JP6574360B2 - Determination of crocetin - Google Patents

Description

  The present invention relates to a method for quantifying crocetin.

  Crocetin, a kind of carotenoid, has various physiological functions such as antioxidant action, eye strain, and improvement of sleep disorders, so it is used as a material for health foods and supplements, as well as food and drink such as drinks and jellies. Also included in the product. Therefore, it is necessary to quantify the content of crocetin for quality control of products containing crocetin.

  As a method for quantifying carotenoids, a direct quantification method for analysis by high performance liquid chromatography (HPLC) has been performed (see Patent Document 1). However, this method has a problem in convenience because accurate quantification cannot be performed unless a high-purity carotenoid commercially available as a standard product is available, and a dedicated analytical instrument is required.

  On the other hand, an indirect quantification method is also performed in which the absorbance of a carotenoid-containing sample is measured using a spectrophotometer and the carotenoid content of the sample is calculated from the absorbance coefficient of the carotenoid reported in the literature (Patent Literature). 2). However, this method tends to require a numerical value higher than the actual carotenoid content due to light absorption by substances other than carotenoids contained in the sample, and accurate quantification is relatively difficult.

  The problems in the direct quantification method and indirect quantification method are strongly desired to be solved particularly in the quantification of crocetin among carotenoids.

JP 2011-127905 A (Claim 1) JP 2014-110765 A (paragraph paragraphs 0058 to 0059)

  An object of the present invention is to provide a method for quantifying crocetin that allows simple and accurate quantification without using a standard crocetin.

  As a result of diligent studies on the above problems, the present inventors have dissolved a composition containing crocetin in an alkaline aqueous solution to remove insoluble matters, and then used the anion exchange resin and the cation exchange resin. It was found that the accuracy of quantification of crocetin by absorbance measurement is improved by processing with the use of this, and the present invention has been made based on this finding.

That is, this invention consists of following (1)-(6).
(1) A method for quantitatively determining crocetin, comprising sequentially performing the following steps (A) to (H).
(A): Step of dissolving crocetin-containing composition in alkaline aqueous solution (B): Step of removing insoluble matter contained in solution obtained in step (A) (C): Insoluble in step (B) Step (D) in which the dissolved solution from which substances have been removed is brought into contact with an anion exchange resin: Step (E) in which the anion exchange resin after step (C) is washed with an alkaline aqueous solution Step (F) of washing the resin with an organic solvent: A step of eluting the components adsorbed on the anion exchange resin after the step (E) with a highly polar organic solvent containing an acid agent and collecting the eluate ( G): The eluate recovered in step (F) is brought into contact with a cation exchange resin, and the non-adsorbed fraction is recovered (H): the recovered liquid of the non-adsorbed fraction obtained in step (G) Step of measuring absorbance derived from crocetin (2) alkaline water The liquid is one or more selected from a sodium hydroxide aqueous solution, a potassium hydroxide aqueous solution, a calcium hydroxide aqueous solution, a sodium carbonate aqueous solution, a sodium hydrogen carbonate aqueous solution, a sodium tetraborate aqueous solution, and an ammonia aqueous solution. The method for quantifying crocetin according to 1).
(3) The method for quantifying crocetin according to (1) above, wherein the acid agent is at least one selected from formic acid, phosphoric acid, hydrochloric acid, acetic acid, trifluoroacetic acid and citric acid.
(4) The method for quantifying crocetin according to (1) above, wherein the organic solvent is at least one selected from methanol, ethanol, propanol, butanol, acetone, pyridine, dimethylsulfoxide and dimethylformamide.
(5) The crocetin quantification method according to (1) above, wherein the highly polar organic solvent is at least one selected from methanol, ethanol, isopropanol, dimethyl sulfoxide, dimethylformamide, pyridine and acetone.
(6) The method for quantifying crocetin according to any one of (1) to (5), wherein the composition containing crocetin is a food or drink or a pharmaceutical product.

  According to the quantification method of the present invention, accurate and accurate quantification of crocetin is possible without using a standard crocetin standard.

