 50:50) to isolate 60 mg of the tertiary kerelactone ester compound.

[0092] The fraction containing the 4th to 6th kerelactone ester compounds was subjected to preparative HPLC (column: Develosil RPAQUEOUS-AR-5 (Nomura Chemical Co., Ltd.), acetonitrile:methanol:water = 40:10:50) to obtain a crude product containing the 4th and 5th kerelactone ester compounds, and 130 mg of the 6th kerelactone ester compound. The crude product was subjected to recycled preparative HPLC (column: JAIGEL-GS310 (Nippon Analytical Industry Co., Ltd.) x 2, MeOH) to obtain 109 mg of the 4th kerelactone ester compound. The crude product was then subjected to recrystallization (methanol-water mixture) to obtain 150 mg of the 5th kerelactone ester compound.

[0093] (2) Preparation of NMR sample solutions for NMR 1 to 7 Hyuganin D (first kerelactone ester compound), cis-3'-acetyl-4'-tigroylkeractone (second kerelactone ester compound), poisedanocoumarin III (third kerelactone ester compound), isosamidin (fourth kerelactone ester compound), trans-3'-acetyl-4'-senesioylkeractone (fifth kerelactone ester compound), pterixin (sixth kerelactone ester compound), and 7-ethoxy-4-methylcoumarin (standard substance for quantification) were each weighed in 1 to 5 mg in a microbalance, along with approximately 2 mg of DSS-d6 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) as a standard substance for purity calculation. These were then dissolved in approximately 0.75 mL of DMSO-d6 (manufactured by Merck) to prepare the NMR sample solutions for the first to seventh spectra.

[0094] (3) Quantitative NMR measurement The above NMR sample solutions 1 to 7 are prepared under the following conditions: 1 NMR spectra were obtained by 1H-qNMR measurement. From the obtained NMR spectra, the signal areas A1-A6,A of the 1st to 6th keratone ester compounds, the quantitative standard, and DSS-d6 were analyzed using NMR analysis software (VnmrJ version 3.2, Varian). S ,A IS And each signal area A1~A6,A S ,A IS The proton number derived from H1~H6,H S ,H IS The following formulas (3-1) to (3-6) and (4) were used to determine the purity P1 to P6 of the first to sixth kerelactone ester compounds and the purity P of the standard substance for quantification. S The result was calculated and is shown in Table 1.

[0095] < 1 H-qNMR measurement conditions > NMR device: Varian NMR System 500, manufactured by Varian) Observation center and width: δ5 ppm ± 20 ppm Import time: 4.0 seconds Pulse angle: 90° Delay time: 60 seconds Total number of times: 8 Measurement temperature: 40℃ Dummy scan: 2 times Spinning: Off 13 C decoupling: on

[0096]

number

[0097]

number

[0098] In the above formulas (3-1) to (3-6) and formula (4), P1 to P6 represent the "purity of the first to sixth kerelactone ester compounds," and P S This represents the "purity of the standard substance for quantitative analysis," and P IS A represents the "purity of DSS-d6", A1 to A6 represent the "signal area of ​​the 1st to 6th kerlactone ester compounds in the 1st to 6th NMR spectra", and A S represents the "signal area of ​​the quantitative standard in the 7th NMR spectrum," and A IS H represents the signal area of ​​DSS-d6 in each NMR spectrum, H1~H6 represents the number of protons derived from the signal areas A1~A6 of the 1st~6th keralactone ester compounds in the 1st~6th NMR spectra, H S This is "Signal area A of the quantitative standard in the 7th NMR spectrum S It represents the number of protons derived from H IS "The signal area A of DSS-d6 in each NMR spectrum" IS M represents the number of protons derived from, and M1 to M6 represent the molecular weight of the first to sixth kerelactone ester compounds. S represents the "molecular weight of the standard substance for quantitative analysis," and M ISW represents the molecular weight of DSS-d6, W1 to W6 represent the weighed values ​​(mg) of the first to sixth keralactone ester compounds during the preparation of the first to sixth NMR sample solutions, and W S This represents the "weighing value (mg) of the quantitative standard substance during the preparation of the 7th NMR sample solution," and W IS This represents the "weighing value (mg) of DSS-d6 during the preparation of the 1st to 7th NMR sample solutions."

