WO2021114218A1 - 类菌胞素氨基酸Porphyra-334和Shinorine以及从海藻中提取Porphyra-334和Shinorine的方法 - Google Patents

类菌胞素氨基酸Porphyra-334和Shinorine以及从海藻中提取Porphyra-334和Shinorine的方法 Download PDF

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WO2021114218A1
WO2021114218A1 PCT/CN2019/125103 CN2019125103W WO2021114218A1 WO 2021114218 A1 WO2021114218 A1 WO 2021114218A1 CN 2019125103 W CN2019125103 W CN 2019125103W WO 2021114218 A1 WO2021114218 A1 WO 2021114218A1
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porphyra
shinorine
extract
chromatography
mixed
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French (fr)
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乐占线
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乐占线
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/02—Algae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/02—Algae
    • A61K36/04—Rhodophycota or rhodophyta (red algae), e.g. Porphyra
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C249/00—Preparation of compounds containing nitrogen atoms doubly-bound to a carbon skeleton
    • C07C249/02—Preparation of compounds containing nitrogen atoms doubly-bound to a carbon skeleton of compounds containing imino groups
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/10—Process efficiency

Definitions

  • the present invention relates to the field of biotechnology, in particular to the bacterin-like amino acids Porphyra-334 and Shinorine and a method for extracting Porphyra-334 and Shinorine from seaweed.
  • the ultraviolet rays reaching the surface of the earth are increasing day by day. Excessive ultraviolet radiation will accelerate the photoaging of the skin and pose a serious threat to human skin health.
  • the ultraviolet rays that can reach the surface of the earth are mainly in the medium-wave band (280-320nm) and long-wavelength (320-400nm) regions. Therefore, the sunscreen cosmetics developed to resist ultraviolet rays at home and abroad are mostly aimed at combating the medium-wave band (UVB area) and long-range. Wave band (UVA area) ultraviolet rays.
  • UVA area has a slower effect than the medium wavelength
  • UVA area has a slower effect than the medium wavelength
  • UV rays in this wavelength can directly reach the dermis of the skin, causing blood vessel damage and the invasion of surrounding inflammatory cells, leading to skin pigmentation. After long-term accumulation, it leads to the skin. Irreversible aging.
  • Mycosporine-like amino acids are water-soluble active substances formed by condensation of various types of amino acids with cyclohexenone as the basic skeleton.
  • mycotinoid amino acids are widely distributed in aquatic organisms, especially in Rhodophyceae algae.
  • the content of mycotinoid amino acids in Porphyra yezoensis is as high as 9.065% of the weight of dry algae.
  • 29 kinds of cytokinin-like amino acids have been extracted, isolated and identified from nature.
  • researches on the source, distribution, extraction and purification methods, analysis and detection methods, biological activity and application of bacterin-like amino acids in the field of cosmetics have been research hotspots.
  • mycocytin-like amino acids have unique activity and are shown in UV protection
  • a rare active advantage, successful application in the cosmetics field will bring huge economic value.
  • mycosporin-like amino acids are a class of active substances with high added value, because of their strong water solubility, it is difficult to extract and isolate high-purity mycosporin-like amino acids, which limits the wide application of mycosporin-like amino acids.
  • the inventor provides a method for extracting the cytokinin-like amino acids Porphyra-334 and Shinorine from seaweed, and the method includes the steps:
  • the seaweed is washed and drained, then crushed, and then the crushed seaweed is soaked in a sodium chloride solution with a concentration of 0.5-10.5% for 1-2 hours and then taken out to obtain the first extract;
  • the seaweed residue is soaked in an ethanol solution with a concentration of 60-80% for 2-3 hours and then removed to obtain a secondary extract; the first extract and the secondary extract are mixed to obtain a mixed extract ;
  • the mass ratio of the crushed seaweed, sodium chloride solution, and ethanol solution is 1:8-15:5-15;
  • Flocculation Add chitosan to the mixed extract, stir it evenly, perform flocculation for 50-70 minutes, and separate solid and liquid to obtain a de-impurity extract; the quality of the mixed extract and the chitosan The ratio is: 10000:3-10;
  • Filtration filtering the de-impurity extract with an ultrafiltration membrane to obtain a filtrate; the filtrate is concentrated with a nanofiltration membrane to obtain a concentrated solution with a volume of 5%-10% of the filtrate;
  • the eluent is an organic solvent and has a pH of 3.5 -4.5 is configured with a buffer solution, the organic solvent includes one or more of methanol, ethanol, isopropanol, acetonitrile and acetone;
  • Porphyra-334 concentration the Porphyra-334 purified solution is concentrated by membrane, and the resulting concentrate is washed with water to obtain a Porphyra-334 concentrate.
  • the Porphyra-334 concentrate is added to 3-8 times the volume of acetone and heated to After 45-55°C, stop heating, lower the temperature to crystallize, and lower the temperature to 4°C to precipitate a white solid. Heat and dry the white solid at 45-55°C to obtain Porphyra-334 amorphous crystals;
  • Shinorine concentration the Shinorine purified solution is concentrated by membrane, and the obtained concentrate is washed with water to obtain a Shinorine-containing concentrate. Add 3-8 times the volume of ethanol or acetone to the Shinorine-containing concentrate and heat to 45-55°C, The heating was stopped, the temperature was lowered to crystallize, and the temperature was lowered to 4°C to precipitate a white solid. The white solid was heated and dried at 45-55°C to obtain Shinorine amorphous crystals.
  • the inventor provides the cytokinin amino acid Porphyra-334, which is prepared according to the method described in the first aspect of the present invention.
  • the inventor provides Shinorine, the cytokinin amino acid, which is characterized in that the cytokinin amino acid Shinorine is prepared according to the method described in the first aspect of the present invention.
  • the above-mentioned technical scheme adopts the extraction of Rhodophyta seaweed, membrane filtration, ODS mixed column chromatography, concentration and crystallization, and can obtain Porphyra-334 crystals with a purity of more than 97% and 95%. Shinorine crystals above.
  • the technical solution can effectively remove impurities in the seaweed, the yield is 20-30% higher than that of the traditional ion exchange resin, the process waste water is less, and it can be used for large-scale industrial production. The whole process is continuous and convenient, and the operation time is fast. Unlike conventional vacuum heating and concentration, it will affect the stability of the product, and the pigment produced by oxidation will affect the quality and interfere with the quality of the product.
  • the adsorption of chitosan removes most of the metal ions, impurity proteins and heteropolysaccharide components, and the mixed chromatography can effectively solve the shortcomings of removing small molecular sugars on ordinary chromatography columns due to the small gap of polar MAAs. This enables Shinorine and Porphyra-334 in MAAs to be effectively purified to obtain high-purity single components.
  • Fig. 3 is an NMR-H spectrum chart of the mixed extract of Red Caulis in Example 1.
  • Fig. 4 is the NMR-C spectrum of the mixed extract of red hairpin in Example 1.
  • Fig. 5 is an NMR-DEPT spectrum chart of the mixed extract of Red Caulis in Example 1.
  • Fig. 8 is an HPLC chart of the amorphous crystal of Shinorine in Example 1, and Agilent ZORBAX SB-C18 column conditions.
  • FIG. 9 is an NMR-H spectrum of the amorphous crystal of Porphyra-334 in Example 1.
  • FIG. 10 is an NMR-C spectrum of the amorphous crystal of Porphyra-334 in Example 1.
  • FIG. 11 is an NMR-DEPT spectrum of the amorphous crystal of Porphyra-334 in Example 1.
  • FIG. 12 is a 200-fold microscope image of the amorphous crystal of Porphyra-334 in Example 1.
  • FIG. 13 is a 200-times microscope image of the Shinorine amorphous crystal of Example 1.
  • Figure 14 is the HPLC detection diagram of the mixed extract of Porphyra yezoensis in Example 2, under Kromasil 100-5C18 column conditions.
  • Figure 15 is the HPLC detection diagram of the mixed extract of Porphyra haitanensis in Example 3, and Agilent ZORBAX SB-C18 column conditions.
  • Figure 16 is a graph showing the flocculation of chitosan in the mixed extract of Porphyra haitanensis in Example 3.
  • Figure 17 is an HPLC detection diagram of the mixed extract of Asparagus vulgare in Example 4, and Agilent ZORBAX SB-C18 column conditions.
  • Rhodophyta algae most of them are multicellular algae, a few are single-celled algae; the algae are purple, rose red, dark red and other colors.
