CN114214377A - Phosphatidyl-agaropectin oligosaccharide and preparation method thereof - Google Patents

Phosphatidyl-agaropectin oligosaccharide and preparation method thereof Download PDF

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CN114214377A
CN114214377A CN202111600499.6A CN202111600499A CN114214377A CN 114214377 A CN114214377 A CN 114214377A CN 202111600499 A CN202111600499 A CN 202111600499A CN 114214377 A CN114214377 A CN 114214377A
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phosphatidyl
oligosaccharide
agaropectin
agar
agar oligosaccharide
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毛相朝
张海洋
吴虹艳
孙建安
贺晨曦
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Ocean University of China
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Abstract

The invention discloses a phosphatidyl-agar oligosaccharide which is prepared by the following method: phosphatidyl choline and agar oligosaccharide are catalyzed by phospholipase D in a two-phase reaction system to generate transphosphatidylation action, and the phosphatidyl-agar oligosaccharide is synthesized; the agar oligosaccharide is selected from D-galactose and neoagarobiose; the phospholipase D is selected from PLDr 34. The phosphatidyl-agar oligosaccharide is a novel phosphatidyl glycoside, phospholipid can be used for preparing liposome materials, and a transesterification product has an encapsulation function, so that the phosphatidyl-agar oligosaccharide has good application prospects in the aspects of encapsulation and transportation, and can be used as an active substance to be applied to the aspects of liposome preparation, drug delivery and the like. The invention explores the generation conditions of the transphosphatidylation of phosphatidylcholine and agar oligosaccharide, synthesizes novel phosphatidyl glycoside, provides reasonable guidance for the synthesis of other phosphatidyl glycoside, and has certain research prospect.

