Method for producing codium cylindricum extract

Abstract

To provide a method for producing Codium cylindricum extract with increased content of siphonaxanthin.SOLUTION: The present production method is a method for producing Codium cylindricum extract containing siphonaxanthin, comprising an extraction step in which Codium cylindricum is extracted to obtain crude extract containing siphonaxanthin and its esterified form, which is siphonein; a saponification step in which the solution of the resulting crude extract is treated with an alkali to saponify siphonein; and a removal step performed after the saponification step to remove the insoluble matter, wherein the removal step comprises a procedure of adding an aprotic polar solvent to the reaction solution neutralized with acid in the saponification step, and removing the resulting insoluble matter.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a method for producing Nagamil extract, and more particularly to a method for producing Nagamil extract having an increased siphonaxanthin content. [Background technology]

[0002] Some green algae, such as Caulerpa lentillifera (known as sea grapes), Codium fragile, and Codium cylindricum, contain siphonaxanthin (SPX), a type of carotenoid pigment.

[0003] While siphonaxanthin is found in some green algae, it is not found in higher plants such as land plants. Green algae that grow on the seafloor can only convert some wavelengths of sunlight into energy, so it is thought that the light-harvesting function of siphonaxanthin enables efficient photosynthesis.

[0004] There have been several reports on the physiological functions of siphonaxanthin. For example, Patent Document 1 reports that siphonaxanthin exhibits high antitumor activity at low concentrations compared to fucoxanthin, which is the same carotenoid pigment. Patent Document 2 reports that siphonaxanthin has a blood lipid-reducing effect. Siphonaxanthin, which has such high physiological activity, has attracted attention in the fields of food, medicine, cosmetics, and the like.

[0005] However, since the resource amount of biological species that contain siphonaxanthin is unknown, and furthermore, the amount of siphonaxanthin contained in the green algae themselves is very small, siphonaxanthin has not yet been utilized industrially.

[0006] Nagamil is said to contain more siphonaxanthin than other seaweeds, but the amount of siphonaxanthin contained in the whole alga is very small (about 10 mg per 100 g of algae weight), and there are still problems in utilizing Nagamil and the siphonaxanthin contained in it in an advanced manner. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2009-227642 A [Patent Document 2] JP 2016-210745 A Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention has been made in consideration of the above circumstances, and aims to provide a method for producing Nagamil extract having an increased siphonaxanthin content. [Means for solving the problem]

[0009] The present inventors have noted that Nagamil contains siphonein (hereinafter also referred to as SPN), an ester of siphonaxanthin, as a compound with a structure similar to siphonaxanthin, and that no physiological activity like siphonaxanthin has been reported for SPN, and have discovered that, rather than simply extracting and purifying siphonaxanthin contained in Nagamil, it is possible to obtain a Nagamil extract with an increased siphonaxanthin content by converting siphonein, which is also contained in Nagamil like siphonaxanthin, to siphonaxanthin through saponification treatment. That is, the present invention has the following configuration.

[0010] The method for producing Nagamil extract of the present invention is a method for producing Nagamil extract containing siphonaxanthin, and is characterized by having an extraction step of extracting Nagamil to obtain a crude extract containing siphonaxanthin and its ester, siphonein, and a saponification step of treating the obtained solution of the crude extract with alkali to saponify the siphonein.

[0011] The saponification step is characterized in that the crude extract is reacted in the solution at a temperature of 70° C. to 90° C. for 40 minutes to 3 hours. The saponification step is also characterized in that the solid content of the crude extract in the solution is 0.3% or more and less than 2%.

[0012] In the extraction step, the Nagamil is extracted with acetone or aqueous ethanol.