  • the majority of Rhodophyta seaweed grows in the ocean, with a wide distribution and many types. According to statistics, there are about 558 genera and more than 3740 species.
  • Rhodophyta include: Bangia atropurpurea, Pyropia haitanensis, Pyropia yezoensis, Pyropia suborbiculata, Pyropia crispata, Pyropia dentata, Glopeliopeltis furcata, Glomus tenella Red Plocamium telfariae, Chondrus crispus, Chondrus nipponicus, Chondrus verrucosus, Ceramium kondoi, Ceramium paniculatum, Campylaephora crassa, Gicloupia Gelidium amansii, Gelidium pacificum, Gelidium crinale, Gelidium vagum, Hypnea cervicornis, Gracilaria bailiniae, Gracilaria vermiculophylla, Gracilaria vermiculophylla, Gracilaria vermiculophylla Gracilaria tenuistipitata, Gracilaria tenuistipitata var
  • a method for extracting cytokinin-like amino acids Porphyra-334 and Shinorine from seaweed comprising the steps:
  • the seaweed is washed and drained, then crushed, and then the crushed seaweed is soaked in a sodium chloride solution with a concentration of 0.5-10.5% for 1-2 hours and then taken out to obtain the first extract;
  • the seaweed residue is soaked in an ethanol solution with a concentration of 60-80% for 2-3 hours and then removed to obtain a secondary extract; the first extract and the secondary extract are mixed to obtain a mixed extract ;
  • the mass ratio of the crushed seaweed, sodium chloride solution, and ethanol solution is 1:8-15:5-15;
  • Flocculation Add chitosan to the mixed extract, stir it evenly, perform flocculation for 50-70 minutes, and separate solid and liquid to obtain a de-impurity extract; the quality of the mixed extract and the chitosan The ratio is: 10000:3-10;
  • Filtration filtering the de-impurity extract with an ultrafiltration membrane to obtain a filtrate; the filtrate is concentrated with a nanofiltration membrane to obtain a concentrated solution with a volume of 5%-10% of the filtrate;
  • the eluent is an organic solvent and has a pH of 3.5 -4.5 is configured with a buffer solution, the organic solvent includes one or more of methanol, ethanol, isopropanol, acetonitrile and acetone;
  • Porphyra-334 concentration the Porphyra-334 purified solution is concentrated by membrane, and the resulting concentrate is washed with water to obtain a Porphyra-334 concentrate.
  • the Porphyra-334 concentrate is added to 3-8 times the volume of acetone and heated to After 45-55°C, stop heating, lower the temperature to crystallize, and lower the temperature to 4°C to precipitate a white solid. Heat and dry the white solid at 45-55°C to obtain Porphyra-334 amorphous crystals;
  • Shinorine concentration the Shinorine purified solution is concentrated by membrane, and the obtained concentrate is washed with water to obtain a Shinorine-containing concentrate. Add 3-8 times the volume of ethanol or acetone to the Shinorine-containing concentrate and heat to 45-55°C, The heating was stopped, the temperature was lowered to crystallize, and the temperature was lowered to 4°C to precipitate a white solid. The white solid was heated and dried at 45-55°C to obtain Shinorine amorphous crystals.
  • the technical solution can effectively remove impurities in the seaweed, the yield is 20-30% higher than that of the traditional ion exchange resin, the process waste water is less, and it can be used for large-scale industrial production.
  • the whole process is continuous and convenient, and the operation time is fast.
  • it will affect the stability of the product, and the pigment produced by oxidation will affect the quality and interfere with the quality of the product.
  • the adsorption of chitosan removes most of the metal ions, impurity proteins and heteropolysaccharide components, and the mixed chromatography can effectively solve the shortcomings of removing small molecular sugars on ordinary chromatography columns due to the small gap of polar MAAs. This enables Shinorine and Porphyra-334 in MAAs to be effectively purified to obtain high-purity single components.
  • seaweed is Rhodophyta seaweed.
  • the ultrafiltration membrane is made of polyethersulfone with a pore selectivity of 2500 Dal.
  • the mixed chromatography column includes an upper part and a lower part, the upper chromatography filler is silica gel, and the lower filler is octadecylsilane bonded silica gel.
  • the upper packing is silica gel, which can effectively separate and remove pigments and effective impurities, and at the same time can protect the service life of ODS materials.
  • silica gel materials have adsorbed a lot of pigment impurities after 5 batches of purification, and the column efficiency deteriorates---elution The color of the liquid deepens.
  • the 80-90% ethanol eluent can be reversed from the bottom of the ODS material part of the chromatography column in the opposite direction to activate the elution chromatography column.
  • the sieve plate between the ODS layer filler and the silica gel layer can prevent the ODS material from floating up directly. Rinse the silica gel layer filler.
  • the separation and purification of conventional natural products using a combination of silica gel and ODS is rarely such an operation.
  • the inventor combined the characteristics of Shinorine and Porphyra-334 products, and discovered through process development and innovation that the solution
  • the chromatographic method will be more convenient and faster than the traditional ion exchange resin chromatography + gel chromatography (laboratory scale) operation, while the yield is improved, and the amount of waste water can be reduced by more than 50%.
  • the organic solvent used in production Part of the wastewater can be recycled and processed.
  • the wastewater has good biodegradability, while traditional ion exchange resins contain high salinity, the wastewater has poor biochemistry, and the service life of ion exchange resins is limited during the purification and use of complex natural products. More solid wastes are generated.
  • the height ratio of the upper part to the lower part of the mixed chromatography column is 4-5:5-6, and the diameter-to-height ratio of the mixed chromatography column is 1:5-10.
  • the organic solvent is methanol.
  • the predominant order of organic solvents is methanol>ethanol>isopropanol>acetonitrile>acetone.
  • the best elution effect is 2-6% methanol;
  • the buffer solution is an ammonium acetate buffer solution with a concentration of 0.05-0.1 mol/L, and acetic acid adjusts the pH of the ammonium acetate buffer solution to 3.5-4.5.
  • the buffer solution can also be sodium acetate, phosphoric acid+sodium dihydrogen phosphate buffer system, or acid water adjusted directly with acetic acid, but experiments show that the above buffer conditions have poor separation of impurities, and the pigments are easy to follow directly, and the colors are more comparable. Deep, it will affect the purity of the product.
  • the ammonium acetate buffer solution with a concentration of 0.05-0.1 mol/L and a pH of 3.5-4.5 has the best elution effect, high separation of impurities, and high product purity.
  • the concentration membrane adopts a polyethersulfone nanofiltration membrane with a pore selectivity of 300Dal.
  • nanofiltration membranes can remove inorganic salt components such as sodium chloride in seawater.
  • the temperature is reduced from 45-55°C to 4°C within 4-10 hours.
  • the liquid mycotinoid amino acid is prone to oxidation, the pigment is deepened, and it is unstable in an alkaline environment and relatively stable in an acidic environment. From the structure of the cytokinin amino acid and the stability experiment of the pure Shinorine and Porphyra-334 components, it is found that the product pigment will deepen and the components will be unstable when placed in a higher temperature environment, so the crystallization process is not stable.
  • the temperature is controlled below 55°C.
  • Chromatographic column Kromasil 100-5C18, 5 ⁇ m, 4.6*250mm, mobile phase: 0.1% acetic acid water, flow rate 0.8ml/min, column temperature 40°C;
  • the above materials are extracted using the extraction method of "Extraction of MAAs Porphyra-334 and its new functions" (the steps are: take 10g of sample and subject it to liquid nitrogen freezing and crushing, and then dry it in a vacuum freeze dryer. Calculate the weight loss, then grind it into a fine powder sample to be measured, weigh it during the measurement, dissolve the quantitative liquid with ultrasound and filter to remove the solids, filter through a 0.2um nylon filter membrane, HPLC detection, calculation), UV 334nm detection The content ratio determined by HPLC.
  • Example 1 Extraction of cytokines Porphyra-334 and Shinorine from red hairpin
  • Extraction Wash the red hair from the sea water with clean sea water to wash the sediment, drain and pulverize, soak the smashed red hair in 8 times the weight of 5% sodium chloride solution for 1 hour and then fish. To obtain the first extract; soak the scraped residue with 10 times the weight of 70% ethanol solution for 3 hours and then remove it to obtain the second extract; the first extract and the second extract are processed Mix to obtain a mixed extract.
  • impurities and insolubles can be removed.
