WO2021132764A1 - Method for separating and purifying fucoxanthin from diatoms - Google Patents

Abstract

The present invention relates to a method for separating and purifying fucoxanthin from diatoms, and provides the optimal method for separating and purifying fucoxanthin from adhesive diatoms collected from Yong Am sea water on the South Korean island of Jeju. Highly pure fucoxanthin, separated and purified from the adhesive diatoms cultured at a large scale, can be used and applied in a variety of industrial fields such as medicine and cosmetics.

Description

Separation and purification method of fucoxanthin derived from diatoms

The present invention relates to a method for separating and purifying diatom-derived fucoxanthin, and more particularly, to an optimal method for separating and purifying fucoxanthin from adherent diatoms taken together with Jeju lava seawater.

Lava seawater is seawater that penetrates the basalt layer of Jeju Island and flows underground. Lava seawater is found mainly in the western and eastern parts of Jeju Island and contains high concentrations of minerals. In addition, while general seawater is exposed to unstable environments such as domestic sewage, industrial wastewater, and port pollution, it takes a lot of money to process industrial materials, whereas lava seawater undergoes natural purification and filtration by volcanic bedrock to block heavy metal adsorption and harmful substances. Therefore, it secures safety, stability, and economic feasibility, and the cost of water intake is incomparably lower than that of deep sea water taken from the deep sea.

The inventor of the present application confirmed that adherent diatoms were taken together with lava seawater in the process of aquaculture using Jeju lava seawater, and established a method of mass-cultivating these adherent diatoms dominantly. Adherent diatoms can be used as a valuable resource as an alternative energy source for feed in the aquaculture industry or as a basic material in various industrial fields such as medicine, environment, and life industry.

On the other hand, the carotenoid market is classified into synthetic carotenoids and carotenoids derived from natural products, and is growing steadily. The global carotenoid market was valued at USD 1.5 billion in 2014 and is expected to increase to approximately USD 1.8 billion in 2019. In particular, the value of xanthophyll-based carotenoids such as lutein, canthaxanthin, and astaxanthin is increasing, and they are mainly used as natural pigments, antioxidants, vitamin A precursors, and aquaculture feed additives.

Fucoxanthin refers to a kind of carotenoid pigment that is mainly present in brown algae such as seaweed, kelp, motherwort, and turmeric. Brown algae are brown because their unique photosynthetic pigment called fucoxanthin is relatively higher than that of chlorophyll. It assists photosynthesis by capturing light energy and delivering about 80% of it to chlorophyll. The captured light energy absorbs light with a wavelength of 450 to 540 nm on the spectrum, and absorbs the most light with a wavelength of 510 to 525 nm, and is known to have various effects such as anti-obesity, anti-inflammatory, skin protection, and anti-aging.

We provide an optimal method for separating and purifying fucoxanthin from adherent diatoms taken from lava seawater in Jeju, Korea.

In the present invention in order to solve the above problems, (S1) agitating the diatoms collected by mass culture in 70% alcohol; (S2) loading and filtering on a filter bed pre-coated with diatomaceous earth; (S3) fractionating and concentrating the filtered extract; (S4) separating and purifying fucoxanthin from the concentrated extract; And (S5) provides a separation and purification method of diatom-derived fucoxanthin comprising the step of storing the separated and purified fucoxanthin in a dark room.

In the present invention, the diatoms may be adherent diatoms that are taken together with Jeju lava seawater.

In the present invention, the adherent diatoms are Melosira nummuloides, Achnanthes brevipes var. It may be at least one species selected from the group consisting of intermedia, Achnanthes sancti-pauli, Achnanthes brevipes, or Melosira octogona.

In the present invention, the diatoms may be diatoms collected from a bottom diatom culture.

In the present invention, step (S1) may be characterized in that it is treated with an alcohol amount of 7 times (v/v) compared to the raw material for 1 day.

In the present invention, it may further include the step of treating plasma for 25 minutes in step (S1).

In the present invention, (S4) the step of separating and purifying fucoxanthin from the concentrated extract may be characterized in that it is performed by silica gel column chromatography.

In the present invention, the elution solvent used for the silica gel column chromatography may be characterized in that the ratio of N-hexane to acetone is 7:3.

The present invention provides an optimal method for separating and purifying fucoxanthin from adherent diatoms taken from lava seawater in Jeju, Korea. Using high-purity fucoxanthin separated and purified from adherent diatoms cultured in large quantities, medical and cosmetic It can be used in various industrial fields such as fields.

