JP7458046B2 - Method for producing apocarotenoids - Google Patents

Description

The present invention relates to a method for producing apocarotenoids.

Carotenoids are natural yellow-orange-red pigments produced by all photosynthetic organisms, as well as some bacteria, fungi, and yeasts. Most of them are fat-soluble C40 pigments consisting of an isoprene skeleton with 8 carbon atoms (C5) (Non-Patent Documents 1, 2). To date, more than 750 carotenoids have been isolated and identified from nature. Meanwhile, the orange pigments of saffron (the stigma of Crocus sativus , a monocotyledonous order, Asparagales, family Iridaceae) and gardenia (the fruit of Gardenia jasminoides, a dicotyledonous order, Rubiaceae, family Gentianales ), which are important in the food industry, consist of water-soluble carotenoids (apocarotenoids) such as C20 crocetin and crocin (crocetin esterified with two molecules of D-glucose at each end), and are used as food additives, functional foods, and pharmaceutical raw materials. The petals of yellow Crocus plants also contain crocetin glycosides (Non-Patent Document 3). It has also been reported that the orange petals (flowering period: July to September) of Crocosmia (Iridaceae, Crocosmia x crocosmiiflora ) and the orange petals (flowering period: May to June) of California poppy (Dicotyledonous plant class, Ranunculales, Papaveraceae, Eschsholtzia californica ) contain crocin and crocetin, respectively (Non-Patent Document 2). Crocosmia means "saffron scent" in Latin.

Freesia (Iridaceae, Freesia refracta ) is a flowering plant native to the Cape region of South Africa, and is also known by other names such as Asagizuinen, Kabusuinen, and Kosetsuran. Many horticultural varieties ( Freesia x hybrida ) have been bred. Freesia has a wide range of flower colors, including white, yellow, orange, red, pink, purple, and blue (flowering season: March to April), but 78% of freesias sold in Japan are yellow (Non-Patent Document 4).

``Airy Flora'' is an original variety of freesia ( Freesia x hybrida ) from Ishikawa Prefecture. Airy flora is characterized by its wide variety of flower colors, and there are currently 10 varieties in circulation, each with its own unique flower color. There are varieties with various flower colors including pale purple, red, orange, yellow, and white. Note that Airy Flora f2 strain ('Ishikawa f2-go') is a yellow-flowered variety.

Norihiko Misawa, Bioengineering 93: 403-406, 2015 AkemiOhmiya, JARQ 45: 163-171, 2011 A. Rubio Moraga et al, Crocins with High Levels of Sugar ConjugationContribute to the Yellow Colors of Early-Spring Flowering CrocusTepals. PLoS ONE 8(9): e71946, 2013. https://doi.org/10.1371/journal.pone. 0071946 Takeshi Honzu, Changes in breeding, cultivation technology, and production in freesia, Ibaraki National Agriculture and Food Research Institute Research Report 15: 1-31, 2015

The problem to be solved by the present invention is to provide a method for producing crocetin glycoside, an apocarotenoid that is important in the food industry and expected to be useful for human health, using plants. be.

The present inventors conducted intensive research to solve the above problems, and discovered that freesias with yellow petals, such as the yellow-flowered "Airleaf Flora" variety "Ishikawa f2" and the versatile yellow-flowered variety "Aladdin," produce the crocetin glycoside crocetin neapolitanosyl ester [a water-soluble apocarotenoid in which three molecules of D-glucose are ester-bonded to one side of crocetin: an apocarotenoid represented by formula (1)] and crocetin di-neapolitanosyl ester [a water-soluble apocarotenoid in which three molecules of D-glucose are ester-bonded to both sides of crocetin: an apocarotenoid represented by formula (2)] as yellow pigments in their petals, thereby solving the problem.

It was previously unknown that freesia produces crocetin glycosides. Yellow freesia is the only plant material that produces crocetin neapolitanosyl ester or crocetin di-neapolitanosyl ester as the main component. Furthermore, this is the first report of the isolation of crocetin neapolitanosyl ester from a plant in nature.