[0013] The above-mentioned production method includes a removing step of removing insoluble matter after the saponification step, the removing step including a step of adding an aprotic polar solvent to the reaction liquid neutralized with an acid in the saponification step and removing the resulting insoluble matter, and further includes a step of adding a protic polar solvent to the concentrated residue of the step and removing the resulting insoluble matter. Effect of the Invention

[0014] The method for producing Nagamil extract of the present invention comprises obtaining a crude extract containing siphonaxanthin and its ester siphonein from Nagamil, treating the solution of the crude extract with alkali to saponify siphonein, and converting the siphonein contained in the crude extract to siphonaxanthin, thereby obtaining a Nagamil extract with an increased siphonaxanthin content. This Nagamil extract contains siphonaxanthin with high physiological activity and can be used as a material in the fields of food, medicine, and cosmetics, or as a raw material for obtaining purified siphonaxanthin.

[0015] The saponification step is carried out in a solution of the crude extract at a temperature of 70°C to 90°C for 40 minutes to 3 hours, so that the conversion of siphonein to siphonaxanthin is efficiently carried out while the decomposition of siphonaxanthin itself is suppressed, which contributes to an increase in the siphonaxanthin content in the Nagamil extract. In addition, in the saponification step, the solids concentration of the crude extract in the solution is 0.3% or more and less than 2%, so that the saponification reaction can be efficiently carried out while suppressing the decomposition of siphonaxanthin.

[0016] In the above extraction process, Nagamil is extracted using acetone or aqueous ethanol, which allows the polar compounds siphonaxanthin and siphonein to be extracted efficiently and is also safe from the standpoint of application to human skin, etc.

[0017] The above-mentioned manufacturing method includes a step of adding an aprotic polar solvent to the reaction liquid neutralized with acid in the saponification step and removing the resulting insoluble matter, so that saponified products and the like can be removed from the reaction mixture after the saponification reaction, and Nagamil extract with increased purity of siphonaxanthin is obtained. [Brief description of the drawings]

[0018] [Figure 1] This is a photo of raw Nagamil algae. [Diagram 2] 1 is a schematic diagram showing an example of a process for producing Nagamil extract. [Diagram 3] 1 is a graph showing the results of investigation into the extraction process of Test Example 1. [Figure 4] 1 is a graph showing the analytical results of Nagamil extract and the like in Test Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The Nagamil extract produced in the present invention is made from Nagamil. Nagamil (Codium cylindricum) is a type of green algae belonging to the Codiales and Codiaceae families. Nagamil grows in the bathyal zone (subtidal zone) of an inlet close to the open sea, and its habitat is the central to southern Pacific coast of Honshu, Shikoku, Kyushu, etc.

[0020] As shown in the photograph in Figure 1, the algal body of Nagamil is a long, thin, string-like, branched morphology that is 0.5 to 3.0 m or more in length. Currently, Nagamil is used as food in limited areas such as Gokasho Bay in Mie Prefecture, but is hardly used as a fishery resource.

[0021] Fig. 2 shows a schematic diagram of an example of the process for producing Nagamil extract of the present invention. The process for producing the present invention includes at least (a) an extraction process and (b) a saponification process, and further includes (c) a removal process in Fig. 2. Each process will be described in detail below.

[0022] [(a) Extraction process] In this process, Nagamil is extracted to obtain a crude extract containing siphonaxanthin and its ester, siphonein.

[0023] The raw material is Nagamil algae. The Nagamil algae may be in its raw (raw) state, but it is preferable to use the dried algae. The drying method is not particularly limited, and natural drying, heat drying, freeze drying, etc. can be used. From the viewpoint of extraction efficiency, it is preferable to use the dried algae in powder form. For example, a well-known crushing device such as a crusher can be used to crush the algae into powder form.

[0024] The extraction method in the extraction step is not particularly limited, but for example, solvent extraction can be performed. Examples of the extraction solvent include water, organic solvents, and mixtures thereof. Examples of the organic solvent include alcohol-based solvents such as monohydric alcohol solvents having 1 to 4 carbon atoms, such as methanol, ethanol, and isopropanol, and polyhydric alcohol solvents, such as 1,3-butylene glycol and glycerin; ketone-based solvents, such as acetone and methyl ethyl ketone; ester-based solvents, such as ethyl acetate; hydrocarbon-based solvents, such as hexane; ether-based solvents, such as diethyl ether; and halogenated hydrocarbon-based solvents, such as chloroform. These extraction solvents may be used alone or in combination of two or more. In the present invention, the water-containing alcohol refers to a mixture of water and an alcohol-based solvent.