  • Sodium chloride can be used for extraction to obtain more than 40% of the effective ingredients, and after the second ethanol soaking and lifting, about 50% of the total effective ingredients can be obtained.
  • This extraction method can effectively prevent the extraction of polysaccharides and colloidal impurities at the same time.
  • the extract is mixed, and the alcohol extract will also precipitate and remove part of the polysaccharides and colloids dissolved in the water phase. .
  • the two main substances of MAAs, Shinorine and Porphyra-334, are schematic diagrams of the purity under UV HPLC conditions. As can be seen from the figure, Shinorine accounts for 7.0% of MAAs and Porphyra-334 accounts for 89.8% of MAAs.
  • Fig. 3 is an NMR-H spectrum chart of the mixed extract of Red Caulis in Example 1.
  • Fig. 4 is the NMR-C spectrum of the mixed extract of red hairpin in Example 1.
  • Fig. 5 is an NMR-DEPT spectrum chart of the mixed extract of Red Caulis in Example 1.
  • the mixed extract contains characteristic peaks of MAAs, which also indicates that the nuclear magnetic peaks are more complex and contain interference impurities such as sugars.
  • Flocculation Add chitosan to the mixed extract, stir it evenly, perform flocculation for 60 minutes, and separate solid and liquid to obtain an extract after impurity removal; the mass ratio of the mixed extract to the chitosan is 10000:5;
  • the macromolecular proteins, polysaccharides and pigments contained in the mixed extract will be flocculated and removed during this process.
  • the mixed extract is filtered with a 2500Dal polyethersulfone ultrafiltration membrane to obtain a filtrate; the filtrate is concentrated with a 300Dal polyethersulfone nanofiltration membrane to obtain 5%-10% of the volume of the filtrate
  • the ultrafiltration membrane removes the macromolecule part above 2500Dal, and the nanofiltration membrane can remove the inorganic salt components such as sodium chloride in the seawater.
  • the mixed chromatography column includes an upper part and a lower part, the upper chromatography filler is silica gel, the lower filler is octadecylsilane bonded silica gel, the height ratio of the upper part and the lower part is 5:5, and the mixed chromatography column The ratio of diameter to height is 1:8.
  • the HPLC purity of Porphyra-334 is 97%.
  • Porphyra-334 Concentrate the purified solution of Porphyra-334 with a 300Dal nanofiltration membrane. The resulting concentrate is washed with water to obtain a concentrate containing Porphyra-334. The concentrate containing Porphyra-334 is heated and added to 5 times the volume while stirring. After heating the acetone to 50°C, stop heating, continue to stir and reduce the temperature to crystallize. The temperature is lowered to 4°C within 8 hours, and a white solid is precipitated. The solid is separated from the solid and liquid. The white solid is heated and dried at 45-55°C to obtain Porphyra- 334 amorphous crystals.
  • FIG. 9 is an NMR-H spectrum of the amorphous crystal of Porphyra-334 in Example 1.
  • FIG. 10 is an NMR-C spectrum of the amorphous crystal of Porphyra-334 in Example 1.
  • FIG. 11 is an NMR-DEPT spectrum of the amorphous crystal of Porphyra-334 in Example 1.
  • Figure 12 is a 200-fold microscope image of the amorphous crystal of Porphyra-334 of Example 1. It can be seen from the figure that the crystal is in an amorphous state, and it is easy to absorb moisture and dissolve when placed in a humid environment during microscope observation.
  • Shinorine concentration The Shinorine purified solution is concentrated with a 300Dal nanofiltration membrane, and the resulting concentrate is washed with water to obtain a Shinorine-containing concentrate.
  • the Shinorine-containing concentrate is heated while stirring and adding 5 times the volume of acetone, and then heated to 50°C. , Stop heating, continue to stir and lower the temperature to crystallize, the temperature is lowered to 4°C within 10 hours, a white solid is precipitated, and solid-liquid separation is obtained.
  • the white solid is heated and dried at 45-55°C to obtain Shinorine amorphous crystals.
  • Fig. 13 is a 200-times microscope image of the amorphous Shinorine crystal of Example 1. It can be seen from the figure that Shinorine crystallizes in an amorphous state, and it is easy to absorb moisture and dissolve when placed in a humid environment during microscope observation.
  • Porphyra yezoensis picked up from seawater is washed with seawater and drained, and then smashed.
  • the crushed Porphyra yezoensis is soaked in a 3% sodium chloride solution for 2 hours and then taken out to obtain the first time Extraction liquid;
  • the removed Porphyra yezoensis residues are soaked in a 60% ethanol solution for 2-3 hours and then removed to obtain a secondary extract; the first extract and the second extract
  • the mixing is performed to obtain a mixed extract; the mass ratio of the crushed Porphyra yezoensis, the sodium chloride solution, and the ethanol solution is 1:10:10.
  • Flocculation Add chitosan to the mixed extract, stir it evenly, perform flocculation for 50 minutes, and separate solid and liquid to obtain an extract after impurity removal; the mass ratio of the mixed extract to the chitosan is 10000:3;
  • the mixed extract is filtered with a 2500Dal polyethersulfone ultrafiltration membrane to obtain a filtrate; the filtrate is concentrated with a 300Dal polyethersulfone nanofiltration membrane to obtain 5%-10% of the volume of the filtrate Concentrate;
  • the mixed chromatography column comprises an upper part and a lower part, the upper chromatography filler is silica gel, the lower filler is octadecylsilane bonded silica gel, the height ratio of the upper part and the lower part is 4:6, the mixed chromatography column The ratio of diameter to height is 1:10.
  • Concentration of Porphyra-334 Concentrate the purified solution of Porphyra-334 with a 300Dal nanofiltration membrane, and wash the resulting concentrate with water to obtain a concentrate containing Porphyra-334.
  • the concentrate containing Porphyra-334 is heated and added to 3 times the volume while stirring. After heating the acetone to 45°C, stop heating, continue to stir and lower the temperature to crystallize. The temperature is lowered to 4°C within 12 hours, and a white solid is precipitated. The solid is separated from the solid and liquid. The white solid is heated and dried at 45-55°C to obtain Porphyra- 334 amorphous crystals;
  • Shinorine concentration The Shinorine purified solution is concentrated with a 300Dal nanofiltration membrane, and the resulting concentrate is washed with water to obtain a Shinorine-containing concentrate.
  • the Shinorine-containing concentrate is heated while stirring and adding 8 times the volume of acetone, and then heated to 55°C , Stop heating, continue to stir and lower the temperature to crystallize, the temperature is lowered to 4°C within 4 hours, a white solid is precipitated, and solid-liquid separation obtains a solid.
  • the white solid is heated and dried at 45-55°C to obtain Shinorine amorphous crystals.
  • Porphyra haitanensis picked up from the sea water, washed with sea water, and dried, and then smashed.
  • the crushed Porphyra haitanensis is soaked in a 10.5% sodium chloride solution for 1 hour and then taken out to obtain the first extraction.
  • the extracted seaweed residue is soaked in an ethanol solution with a concentration of 80% for 2 hours and then removed to obtain a secondary extract; the first extract and the second extract are mixed to obtain a mixed extract Extract; the mass ratio of the crushed seaweed, sodium chloride solution, and ethanol solution is 1:8:15.
  • the two main substances of MAAs, Shinorine and Porphyra-334, are schematic diagrams of the purity under UV HPLC conditions. Shinorine accounts for 33.9% of MAAs and Porphyra-334 accounts for 63.9% of MAAs.
  • Flocculation Add chitosan to the mixed extract, stir it evenly, perform flocculation for 70 minutes, and separate solid and liquid to obtain a de-impurity extract; the mass ratio of the mixed extract to the chitosan is : 10000:3;
  • the mixed extract is filtered with a 2500Dal polyethersulfone ultrafiltration membrane to obtain a filtrate; the filtrate is concentrated with a 300Dal polyethersulfone nanofiltration membrane to obtain 5%-10% of the volume of the filtrate Concentrate;
  • Figure 16 shows the phenomenon of chitosan flocculation in the mixed extract of Porphyra chinensis in Example 3. From the figure, it can be seen that the protein and pigments are flocculated, and the liquid presents a clear tendency.