The invention is as follows.
1. A method for producing an apocarotenoid, comprising a step of extracting an apocarotenoid represented by formula (1) from an apocarotenoid-producing plant.
2. A method for producing an apocarotenoid, comprising a step of extracting an apocarotenoid represented by formula (2a) from an apocarotenoid-producing plant.
(In the formula, R is represented by formula (2b).)
3. 3. The method for producing apocarotenoid according to item 1 or 2, further comprising the step of decomposing the extracted apocarotenoid to obtain the apocarotenoid represented by formula (3).
4. 4. The production method according to any of the preceding items 1 to 3, wherein the apocarotenoid-producing plant is a Freesia plant.
5. 4. The production method according to item 4, wherein the freesia plant is yellow-flowered freesia.
6. 4. The production method according to item 4, wherein the Freesia plant is Airy Flora or Aladdin.
7. A pigment composition or food composition containing an apocarotenoid obtained by the method according to any one of items 1 to 6 above.

According to the present invention, a method for producing an apocarotenoid represented by formula (1), formula (2), or formula (3) can be provided.

FIG. 2 is a diagram showing the results of HPLC analysis of an extract using a CH ₂ Cl ₂ -MeOH (1:1) solution. It is a figure showing the HPLC analysis result of the extract by 100% MeOH solution. It is a figure showing the HPLC analysis result of the extract by 80% MeOH solution. It is a figure showing the HPLC analysis result of the extract by 50% MeOH solution. It is a figure showing the HPLC analysis result of 100% MeOH fraction of HP20. It is an HPLC fractionation diagram of Freesia 100% MeOH fraction. It is a figure showing the HPLC analysis result of 50% MeOH fraction of HP20. It is an HPLC fractionation diagram of Freesia 50% MeOH fraction. ¹ H NMR spectrum of crocetin neapolitanosyl ester (compound 1) in CD ₃ OD. ¹³ C NMR spectrum (in CD ₃ OD) of crocetin neapolitanosyl ester (compound 1). ^{1H- 1H} DQF COSY spectrum of compound 1 (in _CD3OD ). HMQC spectrum of compound 1 (in _CD3OD ). HMBC spectrum of compound 1 (in _CD3OD ). ¹ H NMR spectrum of crocetin (compound 3) in CD ₃ OD. ¹³ C NMR spectrum of crocetin (compound 3) (in CD ₃ OD). ¹³ CNMR spectrum (in CD ₃ OD) of crocetin di-neapolitanosyl ester (compound 2).

The present invention relates to a method for producing apocarotenoids, particularly apocarotenoids represented by formula (1), formula (2), or formula (3). The production method of the present invention can be produced by extracting the apocarotenoid from an apocarotenoid-producing plant (particularly Freesia).
The present invention will be explained in detail below.

(apocarotenoid)
The apocarotenoids of the present invention are the following apocarotenoids. These apocarotenoids are known to be difficult to synthesize.

{crocetin neapolitanosyl ester}
Crocetin neapolitanosyl ester is obtained as compound 1 in the examples below and has a structure represented by formula (1).

{crocetindi-neapolitanosyl ester}
Crocetin dineapolitanosyl ester is obtained as compound 2 in the following examples and has a structure represented by formula (2a).

(In the formula, R is represented by formula (2b).)

{crocetin}
Crocetin is obtained as compound 3 in the examples below and has a structure represented by formula (3). Crocetin can also be obtained by decomposing (particularly by acid hydrolysis) crocetin neapolitanosyl ester or crocetin dineapolitanosyl ester.

(Apocarotenoid-producing plants)
The apocarotenoids of the present invention are obtained from apocarotenoid-producing plants.
The apocarotenoid-producing plant is not particularly limited as long as it is a plant capable of producing the apocarotenoid of the present invention, but examples thereof include Iridaceae plants such as freesia, Papaveraceae plants, Rubiaceae plants, etc., and freesia and yellow flower plants are preferred, with yellow freesia being more preferred. Examples of yellow freesia include 'Ishikawa f2' and Aladdin.