[0025] As the extraction solvent, it is preferable to use acetone or aqueous ethanol in order to efficiently extract siphonaxanthin and siphonein and in consideration of safety to the skin, etc. The ethanol concentration in the aqueous ethanol is, for example, 10 v / v% to 99.5 v / v%, preferably 50 v / v% to 80 v / v%, and may be 60 v / v% or more.

[0026] Nagamil is immersed in the above extraction solvent and allowed to stand or stir at room temperature or under heat to elute the components contained in Nagamil (including siphonaxanthin and siphonein). Thereafter, the extraction residue can be removed by centrifugation or filtration as necessary to obtain a crude extract.

[0027] The amount of the solvent used for extraction is not particularly limited, but is, for example, 10 to 100 times, and preferably 10 to 60 times, the dry weight of Nagamil.

[0028] The extraction conditions are not particularly limited, but the extraction temperature is appropriately set within the range of, for example, 20° C. to 100° C., preferably 20° C. to 80° C., and may be 20° C. to 50° C. The extraction time is appropriately set within the range of 10 minutes to 24 hours, preferably 1 hour to 12 hours. The number of extractions may be one, but it is preferable to perform multiple extractions (preferably 2 to 3 times) in order to improve the extraction efficiency, as shown in the examples described later.

[0029] The extraction method in the extraction step is not limited to solvent extraction, and may be a method using a supercritical fluid such as water or carbon dioxide.

[0030] The crude extract contains siphonaxanthin and its ester, siphonein. Siphonaxanthin is a compound represented by the following formula (1), and its molecular formula is C 40 H 56 Siphonaxanthin has a characteristic chemical structure with a keto group and a hydroxyl group in the basic skeleton, as well as a hydroxyl group in the cyclohexene ring.

[0031] [ka]

[0032] Siphonein is a compound represented by the following formula (2): Siphonein is an ester of siphonaxanthin, specifically, a fatty acid is bound to the CH2-OH group of siphonaxanthin.

[0033] [ka]

[0034] The presence of siphonaxanthin and siphonein in the crude extract can be confirmed, for example, by TLC or HPLC analysis, as described in detail below in the Examples section.

[0035] In the extraction process, the crude extract may be obtained in the form of a solution, or may be obtained as a concentrate (evaporation residue) obtained by concentrating the solution.

[0036] [(b) Saponification step] In this step, the solution of the crude extract obtained in the above extraction step is treated with an alkali to saponify siphonein. As the solution of the crude extract, the crude extract obtained in the above extraction step in solution state may be used as it is, or a concentrate of the crude extract may be dissolved in water or the like to be used.

[0037] The alkali used in the saponification reaction is not particularly limited, and examples thereof include hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metals such as magnesium and calcium; carbonates of alkali metals; carbonates of alkaline earth metals; hydrogen carbonates of alkali metals; and hydrogen carbonates of alkaline earth metals. Preferred are hydroxides of alkali metals. These alkalis may be added as solids or as solutions such as aqueous solutions.

[0038] The amount of alkali to be added to the solution of the crude extract is not particularly limited, but based on the weight of the solid content (evaporation residue) of the crude extract, the amount of alkali is, for example, 0.1 to 10 times, preferably 0.3 to 3 times, and may be less than 1. The alkali is added so that the concentration of the alkali (by weight) in the solution of the crude extract is, for example, 0.1% to 10%, preferably 0.3% to 3%.

[0039] The solvent used in the saponification reaction is not particularly limited, and the solvents listed in the above extraction step can be used appropriately. Among them, water or a mixture of water and an organic solvent is preferable, and in the case of a mixture, it is preferable to use an alcohol solvent or a ketone solvent as the organic solvent.

[0040] The reaction temperature in the saponification reaction is appropriately set, for example, in the range of 20° C. to 100° C., preferably 65° C. to 100° C., and more preferably 70° C. to 90° C. The reaction time is appropriately set, for example, in the range of 10 minutes to 5 hours, and preferably 40 minutes to 3 hours. Carrying out the saponification reaction at a higher temperature for a longer period of time may lead to decomposition of siphonaxanthin.