  • Porphyra-334 Concentrate the purified solution of Porphyra-334 with a 300Dal nanofiltration membrane. The resulting concentrate is washed with water to obtain a concentrate containing Porphyra-334. The concentrate containing Porphyra-334 is heated and added to 5 times the volume while stirring. After heating the acetone to 55°C, stop heating, continue to stir and lower the temperature to crystallize, cool to 4°C, a white solid precipitates, solid-liquid separation obtains a solid, the white solid is heated and dried at 45-55°C to obtain Porphyra-334 amorphous crystal Thing
  • Shinorine concentration The Shinorine purified solution is concentrated with a 300Dal nanofiltration membrane, and the resulting concentrate is washed with water to obtain a Shinorine-containing concentrate.
  • the Shinorine-containing concentrate is heated while stirring and adding 3 times the volume of acetone, and then heated to 45°C. , Stop heating, continue to stir and lower the temperature to crystallize, lower the temperature to 4°C, a white solid is precipitated, solid-liquid separation obtains a solid, the white solid is heated and dried at 45-55°C to obtain Shinorine amorphous crystals.
  • the asparagus picked up from the seawater is washed with seawater and the sediment is drained, then crushed, crushed into slags with a width of 1-5mm, and then the crushed asparagus is treated with a concentration of 0.5% chlorine. Soaked in sodium chloride solution for 2 hours and then fished out to obtain the first extract; the extracted asparagus residues were soaked in a 60% ethanol solution for 3 hours and then removed to obtain the second extract; The first extract and the second extract are mixed to obtain a mixed extract; the mass ratio of the crushed asparagus, sodium chloride solution, and ethanol solution is 1:10:15;
  • Flocculation Add chitosan to the mixed extract, stir it evenly, perform flocculation for 70 minutes, and separate solid and liquid to obtain a de-impurity extract; the mass ratio of the mixed extract to the chitosan is 10000:3;
  • the mixed extract is filtered with a 2500Dal polyethersulfone ultrafiltration membrane to obtain a filtrate; the filtrate is concentrated with a 300Dal polyethersulfone nanofiltration membrane to obtain 5%-10% of the volume of the filtrate Concentrate;
  • the mixed chromatography column includes an upper part and a lower part.
  • the upper chromatography filler is silica gel
  • the lower filler is octadecylsilane bonded silica gel
  • the height ratio between the upper part and the lower part is 4:5.
  • the mixed chromatography column The ratio of diameter to height is 1:5.
  • Shinorine concentration the Shinorine purified solution is concentrated with a 300Dal nanofiltration membrane, and the resulting concentrate is washed with water to obtain a Shinorine-containing concentrate.
  • the Shinorine-containing concentrate is heated while stirring, adding 3-8 times the volume of ethanol or acetone, and heating After reaching 45-55°C, stop heating, continue to stir and lower the temperature to crystallize. In 4 hours, the temperature is lowered to 4°C, a white solid is precipitated, and solid-liquid separation is obtained. The white solid is heated and dried at 45-55°C to obtain Shinorine amorphous crystals. Things.
  • the present invention does not exclude the technical application of extracting MAAs and separating and purifying the red algae material after the dry product is re-foamed.

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Abstract

提供了类菌胞素氨基酸Porphyra-334和Shinorine以及从海藻中提取Porphyra-334和Shinorine的方法，所述的方法包括以下步骤：浸提、絮凝、过滤、层析、Porphyra-334浓缩、Shinorine浓缩。上述方法可得到纯度可达到97％以上的Porphyra-334晶体和95％以上的Shinorine晶体。该方法能有效去除海藻中杂质，收率比传统的离子交换树脂高20-30％，工艺废水较少，可用于大规模工业化生产。整个工艺连续便捷，操作时间快速。

Description