Description

Phosphatidyl-agaropectin oligosaccharide and preparation method thereof
Technical Field
The invention relates to a phosphatidyl-agaropectin oligosaccharide and a preparation method thereof, belonging to the technical field of phosphatidyl glycoside.
Background
The sea is a treasure house and is a potential source of a plurality of natural bioactive substances. In all tissues, phospholipids, which are basic substances of life, are composed of a hydrophilic head group and a hydrophobic tail group, and are classified into glycerophospholipids and sphingolipids according to their connection modes, wherein the content of Phosphatidylcholine (PC) in the glycerophospholipids is most abundant.
Phospholipids can not only participate in the formation of cell membranes and maintain their biological functionality, but also play an important role in cell micelles and organelles, for example, part of phospholipids are precursors of various anti-inflammatory compounds, can regulate systemic inflammation, and provide protection against chronic diseases. Phospholipids can be modified under various conditions such as hydrogenation, acetylation, sulfonation and enzymatic modification, and the first three chemical modification methods are applied to the improvement of physical properties of membranes, and the enzymatic modification is modification of hydrophilic head groups or hydrophobic fatty acyl chains, such as phospholipase D (PLD) mediated transphosphatidylation. Multiple studies in animal models have shown that modified phospholipids have higher bioavailability and bioactivity, especially in terms of their effect on plasma and liver lipid levels.
One important product after phospholipid modification is phosphatidyl glycoside, and most of the glycosylated phospholipids are rich in lipid rafts or microdomains and play an important role in various cellular processes. In rodent brain, expression of phosphatidylglucoside (PtdGlc) is developmentally regulated. PtdGlc is most strongly expressed in radial glia in early rat brain development and is considered to be a good cell surface marker for stem cells. Lysophosphatidylglucoside (LPGlc) can act as a guide wire for extending axons during central nervous system development by activating the AG-like protein-coupled receptor (GPR) 55 of spinal sensory axons.
At present, most of the research on the aspect of phosphatidyl glycoside focuses on glucose, fructose, mannose and raffinose, and no deep research on agar oligosaccharide exists.
Agar is used as a raw material to prepare the marine functional oligosaccharide with the polymerization degree of 2-20. The preparation method of the agar oligosaccharide mainly comprises a chemical degradation method and a biological enzyme degradation method. Chemical methods are applied more, but still have some disadvantages, such as non-uniform product composition, complicated operation, easy environmental pollution, etc. The biological enzyme degradation method is to adopt specific agarase to hydrolyze glycosidic bonds on agar sugar chains so as to obtain specific agar oligosaccharides, and has the advantages of high catalytic efficiency, good product specificity, mild reaction conditions, no pollution and the like.
Disclosure of Invention
Aiming at the prior art, the invention provides a novel phosphatidyl glycoside-phosphatidyl-agaropectin oligosaccharide and a preparation method thereof. The preparation method has the advantages of short reaction route, simple steps, high yield, no toxic chemicals involved in the synthesis process and high stability of the transesterification product.
The invention is realized by the following technical scheme:
a phosphatidyl-agar oligosaccharide is prepared by the following method: phosphatidyl choline and agar oligosaccharide are catalyzed by phospholipase D in a two-phase reaction system to generate transphosphatidylation action, and the phosphatidyl-agar oligosaccharide is synthesized; the agar oligosaccharide is selected from D-galactose and neoagarobiose; the phospholipase D is selected from PLDr 34.
Further, the fatty acid chains on the phosphatidylcholine are palmitic acid C16:0 and linoleic acid C18:2, respectively.
Further, the phosphatidylcholine is dissolved in an organic solvent to serve as an organic phase; the organic solvent is selected from methyl ether, ethyl ether, cyclopentyl methyl ether, ethyl acetate, butyl acetate or ethyl butyrate; the concentration of the phosphatidylcholine is 10-100 mg/mL.
Further, the agar oligosaccharides are dissolved in an aqueous solution as an aqueous phase; the water solution is selected from citric acid-sodium citrate buffer solution with the pH value of 4.0-6.0.
The agar oligosaccharide can be prepared by a biological enzymolysis method or a chemical degradation method.
Furthermore, the neoagarobiose is prepared by degrading agarose by beta-agarase AgWH50B (Access Number: KY 417136) and/or beta-agarase AgWH50C (Access Number: KC 913197) obtained by screening in the subject group of the inventor of the invention. The specific preparation method can be as follows: taking 1% agarose solution (g/ml) as a substrate, adding 2.0 g of AgWH50B crude enzyme, carrying out water bath reaction at 37 ℃ for 12 h, carrying out boiling water bath for 10 min, then adding 1.5 g of AgWH50C crude enzyme, carrying out water bath reaction at 37 ℃ for 12 h, carrying out boiling water bath for 10 min, centrifuging, taking supernatant, concentrating and drying to obtain the neoagarobiose. The neoagarotetraose can be prepared by the following method: taking a 1-2% agarose solution (g/ml) as a substrate, adding 2.0 g AgWH50B crude enzyme, reacting in a water bath at 37 ℃ for 12 h, boiling for 10 min, centrifuging to obtain a supernatant, concentrating and drying to obtain the neoagarotetraose.
Further, in the two-phase reaction system, the molar ratio of the phosphatidylcholine to the agar oligosaccharide is 1: 45-55, preferably 1: 50; the volume ratio of the organic phase to the aqueous phase is 1: 0.8-1.2, preferably 1: 1; the enzyme adding amount of the phospholipase D is 1.0-1.5U, and 1.4U is preferred.
Further, the specific reaction conditions for catalyzing transphosphatidylation are as follows: the reaction system is subjected to water bath reaction at 37-42 ℃ for 8-12 h; and centrifuging to take the supernatant after the reaction is finished, dissolving the product phosphatidyl-agar oligosaccharide in an upper organic phase, and blowing nitrogen to obtain the phosphatidyl-agar oligosaccharide.
The phosphatidyl-agar oligosaccharide is a novel phosphatidyl glycoside, phospholipid can be used for preparing liposome materials, and a transesterification product has an encapsulation function, so that the phosphatidyl-agar oligosaccharide has a good application prospect in the aspects of encapsulation and transportation, and can be used as an active substance (such as a drug carrier, a liposome membrane material and the like) to be applied to the aspects of liposome preparation, drug delivery and the like.
The invention explores the occurrence conditions of the transphosphatidylation of phosphatidylcholine and agar oligosaccharide, and the screened agar oligosaccharide with higher substrate preference is connected to the phosphatidylcholine through the transphosphatidylation, so that a novel phosphatidyl glycoside, namely phosphatidyl-agar oligosaccharide, is synthesized, reasonable guidance is provided for the synthesis of other phosphatidyl glycosides, and the method has a certain research prospect. The preparation method of the invention is a double-enzyme synthesis method, which greatly reduces the production cost.
The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art.
Drawings
