Method for purifying fucoxanthin by column chromatography
The crude algae extract was purified by column chromatography using a mixed petroleum ether-ethanol solvent, which solved the problem of low purity and high separation cost in the prior art, and achieved a high purity, low cost and environmentally friendly fucoxanthin separation effect.
Patent Information
- Application Number
- CN202510036886.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-09
- Publication Date
- 2025-05-27
AI Technical Summary
The prior art is difficult to efficiently obtain high-purity fucoxanthin, and the separation cost is high and the environmental pollution is severe, which cannot meet the needs of the domestic market.
The crude algae extract was purified by column chromatography, and the silica gel column chromatography was used to purify the crude algae extract in one step using petroleum ether-ethanol mixed solvent (volume ratio of 5:1).
The separation of high-purity fucoxanthin is achieved, which reduces the separation cost, is simple to operate, is suitable for industrial production, and is safer and will not cause harm to human health.
Smart Images
Figure CN120040386A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of separation and purification, and particularly relates to a method for purifying fucoxanthin by column chromatography. Background Art
[0002] Fucoxanthin is a fat-soluble yellow-brown pigment, mainly present in brown algae and certain diatoms, and is the main component of carotenoids in brown algae, with a maximum absorption wavelength of 450 nm. Fucoxanthin contains structures such as allene bonds, 5,6-epoxy groups, conjugated carbonyl groups, and acetyl groups. Factors such as pH value, light, and temperature all affect its stability, and its chemical properties are unstable.
[0003] At present, many foreign enterprises and research institutions have applied fucoxanthin to the development and research of new drugs. Japan has also launched skin care products with fucoxanthin as an additive and obtained a high market share. However, at present, there are mainly two problems in the fucoxanthin market: one is that the domestic and foreign markets for high-purity fucoxanthin are still vacant. And in China, high-purity fucoxanthin mainly still relies on foreign imports; the other is that the separation cost is too high and the environmental pollution is serious during the separation process. Therefore, there is an urgent need for a simple, economical and environmentally friendly separation and purification technology to meet the domestic market demand for fucoxanthin.
[0004] There are currently records on the separation and purification of fucoxanthin by silica gel column, and the eluents used include a mixed solvent of petroleum ether - ethyl acetate or a mixed solvent of petroleum ether - acetone. However, the purity of fucoxanthin obtained by the above silica gel column separation and purification method is not high enough. Summary of the Invention
[0005] In view of this, the purpose of the present invention is to provide a method for purifying fucoxanthin by column chromatography. The method provided by the present invention obtains fucoxanthin with high purity.
[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:
[0007] The present invention provides a method for purifying fucoxanthin by column chromatography, including the following steps: subjecting the crude extract of algae to silica gel column chromatography purification to obtain fucoxanthin; the eluent used for the silica gel column chromatography purification is a mixed solvent of petroleum ether - ethanol, and the volume ratio of petroleum ether to ethanol in the mixed solvent of petroleum ether - ethanol is 5:1.
[0008] Preferably, the particle size of the silica gel used for the silica gel column chromatography purification is 200 - 300 mesh.
[0009] Preferably, the sample loading method for the silica gel column chromatography purification includes dry sample loading.
[0010] Preferably, the preparation method of the sample components for dry sample loading comprises the following steps: dissolving the crude algal extract to obtain a crude extract solution; mixing the crude extract solution with silica gel and then drying.
[0011] Preferably, the solvent used for dissolution is petroleum ether and ethanol, and the volume ratio of petroleum ether to ethanol is 1:1.
[0012] Preferably, the mass ratio of the crude algal extract to silica gel in the sample components is 1:1.5 - 2.
[0013] Preferably, the particle size of the silica gel is 100 - 200 mesh.
[0014] Preferably, the preparation method of the crude algal extract comprises the following steps: freeze-drying and pulverizing a sample containing fucoxanthin to obtain a crude algal extract.
[0015] Preferably, the sample containing fucoxanthin includes an algal sample.
[0016] Preferably, the temperature of the freeze-drying is -60 to -40 °C.