[0099] [Test Example 3] Calculation of Relative Molar Sensitivity By dissolving the first and fourth kerelactone ester compounds, whose purity was calculated, in solution A, which is obtained by dissolving 7-ethoxy-4-methylcoumarin, used as a quantitative standard substance whose purity was calculated in Test Example 2, in methanol, the molar concentrations of the six compounds (518 × 10) can be determined. -6 mol / L, 414 × 10 -6 mol / L, 311 × 10 -6 mol / L, 207 × 10 -6 mol / L, 10⁴ × 10⁴ -6 mol / L, 2.59 × 10 -6 A solution of the first and second LC samples at molar concentrations (518 × 10⁻¹⁰ mol / L) was prepared. Similarly, the second and fifth kerelactone ester compounds, whose purity had been calculated, were dissolved in the above solution A to obtain six different molar concentrations (518 × 10⁻¹⁰ mol / L). -6 mol / L, 414 × 10 -6 mol / L, 311 × 10 -6 mol / L, 207 × 10 -6 mol / L, 10⁴ × 10⁴ -6 mol / L, 2.59 × 10 -6 A 2-2LC sample solution with a molar concentration of (518 × 10) was prepared, and the 3rd and 6th kerelactone ester compounds, whose purity had been calculated, were dissolved in the above solution A, thereby obtaining six types of molar concentrations (518 × 10). -6 mol / L, 414 × 10 -6 mol / L, 311 × 10 -6 mol / L, 207 × 10 -6 mol / L, 10⁴ × 10⁴ -6 mol / L, 2.59 × 10 -6A 3-2LC sample solution (mol / L) was prepared.

[0100] Three parallel LC sample solutions of various concentrations (1st-2nd to 3rd-2nd) were prepared and subjected to liquid chromatography treatment under conditions 1 and 2 below to obtain LC chromatograms.

[0101] <Liquid chromatography conditions 1> Liquid chromatograph system: 1260 Infinity II Prime LC (manufactured by Agilent Technologies) Detector: Diode array detector G7117C (Detection wavelength: 320nm, manufactured by Agilent Technologies) Injection volume: 15μL Column: SunShell C30 (2.6μm, 3.0mm x 100mm, manufactured by Chromanic Technologies) Column temperature: 40℃ Mobile phase: Water: Trifluoroacetic acid: Methanol: Tetrahydrofuran = 400:0.2:50:55 (volume ratio) Flow rate: 1.0mL / min Analysis time: 50min

[0102] <Liquid chromatography conditions 2> Liquid chromatograph: Prominence (manufactured by Shimadzu Corporation) Detector: UV detector SPD-20A (Detection wavelength: 320nm, manufactured by Shimadzu Corporation) Injection volume: 15μL Column: SunShell C30 (2.6μm, 3.0mm x 100mm, manufactured by Chromanic Technologies) Column temperature: 40℃ Mobile phase: Water: Trifluoroacetic acid: Methanol: Tetrahydrofuran = 400:0.2:50:55 (volume ratio) Flow rate: 0.5mL / min Analysis time (Run time): 100min