类菌胞素氨基酸Porphyra-334和Shinorine以及从海藻中提取Porphyra-334和Shinorine的方法 技术领域
本发明涉及生物技术领域，特别涉及类菌胞素氨基酸Porphyra-334和Shinorine以及从海藻中提取Porphyra-334和Shinorine的方法。
背景技术
近年来，由于臭氧层破环现象严重，到达地球表面的紫外线日渐增加，过量的紫外线照射会加速皮肤的光老化，给人类皮肤健康造成严重威胁。而能够到达地球表面的紫外线主要为中波段(280-320nm)和长波段(320-400nm)区域，因此，国内外抵抗紫外线而研发的防晒类化妆品多针对于对抗中波段(UVB区)和长波段(UVA区)紫外线。研究发现，中波段紫外线透射能力强，作用效果快，可在短时间内破坏皮肤细胞内的核酸及蛋白，导致皮肤出现红肿、晒斑、炎症等现象。因此，对中波段紫外线的防护已成为防晒产品开发的重点。然而，长波段(UVA区)虽然相对中波段作用效果缓慢，但该波段紫外线能够直接到达皮肤的真皮层，致使血管损伤及其周围炎症细胞的侵袭，导致皮肤色素沉积，经长期积累，导致皮肤不可逆性衰老。
目前，市面上开发生产的防晒化妆品多存在作用效果单一、预防波段阈值较窄的特点，而针对于全波段的紫外防护产品又存在化学防护剂添加量超标。所以，针对于不同波段紫外线对皮肤损伤的特点，研发针对于紫外全波段、天然植物中提取UV防护剂已成为研究的重点。
类菌胞素氨基酸(mycosporine-like amino acids，MAAs)是一类以环己烯酮为基本骨架，与多种类型氨基酸通过缩合作用而形成的水溶性活性物质。在自然界中，类菌胞素氨基酸广泛分布于水生生物中，尤其在红藻纲藻类中含量丰富，如条斑紫菜中的类菌胞素氨基酸含量高达干藻重量的9.065％。目前，从自然界中提取、分离并鉴定类菌胞素氨基酸类成分共29种。近年来，针对类菌胞素氨基酸的来源、分布、提取与纯化方法、分析检测方法、生物活性及其在化妆品领域应用等方面所进行的研究一直是研究热点。一方面是因为水生生物资源丰富，尤其是红藻的养殖资源充足，为类菌胞素氨基酸的研究提供了充沛的原料来源；另一方面，类菌胞素氨基酸活性独特，在紫外线防护方面展现出罕见的活性优势，在化妆品领域的成功应用将带来巨大的经济价值。尽管类菌胞素氨基酸是一类具有高附加值的活性物质，但因其水溶性强，提取分离获得高纯度类菌胞素氨基酸较为困难，使得类菌胞素氨基酸的广泛应用受到了限制。
目前国内专门对类菌胞素氨基酸的提取和纯化成单组份的研究涉及很少，并且纯度不高。国内外研究MAAs的文献较多，但是如何快速有效的得到MAAs以及其中的单组分，以便提供更多的原料成分，进行深入的药理研究，只有实验室研发的高效液相色谱法小量制备单组分研究，未见到工业放大生产的工艺，对于工业化需求量大，需要大量制备产品 工艺。而且目前在提纯过程中普遍使用的离子交换树脂吸附法，会产生大量废水，生产过程对环境的影响较大。
在论文《类菌胞素氨基酸(MAAs)Porphyra-334的提取及其新功能研究》和专利：一种紫菜类菌胞素氨基酸Porphyra-334的制备方法(申请号201210114628.5)使用了常规的离子交换树脂方法，而MAAs是属于易氧化的极性水溶性化合物，稳定性相对较差，离子交换法局部强酸碱条件对产品的稳定和收率均很难做到很好的规模化生产。
发明内容
为此，需要提供一种适合工业化生产、操作简单的，收率较高，得到Porphyra-334和Shinorine的纯度较高的方法。
本发明的第一方面，发明人提供了一种从海藻中提取类菌胞素氨基酸Porphyra-334和Shinorine的方法，所述的方法包括步骤：
浸提：将海藻洗净沥干后进行粉碎，然后将粉碎后的海藻在浓度为0.5-10.5％的氯化钠溶液中浸泡1-2h后捞出，得到首次浸提液；将捞出的海藻碎渣再用浓度60-80％的乙醇溶液中浸泡2-3h后捞出，得到二次浸提液；将所述首次浸提液和二次浸提液进行混合，得到混合浸提液；所述粉碎后的海藻、氯化钠溶液、乙醇溶液的质量比为1:8-15:5-15；
絮凝：在混合浸提液中加入壳聚糖，搅拌均匀，进行50-70分钟絮凝后，固液分离，得到除杂后浸提液；所述混合浸提液与所述壳聚糖的质量比为：10000:3-10；
过滤：将所述除杂后浸提液用超滤膜进行过滤，得到过滤液；所述过滤液用纳滤膜进行浓缩，得到5％-10％过滤液体积的浓缩液；
层析：将浓缩液上样ODS混合层析柱进行层析，洗脱液冲洗，HPLC跟踪监测，分别得到Porphyra-334纯化液和Shinorine纯化液；所述洗脱液为有机溶剂和pH为3.5-4.5缓冲溶液配置而成，所述有机溶剂包括甲醇、乙醇、异丙醇、乙腈以及丙酮中的一种或多种；
Porphyra-334浓缩：将所述Porphyra-334纯化液进行膜浓缩，所得的浓缩物进行水洗，得到含Porphyra-334浓缩物，将含Porphyra-334浓缩物加入3-8倍体积的丙酮，加热至45-55℃后，停止加热，降温结晶，降温至4℃，析出白色固体，将白色固体在45-55℃加热干燥，得到Porphyra-334无定型结晶物；
Shinorine浓缩：将所述Shinorine纯化液进行膜浓缩，所得的浓缩物进行水洗，得到含Shinorine浓缩物，将含Shinorine浓缩物加入3-8倍体积的乙醇或丙酮，加热至45-55℃后，停止加热，降温结晶，降温至4℃，析出白色固体，将白色固体45-55℃加热干燥，得到Shinorine无定型结晶物。
本发明的第二个方面，发明人提供了类菌胞素氨基酸Porphyra-334，所述类菌胞素氨基酸Porphyra-334根据本发明第一方面所述的方法进行制备。
本发明的第三个方面，发明人提供了类菌胞素氨基酸Shinorine，其特征在于，所述类菌胞素氨基酸Shinorine根据本发明第一方面所述的方法进行制备。
区别于现有技术，上述技术方案采用了通过对红藻门海藻进行浸提，膜过滤、ODS混 合柱层析、浓缩和结晶，可得到纯度可达到97％以上的Porphyra-334晶体和95％以上的Shinorine晶体。该技术方案能有效去除海藻中杂质，收率比传统的离子交换树脂高20-30％，工艺废水较少，可用于大规模工业化生产。整个工艺连续便捷，操作时间快速，不同于常规的减压加热浓缩会对产品稳定性造成影响，氧化产生色素造成品质影响干扰，对产品的质量起到保障作用。通过壳聚糖吸附去除大多数的金属离子和杂蛋白以及杂多糖成分，通过混合层析能有效解决去除小分子糖在普通层析柱上因极性MAAs差距小不好分离去除的缺点，从而使得MAAs中的Shinorine和Porphyra-334能有效纯化得到高纯度的单组份。
附图说明
图1为实施例1红毛菜浸提液的HPLC检测图A，Kromasil 100-5C18柱条件，RTShinorine＝3.8min，RT Porphyra-334＝5.7min。
图2为实施例1红毛菜浸提液的HPLC检测图B，Agilent ZORBAX SB-C18柱条件，RTShinorine＝3.7min，RT Porphyra-334＝4.5min。
图3为实施例1红毛菜混合浸提液NMR-H谱图。
图4为实施例1红毛菜混合浸提液NMR-C谱图。
图5为实施例1红毛菜混合浸提液NMR-DEPT谱图。
图6为实施例1红毛菜浓缩液经过层析后得到的洗脱液HPLC检测图(HPLC跟踪监测，跟踪样品1)，Kromasil 100-5C18柱条件，RT Porphyra-334＝5.8min。
图7为实施例1Porphyra-334无定型结晶物的HPLC图，Agilent ZORBAX SB-C18柱条件，RT Porphyra-334＝4.5min。
图8为实施例1Shinorine无定型结晶物的HPLC图，Agilent ZORBAX SB-C18柱条件。
图9为实施例1的Porphyra-334无定型结晶物的NMR-H谱图。
图10为实施例1的Porphyra-334无定型结晶物的NMR-C谱图。
图11为实施例1的Porphyra-334无定型结晶物的NMR-DEPT谱图。
图12为实施例1的Porphyra-334无定型结晶物的200倍显微镜图。
图13为实施例1的Shinorine无定型结晶物的200倍显微镜图。
图14为实施例2中条斑紫菜混合浸提液HPLC检测图，Kromasil 100-5C18柱条件。
图15为实施例3中坛紫菜混合浸提液HPLC检测图，Agilent ZORBAX SB-C18柱条件。
图16为实施例3中坛紫菜混合浸提液经过壳聚糖絮凝现象图。
图17为实施例4中龙须菜混合浸提液HPLC检测图，Agilent ZORBAX SB-C18柱条件。
具体实施方式
为详细说明技术方案的技术内容、构造特征、所实现目的及效果，以下结合具体实施例并配合附图详予说明。