[0017] The present invention provides a method for purifying fucoxanthin by column chromatography, comprising the following steps: subjecting a crude algal extract to silica gel column chromatography purification to obtain fucoxanthin; the eluent used for the silica gel column chromatography purification is a petroleum ether-ethanol mixed solvent, and the volume ratio of petroleum ether to ethanol in the petroleum ether-ethanol mixed solvent is 5:1. The present invention uses a petroleum ether-ethanol mixed solvent with a volume ratio of 5:1 as the eluent, and adopts an isocratic elution method to perform one-step purification of the crude algal extract by silica gel column chromatography, thereby obtaining high-purity fucoxanthin, with low separation cost, simple operation, and being suitable for industrial production.
[0018] Moreover, when fucoxanthin is used in the cosmetics industry, if the residual concentrations of ethyl acetate and acetone are too high, it will have an impact on human health. Long-term exposure of the human body to an ethyl acetate environment may cause symptoms such as dry skin, redness, itching, dizziness, nausea, and throat irritation, and may seriously cause nerve damage. Compared with ethyl acetate and acetone, the fucoxanthin purified by using petroleum ether-ethanol as the eluent in the present invention can be applied to the cosmetics industry with high safety. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 It is a process diagram of fucoxanthin column chromatography separation in Example 1;
[0020] Figure 2 It is a color result diagram of each component in Example 1;
[0021] Figure 3 It is a spectral scan diagram of Component 3 and the fucoxanthin competitor in Example 1;
[0022] Figure 4 It is the HPLC analysis chart of Component 3 in Example 1;
[0023] Figure 5 It is the HPLC analysis chart of fucoxanthin competitors. Detailed implementation manners
[0024] The present invention provides a method for purifying fucoxanthin by column chromatography, which includes the following steps: subjecting the crude algal extract to silica gel column chromatography purification to obtain fucoxanthin; the eluent used in the silica gel column chromatography purification is a petroleum ether-ethanol mixed solvent, and the volume ratio of petroleum ether to ethanol in the petroleum ether-ethanol mixed solvent is 5:1.
[0025] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.
[0026] In the present invention, the particle size of the silica gel used in the silica gel column chromatography purification is preferably 200 - 300 mesh.
[0027] In the present invention, the sample loading method of the silica gel column chromatography purification includes dry sample loading. In the present invention, the preparation method of the sample loading component in the dry sample loading includes the following steps: dissolving the crude algal extract to obtain a crude extract solution; mixing the crude extract solution with silica gel and then drying.
[0028] In the present invention, the solvents used for dissolution are preferably petroleum ether and ethanol, and the volume ratio of petroleum ether to ethanol is preferably 1:1; the present invention has no special limitation on the dosage of the solvents, and it is only necessary to be able to dissolve the crude algal extract.
[0029] In the present invention, the mass ratio of the crude algal extract to silica gel in the sample loading component is preferably 1:1.5 - 2, and in specific embodiments, it can be 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2; the particle size of the silica gel is preferably 100 - 200 mesh.
[0030] In the present invention, the preparation method of the crude algal extract preferably includes the following steps: freeze-drying and then pulverizing the sample containing fucoxanthin to obtain the crude algal extract. In the present invention, the sample containing fucoxanthin preferably includes algal samples, and the present invention has no special limitation on the algal samples, as long as they contain fucoxanthin, specifically such as diatoms, and the diatoms preferably include Antarctic diatoms and / or Phaeodactylum tricornutum. In the present invention, the pulverization preferably includes grinding. In the present invention, the temperature of the freeze-drying is preferably -60 to -40 °C, and in specific embodiments, it can be -60 °C, -55 °C, -50 °C, -45 °C or -40 °C; the present invention has no special limitation on the freeze-drying time, and freeze-drying is sufficient.
[0031] The present invention uses a petroleum ether-ethanol mixed solvent with a volume ratio of 5:1 as the eluent, and adopts an isocratic elution method to perform one-step purification of the crude algal extract by silica gel column chromatography, so as to obtain high-purity fucoxanthin. The separation cost is low, the operation is simple, and it is suitable for industrial production.