(Method for producing apocarotenoid of the present invention)
The method for producing apocarotenoids of the present invention can employ a known method for extracting useful compounds (especially carotenoids) from plants. For example, the following can be exemplified.
After collecting all or a part of the plant body, preferably the petals, of the apocarotenoid-producing plant, freeze-drying as necessary, and crushing the freeze-dried product, and then extracting with an extraction solvent or apocarotenoids extracted with an extraction solvent. obtained by decomposing (especially acid hydrolysis).
Extraction solvents include, for example, alcohols such as methanol, ethanol, propanol, and butanol, hydroalcohols prepared by mixing these alcohols and water in any proportion, and glycols such as 1,3-butylene glycol, glycerin, and propylene glycol. Or hydrous glycols made by mixing these glycols and water in any proportion, esters such as ethyl acetate and butyl acetate, ethers such as ethyl ether, propyl ether, isopropyl ether, tetrahydrofuran, dioxane, dichloromethane, methylene chloride, chloroform, etc. halogenated hydrocarbons such as, polar organic solvents such as acetone, nonpolar organic solvents such as hexane, cyclohexane, petroleum ether, etc. can be used. Further, these solvents can be used alone or as a mixed solvent of two or more. Preferred extraction solvents include a mixed solvent of dichloromethane and methanol, aqueous methanol, and the like.
After obtaining the extract by the above method, add water (H ₂ O), methanol (MeOH), ethanol (EtOH), propanol, butanol, chloroform, dichloromethane (CH ₂ Cl ₂ ), ethyl acetate (EtOAc) as necessary. ), toluene, hexane, benzene, and other organic solvents, and the active fraction is extracted from the resulting extract by a two-phase partition operation using two types of organic solvents that are immiscible with each other (e.g., water in a two-phase partition of ethyl acetate/water). phase fraction) can be fractionated.
Furthermore, if necessary, separation and purification means using glass tube column chromatography such as silica gel chromatography, hydrophobic chromatography, reversed phase chromatography, gel filtration chromatography, ion exchange chromatography, or high performance liquid chromatography in an appropriate solvent may be used. It is also possible to purify one type or a combination of two or more types.

For example, the apocarotenoid of the present invention can be obtained through the step of collecting a part of an apocarotenoid-producing plant, such as a freesia petal, crushing it, adding water or the above-mentioned solvent, and performing centrifugation to collect the supernatant. The supernatant can also be obtained by performing this procedure one or more times.

More specifically, an example of a method for extracting crocetin neapolitanosyl ester (and crocetin) is as follows.
Compulsory cultivation is performed in a greenhouse, and petals are collected one by one from flowering freesia 'Ishikawa F2' and frozen in a -80℃ freezer.
The petals were freeze-dried for 3 days, turned into powder using a mixer, and extracted sequentially with CH ₂ Cl ₂ -MeOH (1:1), 100% MeOH, 80% MeOH, and 50% MeOH (each solvent The yellow pigment is extracted with a solvent.
Next, the extract containing the yellow pigment is concentrated to a small volume to remove the organic solvent, and then partitioned into two phases using EtOAc/H ₂ O. Since the yellow pigment is distributed into the aqueous phase, the aqueous phase is concentrated to half its volume to remove EtOAc and then adsorbed onto an HP20 column. After washing the HP20 column with water and 50% MeOH, the yellow dye is eluted with 100% MeOH.
After concentrating the eluate to dryness, a pure yellow pigment is isolated by reverse phase (C30) HPLC fractionation (developing solvent: 30% CH ₃ CN + 0.1% TFA).

(How to confirm each compound)
If necessary, perform various NMR ( ¹ H, ¹³ C, DQF COSY, HMBC, HMQC, NOESY) and HRESI-MS ((+) or (-)) measurements on the obtained pure product.
Next, hydrolyze into aglycones and sugars using 2N HCl. Aglycones and sugars are each purified by two-phase partitioning of EtOAc/H ₂ O (EtOAc phase: aglycone, aqueous phase: sugar).
If necessary, the aglycone is subjected to various NMR analyses, and the sugar is measured by optical rotation and ¹ H NMR to identify the obtained dye.

(Apocarotenoid obtained by the production method of the present invention)
Apocarotenoids obtained by the production method of the present invention can be used as pigments, foods, functional foods, pharmaceutical raw materials, etc.

The present invention will be explained in detail by giving specific examples below, but the present invention is not limited to these examples.