红藻门海藻，绝大多数为多细胞体、极少数为单细胞体的藻类；其藻体为紫红、玫瑰 红、暗红等色。红藻门海藻绝大部分生长于海洋中，分布广，种类多，据统计约有558属、3740余种。红藻门包括：红毛菜Bangia atropurpurea，坛紫菜Pyropia haitanensis，条斑紫菜Pyropia yezoensis，圆紫菜Pyropia suborbiculata，绉紫菜Pyropia crispata，长紫菜Pyropia dentata，海萝Gloiopeltis furcata，鹿角海萝Gloiopeltis tenax，海头红Plocamium telfariae，皱波角叉菜Chondrus crispus，日本角叉菜Chondrus nipponicus，异色角叉菜Chondrus verrucosus，三叉仙菜Ceramium kondoi，圆锥仙菜Ceramium paniculatum，凝菜Campylaephora crassa，蜈蚣菜Grateloupia filicina，石花菜Gelidium amansii，大石花菜Gelidium pacificum，细毛石花菜Gelidium crinale，异形石花菜Gelidium vagum，鹿角沙菜Hypnea cervicornis，异枝江蓠Gracilaria bailiniae，真江蓠Gracilaria vermiculophylla，脆江蓠Gracilaria chouae，细基江蓠Gracilaria tenuistipitata，细基江蓠繁枝变种Gracilaria tenuistipitata var.liui，扁江蓠Gracilaria textorii，芋根江蓠Gracilaria blodgettii，帚状江蓠Gracilaria edulis，缢红江蓠Gracilaria Salicornia，智利江蓠Gracilaria chilensis，刺边江蓠Gracilaria spinulosa，龙须菜Gracilaria lemaneiformis。
本发明的第一方面，一种从海藻中提取类菌胞素氨基酸Porphyra-334和Shinorine的方法，所述的方法包括步骤：
浸提：将海藻洗净沥干后进行粉碎，然后将粉碎后的海藻在浓度为0.5-10.5％的氯化钠溶液中浸泡1-2h后捞出，得到首次浸提液；将捞出的海藻碎渣再用浓度60-80％的乙醇溶液中浸泡2-3h后捞出，得到二次浸提液；将所述首次浸提液和二次浸提液进行混合，得到混合浸提液；所述粉碎后的海藻、氯化钠溶液、乙醇溶液的质量比为1:8-15:5-15；
絮凝：在混合浸提液中加入壳聚糖，搅拌均匀，进行50-70分钟絮凝后，固液分离，得到除杂后浸提液；所述混合浸提液与所述壳聚糖的质量比为：10000:3-10；
过滤：将所述除杂后浸提液用超滤膜进行过滤，得到过滤液；所述过滤液用纳滤膜进行浓缩，得到5％-10％过滤液体积的浓缩液；
层析：将浓缩液上样ODS混合层析柱进行层析，洗脱液冲洗，HPLC跟踪监测，分别得到Porphyra-334纯化液和Shinorine纯化液；所述洗脱液为有机溶剂和pH为3.5-4.5缓冲溶液配置而成，所述有机溶剂包括甲醇、乙醇、异丙醇、乙腈以及丙酮中的一种或多种；
Porphyra-334浓缩：将所述Porphyra-334纯化液进行膜浓缩，所得的浓缩物进行水洗，得到含Porphyra-334浓缩物，将含Porphyra-334浓缩物加入3-8倍体积的丙酮，加热至45-55℃后，停止加热，降温结晶，降温至4℃，析出白色固体，将白色固体在45-55℃加热干燥，得到Porphyra-334无定型结晶物；
Shinorine浓缩：将所述Shinorine纯化液进行膜浓缩，所得的浓缩物进行水洗，得到含Shinorine浓缩物，将含Shinorine浓缩物加入3-8倍体积的乙醇或丙酮，加热至45-55℃后，停止加热，降温结晶，降温至4℃，析出白色固体，将白色固体45-55℃加热干燥，得到Shinorine无定型结晶物。
该技术方案能有效去除海藻中杂质，收率比传统的离子交换树脂高20-30％，工艺废水 较少，可用于大规模工业化生产。整个工艺连续便捷，操作时间快速，不同于常规的减压加热浓缩会对产品稳定性造成影响，氧化产生色素造成品质影响干扰，对产品的质量起到保障作用。通过壳聚糖吸附去除大多数的金属离子和杂蛋白以及杂多糖成分，通过混合层析能有效解决去除小分子糖在普通层析柱上因极性MAAs差距小不好分离去除的缺点，从而使得MAAs中的Shinorine和Porphyra-334能有效纯化得到高纯度的单组份。
进一步地，所述海藻为红藻门海藻。
进一步地，所述过滤步骤，所述过滤步骤，所述超滤膜为聚醚砜材质，孔隙选择性2500Dal。
进一步地，所述层析步骤，所述混合层析柱的包括上部和下部，所述上部层析填料为硅胶，所述下部填料为十八烷基硅烷键合硅胶。
上部填料为硅胶可有效分离去除色素和有效的杂质，同时可以起到保护ODS材料使用寿命，硅胶材料一般使用5批次纯化左右就已经吸附很多色素杂质，同时柱效变差---洗脱液颜色加深。可以采取相反方向从ODS材料部分的层析柱底部逆向进80-90％乙醇洗脱液，进行活化洗脱层析柱，ODS层填料与硅胶层之间的筛板可以避免ODS材料上浮，直接冲洗掉硅胶层填料，使用时新的批次只需要新装填上部的硅胶层部分，这样可以避免色素从硅胶层洗到ODS层上，减少洗脱液用量，延长ODS填料层的使用寿命。
同时，用硅胶和ODS结合使用的方式在常规天然产物的分离纯化很少这么操作，发明人经过长期的实验后，结合Shinorine和Porphyra-334产品的特性，通过工艺开发和创新发现，本方案的层析方法会比传统的离子交换树脂层析+凝胶层析(实验室规模)操作上来的方便，快速，同时收率提高，而且废水量可以少50％以上，此外生产上使用的有机溶剂部分可以通过回收进行套用处理，废水可生化性好，而传统的离子交换树脂含有较高盐度，废水可生化较差，并且对于复杂的天然产物纯化使用过程中离子交换树脂的使用寿命有限，产生的固体废弃物较多。
进一步地，所述层析步骤，所述混合层析柱的上部与下部的高度比为4-5:5-6，所述混合层析柱的径高比为1:5-10。
进一步地，所述层析步骤，所述有机溶剂为甲醇。有机溶剂的优势顺序是甲醇＞乙醇＞异丙醇＞乙腈＞丙酮。用甲醇2-6％洗脱效果最好；
进一步地，所述缓冲溶液为浓度为0.05-0.1mol/L的乙酸铵缓冲溶液，乙酸调节乙酸铵缓冲溶液的pH为3.5-4.5。
缓冲液也可以用乙酸钠，磷酸+磷酸二氢钠缓冲体系，或者直接用乙酸调节的酸水，但实验表明以上缓冲条件，对于杂质的分离度不好，同时色素容易直接跟出，颜色比较深，会影响产物的纯度。而浓度为0.05-0.1mol/L，pH为3.5-4.5的乙酸铵缓冲溶液的洗脱效果最好，对杂质的分离度高，产物的纯度高。
进一步地，所述Porphyra-334浓缩步骤和Shinorine浓缩步骤，浓缩膜采用孔隙选择性300Dal的聚醚砜材质的纳滤膜。
采用纳滤膜可去除了海水中的氯化钠等无机盐成分。
进一步地，所述Porphyra-334浓缩和Shinorine浓缩步骤中，在4-10小时内，温度由45-55℃降低到4℃。液体的类菌胞素氨基酸容易发生氧化，色素加深，其在碱性环境不稳定，在酸性环境下相对稳定。从类菌胞素氨基酸结构，以及得到纯品的Shinorine和Porphyra-334组分进行的稳定性实验发现，在温度较高的环境放置，产品色素加深，成分发生不稳定的氧化，因此结晶过程的温度均控制在55℃以下。
本实施方式中，液相色谱条件HPLC-UV：检测波长：334nm；
色谱柱：Kromasil 100-5C18，5μm，4.6*250mm，流动相：0.1％乙酸水，流速0.8ml/min，柱温40℃；
Agilent ZORBAX SB-C18，5μm，4.6*250mm，流动相：30ml甲醇+970ml0.1％乙酸水，流速0.8ml/min，柱温40℃；
NMR条件：BRUKER 400M，氘水。
其中：实施例1材料红毛菜中Porphyra-334和Shinorine的含量分别占MAAs比例为：88.3％和10.0％；MAAs占提取物中的比例约5.5％。
在实施例2材料条斑紫菜中Porphyra-334和Shinorine的含量分别占MAAs比例为：63.1％和36.3％。MAAs占总提取物中的比例约4.9％。
以上材料采用《类菌胞素氨基酸(MAAs)Porphyra-334的提取及其新功能研究》提取方法(步骤为：取10g样品经过液氮冷冻粉碎前处理，然后在真空冷冻干燥机中进行干燥，计算失重，然后研磨成微细粉末状样品待测，测定的时候称量，用定量液体经过超声溶解后过滤去除固体，检测时过0.2um尼龙滤膜过滤，HPLC检测，计算)，紫外334nm检测下HPLC测定的含量比例。
实施例1：红毛菜中提取类菌胞素氨基酸Porphyra-334和Shinorine
浸提：将从海水中捞起的红毛菜用干净海水洗净泥沙，沥干后进行粉碎，将粉碎后的红毛菜在8倍重量的5％氯化钠溶液中浸泡1h后捞出，得到首次浸提液；将捞出的碎渣用10倍重量的70％乙醇溶液浸泡3h后捞出，得到二次浸提液；将所述首次浸提液和二次浸提液进行混合，得到混合浸提液。