[0103] Then, regression analysis was performed with the peak areas of the 1st to 6th keralactone ester compounds calculated from each LC chromatogram as the dependent variable, and the molar concentrations (mol / L) of the 1st to 6th keralactone ester compounds and the quantitative standard substance based on the above purity in each of the 1st-2nd to 3rd-2nd LC sample solutions as independent variables, with the constant term set to 0. Regression lines (1st to 6th calibration curves) for the 1st to 6th keralactone ester compounds and the regression line (standard calibration curve) for the quantitative standard substance were created, and the slopes S1 to S6 and S1 of each regression line (each calibration curve) were determined. S The following was calculated. Next, the relative molar sensitivities RMS1 to RMS6 of each of the 1st to 6th keralactone ester compounds were used, and the slope S of the standard calibration curve was calculated. S The ratios of the slopes S1 to S6 of the 1st to 6th calibration curves were calculated using the following formulas (a) to (f). The relative molar sensitivity of the 1st to 6th keralactone ester compounds was calculated by averaging the results of a total of six trials in which the 1st-2nd to 3rd-2nd LC sample solutions, prepared in three parallel rows, were treated under liquid chromatography conditions 1 and 2, respectively. The results are shown in Table 1. RMS1 = S1 / S S …(a) RMS2 = S2 / S S …(b) RMS3 = S3 / S S …(c) RMS4 = S4 / S S …(d) RMS5 = S5 / S S …(e) RMS6 = S6 / S S …(f)

[0104] [Test Example 4] Determination of keryllactone ester compounds in Chomeiso extract preparations by preparation of quantitative sample solutions The Chomeiso extract preparation was suspended in water, then diatomaceous earth (Celite 545) was added and suspended again, and methanol was added to obtain a suspension. This suspension was filtered to prepare a sample solution for quantitative analysis. In addition, the quantitative standard substance whose purity was determined in Test Example 2 was dissolved in methanol, diluted with water, filtered through a membrane filter, and a quantitative standard solution was prepared.

[0105] For both the quantitative sample solution and the quantitative standard solution, a quantitative chromatogram and a standard chromatogram were obtained by subjecting them to liquid chromatography under the above conditions. A regression analysis was performed with the peak area of ​​the quantitative standard substance obtained from the standard chromatogram as the dependent variable, and the molar concentration (mol / L) of the quantitative standard substance in the quantitative standard solution based on the above purity as the independent variable, with a constant term of 0. A regression line (quantitative calibration curve) for the quantitative standard substance was created, and the slope S of each regression line (quantitative calibration curve) was calculated. S ' was calculated.

[0106] From the quantitative chromatograms obtained as described above, the peak areas a1 to a6 of the first to sixth kerelactone ester compounds were determined, and the slope S was calculated. S From the peak areas a1 to a6, the respective content Ct1 to Ct6 (g / g) of the first to sixth keralactone ester compounds per 1g of Chomeiso extract was calculated using the following formulas (1-1) to (1-6) and formulas (2-1) to (2-6). The results are shown in Table 1.

[0107]

number

[0108]

number

[0109] In the above formulas (1-1) to (1-6) and formulas (2-1) to (2-6), Ct1 is the "content of the first keralactone ester compound per 1 g of Chomeiso extract (g / g)", Ct2 is the "content of the second keralactone ester compound per 1 g of Chomeiso extract (g / g)", Ct3 is the "content of the third keralactone ester compound per 1 g of Chomeiso extract (g / g)", Ct4 is the "content of the fourth keralactone ester compound per 1 g of Chomeiso extract (g / g)", Ct5 is the "content of the fifth keralactone ester compound per 1 g of Chomeiso extract (g / g)", Ct6 is the "content of the sixth keralactone ester compound per 1 g of Chomeiso extract (g / g)", C1 is the "molar concentration of the first keralactone ester compound in the quantitative sample solution (mol / L)", and C2 is the "molar concentration of the second keralactone ester compound in the quantitative sample solution C3 is "Molar concentration (mol / L) of the ester compound," C4 is "Molar concentration (mol / L) of the third kerelactone ester compound in the quantitative sample solution," C5 is "Molar concentration (mol / L) of the fourth kerelactone ester compound in the quantitative sample solution," C6 is "Molar concentration (mol / L) of the sixth kerelactone ester compound in the quantitative sample solution," M1 is "Molecular weight of the first kerelactone ester compound," M2 is "Molecular weight of the second kerelactone ester compound," M3 is "Molecular weight of the third kerelactone ester compound," M4 is "Molecular weight of the fourth kerelactone ester compound," M5 is "Molecular weight of the fifth kerelactone ester compound," M6 is "Molecular weight of the sixth kerelactone ester compound," C Ea1 represents the concentration (mg / mL) of the Chomeiso extract preparation in the quantitative sample solution, a2 represents the peak area of ​​the first kerelactone ester compound in the quantitative chromatogram, a3 represents the peak area of ​​the second kerelactone ester compound in the quantitative chromatogram, a4 represents the peak area of ​​the third kerelactone ester compound in the quantitative chromatogram, a5 represents the peak area of ​​the fifth kerelactone ester compound in the quantitative chromatogram, and a6 represents the peak area of ​​the sixth kerelactone ester compound in the quantitative chromatogram.