  In the present invention, crocetin refers to a formula

で表される化合物である。このクロセチンは、通常、カロテノイド系の黄色色素であるクロシン（クロセチンのジゲンチオビオースエステル）を加水分解することにより得られる。クロシンは、アカネ科クチナシ（Ｇａｒｄｅｎｉａ ａｕｇｕｓｔａ ＭＥＲＲＩＬ ｖａｒ．ｇｒａｎｄｉｆｌｏｒａ ＨＯＲＴ．，Ｇａｒｄｅｎｉａ ｊａｓｍｉｎｏｉｄｅｓ ＥＬＬＩＳ）の果実、サフランの柱頭の乾燥物等に含まれるが、クロシンを得るための工業的原料としてはクチナシの果実が好ましく用いられる。

It is a compound represented by these. This crocetin is usually obtained by hydrolyzing crocin (a digentiobiose ester of crocetin), which is a carotenoid yellow pigment. Crocin is contained in the fruits of Rubiaceae gardenia (Gardenia augusta MERRIL var.grandiflora HORT., Gardenia jasminoides ELLIS), dried saffron stigma, etc., but as an industrial raw material for obtaining crocin, gardenia Used.

  The method for quantifying crocetin according to the present invention is characterized in that the following steps (A) to (F) are sequentially performed on a composition containing crocetin. Hereinafter, each step will be described.

[Step (A)]
Step (A) is a step of dissolving a composition containing crocetin (hereinafter also simply referred to as “sample”) in an alkaline aqueous solution. The alkaline aqueous solution is not particularly limited as long as it is a pH 8.0 or higher solution. For example, sodium hydroxide aqueous solution, potassium hydroxide aqueous solution, calcium hydroxide aqueous solution, sodium carbonate aqueous solution, sodium hydrogen carbonate aqueous solution, sodium tetraborate 1 or more types chosen from aqueous solution and aqueous ammonia solution are mentioned. There are no particular limitations on the dissolution method, and various methods such as dissolution by stirring, dissolution by ultrasonic irradiation, and dissolution by shaking can be used, but dissolution by ultrasonic irradiation is preferably used. In addition, in the case of a sample that is solid at room temperature and generates fluidity by heating (for example, jelly), the sample may be appropriately heated in advance in order to increase the solubility. The density | concentration of alkaline aqueous solution is 0.001-2M normally, Preferably it is 0.05M-1M. Although there is no restriction | limiting in particular in the usage-amount of alkaline aqueous solution, It is 200-100000 mass parts normally with respect to 100 mass parts of samples, Preferably it is 500-20000 mass parts. Moreover, although there is no restriction | limiting in particular in dissolution temperature, Usually, 10-90 degreeC, Preferably it is 20-80 degreeC.

[Step (B)]
Step (B) is a step of removing insoluble matters (that is, components other than crocetin (contaminating components) in the sample and not dissolved in the alkaline aqueous solution) contained in the solution obtained in step (A). is there. The method for removing the insoluble matter is not particularly limited, and for example, a solid-liquid separation method known per se such as centrifugation or filtration can be used, but filtration is preferably used. Although there is no restriction | limiting in particular in the filtration method, For example, it can filter by a conventional method using a filter and a conventional apparatus. As the filter, for example, a syringe filter is preferably used, and more specifically, a hydrophilic syringe filter such as Polyvinylidene difluoride (PVDF) or Cellulose Acetate is preferably used. In addition, it is preferable to use a syringe filter having a two-layer structure in order to prevent clogging due to insoluble matter. The insoluble matter to be removed in the step (B) varies depending on the composition of the sample and the like, and is not uniform. For example, carotenoids such as β-carotene, lycopene, astaxanthin, lutein, β-cryptoxanthin, zeaxanthin, capsanthin and the like The ester body, ubiquinone, etc. are mentioned.

[Step (C)]
Step (C) is a step of bringing the solution from which insolubles have been removed in step (B) into contact with the anion exchange resin. In step (C), crocetin present in the solution from which insolubles have been removed in step (B) is adsorbed to the anion exchange resin, and crocetin is separated from the solution. The contacting method is not particularly limited as long as it can adsorb crocetin present in the solution from which insoluble matters have been removed in step (B) to the anion exchange resin. For example, batch method, column packing The method is preferably a method in which the solution is passed through a solid phase extraction column filled with an anion exchange resin, and crocetin in the solution is adsorbed on the solid phase. In addition, in this method, there is no restriction | limiting in particular in a liquid flow method, per se well-known liquid flow means, such as a natural fall method, a pressurization method, and a pressure reduction method, can be used. Further, in this method, before passing the solution from which insoluble matters have been removed in step (B), methanol and an alkaline aqueous solution are passed through a column filled with an anion exchange resin in advance. Is preferably conditioned. As the alkaline aqueous solution used for conditioning, any alkaline aqueous solution that can be used in the step (A) can be preferably used.