1 is a standard solution 1 in which the raw material of diatomaceous material is stirred in alcohol, loaded on a filter bed pre-coated with diatomaceous earth and filtered, and fucoxanthin purified by silica gel column chromatography is dissolved in methanol. HPLC analysis for

Figure 2 is HPLC for the mother liquid of standard solution 2, in which the raw material of diatomaceous material was stirred in alcohol, loaded on a filter bed pre-coated with diatomaceous earth, filtered, and stored at -20°C for two days for recrystallization. analysis is shown.

Figure 3 is HPLC for the crystallization of standard solution 2, in which the raw material of diatomaceous material is stirred in alcohol, loaded on a filter bed pre-coated with diatomaceous earth, filtered, and stored at -20°C for two days for recrystallization. analysis is shown.

Figure 4 shows the HPLC analysis of the standard solution 3 prepared in the same manner as the standard solution 1, but without the desalting reaction.

Figure 5 shows the HPLC analysis of the standard solution 3 prepared in the same manner as the standard solution 1, but subjected to a desalting reaction.

6 shows the HPLC analysis of the standard solution 4 prepared in the same manner as the standard solution 1, but exposed to sunlight.

7 shows the HPLC quantitative analysis of Sample No 4 of Table 1.

8 shows the HPLC quantitative analysis of Sample No 5 of Table 1.

9 shows the HPLC quantitative analysis of Sample No 6 of Table 1.

Figure 10 shows a schematic diagram of the fucoxanthin separation and purification process.

Figure 11 shows the fucoxanthin separation and purification process by silica gel column chromatography (silica gel column chromatography).

Figure 12 shows the results of TLC (Thin Layer Chromatography) of the fraction containing fucoxanthin.

13 shows the concentration process of fucoxanthin.

14 shows the creation of a standard calibration curve using standard fucoxanthin (purchased from Sigma Aldrich).

15 shows HPLC quantitative analysis of 60% pure fucoxanthin finally obtained through fractionation/concentration process from 70% alcohol extract of diatom powder.

16 shows the diatomaceous earth precoat filtration and recrystallization process.

17 shows a microscopic observation photograph (×400) of the filtration process and recrystallization.

18 shows the HPLC quantitative analysis of fucoxanthin subjected to recrystallization.

Figure 19 shows the HPLC quantitative analysis of Sample No 1 of Table 3.

20 shows the HPLC quantitative analysis of Sample No 3 of Table 3.

Figure 21 shows the HPLC quantitative analysis of Sample No 15 of Table 3.

Figure 22 shows the HPLC quantitative analysis of Sample No 16 of Table 3.

23 shows a powder formulation of fucoxanthin.

Figure 24 shows the HPLC quantitative analysis of Sample No 8 of Table 3.

25 shows the HPLC quantitative analysis of Sample No 9 of Table 3.

26 shows the HPLC quantitative analysis of Sample No 10 of Table 3.

27 shows the HPLC quantitative analysis of Sample No 11 of Table 3.

28 to 47 show HPLC quantitative analysis of samples 1 to 20 of Table 6.

As an embodiment of the present invention, (S1) agitating the diatoms collected by mass culture in 70% alcohol; (S2) loading and filtering on a filter bed pre-coated with diatomaceous earth; (S3) fractionating and concentrating the filtered extract; (S4) separating and purifying fucoxanthin from the concentrated extract; And (S5) provides a separation and purification method of diatom-derived fucoxanthin comprising the step of storing the separated and purified fucoxanthin in a dark room.

In another embodiment according to the present invention, the diatoms may be adherent diatoms that are taken together with Jeju lava seawater.

In another embodiment according to the present invention, the adherent diatom is Melosira nummuloides, Achnanthes brevipes var. It may be at least one species selected from the group consisting of intermedia, Achnanthes sancti-pauli, Achnanthes brevipes, or Melosira octogona.

In another embodiment according to the present invention, the diatoms may be diatoms collected from a bottom diatom culture.

In another embodiment according to the present invention, step (S1) may be characterized in that it is treated with an alcohol amount of 7 times (v/v) compared to the raw material for 1 day.

In another embodiment according to the present invention, it may further include the step of treating plasma for 25 minutes in step (S1).

In another embodiment according to the present invention, (S4) the step of separating and purifying fucoxanthin from the concentrated extract may be characterized in that it is performed by silica gel column chromatography.

In another embodiment of the present invention, the elution solvent used for the silica gel column chromatography may be characterized in that the ratio of N-hexane to acetone is 7:3.