[0110] [Test Example 5] Determination of keryllactone ester compounds in Chomeiso extract preparations by preparation of standard mixed quantitative sample solution After suspending the Chomeiso extract preparation in water, methanol was added to obtain a suspension in a methanol:water ratio of 3:7. The suspension was passed through a solid-phase extraction cartridge (Sep-pak Plus tC18 (Waters)) conditioned in the order of methanol and water, then water and aqueous methanol (methanol:water = 1:1) were passed through, and the eluent was discarded. Next, methanol was passed through, and the eluent was collected. To this eluent, a methanol solution of the quantitative standard substance whose purity was determined in Test Example 2 was added, then water was added to dilute it, and the solution was filtered through a membrane filter to prepare a standard mixed quantitative sample solution.

[0111] A standard mixed quantitative chromatogram was obtained by subjecting the sample solution to liquid chromatography under the above conditions. A regression analysis was performed with the peak area of ​​the quantitative standard substance obtained from the standard mixed quantitative chromatogram as the dependent variable, and the molar concentration (mol / L) of the quantitative standard substance in the standard mixed quantitative standard solution based on the above purity as the independent variable, with a constant term of 0. A regression line (quantitative calibration curve) for the quantitative standard substance was created, and the slope S of each regression line (quantitative calibration curve) was calculated. S ' was calculated.

[0112] From the standard mixed quantitative chromatogram obtained as described above, the peak areas a1 to a6 of the first to sixth kerlactone ester compounds were determined, and the slope S was calculated. S From the peak areas a1 to a6, the respective content Ct1 to Ct6 (g / g) of the first to sixth keralactone ester compounds per 1g of Chomeiso extract was calculated using the following formulas (1-1) to (1-6) and formulas (2-1) to (2-6). The results are shown in Table 1.

[0113]

number

[0114]

number

[0115] In the above formulas (1-1) to (1-6) and formulas (2-1) to (2-6), Ct1 is the "content of the first keralactone ester compound per 1 g of Chomeiso extract (g / g)", Ct2 is the "content of the second keralactone ester compound per 1 g of Chomeiso extract (g / g)", Ct3 is the "content of the third keralactone ester compound per 1 g of Chomeiso extract (g / g)", Ct4 is the "content of the fourth keralactone ester compound per 1 g of Chomeiso extract (g / g)", Ct5 is the "content of the fifth keralactone ester compound per 1 g of Chomeiso extract (g / g)", Ct6 is the "content of the sixth keralactone ester compound per 1 g of Chomeiso extract (g / g)", C1 is the "molar concentration of the first keralactone ester compound in the quantitative sample solution (mol / L)", and C2 is the "molar concentration of the second keralactone ester compound in the quantitative sample solution C3 is "Molar concentration (mol / L) of the ester compound," C4 is "Molar concentration (mol / L) of the third kerelactone ester compound in the quantitative sample solution," C5 is "Molar concentration (mol / L) of the fourth kerelactone ester compound in the quantitative sample solution," C6 is "Molar concentration (mol / L) of the sixth kerelactone ester compound in the quantitative sample solution," M1 is "Molecular weight of the first kerelactone ester compound," M2 is "Molecular weight of the second kerelactone ester compound," M3 is "Molecular weight of the third kerelactone ester compound," M4 is "Molecular weight of the fourth kerelactone ester compound," M5 is "Molecular weight of the fifth kerelactone ester compound," M6 is "Molecular weight of the sixth kerelactone ester compound," C Ea1 represents the concentration (mg / mL) of the Chomeiso extract preparation in the quantitative sample solution, a2 represents the peak area of ​​the first kerelactone ester compound in the quantitative chromatogram, a3 represents the peak area of ​​the second kerelactone ester compound in the quantitative chromatogram, a4 represents the peak area of ​​the third kerelactone ester compound in the quantitative chromatogram, a5 represents the peak area of ​​the fifth kerelactone ester compound in the quantitative chromatogram, and a6 represents the peak area of ​​the sixth kerelactone ester compound in the quantitative chromatogram.