  The anion exchange resin used in the step (C) is not particularly limited as long as it can adsorb crocetin. For example, a resin capable of adsorbing a compound having a carboxyl group can be used. As such an anion exchange resin, for example, Oasis MAX (trade name: manufactured by Nippon Waters Co., Ltd.) and the like are commercially produced and sold, and can be used in the present invention. The amount of the anion exchange resin used in the step (C) is usually 5 to 200 parts by volume, more preferably 10 to 40 parts by volume with respect to 100 parts by volume of the solution from which insolubles have been removed in the step (B). It is preferable that

[Step (D)]
Step (D) is a step of washing the anion exchange resin after step (C) with an alkaline aqueous solution. By the step (D), the basic highly polar component adsorbed on the anion exchange resin in the step (C) is mainly removed. The washing method is not particularly limited as long as such a component can be removed. For example, a natural drop method or a pressure method is applied to a solid phase extraction column filled with an anion exchange resin after step (C). Examples thereof include a method in which an alkaline aqueous solution is passed using a known permeation means such as a decompression method and the passing solution is discarded.

  The alkaline aqueous solution used in the step (D) is not particularly limited as long as it is a pH 8.0 or higher solution. For example, a sodium hydroxide aqueous solution, a potassium hydroxide aqueous solution, a calcium hydroxide aqueous solution, a sodium carbonate aqueous solution, a hydrogen carbonate Examples thereof include an aqueous sodium solution, an aqueous sodium tetraborate solution, and an aqueous ammonia solution. The density | concentration of alkaline aqueous solution is 0.001-2M normally, Preferably it is 0.05-1M. Although there is no restriction | limiting in particular in the usage-amount of alkaline aqueous solution, It is 200-2000 volume parts normally with respect to 100 volume parts of anion exchange resin after a process (C), Preferably it is 500-1500 volume parts. In addition, although the component removed by process (D) changes with the compounding composition of a sample, etc., it is not uniform, For example, anthocyanins, polyphenols, etc. are mentioned.

[Step (E)]
Step (E) is a step of washing the anion exchange resin after step (D) with an organic solvent. By the step (E), contaminant components that could not be sufficiently removed in the step (B) are removed. The washing method is not particularly limited as long as it is a method capable of removing such contaminant components. For example, an organic solvent is passed through a solid phase extraction column filled with an anion exchange resin after step (D), There is a method of discarding the passing liquid. In this method, the liquid passing method is not particularly limited, and per se known liquid passing means such as a natural dropping method, a pressurizing method, and a depressurizing method can be used.

  The organic solvent used in the step (E) is not particularly limited, but for example, an organic solvent that is miscible with both water and methanol and can dissolve a fat-soluble substance is preferable. Examples thereof include one or more selected from methanol, ethanol, propanol, butanol, acetone, pyridine, dimethyl sulfoxide, and dimethylformamide. Although there is no restriction | limiting in particular in the usage-amount of an organic solvent, It is 200-10000 volume parts normally with respect to 100 volume parts of anion exchange resins after a process (C), Preferably it is 500-2000 volume parts. The components to be removed in step (E) vary depending on the composition of the sample, etc., and are not uniform. For example, carotenoids such as β-carotene, lycopene, astaxanthin, lutein, β-cryptoxanthin, zeaxanthin, capsanthin, etc. And its ester, ubiquinone and the like.

[Step (F)]
Step (F) is a step of eluting the component adsorbed on the anion exchange resin after step (E) with a highly polar organic solvent containing an acid agent, and collecting the eluate. By the step (F), crocetin adsorbed on the anion exchange resin can be eluted and recovered. The elution method is not particularly limited as long as it can elute crocetin adsorbed on the anion exchange resin. For example, the solid phase extraction column packed with the anion exchange resin after step (E) contains an acid agent. And a method of passing the highly polar organic solvent and recovering the passing solution. In this method, the liquid passing method is not particularly limited, and per se known liquid passing means such as a natural dropping method, a pressurizing method, and a depressurizing method can be used.

  The acid agent contained in the highly polar organic solvent in the step (F) is a substance having 1 g or more dissolved in 1 L of ion-exchanged water at 20 ° C. and a pH of 5 or less at 20 ° C. at a concentration of 1 g / 1 L, It is soluble in highly polar organic solvents. Examples of such an acid agent include one or more selected from formic acid, phosphoric acid, hydrochloric acid, acetic acid, trifluoroacetic acid, and citric acid. Among these, formic acid or phosphoric acid is preferably used from the viewpoint of quantitative accuracy. .