[0041] In the saponification step, the solid content concentration (weight basis) of the crude extract in the solution is, for example, 0.1% to 10%. It is believed that increasing the solid content concentration will increase the reaction rate of saponification, but on the other hand, there is a risk of decomposing siphonaxanthin, so the solid content concentration is preferably 0.3% or more and less than 2%.

[0042] This saponification reaction converts siphonein (SPN) in the crude extract to siphonaxanthin (SPX), which can be confirmed by TLC or HPLC analysis.

[0043] In the saponification step, an acid such as hydrochloric acid or sulfuric acid is added to the reaction mixture after the saponification reaction to neutralize it to about pH 7.

[0044] [(c) Removal process] In this step, after the saponification step, insoluble matter is removed. This removal step includes at least one of a step (c1) of removing saponified matter and a step (c2) of removing low-polarity components, and may include, for example, only the step (c1), or may include both the step (c1) and the step (c2).

[0045] For example, in the step (c1) of removing the saponified product, an equivalent or more amount of organic solvent is added to the reaction solution neutralized with an acid in the saponification step to precipitate the saponified product, and the saponified product (insoluble matter) is removed by filtration or the like. As the organic solvent, it is preferable to use an aprotic polar solvent such as a ketone solvent, an ester solvent, or an ether solvent, for example, acetone. After removing the insoluble matter, the solvent is distilled off to obtain a concentrated residue. The same organic solvent may be further added to this concentrated residue and the operation may be repeated.

[0046] For example, in the step (c2) of removing the low polarity components, for example, an equivalent or greater amount of a solvent (a solvent different from that in step (c1)) is added to the concentrated residue of the above step (c1) to precipitate the low polarity components, and the low polarity components (insoluble matter) are removed by filtration or the like. As the solvent, it is preferable to use a protic polar solvent such as water-containing alcohol (e.g., 60 v / v % ethanol). After removing the insoluble matter, the solvent is distilled off to obtain a concentrated residue. The same solvent may be further added to this concentrated residue and the operation may be repeated.

[0047] The Nagamil extract of the present invention contains more siphonaxanthin than the crude extract, and also contains other components derived from Nagamil, but does not contain siphonanein. The siphonaxanthin concentration of the Nagamil extract is preferably 50 nmol / mL or more, and may be 80 nmol / mL or more. In addition, compared to the crude extract, the Nagamil extract has a reduced content of other components derived from Nagamil, and the purity of siphonaxanthin is also increased. For example, when the Nagamil extract is analyzed under the HPLC conditions described below, no detection peak is observed between retention times of 19 minutes and 21 minutes.

[0048] The Nagamil extract of the present invention can be obtained, for example, by concentrating the reaction solution after the (b) saponification step or the filtrate after the (c) removal step. The concentrated product can also be adjusted in concentration as necessary to obtain a solution. Obtaining the Nagamil extract as a solution provides excellent handling and storage stability.

[0049] The Nagamil extract of the present invention has an increased content of siphonaxanthin, which exhibits various physiologically active effects, and therefore can be used in foods and beverages, supplements, medicines, cosmetics, etc., which are intended to exhibit such physiologically active effects, and specifically can be used as a material to be blended in them. The Nagamil extract of the present invention can also be used as a raw material for obtaining siphonaxanthin purified by a column or the like. EXAMPLES

[0050] [Test Example 1. Study of the extraction process] (a) Extraction solvent The Bligh & Dyer method is generally known for quantifying siphonaxanthin in algae. However, this method uses solvents such as chloroform and methanol for extraction, which may affect the skin. Taking this into consideration, 95v / v% aqueous ethanol or acetone was used.

[0051] 40 g of Nagamil dry powder was taken, and 30 times the amount of solvent (1200 g) was added, which is the amount that the Nagamil dry powder was fully immersed in, and the mixture was stirred overnight, and then the filtrate was collected by suction filtration. The filtrate was concentrated and dried using an evaporator, and then the volume was adjusted to 250 mL with each solvent, and the siphonaxanthin concentration was measured by HPLC under the following conditions. The results are shown in Figure 3 (a).