Hereinafter, the present invention will be described in more detail through specific examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1. Optimization of fucoxanthin separation and purification method

The separation and purification method was optimized by measuring the change in the content of fucoxanthin according to the separation and purification method. To this end, 50 g of diatoms in wet state were first put and dried in a vacuum dryer for 48 hours, and thereafter, the change in the content of fucoxanthin by various processes was measured using HPLC.

First, standard solution 1 was stirred for 2 hours in 70% alcohol with diatoms, and then loaded into a filter bed pre-coated with diatomaceous earth, filtered and concentrated under reduced pressure. Accordingly, 1.2 g of the sample was weighed with an electronic balance and subjected to silica gel column chromatography (Φ 3.5 cm x 15 cm), and as an elution solvent, N-Hexane: Acetone = 70: 30 was flowed. The separated and purified fucoxanthin was dissolved in methanol to prepare a standard solution 1 having a concentration of 0.1%. After that, standard solution 1 was dispensed in a transparent container and the content of fucoxanthin was measured by HPLC. There was no color change and fucoxanthin content was calculated based on the peak area value. When compared with the following standard solution 2, it was found that the content of fucoxanthin was high, and the other components were removed to show a purity of 90% (FIG. 1).

Next, standard solution 2 was prepared through a recrystallization process. Dissolving a solute in a solvent at a higher temperature can dissolve a larger amount of the solute than at a lower temperature. In this dissolved state, if the temperature is lowered again slowly, the solubility decreases, so the solute is precipitated again to form crystals, which is called recrystallization. In the present invention, after stirring and filtering 5 g of raw diatoms in 70% alcohol for 2 hours, the filtered sample for recrystallization experiment was stored at -20°C for two days to prepare standard solution 2. After that, standard solution 2 was dispensed in a transparent container and the content of fucoxanthin was measured by HPLC. There was no color change and fucoxanthin content was calculated based on the peak area value. Compared with standard solution 1, the chlorophyll component was not removed, so that the crystal sample had a purity of 75%, and the mother solution sample had a purity of 61% ( FIGS. 2 and 3 ).

Standard solution 3 proceeded in the same manner as standard solution 1 except for adding the desalting process for raw diatoms, and the effect of the desalting process was verified. Standard solution 3 was dispensed in a transparent container and the content of fucoxanthin was measured by HPLC. There was no color change and fucoxanthin content was calculated based on the peak area value. The observed fucoxanthin content was lowered to a level of 21% compared to standard solution 1 when desalting was performed, and was lowered to a level of 17% when desalting was not performed ( FIGS. 4 and 5 ).

The following standard solution 4 verified the stability of fucoxanthin by changing the content of fucoxanthin when exposed to sunlight. Standard solution 4 was dispensed in a transparent container and the content of fucoxanthin was measured by HPLC. There was no color change and fucoxanthin content was calculated based on the peak area value. As a result of observing the content and purity of fucoxanthin after being left in sunlight for a week, the content decreased by about 82%, and the purity decreased from 90% to 55% (FIG. 6).

In conclusion, as a result of HPLC analysis of a fucoxanthin preparation dissolved in methanol at a concentration of 0.1%, standard solution 1 was found to be excellent in content and purity. When silica gel column chromatography was not performed, separation and purification were not performed properly. Thus, it was found that the content and purity of fucoxanthin were lowered. In addition, it was confirmed that the desalting process did not significantly affect the fucoxanthin content and purity, and the recrystallization process also showed a somewhat lower purity and content of fucoxanthin compared with the standard solution 1. Furthermore, it was confirmed in Standard Solution 4 that fucoxanthin was sensitive to sunlight exposure.

Example 2. Determination of a suitable solvent for fucoxanthin extraction

In the separation of fucoxanthin, the solvent extraction of the same type of microalgal powder showed the highest extraction yield when alcohol was used, and other extraction methods (Sohxlet method, ultrasonic extraction method, subcritical extraction method, etc.) 70% alcohol was used as the extraction solvent in consideration of the result that it was not significantly different from the use of alcohol and the fact that it can be used in food.

However, since the amount of extraction solvent used is an important factor in determining the production cost in the future industrialization process, an appropriate solvent ratio compared to the raw material for diatoms was first tested. 10 times 70% alcohol was stirred in 100 g of diatom powder (sample Nos. 1, 2, 3), and 10 times, 20 times, and 30 times 70% alcohol was stirred in 50 g (No. 4, 5, 6), respectively. The extraction efficiencies were compared and shown (Table 1, FIGS. 7 to 9).