  The highly polar organic solvent used in the step (F) is not particularly limited as long as it is a relatively highly polar organic solvent and can dissolve the acid agent. For example, methanol, ethanol, isopropanol, One or more selected from dimethyl sulfoxide, dimethylformamide, pyridine and acetone can be used, and among them, methanol, ethanol or acetone is preferably used from the viewpoint of quantitative accuracy. The concentration of the acid agent contained in the highly polar organic solvent is usually 0.05 to 5% (w / v), preferably 0.1 to 4% (w / v).

  Although there is no restriction | limiting in particular in the usage-amount of the highly polar organic solvent containing an acid agent in a process (F), Usually, 200-2000 volume parts with respect to 100 volume parts of anion exchange resins after a process (E), Preferably Is 500-1500 parts by volume.

[Step (G)]
Step (G) is a step of bringing the eluate collected in step (F) into contact with a cation exchange resin and collecting the non-adsorbed fraction. By the step (G), components that are not sufficiently removed in the step (D) (for example, anthocyanins and polyphenols) are adsorbed on the cation exchange resin and removed. As a method for bringing the eluate collected in the step (F) into contact with a cation exchange resin and collecting the non-adsorbed fraction, a method that can remove components that have not been sufficiently removed in the step (D) is particularly useful. Although there is no limitation, for example, a batch method, a column packing method and the like can be mentioned. Preferably, there is a method in which the eluate is passed through a solid phase extraction column packed with a cation exchange resin and the passing solution is recovered. Can be mentioned. In this method, the liquid passing method is not particularly limited, and per se known liquid passing means such as a natural dropping method, a pressurizing method, and a depressurizing method can be used. In this method, before the eluate collected in step (F) is passed through a solid phase extraction column packed with a cation exchange resin, the column is used in advance in step (F). It is preferable to perform conditioning by passing a “high polarity organic solvent containing an acid agent” or the like. In this method, after passing the eluate collected in the step (F) through a solid phase extraction column packed with a cation exchange resin, the “highly polar organic acid containing acid agent” used in the step (F) is used. It is preferable from the viewpoint of increasing the recovery rate of crocetin and increasing the accuracy of quantification, by passing an appropriate amount of “solvent” and further recovering the passing solution as a non-adsorbed fraction.

  The cation exchange resin used in step (G) is not particularly limited as long as it can adsorb components that have not been sufficiently removed in step (D). For example, Oasis MCX (trade name: Nippon Waters Co., Ltd.) Etc.) are commercially available and can be preferably used in the present invention. Although there is no restriction | limiting in particular in the usage-amount of the cation exchange resin in a process (G), 5-50 volume parts normally with respect to 100 volume parts of eluates collect | recovered at the process (F), Preferably 5-20 volume parts, More preferably, it is 5-10 volume parts.

[Step (H)]
Step (H) is a step of measuring the absorbance derived from crocetin for the recovered liquid of the non-adsorbed fraction obtained in step (G). Specifically, the absorbance derived from crocetin (for example, absorbance at a wavelength of 410 to 430 nm, preferably absorbance at a wavelength of 420 nm) is measured by using a known absorbance measuring means such as an ultraviolet / visible spectrophotometer. can do. In step (H), it is preferable to appropriately adjust the recovered liquid of the non-adsorbed fraction obtained in step (G) to a certain volume using an alkaline aqueous solution in advance before measuring the absorbance. By such adjustment, it is possible to adjust the measured absorbance used for the calculation of the crocetin content described later to be in the range of 0.1 to 1.0. As the alkaline aqueous solution, for example, an alkaline buffer solution having a pH of 8 or more is preferably used. Among them, Kolthoff's buffer solution (50 mM Na ₂ CO _{3) in which} the molar extinction coefficient of crocetin and the color value of pure crocetin are known. The use of −50 mM Na ₂ B ₄ O ₇ ; pH 10.0) is more preferred.