1-1 Cultivation of Freesia and Extraction of Yellow Pigment As yellow-flowered varieties of Freesia (Freesia there was. 'Ishikawa F2' was produced by forced cultivation in the greenhouse of the Ishikawa Prefectural Agriculture and Forestry Research Center, and flower petals were used. ``Aladdin'' uses petals collected from ``Freesia Bouquet 3L'' purchased from flower gift specialty store Edelweiss. Since the yellow pigments extracted from both were composed of exactly the same compound, only the example of 'Ishikawa f2' will be shown below.
Petals were collected one by one from 'Ishikawa F2' and frozen in a -80°C freezer. Frozen 'Ishikawa F2' petals (179.3 g) were freeze-dried for 72 hours to obtain dry Freesia (18.6 g). The obtained dry Freesia was pulverized for 1 minute using a mixer, 1 L of CH ₂ Cl ₂ -MeOH (1:1) solution was added, and the mixture was stirred and extracted at room temperature for 30 minutes with the lights off, followed by vacuum filtration. Next, 1 L of 100% MeOH was added to the filtration residue and solvent extraction was performed in the same manner, followed by vacuum filtration, and 1 L of 80% MeOH and 1 L of 50% MeOH were further added to the filtration residue to perform the same extraction.
As a result of the above extraction, all the yellow pigment contained in the petals was extracted, so the residue that remained at the end was almost white.
The four obtained filtrates were analyzed in 30 μL portions each under the HPLC analysis conditions shown below without being concentrated. The results for CH ₂ Cl ₂ -MeOH (1:1) solution are shown in Figure 1, the results for 100% MeOH solution in Figure 2, the results for 80% MeOH solution in Figure 3, and the results for 50% MeOH solution in Figure 4.
(HPLC analysis conditions)
Column: CAPCELL PAK (SHISEIDO) 4.6 mm×100 mm
Flow rate: 1.0 mL/min
Solvent: A solution 5% CH ₃ CN + 20 mM H ₂ PO ₄ , B solution 95% CH ₃ CN + 20 mM H ₂ PO ₄
0→3 min: A liquid 100%, 3→20 min: A liquid 100% → B liquid 100%, 20→30 min: A liquid 100%
Detection: DAD (200-600 nm)

As a result of this analysis, in the CH ₂ Cl ₂ -MeOH (1 : 1) and 100% MeOH extracts, there is a compound (compound 1) that has a maximum absorption at 434.3 nm with a retention time of 12.5 minutes, which is surrounded by an oval ( 1 and 2), the 50% MeOH extract contains a compound (compound 2) with maximum absorption at 439.9 nm, which is surrounded by a rectangle (Fig. 4), and the 80% MeOH extract contains compound 1 and compound 2. It was found that both were included (Figure 3).