[0116] [Reference Test Example 1] Determination of kerelactone ester compounds based on a calibration curve The content (mass %) of each of the first to sixth kerelactone ester compounds in the Chomeiso extract preparation was calculated from the peak areas a1 to a6 of the quantitative chromatograms obtained in Test Example 4 and the calibration curves for the first to sixth kerelactone ester compounds. The results are shown in Table 1.

[0117] [Table 1]

[0118] As is clear from the results shown in Table 1, the mass %) content of kerelactone ester compounds in the Chomeiso extract preparations, calculated as in Test Examples 2-5, was found to be equivalent to the mass %) content of kerelactone ester compounds calculated in Reference Test Example 1. From these results, it was found that if the relative molar sensitivity RMS of each kerelactone ester compound contained in the plant extract is determined in advance, each kerelactone ester compound can be quantified with high accuracy by liquid chromatography using 7-ethoxy-4-methylcoumarin as a standard substance.

Claims

1. A method for quantifying at least one kerlactone ester compound contained in an extract from a plant, A purity calculation step is performed to calculate the purity of the kerelactone ester compound and the quantitative standard substance using quantitative nuclear magnetic resonance spectroscopy (qNMR), A relative molar sensitivity acquisition step to obtain the relative molar sensitivity of the kerelactone ester compound whose purity has been calculated with respect to the quantitative standard substance whose purity has been calculated, A quantitative step of calculating the content of the kerelactone ester compound in the extract from a quantitative chromatogram obtained by treating a quantitative sample solution containing the extract with liquid chromatography, a standard chromatogram obtained by treating a quantitative standard solution containing the quantitative standard substance whose purity has been calculated with liquid chromatography, or a standard mixed quantitative chromatogram obtained by treating a standard mixed quantitative sample solution containing the extract and the quantitative standard substance whose purity has been calculated with liquid chromatography. It has, The aforementioned relative molar sensitivity acquisition step is, A step of preparing an LC sample solution containing the kerelactone ester compound and the quantitative standard substance at at least one molar concentration, The process involves obtaining an LC chromatogram by treating the various LC sample solutions mentioned above with liquid chromatography, A step of calculating the slope of each calibration curve that shows the relationship between the molar concentrations of the kerelactone ester compounds and the quantitative standard substances in the various LC sample solutions, based on their respective purity, and the peak areas of the kerelactone ester compounds and the quantitative standard substances in the LC chromatogram. A step of calculating the ratio of the slope of the calibration curve of the kerelactone esters to the slope of the calibration curve of the quantitative standard substance as the relative molar sensitivity of the kerelactone ester compound. Includes, The aforementioned quantitative process is: A step of creating a standard calibration curve showing the relationship between the peak area of ​​the at least one quantitative standard substance whose purity has been calculated, obtained from the standard chromatogram obtained by treating the at least one quantitative standard solution containing the quantitative standard substance whose purity has been calculated at at least one molar concentration with the liquid chromatography, or the peak area of ​​the at least one quantitative standard substance whose purity has been calculated, obtained from the standard mixed quantitative chromatogram obtained by treating the standard mixed quantitative sample solution containing the extract and the quantitative standard substance whose purity has been calculated at a predetermined molar concentration with the liquid chromatography, and the molar concentration of the quantitative standard substance based on its purity in each of the at least one quantitative standard solution. A step of calculating the content of each of the kerelactone ester compounds in the extract from the peak area of ​​the kerelactone ester compounds obtained from the quantitative chromatogram obtained by treating the quantitative sample solution containing the extract by liquid chromatography, or from the peak area of ​​the kerelactone ester compounds obtained from the standard mixed quantitative chromatogram obtained by treating the standard mixed quantitative sample solution containing the extract and the quantitative standard substance by liquid chromatography, the slope of the standard calibration curve, and the relative molar sensitivity. Includes, A method for quantifying kerelactone ester compounds, characterized in that the quantitative standard substance is a substance that exhibits an absorption maximum at a wavelength of 305 to 335 nm in the UV spectrum and is a compound shown by the following chemical structural formula (I). 【Chemistry 1】 In the above chemical structural formula (I), R1 represents hydrogen, an alkyl group having 1 to 5 carbon atoms, or an acetyl group; R2 represents hydrogen or a hydroxyl group; R3 represents hydrogen or an alkyl group having 1 to 5 carbon atoms; and R4 represents hydrogen or a 3-methyl-2-butenyl group.