FIG. 1: and (3) a TLC result graph of transesterification reaction of phosphatidylcholine and agar oligosaccharides with different polymerization degrees, wherein PC represents phosphatidylcholine, and 1, 2, 3 and 4 respectively represent transesterification reaction results of PC and D-galactose, neoagarobiose, neoagarotetraose and 1-15 AOS.
FIG. 2: chemical structure of phosphatidylcholine.
FIG. 3: chemical structure diagram of phosphatidyl-D galactose.
FIG. 4: chemical structure diagram of phosphatidyl-neoagarobiose.
FIG. 5: the results of mass spectrometry of phosphatidyl-D galactose are shown schematically.
FIG. 6: the results of mass spectrometry of phosphatidyl-neoagarobiose are shown schematically.
FIG. 7: and (3) a result chart of analyzing the phosphatidyl-agaropectin oligosaccharide by an HPLC-ELSD method.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
The phosphatidylcholine used in the present invention is Soy PC (95%) (SPC, 441601G-50G-I-175, Avanti Polar Lipids).
The 1-15AOS used in the invention is mixed sugar of agar monosaccharide to pentadecaose, and 1-15 agar oligosaccharide (1500 Da, Qingdao Bozhi Hui Virginian Biotech Co., Ltd.).
The phospholipase D used in the invention is PLDr34 (Access Number: MN 604233) (the enzyme is described in another patent application of the applicant of the invention, CN 110564708A, namely the phospholipase shown in SEQ ID NO.1 in the specification of CN 110564708A), and the amino acid sequence of the phospholipase D is shown as follows.
Amino acid sequence of PLDr 34:
MIISFRLSRPARAALICALALTVLPASPATAADAATPHLDAVERTLREVSPGLEGEVWERTAGNRLDAGADDPAGWLLQTPGCWGDAGCRDRVGTRRLLAKMTENISRATRTVDISTLAPFPNGAFQDAIVAGLKSSAARGNKLTVRVLVGAAPIYHMNVLPSKYRDELVAKLGADARNVDLNVASMTTSKTSFSWNHSKLLVVDGQSVITGGINDWKDDYLETAHPVADVDLALRGPAAASAGRYLDELWSWTCQNRNNIAGVWFASSNGTACMPAMAKDTAPAAPPAAPGDVPAIAVGGLGVGIKRSDPSSAFRPTLPSAADTKCVVGLHDNTNADRDYDTVNPEESALRTLISSAKGHIEISQQDVNATCPPLPRYDIRVYDALAARMAAGVKVRIVVSDPANRGAVGSGGYSQIKSLSEISDTLRDRLALLTGDQGAAKATMCSNLQLATFRSSKSPTWADGHPYAQHHKVVSVDDSAFYIGSKNLYPAWLQDFGYIVESPGAAQQLDAQLLSPQWTHSKETATVDYERGLCHI。
experiment 1: substrate preference selection for transphosphatidylation reactions
The method comprises the following steps:
(I) transphosphatidylation
(1) 10 mg of phosphatidylcholine was dissolved in 1 mL of cyclopentyl methyl ether to obtain an organic phase.
(2) 0.12 g D-galactose was dissolved in 1 mL citric acid-sodium citrate buffer (pH 6.0, 0.1M) to give an aqueous phase.
(3) The organic phase and the aqueous phase were mixed at a volume ratio of 1:1, and 1.4U of PLD was added to form a biphasic reaction condition, and the mixture was sealed in a brown vial.
(4) The brown bottle is put into a constant temperature water bath kettle and is subjected to water bath reaction for 10 hours under the conditions of 40 ℃ and 200 r.
(5) After the reaction is finished, centrifuging (8000 r, 5 min) to obtain supernatant A.
And (3) replacing the D-galactose in the step (2) with 0.21 g of neoagarobiose, 0.49 g of neoagarotetraose and 1.5 g of 1-15 agaro-oligosaccharide respectively (under the same conditions), and obtaining a supernatant B, a supernatant C and a supernatant D.
(II) carrying out thin-layer chromatography analysis on the prepared supernatant A, the prepared supernatant B, the prepared supernatant C and the prepared supernatant D:
(1) samples with the volume of a capillary tube of 0.5mm are sampled, and the samples are dried by a blower after each sample sampling.
(2) Placing the silica gel plate with the spotted sample in a spreading cylinder containing a spreading agent, wherein the formula of the spreading agent is chloroform: methanol: glacial acetic acid: water =50:25:6:2 (v: v: v: v).
(3) After about 20 min, the silica gel plate was taken out, dried by a blower, and then placed in an iodine jar, and the experimental results were observed later.
From the thin layer chromatography analysis result diagram (as shown in fig. 1), it can be seen that in the transphosphatidylation reaction taking agar oligosaccharides with different polymerization degrees as raw materials, the transesterification reaction taking neoagarobiose as a main component is more thorough, and the substrate PC is almost completely degraded. The transesterification reaction with D-galactose is relatively thorough, the transesterification reaction with neoagarotetraose is relatively weak, and the mixed agar oligosaccharide has no transesterification reaction. Therefore, the agaro-oligosaccharides obtained by the subsequent transphosphatidylation reaction are determined to be D-galactose and neoagarobiose.
Example 1: synthesis of phosphatidyl-agaropectin oligosaccharide by PLD (pulsed laser deposition) catalytic enzyme method
The method comprises the following steps:
(1) 10 mg of phosphatidylcholine was dissolved in 1 mL of cyclopentyl methyl ether to obtain an organic phase.
(2) 0.12 g D-galactose was dissolved in 1 mL citric acid-sodium citrate buffer (pH 6.0, 0.1M) to give an aqueous phase.
(3) The organic phase and the aqueous phase were mixed at a volume ratio of 1:1, and 1.4U of PLD was added to form a biphasic reaction condition, and the mixture was sealed in a brown vial.
(4) The brown bottle is put into a constant temperature water bath kettle and is subjected to water bath reaction for 10 hours under the conditions of 40 ℃ and 200 r.
(5) After the reaction is finished, centrifuging (8000 r, 5 min) to obtain supernatant, and obtaining the phosphatidyl-agaropectin oligosaccharide dissolved in the organic solvent.
(6) After nitrogen blowing, the high-concentration phosphatidyl-agaropectin oligosaccharide is obtained, and the structural formula is shown in figure 3.
Replacing the D-galactose in the step (2) with 0.21 g of neoagarobiose (under the same other conditions), and preparing the phosphatidyl-neoagarobiose, wherein the structural formula of the phosphatidyl-neoagarobiose is shown in figure 4.
The relative molecular mass of the two phosphatidyl-agaropectides is measured by the MS method, the results are shown in figures 5 and 6, the analysis result is consistent with the calculation result of the corresponding molecular ion peak (M phosphatidyl-D galactose is approximately 851, M phosphatidyl-neoagaropectide is approximately 995), and the mass spectrometry result and the liquid phase product peak result can both indicate that the target phosphatidyl glycoside is generated after transesterification reaction, so that the phosphatidyl-D galactose and the phosphatidyl-neoagaropectide are determined to be prepared.
Experiment 2: key parameter of transphosphatidylation preparation process
The parameters critical to the phospholipid acylation reaction include: in the transesterification, the molar ratio of PC to sugar (1: 20, 1:30, 1:40, 1:50, 1: 60), the amount of PLD added (0.2, 0.6, 1, 1.4, 1.8U), the transesterification time (4, 6, 8, 10, 12 h) and the volume ratio of organic phase to aqueous phase in the biphasic reaction system (1: 3, 1:2, 1:1, 2: 1, 3: 1). Regulating and controlling the 4 factors in the reaction system, detecting the synthesis conversion rate by adopting an HPLC-ELSD method, and finally determining the optimal synthesis condition of the phosphatidyl-agaropectin oligosaccharide. When the molar ratio of PC to sugar is 1:50, the amount of PLD enzyme is 1.4U, the transesterification reaction time is 10 hours, and the volume ratio of the organic phase to the aqueous phase is 1:1, the conversion rate of phosphatidyl-D galactose can reach 85% and the conversion rate of phosphatidyl-neoagarobiose can reach 96% under the optimal reaction conditions (FIG. 7). Experimental results show that when the neoagarobiose is used as a raw material for the transphosphatidylation reaction, the conversion rate of phosphatidyl-neoagarobiose is obviously higher than that of D-galactose.
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.