1-2 Purification of Compound 1 Among the filtrates obtained in 1-1, the CH ₂ Cl ₂ -MeOH (1:1) solution containing a large amount of compound 1 and the 100% MeOH solution were combined and concentrated to dryness under reduced pressure (14.5 g). The dried product was dissolved in 300 mL of EtOAc and 300 mL of distilled water, and the mixture was shaken in a 1 L separatory funnel to perform two-phase partitioning. As a result, most of the yellow pigment was partitioned into the aqueous phase. After that, the EtOAc phase and the aqueous phase were transferred from the separatory funnel to separate Erlenmeyer flasks, and the EtOAc phase was returned to the separatory funnel and 300 mL of water was added, and two-phase partitioning was performed again in the separatory funnel. The two aqueous phases obtained were combined and concentrated to dryness under reduced pressure (13.8 g).
The concentrated and dried aqueous layer was dissolved in 255 mL of distilled water, the pH of which was adjusted to about 4 by adding a few drops of hydrochloric acid, and passed through an HP20 column (50 mm x 130 mm) developed with water, and the yellow pigment was adsorbed by the column. After that, 255 mL of distilled water was passed through the column to completely remove (wash) unadsorbed substances, and then 765 mL (255 mL x 3) of 50% MeOH was passed through the column to elute the adsorbed substances. The eluate (red solution) was collected in one Erlenmeyer flask. Next, the eluate (orange solution) eluted with 765 mL of 100% MeOH was passed through, and finally the eluate (light yellow) eluted with 765 mL of 60% acetone was passed through, each of which was also collected in a single Erlenmeyer flask.
The three fractions obtained (50% MeOH elution fraction, 100% MeOH elution fraction, and 60% acetone elution fraction) were analyzed in 30 μL each under the above HPLC analysis conditions without concentration. As a result, it was found that a peak with a maximum absorption around 450 nm (referred to as compound 1) was detected at 12.5 minutes only in the 100% MeOH fraction (Figure 5).
The obtained 100% MeOH fraction was run under the HPLC separation condition 1 shown below in an attempt to separate compound 1. However, a peak that was not the target carotenoid was detected at a retention time of 47 minutes, and in order to efficiently repeat the separation by HPLC, it was necessary to remove the compound at the peak at 47 minutes (a substance with lower polarity than compound 1). Therefore, the concentrated and dried 100% MeOH fraction (1.1077 g) was completely dissolved once in 6 mL of CH ₂ Cl ₂ -MeOH (1:1), and 15 mL of CH ₂ Cl ₂ was added to this. Compound 1 dissolves in CH ₂ Cl ₂ -MeOH (1:1) but does not dissolve in the solvent resulting from the addition of CH ₂ Cl ₂ (CH ₂ Cl ₂ : MeOH = 6:1), so the resulting precipitate was collected to remove the less polar substance (this operation was performed twice because the supernatant also contained a small amount of compound 1). The two precipitates were combined and concentrated to dryness (914.9 mg). The red mixture obtained by drying was dissolved in 3.0 mL of 50% MeOH and separated by HPLC in 125 μL portions.
The HPLC separation conditions 1 are shown below, and the peaks observed under these conditions are shown in Figure 6.
(HPLC separation condition 1)
Column: Develosil C30-UG-5 10Φ×250 mm
Flow rate: 3.0 mL/min
Solvent: 30% _CH3CN + 0.1% TFA
Detection: DAD (220-600 nm)

When the peak (retention time 17.8 minutes) enclosed in a rectangle shown in Figure 6 was collected and concentrated to dryness, 27.1 mg of pure Compound 1 was obtained.

1-3 Purification of compound 2 Among the filtrate obtained in 1-1, the 80% MeOH solution and the 50% MeOH solution were combined and concentrated to dryness under reduced pressure (12.1 g). The dried product was dissolved in 300 mL of EtOAc and 300 mL of distilled water, and the mixture was shaken in a 1 L separatory funnel to perform two-phase partitioning. As a result, most of the yellow pigment was partitioned into the aqueous phase. After that, the EtOAc phase and the aqueous phase were transferred from the separatory funnel to separate Erlenmeyer flasks, and the EtOAc phase was returned to the separatory funnel and 300 mL of water was added, and two-phase partitioning was performed again in the separatory funnel. The two aqueous phases obtained were combined and concentrated to dryness under reduced pressure (4.82 g).
The concentrated and dried aqueous layer was dissolved in 100 mL of distilled water and passed through a HP20 column (30 mm x 130 mm) developed with water, and the yellow pigment was adsorbed onto the column. After that, 300 mL of distilled water was passed through the column to completely remove (wash) unadsorbed substances, and then 300 mL (100 mL x 3) of 50% MeOH was passed through the column to elute the adsorbed substances. The eluate was collected in one Erlenmeyer flask. Next, the eluate eluted with 300 mL of 100% MeOH was passed through the column, and finally the eluate eluted with 300 mL of 60% acetone was also collected in one Erlenmeyer flask.
The three fractions obtained (50% MeOH elution fraction, 100% MeOH elution fraction, and 60% acetone elution fraction) were analyzed in 30 μL each under the above HPLC analysis conditions without concentration. As a result, it was found that compound 1 and compound 2 were present only in the 50% MeOH fraction (Figure 7).
The resulting 50% MeOH fraction was concentrated to dryness (753.3 mg) and subjected to HPLC separation under the following separation condition 2 to separate compound 2. The compound was dissolved in 14.0 mL of the developing solvent and separated by HPLC in 200 μL portions.
The HPLC separation conditions 2 are described below, and the peaks observed under these conditions are shown in Figure 8.
(HPLC separation conditions 2)
Column: Develosil C30-UG-5 10Φ×250 mm
Flow rate: 3.0 mL/min
Solvent: 20% _CH3CN + 0.1% TFA
Detection: DAD (220-600 nm)

When the peak surrounded by the rectangle shown in Figure 8 (retention time 15.3 minutes) was fractionated and concentrated to dryness, 83.2 mg of pure Compound 2 was obtained.