Next, based on the absorbance measured in the step (H), the crocetin content of the sample can be determined according to a method known per se. For example, since the crocetin in a sample are shifted to the recovered liquid obtained in the step (G), the molar extinction coefficient of the absorbance and crocetin ^{[e.g., 120500mol -1 · L -1 · cm} ( solvent: Kolthoff said buffer )] Or the color value of crocetin pure product [e.g., E ^10% _{1 cm} = 36700 (solvent: Mr. Kolthoff's buffer)] etc., the crocetin content or content of the sample can be determined. More specifically, for example, based on the absorbance derived from crocetin measured in step (H) and the color value of crocetin pure product [E ^10% _{1 cm} = 36700 (solvent: Mr. Kolthoff's buffer)], The crocetin content of the sample can be calculated.
Crocetin content (% by mass) = CV / 36700 × 100
CV: Color value of sample (E ^10% _{1 cm} ) = (10 × A × F) / sample collection amount (g)
A: Absorbance derived from crocetin F: Dilution factor for adjusting the measured absorbance to fall within the range of 0.1 to 1.0

Here, the CV (color value of the sample) in the above formula is measured according to the “color value measurement method” described in “8th Edition Food Additives Official Document” (Japan Food Additives Association). However, in the “color value measurement method”, “usually, the color value is a numerical value (E ¹⁰⁾ measured by measuring the absorbance at the maximum absorption wavelength in the visible part of the colorant solution and converting it to the absorbance of a 10 w / v% solution. ^% _{1 cm} ) ”. However, in the present invention, the color value is determined using not the “absorbance at the maximum absorption wavelength in the visible region” but the absorbance derived from crocetin (for example, absorbance at a wavelength of 410 to 430 nm, preferably absorbance at a wavelength of 420 nm). In that respect, it is different from the definition.

  Further, “F: dilution ratio for adjusting the measured absorbance to fall within the range of 0.1 to 1.0” in the above formula is determined according to the dilution method shown in the “color value measuring method”. For example, when a test solution having an absorbance in the range of 0.1 to 1.0 is prepared by adding a sample to a solvent or the like to 100 mL, the dilution factor is “1”. Also, add a solvent or the like to the sample to make 100 mL, take 2 mL of that, dilute it by adding a solvent or the like to make 50 mL, and prepare a test solution with an absorbance in the range of 0.1 to 1.0. In this case, the dilution factor is “25”.

On the other hand, when the form of the sample is liquid at normal temperature (for example, a beverage) or is liquid (for example, jelly) by heating or the like, the crocetin content of the sample is calculated by the following formula. Is more convenient.
Crocetin content (mass%) = 10 × A × F / 36700
A: Absorbance derived from crocetin F: Dilution factor for adjusting the measured absorbance to fall within the range of 0.1 to 1.0

  There is no particular limitation on the composition containing crocetin, which is the subject of the quantitative method of the present invention. For example, foods and drinks, food and drink materials, quasi drugs, pharmaceuticals, pharmaceutical materials, and quasi drugs containing crocetin. There may be. There are no particular restrictions on components other than crocetin contained in the composition, for example, relatively polar components such as anthocyanins, polyphenols, water-soluble vitamins, β-carotene, lycopene, astaxanthin, lutein, In addition to carotenoids such as β-cryptoxanthin, zeaxanthin and capsanthin and their relatively low polarity components such as ubiquinone, various carriers acceptable for pharmacological or food hygiene such as excipients, Examples of the additives include stabilizers, stabilizers, dispersants, binders, diluents, flavoring agents, sweeteners, flavoring agents, and coloring agents.

  The form of the composition is not particularly limited as long as the effect of the present invention is achieved. For example, tablets, pills, granules, fine granules, chewing agents, liquids, capsules (hard capsules, soft capsules, etc. are filled) And chewable agents.

  Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

[Quantification of crocetin in tablets]
(1) Production of crocetin-containing tablets 1000 g of L-ascorbic acid (manufactured by Eisai Food Chemical Co., Ltd.), 200 g of sodium L-ascorbate (manufactured by Fuso Chemical Industry Co., Ltd.), 193 g of grapefruit powder juice (manufactured by Ogawa Fragrance Co., Ltd.), powdered reduced maltose 4030 g of water candy (trade name: Amarty MR-50; manufactured by Towa Kasei Kogyo Co., Ltd.) and 37 g of crocetin preparation (trade name: clobit P; crocetin content 76.5% by mass; manufactured by Riken Vitamin Co., Ltd.) were mixed. Was passed through a 40-mesh sieve and granulated with a fluid bed granulator using water as a binder. The granulation conditions were as follows: pump 280 g / min, drying 10 minutes, intake air temperature 75 ° C., exhaust gas temperature 40 ° C., cooling 3 minutes, and flow time 50 minutes.
The obtained granulated product was put into a polyethylene bag after removing particles of 20 mesh or less, and glycerin fatty acid ester (trade name: Poem TR-FB; manufactured by Riken Vitamin Co., Ltd.) 240 g, containing β-carotene 300 g of the composition (trade name: Riquetads β-carotene 6CS; manufactured by Riken Vitamin Co., Ltd.) was added and premixed.
Then, after removing particles of 30 mesh or less from the obtained mixture, the mixture was mixed for 10 minutes with a rocking mixer. The mixture was tableted using a rotary tableting machine to obtain crocetin-containing tablets. The tableting conditions were a mortar diameter of 12 mm, three mortars, a rotational speed of 20 rpm, and a tableting pressure of 0.7 to 0.9 kN. The obtained tablet had a weight of 600.0 mg / tablet and a theoretical value of crocetin content of 2.83 mg / tablet.