在浸提过程中，可以去除杂质、不溶物。用氯化钠进行浸提可以得到40％以上有效物质，经过第二次乙醇浸泡提起可以得到总量50％左右的有效成分。该浸提方式同时可以有效阻止多糖类和胶质类杂质的提取，在第二次浸提时混合了提取液，醇提取液还会沉淀去除一部分溶解在水相中的多糖和胶质类。
图1为实施例1红毛菜浸提液的HPLC检测图A，Kromasil 100-5C18柱条件，RTShinorine＝3.7min，RT Porphyra-334＝4.5min。MAAs两个主要物质Shinorine和Porphyra-334在紫外HPLC条件下的纯度示意图，由图可知，其中Shinorine占MAAs的7.0％，Porphyra-334占MAAs的89.8％。
图2为实施例1红毛菜浸提液的HPLC检测图B，Agilent ZORBAX SB-C18柱条件， RTShinorine＝3.8min，RT Porphyra-334＝5.7min。MAAs两个主要物质Shinorine和Porphyra-334在紫外不同色谱分析HPLC条件下的纯度示意图，由图可知，其中Shinorine占MAAs的14.2％，Porphyra-334占MAAs的75.0％。
图3为实施例1红毛菜混合浸提液NMR-H谱图。
图4为实施例1红毛菜混合浸提液NMR-C谱图。
图5为实施例1红毛菜混合浸提液NMR-DEPT谱图。
由图3-图5可知，混合浸提液中含有MAAs特征峰，也表明核磁峰较杂，含有糖类等干扰杂质。
絮凝：在混合浸提液中加入壳聚糖，搅拌均匀，进行60分钟絮凝后，固液分离，得到除杂后浸提液；所述混合浸提液与所述壳聚糖的质量比为10000:5；
混合浸提液中的含有的大分子蛋白质类、多糖和色素等物质在该过程中会被絮凝，去除。
过滤：将所述混合浸提液用2500Dal聚醚砜材质超滤膜进行过滤，得到过滤液；所述过滤液用300Dal聚醚砜材质纳滤膜进行浓缩，得到5％-10％过滤液体积的浓缩液；超滤膜去除2500Dal以上的大分子部分，纳滤膜可去除了海水中的氯化钠等无机盐成分。
层析：将浓缩液上样ODS混合层析柱进行层析，洗脱液冲洗，HPLC跟踪监测，分别得到Porphyra-334纯化液和Shinorine纯化液；所述洗脱液为5％甲醇和95％的pH为3.5-4.5的乙酸铵缓冲溶液(0.08mol/L)配置而成。所述混合层析柱的包括上部和下部，上部层析填料为硅胶，所述下部填料为十八烷基硅烷键合硅胶，上部与下部的高度比为5:5，所述混合层析柱的径高比为1:8。
图6为实施1红毛菜浓缩液经过层析后得到的洗脱液HPLC检测图(HPLC跟踪监测，跟踪样品1)，Kromasil 100-5C18柱条件，RT Porphyra-334＝5.8min，由图可知Porphyra-334的HPLC纯度为97％。
Porphyra-334浓缩：将Porphyra-334纯化液用300Dal的纳滤膜进行浓缩，所得的浓缩物进行水洗，得到含Porphyra-334浓缩物，将含Porphyra-334浓缩物一边加热一边搅拌加入5倍体积的丙酮，加热至50℃后，停止加热，继续搅拌并降温结晶，在8小时内降温至4℃，析出白色固体，固液分离得到固体，将白色固体45-55℃加热干燥，得到Porphyra-334无定型结晶物。
图7为实施例1Porphyra-334无定型结晶物的HPLC图，Agilent ZORBAX SB-C18柱条件，RT Porphyra-334＝4.5min，由图可知Porphyra-334的HPLC纯度为98.7％。
图9为实施例1的Porphyra-334无定型结晶物的NMR-H谱图。
图10为实施例1的Porphyra-334无定型结晶物的NMR-C谱图。
图11为实施例1的Porphyra-334无定型结晶物的NMR-DEPT谱图。
由图9-图11可见，Porphyra-334的MAAs特有结构特征峰，证明所得无定型晶体为Porphyra-334。
图12为实施例1的Porphyra-334无定型结晶物的200倍显微镜图，由图可以看出结晶成无定形状态，在显微镜观察过程中放置潮湿环境就易吸潮溶解。
Shinorine浓缩：将Shinorine纯化液用300Dal的纳滤膜进行浓缩，所得的浓缩物进行水洗，得到含Shinorine浓缩物，将含Shinorine浓缩物一边加热一边搅拌加入5倍体积的丙酮，加热至50℃后，停止加热，继续搅拌并降温结晶，在10小时内降温至4℃，析出白色固体，固液分离得到固体，将白色固体45-55℃加热干燥，得到Shinorine无定型结晶物。
图8为实施例1的Shinorine无定型结晶物的HPLC图，Agilent ZORBAX SB-C18柱条件，RT Shinorine＝3.6min，由图可知Shinorine的HPLC纯度为95.0％。
图13为实施例1的Shinorine无定型结晶物的200倍显微镜图，由图可以看出Shinorine结晶成无定形状态，在显微镜观察过程中放置潮湿环境就易吸潮溶解。
经过实施例1的方案提取一次纯化，纯度97％以上Porphyra-334的收率为81％，纯度为95％以上的Shinorine的收率为92％。
实施例2条斑紫菜中提取类菌胞素氨基酸Porphyra-334和Shinorine
浸提：将从海水中捞起的条斑紫菜海水洗净泥沙沥干后进行粉碎，将粉碎后的条斑紫菜在浓度为3％的氯化钠溶液中浸泡2h后捞出，得到首次浸提液；将捞出的条斑紫菜碎渣再用浓度60％的乙醇溶液中浸泡2-3h后捞出，得到二次浸提液；将所述首次浸提液和二次浸提液进行混合，得到混合浸提液；所述粉碎后的条斑紫菜、氯化钠溶液、乙醇溶液的质量比为1:10:10。
图14为实施例2中条斑紫菜混合浸提液HPLC检测图，Kromasil 100-5C18柱条件，由图可知，RTShinorine＝4.1min，RT Porphyra-334＝5.9min。MAAs两个主要物质Shinorine和Porphyra-334在紫外HPLC条件下的纯度示意图，其中Shinorine占MAAs的36.6％，Porphyra-334占MAAs的61.1％。
絮凝：在混合浸提液中加入壳聚糖，搅拌均匀，进行50分钟絮凝后，固液分离，得到除杂后浸提液；所述混合浸提液与所述壳聚糖的质量比为10000:3；
过滤：将所述混合浸提液用2500Dal聚醚砜材质超滤膜进行过滤，得到过滤液；所述过滤液用300Dal聚醚砜材质纳滤膜进行浓缩，得到5％-10％过滤液体积的浓缩液；
层析：将浓缩液上样ODS混合层析柱进行层析，洗脱液冲洗，HPLC跟踪监测，分别得到Porphyra-334纯化液和Shinorine纯化液；所述洗脱液为6％甲醇和94％的0.1mol/L乙酸铵缓冲溶液(乙酸调节乙酸铵缓冲溶液的pH为3.5-4.5)配置而成。所述混合层析柱的包括上部和下部，上部层析填料为硅胶，所述下部填料为十八烷基硅烷键合硅胶，上部与下部的高度比为4:6，所述混合层析柱的径高比为1:10。
Porphyra-334浓缩：将Porphyra-334纯化液用300Dal的纳滤膜进行浓缩，所得的浓缩物进行水洗，得到含Porphyra-334浓缩物，将含Porphyra-334浓缩物一边加热一边搅拌加入3倍体积的丙酮，加热至45℃后，停止加热，继续搅拌并降温结晶，在12小时内降温 至4℃，析出白色固体，固液分离得到固体，将白色固体45-55℃加热干燥，得到Porphyra-334无定型结晶物；
Shinorine浓缩：将Shinorine纯化液用300Dal的纳滤膜进行浓缩，所得的浓缩物进行水洗，得到含Shinorine浓缩物，将含Shinorine浓缩物一边加热一边搅拌加入8倍体积的丙酮，加热至55℃后，停止加热，继续搅拌并降温结晶，在4小时内降温至4℃，析出白色固体，固液分离得到固体，将白色固体45-55℃加热干燥，得到Shinorine无定型结晶物。
实施例3坛紫菜中提取类菌胞素氨基酸Porphyra-334和Shinorine
浸提：将从海水中捞起的坛紫菜海水洗净泥沙沥干后进行粉碎，将粉碎后的坛紫菜在浓度为10.5％的氯化钠溶液中浸泡1h后捞出，得到首次浸提液；将捞出的海藻碎渣再用浓度80％的乙醇溶液中浸泡2h后捞出，得到二次浸提液；将所述首次浸提液和二次浸提液进行混合，得到混合浸提液；所述粉碎后的海藻、氯化钠溶液、乙醇溶液的质量比为1:8:15。
图15为实施例3中坛紫菜混合浸提液HPLC检测图，Agilent ZORBAX SB-C18柱条件，由图可知，RTShinorine＝3.6min，RT Porphyra-334＝4.5min。MAAs两个主要物质Shinorine和Porphyra-334在紫外HPLC条件下的纯度示意图，其中Shinorine占MAAs的33.9％，Porphyra-334占MAAs的63.9％。
絮凝：在混合浸提液中加入壳聚糖，搅拌均匀，进行70分钟絮凝后，固液分离，得到除杂后浸提液；所述混合浸提液与所述壳聚糖的质量比为：10000:3；
过滤：将所述混合浸提液用2500Dal聚醚砜材质超滤膜进行过滤，得到过滤液；所述过滤液用300Dal聚醚砜材质纳滤膜进行浓缩，得到5％-10％过滤液体积的浓缩液；