1-4 Structure determination of compound 1 The pure compound 1 obtained in 1-2 was dissolved in MeOH to a concentration of 0.1% mg/mL, and HRESI-MS (+) was measured. As a result, a (M+Na) ⁺ ion peak was observed at m/z 837.31834, and the molecular formula of compound 1 was determined to be C ₃₈ H ₅₄ O ₁₉ {calcd for 837.31570 (C ₃₈ H ₅₄ NaO ₁₉ , Δ3.16 ppm) }.
Next, 10 mg of Compound 1 was dissolved in 1 mL of CD ₃ OD, and various NMR spectra were measured. The ¹ H NMR spectrum of Compound 1 is shown in FIG. 9, and ^{the 13} C NMR spectrum is shown in FIG. 10. Next, the structure of Compound 1 was analyzed by measuring and analyzing the 2D NMR spectrum ( ¹ H- ¹ H DQF COSY, HMQC, HMBC) of Compound 1.
^{The 1} H- ¹ H DQF COSY spectrum of Compound 1 is shown in FIG. 11, the HMQC spectrum in FIG. 12, and the HMBC spectrum in FIG. 13.
As a result, the structure of compound 1 was estimated to be crocetin neapolitanosyl ester shown in formula (1). Since the types of constituent sugars and geometric isomerism in the aglycone structure could not be determined in the analysis up to this point, we performed acid hydrolysis of compound 1 to confirm these, and separated the constituent sugars and aglycones by two-layer partitioning of EtOAc/water. Isolated and purified (EtOAc layer aglycone, aqueous layer constituent sugars).
Specifically, 5.0 mg of the pure compound 1 obtained in 1-2 was dispensed into a 25 mL eggplant flask, and 5 mL of 2N HCl was added thereto. The sample was immersed in an oil bath set at 100°C or higher and refluxed for 2 hours with the lights off.
Add 5 mL of EtOAc to the lifted eggplant flask, apply ultrasound to dissolve well, and perform two-phase partitioning. The EtOAc layer and aqueous phase are respectively concentrated. The aglycone (2.3 mg) is extracted from the EtOAc phase, and the sugar is extracted from the aqueous phase. (2.0 mg) of each pure product was obtained.
First, it was determined that the constituent sugar was D-glucose based on the ¹ H NMR spectrum in D ₂ O and the [α] _D value (+68.3°) of the aqueous solution. Furthermore, the aglycon was analyzed by ¹ H NMR spectrum (Figure 14) and ¹³ C NMR spectrum (Figure 15), and was determined to be crocetin (compound 3) shown in formula (3). Based on the analysis results thus far, Compound 1 was confirmed to be crocetin neapolitanosyl ester.
There are no reports that it is contained in natural plants such as saffron and gardenia, and this is the first report of its extraction from the petals of the yellow-flowered freesia variety of the present invention. It was confirmed that yellow-flowered freesia is the only plant material that produces crocetin neapolitanosyl ester as its main component.

1-5 Structure determination of compound 2 The pure compound 2 obtained in 1-3 was dissolved in MeOH to a concentration of 0.1 mg/mL, and HRESI-MS (-) was measured. As a result, a (MH) ^-ion peak was observed at m/z 1299.47367, and the molecular formula of compound 2 was determined to be C ₅₈ H ₈₄ O ₃₄ {calcd for 1299.47367, Δ 2.90 ppm}. This was a molecular formula consistent with the binding of neapolitanosyl ester to the free carboxy group in Compound 1. Therefore, ¹³ C NMR spectrum (in CD ₃ OD) was measured based on the assumption that Compound 2 was crocetin di-neapolitanosyl ester (formula (2a)) having a symmetrical structure. The resulting ¹³ C NMR spectrum (Figure 16). The signals observed in compound 1 are C-8' to C-20', C-1'' to C-6'', C-1''' to C-6''', and C-1' Compound 2 was identified as crocetin di-neapolitanosyl ester (formula (2a)) because it completely matched the signal of the ''' to C-6'''' portion.
There are no reports that it is contained as a main component in natural plants such as saffron and gardenia, and this is the first report of its extraction from the petals of the yellow-flowered freesia variety of the present invention. It was confirmed that yellow-flowered freesia is the only plant material that mainly produces crocetindi-neapolitanosyl ester.