(2) Determination of crocetin (Examples 1 to 4)
One crocetin-containing tablet obtained in (1) was placed in a 100 mL volumetric flask, and about 80 mL of the alkaline aqueous solution described in Table 1 was added thereto. The tablet was dissolved by sonication while heating at 45 ° C. and cooled to room temperature, and then the alkaline aqueous solution shown in Table 1 was added thereto to make up a volume of 100 mL. After stirring the resulting solution, about 10 mL of the solution was filtered with a PVDF hydrophilic syringe filter [product name: 25 mm GD / X filter (PVDF 0.45 μm); manufactured by GE Healthcare Japan, Ltd.]. Insoluble matters contained in the solution were removed. Here, about 5 mL of filtrate that flowed out at the beginning of filtration was discarded, and then about 5 mL of filtrate that flowed out was collected in a test tube.
On the other hand, 1 mL of methanol and 1 mL of alkaline aqueous solution described in Table 1 were passed in order through a solid phase extraction column (trade name: Oasis MAX 3 cc / 60 mg; manufactured by Nihon Waters) filled with an anion exchange resin. After that, 2 mL of the filtrate collected in the test tube was passed through the solid phase extraction column. In addition, the liquid flow rate with respect to the solid phase extraction column (including a solid phase extraction column filled with a cation exchange resin described later) includes the following steps, and was performed at a rate of dropping 1 to 2 drops per second. .
Next, 2 mL of the alkaline aqueous solution described in Table 1 and 2 mL of methanol were passed through the solid phase extraction column filled with the anion exchange resin in this order, and the solid phase of the column was washed. Crocetin was eluted by passing 2.5 mL of a highly polar organic solvent containing the described acid agent. All the eluates obtained were directly passed through a solid phase extraction column (trade name: Oasis MCX 3cc / 60 mg; manufactured by Nihon Waters Co., Ltd.) packed with a cation exchange resin. The solid phase extraction column packed with the cation exchange resin was conditioned by passing 1 mL of a highly polar organic solvent containing the acid agent shown in Table 1 in advance before use.
Subsequently, 1 mL of a highly polar organic solvent containing the acid agent described in Table 1 was passed through the solid phase extraction column packed with the cation exchange resin. The passing liquid (about 3.5 mL) of the solid phase extraction column was collected in a volumetric flask having a total volume of 50 mL. This Kolthoff said buffer; added _{(50mM Na 2 CO 3 -50mM Na} 2 B 4 O 7 pH10.0) was constant volume of 50 mL. After stirring the obtained solution, the absorbance at a wavelength of 420 nm of the solution was measured with an ultraviolet / visible spectrophotometer, and the content of crocetin per tablet (mg / tablet) was determined by the following formula.
Crocetin content (mg / tablet) = 600 × crocetin content (mass%) / 100
Crocetin content (mass%) = tablet color value (E ^10% _{1 cm} ) / 36700 × 100
Tablet color value (E ^10% _{1 cm} ) = 10 × absorbance × 25/600 × 1000

In the above formula, “600” is the weight (mg) per tablet, “36700” is the color value of pure crocetin (E ^10% _{1 cm} = 36700), and “25” is The dilution rate is adjusted so that the measured absorbance falls within the range of 0.1 to 1.0, and “1000” is a conversion value for converting the mass unit from “g” to “mg”.