图16为实施例3中坛紫菜混合浸提液进行壳聚糖絮凝的现象，由图中可以看到蛋白质类和色素等物质被絮凝的现象，液体呈现清澈趋势。
层析：将浓缩液上样ODS混合层析柱进行层析，洗脱液冲洗，HPLC跟踪监测，分别得到Porphyra-334纯化液和Shinorine纯化液；所述洗脱液为5％的乙醇、3％丙酮和92％的pH为3.5-4.5浓度为0.05mol/L的乙酸铵缓冲溶液配制而成。
Porphyra-334浓缩：将Porphyra-334纯化液用300Dal的纳滤膜进行浓缩，所得的浓缩物进行水洗，得到含Porphyra-334浓缩物，将含Porphyra-334浓缩物一边加热一边搅拌加入5倍体积的丙酮，加热至55℃后，停止加热，继续搅拌并降温结晶，降温至4℃，析出白色固体，固液分离得到固体，将白色固体45-55℃加热干燥，得到Porphyra-334无定型结晶物；
Shinorine浓缩：将Shinorine纯化液用300Dal的纳滤膜进行浓缩，所得的浓缩物进行水洗，得到含Shinorine浓缩物，将含Shinorine浓缩物一边加热一边搅拌加入3倍体积的丙酮，加热至45℃后，停止加热，继续搅拌并降温结晶，降温至4℃，析出白色固体，固液分离得到固体，将白色固体45-55℃加热干燥，得到Shinorine无定型结晶物。
实施例4龙须菜中提取类菌胞素氨基酸Porphyra-334和Shinorine
浸提：将从海水中捞起的龙须菜海水洗净泥沙沥干后进行粉碎，粉碎为宽度为1-5mm 的碎渣，然后将粉碎后的龙须菜在浓度为0.5％的氯化钠溶液中浸泡2h后捞出，得到首次浸提液；将捞出的龙须菜碎渣再用浓度60％的乙醇溶液中浸泡3h后捞出，得到二次浸提液；将所述首次浸提液和二次浸提液进行混合，得到混合浸提液；所述粉碎后的龙须菜、氯化钠溶液、乙醇溶液的质量比为1:10:15；
图17为实施例4中龙须菜混合浸提液HPLC检测图，Agilent ZORBAX SB-C18柱条件，由图可知，RTShinorine＝3.6min，RT Porphyra-334＝4.4min。MAAs主要物质Shinorine在紫外HPLC条件下的纯度示意图，其中Shinorine占MAAs的63.0％。
絮凝：在混合浸提液中加入壳聚糖，搅拌均匀，进行70分钟絮凝后，固液分离，得到除杂后浸提液；所述混合浸提液与所述壳聚糖的质量比为10000:3；
过滤：将所述混合浸提液用2500Dal聚醚砜材质超滤膜进行过滤，得到过滤液；所述过滤液用300Dal聚醚砜材质纳滤膜进行浓缩，得到5％-10％过滤液体积的浓缩液；
层析：将浓缩液上样ODS混合层析柱进行层析，洗脱液冲洗，HPLC跟踪监测，分别得到Shinorine纯化液；所述洗脱液为3％异丙醇、5％乙腈和92％pH为3.5的乙酸铵缓冲溶液配置而成。所述混合层析柱的包括上部和下部，上部层析填料为硅胶，所述下部填料为十八烷基硅烷键合硅胶，上部与下部的高度比为4:5，所述混合层析柱的径高比为1:5。
Shinorine浓缩：将Shinorine纯化液用300Dal的纳滤膜进行浓缩，所得的浓缩物进行水洗，得到含Shinorine浓缩物，将含Shinorine浓缩物一边加热一边搅拌加入3-8倍体积的乙醇或丙酮，加热至45-55℃后，停止加热，继续搅拌并降温结晶，在4小时内降温至4℃，析出白色固体，固液分离得到固体，将白色固体45-55℃加热干燥，得到Shinorine无定型结晶物。
综上，经过本技术提取纯化操作，通过浸提、凝絮、过滤去除了大部分的杂质，过滤后得到浓缩液中所含MAAs占原料红藻总MAAs的90-94％，Porphyra-334和Shinorine的纯化得率分别为75％-81％和86％-92％。
目前，文献报道从海藻提取成分常常用干制品，有很多稳定性不好的成分如本案中的类菌胞素氨基酸会在晒制和烘干过程中存在降解的过程，同时产生结构类似的副产物杂质，本案结合实际工作经验选取了新鲜的海藻作为原材料能保障最佳的产品品质和产物收率，同时也不需要经过一些现有技术进行的脱脂操作，减少生产工艺步骤和成本，并且经过提取后的藻渣，进行后期的琼脂等大分子多糖制品的生产制备对其产品质量和品质不会产生影响，能够对海藻资源进行充分的加工和利用。
当然本发明并不排除在干制品经过水复泡后的红藻类材料进行提取MAAs和分离纯化的技术应用。
需要说明的是，在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者终端设备不仅包括那些 要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者终端设备所固有的要素。在没有更多限制的情况下，由语句“包括……”或“包含……”限定的要素，并不排除在包括所述要素的过程、方法、物品或者终端设备中还存在另外的要素。此外，在本文中，“大于”、“小于”、“超过”等理解为不包括本数；“以上”、“以下”、“以内”等理解为包括本数。
需要说明的是，尽管在本文中已经对上述各实施例进行了描述，但并非因此限制本发明的专利保护范围。因此，基于本发明的创新理念，对本文所述实施例进行的变更和修改，或利用本发明说明书及附图内容所作的等效结构或等效流程变换，直接或间接地将以上技术方案运用在其他相关的技术领域，均包括在本发明的专利保护范围之内。

Claims (10)

  1. 一种从海藻中提取类菌胞素氨基酸Porphyra-334和Shinorine的方法，其特征在于，所述的方法包括以下步骤：
    浸提：将海藻洗净沥干后进行粉碎，然后将粉碎后的海藻在浓度为0.5-10.5％的氯化钠溶液中浸泡1-2h后捞出，得到首次浸提液；将捞出的海藻碎渣再用浓度60-80％的乙醇溶液中浸泡2-3h后捞出，得到二次浸提液；将所述首次浸提液和二次浸提液进行混合，得到混合浸提液；所述粉碎后的海藻、氯化钠溶液、乙醇溶液的质量比为1:8-15:5-15；
    絮凝：在混合浸提液中加入壳聚糖，搅拌均匀，进行50-70分钟絮凝后，固液分离，得到除杂后浸提液；所述混合浸提液与所述壳聚糖的质量比为：10000:3-10；
    过滤：将所述除杂后浸提液用超滤膜进行过滤，得到过滤液；所述过滤液用纳滤膜进行浓缩，得到5％-10％过滤液体积的浓缩液；
    层析：将浓缩液上样ODS混合层析柱进行层析，洗脱液冲洗，HPLC跟踪监测，分别得到Porphyra-334纯化液和Shinorine纯化液；所述洗脱液为有机溶剂和pH为3.5-4.5缓冲溶液配置而成，所述有机溶剂包括甲醇、乙醇、异丙醇、乙腈以及丙酮中的一种或多种；
    Porphyra-334浓缩：将所述Porphyra-334纯化液进行膜浓缩，所得的浓缩物进行水洗，得到含Porphyra-334浓缩物，将含Porphyra-334浓缩物加入3-8倍体积的丙酮，加热至45-55℃后，停止加热，降温结晶，降温至4℃，析出白色固体，将白色固体在45-55℃加热干燥，得到Porphyra-334无定型结晶物；
    Shinorine浓缩：将所述Shinorine纯化液进行膜浓缩，所得的浓缩物进行水洗，得到含Shinorine浓缩物，将含Shinorine浓缩物加入3-8倍体积的乙醇或丙酮，加热至45-55℃后，停止加热，降温结晶，降温至4℃，析出白色固体，将白色固体45-55℃加热干燥，得到Shinorine无定型结晶物。
  2. 根据权利要求1所述的方法，其特征在于，所述海藻为红藻门海藻。
  3. 根据权利要求1所述的方法，其特征在于，所述过滤步骤，所述超滤膜为聚醚砜材质，孔隙选择性2500Dal。
  4. 根据权利要求1所述的方法，其特征在于，所述层析步骤，所述混合层析柱的包括上部和下部，所述上部层析填料为硅胶，所述下部填料为十八烷基硅烷键合硅胶。
  5. 根据权利要求4所述的方法，其特征在于，所述层析步骤，所述混合层析柱的上部与下部的高度比为4-5:5-6，所述混合层析柱的径高比为1:5-10。
  6. 根据权利要求1所述的方法，其特征在于，所述层析步骤，所述有机溶剂为甲醇，所述甲醇在洗脱液中的质量百分比为2-6％。
  7. 根据权利要求8所述的方法，其特征在于，所述层析步骤，所述缓冲溶液为浓度为0.05-0.1mol/L的乙酸铵缓冲溶液，采用乙酸调节乙酸铵缓冲溶液的pH为3.5-4.5。
  8. 根据权利要求8所述的方法，其特征在于，所述Porphyra-334浓缩步骤和Shinorine浓缩步骤，浓缩膜采用孔隙选择性300Dal的聚醚砜材质的纳滤膜。
  9. 类菌胞素氨基酸Porphyra-334，其特征在于，所述类菌胞素氨基酸Porphyra-334根 据权利要求1-8任一所述的方法进行制备。
  10. 类菌胞素氨基酸Shinorine，其特征在于，所述类菌胞素氨基酸Shinorine根据权利要求1-8任一所述的方法进行制备。
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