(※ No.1-3; 70% alcohol 10x filtrate, No.4; 70% alcohol 10x filtrate, No.5; 70% alcohol 20x filtrate, No.6; 70% alcohol 30x filtrate)

In the section extracted with 10 times the volume of alcohol, a total of 250 mg, at 20 times, 274 mg, and at 30 times, 260 mg of fucoxanthin were extracted. The extraction efficiency slightly increased as the amount of alcohol increased, but the difference was around 10%, so proper extraction The amount of solvent was carried out on the basis of 10 times 70% alcohol. However, additional experiments were conducted to determine more accurate solvent conditions, and the results are shown in Example 9 below.

Example 3. Separation and purification of fucoxanthin

Silica gel chromatography is used for separation and purification of functional substances such as fucoxanthin. will be. In the present invention, silica gel chromatography was performed under various conditions to efficiently separate and purify fucoxanthin, a target material.

As for the whole process, according to FIG. 10, 70% alcohol in a 10-fold volume was added to the diatom powder, stirred for 2 hours, filtered to obtain each extract, and fractionation process and concentration process were performed, and then separated by silica gel chromatography.

Specifically, the primary alcohol extract obtained by first filtering the extract extracted from 100 g of diatom powder with 70% alcohol at 10 times the volume with 5um filter paper was separated into n-Hexane: 70% alcohol (1:1), and 70% alcohol in the lower layer A fraction was obtained. In order to separate fucoxanthin from the fraction, silica was filled in a column to load a sample, and a gradient (Hexane:Acetone=7:3→6:4→5:5→Acetone) separation process was performed to obtain final fractions. . After that, a fraction (dark orange layer) that is expected to contain fucoxanthin was obtained and TLC (Thin Layer Chromatography) was performed (FIGS. 11 and 12), and as a result, fractions from Fraction No. 1 to No. 9 It was confirmed that fucoxanthin was separated from the , and the concentration process was performed using a high pressure vacuum concentrator (FIG. 13).

Example 4. Quantitative analysis of fucoxanthin using HPLC

First, the standard material fucoxanthin (purchased from Sigma Aldrich) was diluted by concentration to prepare an HPLC standard calibration curve (FIG. 14). For HPLC analysis, Waters 2695 HPLC equipment was used, the column used was Kromasil 100-5-C18 (4.6x250mm, 5um), the detection wavelength was 450 nm, and the solvent was Acetonitrile / water (Table 2).

As a result of quantitative HPLC analysis, 8.7 g of fucoxanthin having a purity of 60% was finally obtained through a fractionation/concentration process from a 70% alcohol extract of 100 g of diatom powder, and as a result, a yield of 5.22% was obtained (FIG. 15).

Example 5. Utility test of recrystallization method

The usefulness of recrystallization of the concentrate to improve the degree of purification and yield of fucoxanthin was studied. Specifically, the primary extract obtained by stirring 100 g of diatomaceous powder with 70% alcohol at 10 times the volume for 2 hours was loaded into a filter bed coated with 50 g of diatomaceous earth to obtain an extract filtrate. The finally obtained concentrate was dissolved in acetone, and the fucoxanthin fraction was sampled by silica gel chromatography, and it was checked by TLC (Thin Layer Chromatography) to confirm that the fucoxanthin was separated (FIG. 16).

Recrystallization was attempted from the fucoxanthin fraction, and the recrystallization process is as follows. The sample was mixed with the same amount of water at 50°C, stirred at 40°C for 2 hours, stored at -20°C overnight, and recrystallized through filtration. The crystals of fucoxanthin were confirmed through microscopic observation (400 magnification) (FIG. 17), and HPLC quantitative analysis was performed under the above conditions (FIG. 18). As a result, although it was possible to concentrate fucoxanthin through recrystallization, it was confirmed that the usefulness was not great for isolating the desired high-purity fucoxanthin.

Example 6. Comparison of fucoxanthin content in various culture/environmental conditions

Changes in fucoxanthin content in diatoms according to the diatom culture environment, sea salt removal, drying type, drying temperature, etc. were comparatively analyzed ( FIGS. 19 to 22 , Table 3).