(3) Determination of crocetin (Comparative Example 1)
One crocetin-containing tablet obtained in (1) was placed in a 100 mL volumetric flask, and about 80 mL of Kolthoff buffer solution (50 mM Na ₂ CO ₃ -50 mM Na ₂ B ₄ O ₇ ; pH 10.0) was added thereto. This was sonicated while heating at 45 ° C. to dissolve the tablet, cooled to room temperature, and then the same Kolthoff buffer solution was added thereto to make a constant volume of 100 mL. After stirring the obtained lysate, 2 mL of the lysate was dispensed into a 50 mL volumetric flask, and the same Kolthoff buffer solution was added thereto to make a constant volume of 50 mL. After stirring the obtained solution, the absorbance at a wavelength of 420 nm of the solution was measured with an ultraviolet / visible spectrophotometer, and the content of crocetin per tablet according to the above formula used in Examples 1 to 4 ( mg / tablet).

(4) Results Crocetin content (mg / tablet) determined by Examples 1-4 and Comparative Example 1 and crocetin recovery based on the theoretical content of crocetin (2.83 mg / tablet) for the content The rate (%) is shown in Table 2.

  As is clear from the results in Table 2, according to the quantification method of the present invention (Examples 1 to 4), crocetin could be quantified with a recovery rate close to 100% compared to the quantification method of Comparative Example 1.

[Quantification of crocetin in beverages]
(1) Production of crocetin-containing beverage Crocetin preparation (trade name: clobit 2.5WD; crocetin content 2.65% by mass; manufactured by Riken Vitamin Co., Ltd.) 33 g, bilberry extract (trade name: bilberry licorice extract; manufactured by Indena Japan) ) 20 g, vitamin E (trade name: Vitamin E-α-50 (A); manufactured by Riken Vitamin Co., Ltd.) 20 g, L-ascorbic acid (manufactured by Eisai Food Chemical Co., Ltd.) 10 g, vitamin mixed powder (trade name: Vitamin Ace Mix) DR-300; manufactured by Riken Vitamin Co., Ltd. 3 g, erythritol (produced by Mitsubishi Chemical Foods Co., Ltd.) 300 g, acesulfame potassium (trade name: Sannet; manufactured by MC Food Specialties Co., Ltd.) 0.5 g, sucralose (trade name: Sun Sweet SU-200 ; Saneigen FFI Co., Ltd.) 0.5g, purified anhydrous citrus 10 g (manufactured by Fuso Chemical Industry Co., Ltd.), 7 g of purified sodium citrate (manufactured by Fuso Chemical Industry Co., Ltd.) and 5 g of grapefruit fragrance (trade name: Grapefruit Micron ZD-4346; manufactured by Takasago Fragrance Co., Ltd.) were stirred and dissolved by adding water. A total amount of 5 L of lysate was obtained. The solution was filled in a brown bottle and sterilized at 80 ° C. for 10 minutes to prepare a crocetin-containing beverage (content 50 mL / book). The obtained beverage had a theoretical value of crocetin content of 8.75 mg / tube.

(2) Determination of crocetin (Examples 5 to 8)
5 mL of the crocetin-containing beverage obtained in (1) was collected in a 25 mL volumetric flask, and 1 M aqueous sodium hydroxide solution was added thereto to make a constant volume of 25 mL. After stirring the obtained solution, about 10 mL of the solution was filtered with a PVDF hydrophilic syringe filter (product name: 25 mm GD / X filter (PVDF 0.45 μm); manufactured by GE Healthcare Japan), and the solution The insoluble matter contained in was removed. Here, about 5 mL of filtrate that flowed out at the beginning of filtration was discarded, and then about 5 mL of filtrate that flowed out was collected in a test tube.
On the other hand, 1 mL of methanol and 1 mL of alkaline aqueous solution described in Table 2 were passed in order through a solid phase extraction column (trade name: Oasis MAX 3 cc / 60 mg; manufactured by Nihon Waters Co., Ltd.) packed with an anion exchange resin. After that, 1 mL of the filtrate collected in the test tube was passed through the solid phase extraction column. In addition, the liquid flow rate with respect to the solid phase extraction column (including a solid phase extraction column filled with a cation exchange resin described later) includes the following steps, and was performed at a rate of dropping 1 to 2 drops per second. .
Next, 1 mL of the alkaline aqueous solution described in Table 2 and 1 mL of methanol were passed through the solid phase extraction column packed with the anion exchange resin in this order, and the solid phase of the column was washed. Crocetin was eluted by passing 2.5 mL of a highly polar organic solvent containing the described acid agent. All the eluates obtained were directly passed through a solid phase extraction column (trade name: Oasis MCX 3cc / 60 mg; manufactured by Nihon Waters Co., Ltd.) packed with a cation exchange resin. The solid phase extraction column packed with the cation exchange resin was conditioned by passing 1 mL of a highly polar organic solvent containing the acid agent shown in Table 2 in advance before use.
Subsequently, 1 mL of a highly polar organic solvent containing an acid agent shown in Table 2 was passed through the solid phase extraction column packed with the cation exchange resin. The passing liquid (about 3.5 mL) of the solid phase extraction column was collected in a volumetric flask having a total volume of 50 mL. This Kolthoff said buffer; added _{(50mM Na 2 CO 3 -50mM Na} 2 B 4 O 7 pH10.0) was constant volume of 50 mL. After stirring the obtained solution, the absorbance at a wavelength of 420 nm of the solution was measured with an ultraviolet / visible spectrophotometer, and the crocetin content (mg / piece) per beverage was determined by the following formula.
Crocetin content (mg / line) = crocetin content (% by mass) × 1000 × content (mL / line) / 100
Crocetin content (mass%) = 10 × absorbance × 250/36700