[0032] Moreover, when fucoxanthin is used in the cosmetics industry, if the residual concentrations of ethyl acetate and acetone are too high, it will have an impact on human health. Long-term exposure of the human body to an ethyl acetate environment may cause symptoms such as dry skin, redness, itching, dizziness, nausea, and throat irritation, and may seriously cause nerve damage. Compared with ethyl acetate and acetone, the petroleum ether-ethanol used in the present invention as the eluent, the purified fucoxanthin can be applied to the cosmetics industry with high safety.
[0033] In order to further illustrate the present invention, the following describes in detail the method for purifying fucoxanthin by column chromatography provided by the present invention in combination with embodiments, but they cannot be understood as limiting the protection scope of the present invention.
[0034] In the following examples, the experimental materials are as follows: silica gel (100-200 mesh, 200-300 mesh); petroleum ether (food grade); ethanol (food grade); methanol (chromatographically pure); syringe filter (nylon 0.22 μm); GF254 thin layer chromatography (TLC) silica gel prefabricated plate. Fucoxanthin competitor, purchased from Hubei Jusheng Technology Co., Ltd. Phaeodactylum tricornutum, Shanghai Guangyu Biotechnology Co., Ltd.
[0035] Experimental instruments: high performance liquid chromatograph (LC-16); analytical liquid chromatography column (Venusil XBP C18: 5 μm, 4.6×250 mm); rotary evaporator (SHZ-D III type); nuclear magnetic resonance spectrometer.
[0036] Example 1
[0037] (1) Separation and purification of fucoxanthin
[0038] The Phaeodactylum tricornutum is freeze-dried at -60 to -40 °C and ground into a powder, which is used as the crude algal extract.
[0039] The crude algal extract is dissolved in ethanol, and the isocratic elution ratio of column chromatography is determined by thin layer chromatography analysis. The developing agents are petroleum ether:ethanol (V / V) = 50:1, 30:1, 20:1, 10:1, 5:1, and 1:1 respectively. Among them, the separation effect is the best when petroleum ether:ethanol (V / V) = 5:1.
[0040] Dissolve 5.0 g of the crude algal extract in a petroleum ether - ethanol (V:V = 1:1) solution. Mix and stir evenly 100 - 200 mesh silica gel with the crude algal extract and air dry to obtain the sample loading component. The mixing mass ratio of the crude algal extract to silica gel is 1:1.5. Mix petroleum ether with 200 - 300 mesh silica gel to moisten, and use the wet method to fill the silica gel column, with a filling height of 13 - 15 cm. Use the dry method to load the sample onto the upper layer of the silica gel column, and use petroleum ether:ethanol (V / V) = 5:1 as the eluent for silica gel column chromatography purification. Collect the eluents of different color bands and concentrate them.
[0041] After silica gel column chromatography purification, thin layer chromatography analysis was carried out on the collected components. The developing agent was petroleum ether:ethanol (V / V) = 5:1, and color development was carried out at wavelengths of 254 nm, 365 nm, and 10 v / v% sulfuric acid - ethanol solution. It was found that the polarities of the components were not very different. Then, ultraviolet full scan, HPLC, nuclear magnetic resonance hydrogen spectrum and other means were used to further verify the purity of the yellow - brown component 3, and the purity was compared with that of the fucoxanthin competitor on the market.
[0042] The schematic diagram of the fucoxanthin separation and purification process is shown in Figure 1 , and a total of 5 components were separated (recorded as component 1, component 2, component 3, component 4, and component 5 in the order of elution), and the colors of each component are shown in Figure 2 . After thin layer chromatography analysis of the components obtained by silica gel column chromatography purification, it was found that fucoxanthin was mainly concentrated in component 3, with a mass of 80.2 mg and a yield of 1.6%.
[0043] (2) Spectral analysis
[0044] Scan component 3 and the fucoxanthin competitor at wavelengths of 350 - 550 nm, as shown in Figure 3 , and determine that the maximum absorption wavelength of component 3 is 450 nm.
[0045] (3) High - performance liquid chromatography analysis
[0046] Using 85 v / v% methanol - aqueous solution as the mobile phase and a wavelength of 450 nm, use high - performance liquid chromatography to analyze the purity of component 3 and the fucoxanthin competitor. Figure 4 This is the HPLC diagram of component 3. Figure 5 This is the HPLC diagram of the fucoxanthin competitor. According to the calculation of the peak area, the purity of the main peak fucoxanthin in component 3 is 94.52%, and the purity of the fucoxanthin competitor is 78.2%. The purity of fucoxanthin in component 3 is much higher than that of the fucoxanthin competitor.