(※ No.1; Bottom diatom/Desalted X/Naturally dry, No.2; Bottom Diatom/Demineralized/60℃ drying, No.3; Floating diatom/Demineralized X/Cold thaw/Naturally dry, No.4; Floating diatom /Desalting / 60℃ drying, No.5; Bottom diatom / Demineralizing 2 / Natural drying, No.7 Floating diatom / 40℃ hot air drying, No.8-11; Beta cyclodextrin formulation powder, No.12-14; Drying Powder/1,3BG Extract/10x,7x,10x, No.15; Fresh Diatom/1,3BG Extract 10x, No.16; Fresh Diatom/Alcoholic Extract 10x)

HPLC analysis was performed under the same conditions as in Example, and dry diatom powder (No. 1 to No. 11) was dissolved in methanol at a concentration of 50 mg/mL, sonicated for 30 minutes, filtered, and then used for analysis. This was repeated 3 times. As a result, in the diatom growth environment, the bottom diatom showed a higher fucoxanthin content than the floating diatom (Table 3, No. 1 & No. 3), and the presence or absence of desalination did not significantly affect the content change. appear.

On the other hand, as a result of analyzing the change in the fucoxanthin content according to the drying temperature, it was found that the fucoxanthin content was significantly reduced in the condition of the drying temperature of 50° C. or higher compared to natural drying (Table 3, No. 1 to No. 5). This result is consistent with the existing research results that fucoxanthin has low thermal stability, and suggests that it is important to set the environment and extraction conditions below 50°C for industrial production of fucoxanthin.

Among the analyzed samples, the diatomite sample that had the highest content of fucoxanthin was an undried diatomite sample. This result indicates that some of the fucoxanthin in the diatom was decomposed during the collection and drying (natural drying or hot air drying) process of the diatom culture ( No. 15, 16). In conclusion, in the first step in establishing the mass extraction process for fucoxanthin, the harvesting/processing step of diatom, it is the most efficient process to directly harvest the raw diatom without going through the drying process and directly enter the extraction process.

Example 7. Formulation of fucoxanthin

To the concentrate obtained by preparing the primary alcohol extract, filtering and concentrating, the same amount of beta-cyclodextrin and 30% increments were added to make a dough, followed by freeze-drying for 72 hours. A powder formulation was prepared by pulverizing the freeze-dried sample, and the content of fucoxanthin in each sample was analyzed (FIGS. 23 to 27, Table 3 No. 8 to 11). As a result of the analysis, there was no significant difference in fucoxanthin content according to the change in the amount of dextrin added, and the highest 316ppm (0.6%) was obtained in the sample (No.8) in which the same amount of dextrin was added to the concentrated solution 5 times more concentrated than other samples. However, compared to other samples, it showed problems in color and smell. In conclusion, it can be confirmed that the most efficient method is to prepare the formulation for food under conditions that minimize the addition of beta-cyclodextrin as an excipient, that is, add dextrin in the same amount as the extract concentrate (1:1).

Example 8. Analysis of fucoxanthin extraction efficiency according to plasma treatment

In extracting fucoxanthin from fresh diatom, the optimal extraction conditions were determined by comparing and analyzing the extraction efficiency of fucoxanthin according to the presence or absence of plasma treatment.

First, extracts were prepared from samples treated with plasma and untreated biodiatoms to compare the extraction yield of fucoxanthin. After adding 20 mL of 70% alcohol to 5 g of biological diatom, plasma was treated for 25 minutes, extracted at 40 ° C for 3 days, the extract was centrifuged at 3500 rpm for 10 minutes to remove the extraction residue, and HPLC analysis was performed using the extract of the upper layer. As a control, a sample that was not subjected to plasma treatment under the same extraction conditions as above was set as a control. Since the extract was small, the extract was obtained after removing the residue by centrifugation without going through a filtration process (Table 4), and the fucoxanthin extraction efficiency according to plasma treatment was reviewed through HPLC analysis (Table 5).

* NP: no plasma treatment, Pla: plasma treatment

According to the table, 1.08 mg was extracted from the sample not treated with plasma, whereas 1.22 mg was extracted from the sample treated with plasma, confirming that the extraction efficiency was increased by about 13% by plasma treatment. This suggests that the plasma treatment of the sample caused a change in the physical properties of the diatom cell wall components, and that the intracellular components were easily eluted.

Example 9. Optimization of fucoxanthin extraction efficiency

In extracting fucoxanthin from fresh diatom, the optimal extraction conditions were determined by comparatively analyzing the extraction efficiency according to the type of solvent used, the amount of solvent, and the extraction time.