In the above formula, “1000” is a conversion value for converting the mass unit from “g” to “mg”, and “36700” is the color value of crocetin pure product (E ^10% _{1 cm} = 36700). “250” is a dilution ratio adjusted so that the measured absorbance falls within the range of 0.1 to 1.0.

(3) Determination of crocetin (Comparative Example 2)
5 mL of the crocetin-containing beverage obtained in (1) is placed in a 25 mL volumetric flask, and Kolthoff's buffer solution (50 mM Na ₂ CO ₃ -50 mM Na ₂ B ₄ O ₇ ; pH 10.0) is added thereto to a constant volume of 25 mL. did. After stirring the obtained solution, 1 mL of the solution was taken into a 50 mL volumetric flask, and the same Kolthoff buffer solution was added thereto to make a constant volume of 50 mL. After stirring the obtained solution, the absorbance at a wavelength of 420 nm of the solution was measured with an ultraviolet / visible spectrophotometer, and the crocetin content per beverage according to the above formulas used in Examples 5 to 8 ( mg / tube).

(4) Results Crocetin content obtained in Examples 5 to 8 and Comparative Example 2 (mg / piece) and crocetin recovery based on the theoretical content of crocetin (8.75 mg / piece) for the content. The rate (%) is shown in Table 4.

  As is clear from the results in Table 4, according to the quantification method of the present invention (Examples 5 to 8), crocetin could be quantified with a recovery rate close to 100% compared to the quantification method of Comparative Example 2.

Claims (6)

A method for quantifying crocetin, comprising sequentially performing the following steps (A) to (H).
(A): Step of dissolving crocetin-containing composition in alkaline aqueous solution (B): Step of removing insoluble matter contained in solution obtained in step (A) (C): Insoluble in step (B) Step (D) in which the dissolved solution from which substances have been removed is brought into contact with an anion exchange resin: Step (E) in which the anion exchange resin after step (C) is washed with an alkaline aqueous solution Step (F) of washing the resin with an organic solvent: A step of eluting the components adsorbed on the anion exchange resin after the step (E) with a highly polar organic solvent containing an acid agent and collecting the eluate ( G): The eluate recovered in step (F) is brought into contact with a cation exchange resin, and the non-adsorbed fraction is recovered (H): the recovered liquid of the non-adsorbed fraction obtained in step (G) , The step of measuring the absorbance derived from crocetin   The alkaline aqueous solution is at least one selected from a sodium hydroxide aqueous solution, a potassium hydroxide aqueous solution, a calcium hydroxide aqueous solution, a sodium carbonate aqueous solution, a sodium hydrogen carbonate aqueous solution, a sodium tetraborate aqueous solution, and an ammonia aqueous solution. Item 2. A method for quantifying crocetin according to Item 1.   The method for quantifying crocetin according to claim 1, wherein the acid agent is at least one selected from formic acid, phosphoric acid, hydrochloric acid, acetic acid, trifluoroacetic acid and citric acid.   The method for quantifying crocetin according to claim 1, wherein the organic solvent is at least one selected from methanol, ethanol, propanol, butanol, acetone, pyridine, dimethylsulfoxide and dimethylformamide.   The method for quantifying crocetin according to claim 1, wherein the highly polar organic solvent is at least one selected from methanol, ethanol, isopropanol, dimethyl sulfoxide, dimethylformamide, pyridine and acetone.   The method for quantifying crocetin according to any one of claims 1 to 5, wherein the composition containing crocetin is a food or drink or a medicine.