[0052] Column: TSK-Gel ODS-80Ts, 4.6 φ x 250 mm Temperature: 40℃ Mobile phase: A; acetonitrile:methanol:water (75:15:10) / 0.1% ammonium acetate B: ethyl acetate:methanol (70:30) / 0.1% ammonium acetate Gradient: 0-5 min; A100%, 5-20 min; A100% → B100%, 20-35 min; B100%, 35-40 min; B100% → A100%, 40-50 min; A100% Flow rate: 0.8 mL / min Detection: 450nm OD

[0053] The retention times of siphonaxanthin and siphonein under the above HPLC analysis conditions are as follows: Siphonaxanthin (SPX): approx. 7 minutes Siphonane (SPN): approx. 18 minutes

[0054] As shown in FIG. 3(a), the siphonaxanthin concentrations (nmol / mL) in the crude extracts in 95 v / v% aqueous ethanol and acetone were 20.3 and 16.6, respectively, and no significant difference was observed between the two solvents.

[0055] (b) Number of samplings 0.1 g of Nagamil dry powder was placed in a stoppered test tube, 3 mL of 95 v / v% aqueous ethanol was added, and the mixture was stirred overnight for extraction. After extraction, the mixture was centrifuged and the supernatant was collected. An additional 3 mL of 95 v / v% aqueous ethanol was added to the residue, and the same procedure was repeated. This was repeated three times. 2 mL of the obtained supernatant was accurately weighed out, methanol was added to make the volume constant to 20 mL, and the siphonaxanthin concentration was measured by HPLC. The extraction efficiency was calculated using the siphonaxanthin content of the Nagamil dry powder extracted by the Bligh & Dyer method. The results are shown in Figure 3 (b).

[0056] As shown in Figure 3(b), the extraction was repeated four times, and the concentration of siphonaxanthin extracted decreased with each iteration, dropping to about 1 / 20 of the first iteration in the third iteration, and in the fourth iteration siphonaxanthin was hardly detected. Based on these results, taking into account the amount of work required for siphonaxanthin recovery and extraction procedures, it is believed that two extractions is the most efficient.

[0057] (c) Ethanol concentration As the extraction solvent, aqueous ethanol concentrations of 60v / v%, 80v / v%, and 95v / v% were used. 0.1g of Nagamil dry powder was placed in a stoppered test tube, 3mL of ethanol was added, and the mixture was stirred overnight for extraction. After extraction, the mixture was centrifuged and the supernatant was collected. An additional 3mL of ethanol was added to the residue, and the same procedure was carried out. 2mL of the obtained supernatant was accurately weighed out, methanol was added, and the volume was adjusted to 20mL, and the siphonaxanthin concentration was measured by HPLC. The extraction efficiency was calculated using the siphonaxanthin content of the Nagamil dry powder extracted by the Bligh & Dyer method. The results are shown in Figure 3(c).

[0058] As shown in Figure 3(c), in a single extraction operation, the extraction efficiency was slightly lower when 60 v / v% aqueous ethanol was used, but when the extraction operation was repeated twice, no difference in extraction efficiency was observed depending on the ethanol concentration. From this result, it is thought that, for example, an ethanol concentration of 60 v / v% or more does not affect the extraction efficiency.

[0059] (d) Condition of algae 10 g of dried algae were either crushed or crushed, and 300 mL of 95 v / v% aqueous ethanol was added to each, followed by stirring overnight for extraction. After extraction, the mixture was centrifuged and the supernatant was collected. The residue was treated in the same manner with 300 mL of 95 v / v% aqueous ethanol. The ethanol was removed from the resulting supernatant, which was then redissolved in acetone and adjusted to a constant volume of 100 mL. An additional 2 mL was accurately weighed out, and acetone was added to adjust the volume to 20 mL, and the siphonaxanthin concentration was measured by HPLC. The extraction efficiency was calculated using the siphonaxanthin content of the Nagamil dry powder extracted by the Bligh & Dyer method. The results are shown in Figure 3(d).