First, in order to determine the amount of extraction solvent and the conditions according to time, the sample amount is 50 g of biological diatom (No. 1~18), and the extraction solvent is 70% alcohol or 1,3BG (Butylene Glycol), and , 3 times, 5 times, and 7 times the amount of the sample (v/v) was set for extraction (Sample name = Alcoholic solvent: A3, A5, A7. BG solvent: B3, B5, B7), and here The extraction time was set to 1 day, 3 days, and 5 days, respectively, and extraction was performed (sample name = 1 day: 1d, 3 days: 3d, 5 days: 5d).

Filtration and centrifugation procedures were as follows. Each extraction sample was filtered with filter paper (Hyundai No. 21: 8-12um, 90mm) and a vacuum pump to filter out residues, and then used for analysis (Table 6, FIGS. 28 to 47). However, since the BG extract has a very high viscosity, filtration takes more than 24 hours, causing variations in the extraction time conditions. Therefore, the samples extracted for 3 and 5 days were centrifuged at 3500 rpm for 10 minutes to obtain an extract (No. 10 to 12). , No. 16-18).

(* A: 70% alcohol, B: 1,3BG, 3/5/7: 3x,5x,7x, d: day, NP: no plasma treatment, Pla: plasma treatment)

(Unit: mg)

First, as a result of analyzing the Fx extraction efficiency according to extraction time using 70% alcohol as a solvent, when extracting 3 times 70% alcohol (150 mL), 13 mg per day, 9 mg for 3 days, 8 mg for 5 days, 1 for 5 times (250 mL) extraction It was found that the Fx extraction efficiency decreased with the passage of time at 29 mg per day, 23 mg per day, 23 mg per day, 19 mg per day, and 7-fold (350 mL), 13 mg per day, 9 mg per day, and 8 mg per day (Table 7). From this, it can be seen that in the 70% alcohol extraction condition, it is most effective to extract for one day at 7 times (v/v) the amount of alcohol compared to the original.

(Unit: mg)

Next, as a result of analyzing the Fx extraction efficiency according to the extraction time with 1,3BG as a solvent, when extracting 3 times (150 mL) 1,3BG, 18 mg per day, 24 mg for 3 days, 28 mg for 5 days, 1 for 5 times (250 mL) extraction 32mg per day, 38mg 3 days, 38mg 5 days, 7 times (350mL) extraction, Fx extraction efficiency increased or equal to 43mg per day, 45mg per day, 45mg per day, and 45mg per day (Table 8). From this result, it can be seen that under the 1,3BG extraction conditions, it is effective to extract for 3 or 5 days with an amount of 1,3BG 7 times (v/v) compared to the original material. However, since the difference between 1 day, 3 days, and 5 days at 7 times the dose is 43 mg and 45 mg, it can be said that it is efficient to extract 7 times (v/v) 1,3 BG for 1 day compared to the raw material in consideration of the time efficiency. have.

Claims (8)

(S1) stirring the diatoms collected by mass culture in 70% alcohol; (S2) loading and filtering on a filter bed pre-coated with diatomaceous earth; (S3) fractionating and concentrating the filtered extract; (S4) separating and purifying fucoxanthin from the concentrated extract; and (S5) step of storing the separated and purified fucoxanthin in a dark room Separation and purification method of fucoxanthin derived from diatoms, characterized in that it consists of The method for separating and purifying diatoms-derived fucoxanthin according to claim 1, wherein the diatoms are adherent diatoms that are taken together with Jeju lava seawater. 3. The method of claim 2, wherein the adherent diatoms are Melosira nummuloides, Achnanthes brevipes var. Intermedia, Achnanthes sancti-pauli, Achnanthes brevipes or Melosira octogona A method for separating and purifying diatom-derived fucoxanthin, characterized in that at least one selected from the group consisting of. [Claim 2] The method for separating and purifying diatoms-derived fucoxanthin according to claim 1, wherein the diatoms are diatoms collected from a bottom diatom culture. According to claim 1, (S1) step, the separation and purification method of diatoms-derived fucoxanthin, characterized in that the treatment is 7 times (v / v) alcohol for 1 day compared to the raw material. [Claim 3] The method for separating and purifying diatom-derived fucoxanthin according to claim 1, further comprising the step of treating plasma for 25 minutes in step (S1). The method of claim 1, wherein (S4) separating and purifying fucoxanthin from the concentrated extract is performed by silica gel column chromatography. The method of claim 7, wherein the elution solvent used for the silica gel column chromatography has a ratio of N-hexane to acetone of 7:3.

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