1-6 Singlet oxygen scavenging activity of compounds 1 and 3 Normally, oxygen present in the atmosphere is called triplet oxygen, which is the most stable and least reactive, but when triplet oxygen receives energy, it generates singlet oxygen, which is an excited oxygen molecule, and singlet oxygen causes spots and wrinkles. Some carotenoid compounds have excellent singlet oxygen scavenging activity, so the activity of compounds 1 and 3 was evaluated.
In the test used, methylene blue acted as a photosensitizer to generate singlet oxygen. The generated singlet oxygen converts linoleic acid into conjugated linoleic acid, which has a light absorption of 235 nm. By measuring the absorbance at 235 nm, the amount of generated conjugated linoleic acid and thus the amount of generated singlet oxygen could be determined.

Singlet oxygen quenching activity test method 80 μL of 0.025 mM methylene blue solution, 100 μL of 0.24 M linoleic acid solution (all EtOH solutions), 40 μL of sample solution (CH ₂ Cl ₂ solution), and 180 μL of EtOH were added to a disposable culture tube (total 400 μL) and stirred well. To prevent evaporation of the sample solution, a marble was placed on the tube and the tube was irradiated with light for 3 hours under a fluorescent lamp. The experiment was carried out in a polystyrene foam box so that the amount of light irradiation was constant, and the distance between the fluorescent lamp stand and the sample was set to about 17 cm. The experiment was carried out in triplicate, and the average value was used as data.
After 3 hours, 120 μL of the reaction solution was transferred to another tube, and 3,480 μL of EtOH was added thereto (= 30-fold dilution), and the absorbance at λ ₂₃₅ nm (Abs ₂₃₅ ) of the diluted solution was measured.
The singlet oxygen quenching rate of the sample was calculated by the formula {100-(S-B2)/(C-B1)}×100.
Here, S (sample) indicates Abs ₂₃₅ when exposed to light and sample was added, C (Control) indicates Abs ₂₃₅ when exposed to light and no sample was added, B1 (Blank1) indicates Abs ₂₃₅ when not exposed to light and no sample was added, and B2 (Blank2) indicates Abs ₂₃₅ when not exposed to light and sample was added. The absorbance of the control was 1.3, and that of the blank was about 0.4.
The evaluation of each compound was performed in single doses for C, B1, and B2, and in triplicate for each concentration for S. Using the obtained data (average absorbance obtained for S), a regression line was obtained with the ordinate representing inhibition percentage and the abscissa representing compound concentration, from which the _IC50 (the concentration that inhibits 50% of linoleic acid oxidation by singlet oxygen) was calculated.
The experiment was carried out in triplicate for the final concentrations of 1 μM, 10 μM, and 100 μM, adjusted based on the molar number of each sample. As a positive control, β-carotene, a carotenoid widely distributed in nature, was used.

Results The IC ₅₀ values (singlet oxygen 50% elimination concentration, μM) of Compound 1 and Compound 3 determined by experiment are shown in Table 1. It was confirmed that all the compounds had singlet oxygen scavenging activity.

Singlet oxygen scavenging activity of compound 1 and compound 3 (IC ₅₀ value)

Claims (4)

A method for producing an apocarotenoid, comprising a step of extracting an apocarotenoid represented by formula (1) from the petals of yellow freesia .
A method for producing an apocarotenoid, comprising a step of extracting an apocarotenoid represented by formula (2a) from the petals of yellow freesia .
(In the formula, R is represented by formula (2b).)
The method for producing apocarotenoid according to claim 1 or 2, further comprising the step of decomposing the extracted apocarotenoid to obtain an apocarotenoid represented by formula (3).
The manufacturing method according to any one of claims 1 to 3, wherein the yellow-flowered Freesia plant is Airy Flora or Aladdin.