[0060] As shown in Figure 3(d), in the case of extraction from crushed algae, approximately 80% of the siphonaxanthin contained in the algae was extracted in the second extraction. In contrast, in the case of extraction from the whole algae, the extraction efficiency of siphonaxanthin was low, and only about 24% was extracted in the second extraction. It was found that the extraction efficiency increased dramatically by crushing the algae.

[0061] [Test Example 2. Examination of the saponification process] (a) Amount of alkali added Potassium hydroxide was used as the alkali. 0.5g, 1g, 2.5g, 5g, and 10g of KOH (potassium hydroxide concentrations 0.1%, 0.2%, 0.5%, 1%, and 2%) were dissolved in 500g of an aqueous solution of Nagamil crude extract with a solid content (evaporation residue) concentration of 1%, and the saponification reaction was carried out at 80°C for 1 hour. After cooling, the solution was neutralized with HCl, and acetone was added to recover the acetone-soluble components. After removing the solvent, the solution was dissolved again in acetone and the volume was adjusted to 500mL. An additional 0.2g was accurately weighed out, acetone was added, the volume was adjusted to 20mL, and the siphonaxanthin concentration was measured by HPLC. The extraction efficiency was calculated by accurately weighing 0.5g of an aqueous solution of Nagamil crude extract with a solid content concentration of 1%, adding acetone, adjusting the volume to 20mL, and measuring the siphonaxanthin concentration by HPLC. An extraction efficiency of more than 100% indicates that the siphonaxanthin concentration has increased compared to before the saponification reaction. The results are shown in the following Table 1. In addition, in the column of the amount of potassium hydroxide added in Table 1, the amount multiplied by the weight of the solid content is also shown.

[0062] [Table 1]

[0063] As shown in Table 1, the extraction efficiency increased depending on the amount of potassium hydroxide added up to a potassium hydroxide concentration of 0.5%. On the other hand, no difference in extraction efficiency was observed at potassium hydroxide concentrations of 1% or more.

[0064] (b) Solids concentration of the crude extract solution 500g of an aqueous solution of Nagamil crude extract with a solid content concentration of 1% was concentrated 2-fold, concentrated 5-fold, and diluted 2-fold (solid content concentrations of 2%, 6%, and 0.5%, respectively), and 2.5g of potassium hydroxide (2.5g per 5g solid weight) was dissolved in each solution, and saponification reaction was carried out at 80°C for 1 hour. The operation after the reaction was carried out in the same manner as in "(a) Amount of alkali added" above, and the extraction efficiency was calculated based on the siphonaxanthin concentration. The results are shown in Table 2 below.

[0065] [Table 2]

[0066] As shown in Table 2, when the solid concentration was 1%, the extraction efficiency was 150%, but as the solid concentration increased, the extraction efficiency decreased. The cause is presumed to be the decomposition of siphonaxanthin. On the other hand, even if the solution was diluted (the solid concentration was reduced), the effect on the extraction efficiency was minor.

[0067] (c) Reaction temperature 2.5 g of potassium hydroxide was dissolved in 500 g of an aqueous solution of Nagamil crude extract with a solid content concentration of 1%, and the saponification reaction was carried out at the boiling temperature (98°C), 80°C, and 60°C for 1 hour. The operation after the reaction was carried out in the same manner as described above in "(a) Amount of alkali added", and the extraction efficiency was calculated based on the siphonaxanthin concentration. The results are shown in Table 3 below.

[0068] [Table 3]

[0069] As shown in Table 3, at the boiling temperature of 98°C, significant foaming occurred due to the soap produced in the saponification reaction, making accurate measurement difficult. At 60°C, the extraction efficiency was significantly lower than at 80°C.