[0047] (4) Fucoxanthin nuclear magnetic spectrum
[0048] Hydrogen spectrum data of component 3: 1 HNMR(400MHz,DMSO - d6 ) δ 7.43 - 7.31 (m, 2H), 7.31 - 7.22 (m, 1H), 6.92 - 6.81 (m, 1H), 6.70 - 6.58 (m, 1H), 2.22 - 2.07 (m, 1H), 2.04 - 1.94 (m, 6H), 1.86 (s, 1H), 1.78 (s, 1H), 1.56 - 1.42 (m, 2H), 1.41 - 1.32 (m, 6H), 1.29 (d, J = 6.8 Hz, 9H), 1.24 (d, J = 9.9 Hz, 27H), 1.10 (s, 2H), 0.99 (s, 1H), 0.94 (s, 1H), 0.87 - 0.82 (m, 7H).
[0049] 1H NMR data of fucoxanthin competitor: 1 1H NMR (400 MHz, DMSO-d 6 ) δ 7.39 (d, J = 11.0 Hz, 1H), 6.92 - 6.56 (m, 5H), 6.50 (d, J = 11.4 Hz, 1H), 6.36 (dd, J = 13.2, 6.1 Hz, 2H), 6.23 - 6.15 (m, 1H), 6.09 (s, 1H), 5.32 - 5.19 (m, 2H), 4.88 (s, 1H), 4.37 (d, J = 5.2 Hz, 1H), 3.47 (s, 1H), 2.20 - 2.04 (m, 4H), 2.01 - 1.92 (m, 10H), 1.89 (d, J = 11.1 Hz, 2H), 1.86 (s, 3H), 1.78 (s, 3H), 1.57 - 1.45 (m, 3H), 1.43 - 1.36 (m, 3H), 1.33 (d, J = 8.9 Hz, 8H), 1.22 (d, J = 9.3 Hz, 6H), 1.17 - 1.00 (m, 10H), 0.99 (s, 3H), 0.94 (s, 4H), 0.83 (s, 3H).
[0050] 1H NMR analysis was performed on Component 3 and the fucoxanthin competitor, and the results showed that the purity of fucoxanthin in Component 3 was higher than that in the competitor.
[0051] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.
Claims
1. A method for purifying fucoxanthin by column chromatography, characterized in that: The following steps are involved: The crude algae extract is purified by silica gel column chromatography to obtain fucoxanthin; the eluent used in the silica gel column chromatography purification is a petroleum ether-ethanol mixed solvent, and the volume ratio of petroleum ether to ethanol in the petroleum ether-ethanol mixed solvent is 5:
1.
2. The method according to claim 1, characterized in that The particle size of the silica gel used in the silica gel column chromatography purification is 200-300 meshes.
3. The method according to claim 1, characterized in that: The sample loading method of the silica gel column chromatography purification includes dry sample loading.
4. The method according to claim 1, characterized in that: The preparation method of the loading component for dry loading comprises the following steps: dissolving the crude algae extract to obtain a crude extract solution; The crude extract solution is mixed with silica gel and then dried.
5. The method according to claim 1, characterized in that The solvents used for the dissolution are petroleum ether and ethanol, and the volume ratio of the petroleum ether to the ethanol is 1:
1.
6. The preparation method according to claim 4 or 5, characterized in that: The mass ratio of the algae crude extract to the silica gel in the loading component is 1:1.5-2.
7. The preparation method according to claim 6, characterized in that: The particle size of the silica gel is 100-200 meshes.
8. The method according to claim 1, characterized in that The method for preparing the algae crude extract comprises the following steps: freeze-drying and then crushing a sample containing fucoxanthin to obtain the algae crude extract.
9. The method according to claim 8, characterized in that The sample containing fucoxanthin includes an algae sample.
10. The method according to claim 8, characterized in that The freeze-drying temperature is -60 to -40°C.