2. The method for quantifying kerelactone ester compounds according to claim 1, characterized in that the aforementioned quantitative standard substance is 7-ethoxy-4-methylcoumarin shown in the following chemical structural formula (II). 【Chemistry 2】

3. The method for quantifying a kerelactone ester compound according to claim 1 or 2, characterized in that, in the quantitative step, the content of the kerelactone ester compound is calculated based on the following formulas (1) and (2). [Math 1] [Math 2] In the above formulas (1) and (2), Ct Ｋ This represents "the content of the kerelactone ester compounds in the extract," and C Ｋ This represents "the molar concentration of the kerelactone ester compound in the quantitative sample solution or standard mixed quantitative sample solution," and M Ｋ represents "the molecular weight of the kerelactone ester compound," and C Ｅ represents "the concentration of the extract in the quantitative sample solution or standard mixed quantitative sample solution," S represents "the slope of the standard calibration curve when the standard calibration curve is prepared using the quantitative standard substance," and a Ｋ This represents "the peak area of ​​the kerelactone ester compound in the quantitative chromatogram or standard mixed quantitative chromatogram," and RMS Ｋ This represents the "relative molar sensitivity of the kerelactone ester compounds."

4. A method for quantifying a kerelactone ester compound according to any one of claims 1 to 3, characterized in that the quantitative nuclear magnetic resonance spectroscopy (qNMR) used to measure the purity of the kerelactone ester compound and the quantitative standard substance in the purity calculation step is a 1H-qNMR measurement method using an internal standard.

5. A method for quantifying kerelactone ester compounds according to any one of claims 1 to 4, characterized in that the plant is a plant belonging to the family Apiaceae, Rubiaceae, or Smilaxaceae.

6. A method for quantifying kerelactone ester compounds according to any one of claims 1 to 5, characterized in that the plant is a plant belonging to the genus Angelica in the family Apiaceae.

7. The method for quantifying kerelactone ester compounds according to claim 6, characterized in that the plant is long-life grass.

8. A method for quantifying kerelactone ester compounds according to any one of claims 1 to 7, characterized in that the kerelactone ester compound is at least one selected from hyuganin D, cis-3'-acetyl-4'-tigloylkhellactone, peucedanocoumarin III, isosamidin, trans-3'-acetyl-4'-senecioylkhellactone, and pteryxin.

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