[0070] (d) Reaction time 3 g of potassium hydroxide was dissolved in 500 g of an aqueous solution of crude extract of Nagamil with a solid content concentration of 1%, and the saponification reaction was carried out at 80°C for 30 minutes, 1 hour, 1.5 hours, and 2 hours. The operation after the reaction was carried out in the same manner as described above in "(a) Amount of alkali added", and the extraction efficiency was calculated based on the siphonaxanthin concentration. The results are shown in Table 4 below.

[0071] [Table 4]

[0072] As shown in Table 4, the extraction efficiency was higher for 1 hour than for 30 minutes, but it was confirmed that the extraction efficiency decreased when the reaction was continued for more than 1 hour. This is presumably due to the decomposition of siphonaxanthin.

[0073] [Test Example 3. Production of Nagamil Extract] A crude extract was obtained by adding 60 times the weight of 95v / v% aqueous ethanol to the dried Nagamil powder. Potassium hydroxide was added to this, and it was heated in a boiling bath for 1 hour, after which HCl was added to neutralize it. The reaction liquid was then mixed with an equal amount of acetone, filtered through filter paper, and the process of concentration and drying was repeated to desalt it, and it was then redissolved in acetone to obtain the Nagamil extract.

[0074] The obtained Nagamil extract was analyzed by TLC and HPLC to determine whether siphonanein (SPN) was converted to siphonaxanthin (SPX), and the siphonaxanthin content per solid was measured and compared with that of the crude extract (before saponification reaction). In the TLC analysis, PR-18 (40 mm x 100 mm) was used as the thin layer, and 95 v / v% aqueous ethanol was used as the developing phase. The HPLC analysis was performed under the conditions described above. The results of each analysis are shown in Figure 4.

[0075] As shown in FIG. 4, it was confirmed that the crude extract (before saponification reaction) contained SPN together with SPX, and the content (corresponding to the area) of SPN was detected to be greater than that of SPX. In contrast, in the Nagamil extract (after saponification reaction), SPN was not detected, and the content of SPX was increased by the corresponding amount (see FIG. 4(c)). From these results, it was found that no siphonein (SPN) remained after the saponification reaction, and it was converted to siphonaxanthin (SPX). In addition, by performing a removal process after the saponification process, the purity of SPX could be increased. As shown in FIG. 4(b), in the HPLC chart of the Nagamil extract, the peaks due to other components derived from Nagamil that were seen in the crude extract disappeared or were reduced. For example, several peaks detected between retention times of 19 minutes and 21 minutes were not detected in the Nagamil extract. In addition, the peak intensity of SPX was the strongest in the Nagamil extract.

[0076] The present invention has established a method for efficiently obtaining siphonaxanthin (SPX) at high concentrations from Nagamil. The Nagamil extract obtained by this manufacturing method has a siphonaxanthin content per solid content that is approximately twice as high as that of the crude Nagamil extract, and this can be a technology for efficiently obtaining highly physiologically active siphonaxanthin (SPX) at high concentrations. Furthermore, according to the present invention, Nagamil has been discovered as a new marine resource, which may lead to advanced use of Nagamil in the future.

Claims

1. A method for producing a Nagamil extract containing siphonaxanthin, A method for producing Nagamil extract, comprising an extraction step of extracting Nagamil to obtain a crude extract containing siphonaxanthin and its ester, siphonein, and a saponification step of treating the obtained solution of the crude extract with alkali to saponify the siphonein.

2. The method for producing Nagamil extract according to claim 1, characterized in that the saponification step comprises reacting the crude extract in the solution at a temperature of 70°C to 90°C for 40 minutes to 3 hours.

3. A method for producing Nagamil extract according to claim 1 or 2, characterized in that in the saponification step, the solids concentration of the crude extract in the solution is 0.3% or more and less than 2%.

4. A method for producing Nagamil extract according to claim 1 or claim 2, characterized in that in the extraction step, the Nagamil is extracted using acetone or aqueous ethanol.

5. The manufacturing method of Nagamil extract described in claim 1 or claim 2, characterized in that the manufacturing method includes a removal step of removing insoluble matter after the saponification step, and the removal step includes a step of adding an aprotic polar solvent to the reaction liquid neutralized with acid in the saponification step and removing the resulting insoluble matter.

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