Claims (10)

1. A preparation method of phosphatidyl-agar oligosaccharide is characterized in that: phosphatidyl choline and agar oligosaccharide are catalyzed by phospholipase D in a two-phase reaction system to generate transphosphatidylation action, and the phosphatidyl-agar oligosaccharide is synthesized; the agar oligosaccharide is selected from D-galactose and neoagarobiose; the phospholipase D is selected from PLDr 34.
2. The method for preparing phosphatidyl-agaropectin oligosaccharide according to claim 1, wherein the method comprises the following steps: the fatty acid chains on the phosphatidylcholine are palmitic acid C16:0 and linoleic acid C18:2 respectively.
3. The method for preparing phosphatidyl-agaropectin oligosaccharide according to claim 1, wherein the method comprises the following steps: dissolving the phosphatidylcholine in an organic solvent to obtain an organic phase; the organic solvent is selected from methyl ether, ethyl ether, cyclopentyl methyl ether, ethyl acetate, butyl acetate or ethyl butyrate; the concentration of the phosphatidylcholine is 10-100 mg/mL.
4. The method for preparing phosphatidyl-agaropectin oligosaccharide according to claim 1, wherein the method comprises the following steps: the agar oligosaccharide is dissolved in an aqueous solution to be used as a water phase; the water solution is selected from citric acid-sodium citrate buffer solution with the pH value of 4.0-6.0.
5. The method for preparing phosphatidyl-agaropectin oligosaccharide according to claim 1, wherein the method comprises the following steps: the neoagarobiose is prepared by degrading agarose by beta agarase AgWH50B and/or beta agarase AgWH 50C.
6. The method for preparing phosphatidyl-agaropectin oligosaccharide according to claim 1, wherein the method comprises the following steps: in the two-phase reaction system, the molar ratio of phosphatidylcholine to agar oligosaccharide is 1: 45-55; the volume ratio of the organic phase to the water phase is 1: 0.8-1.2; the enzyme adding amount of the phospholipase D is 1.0-1.5U.
7. The method for preparing phosphatidyl-agaropectin oligosaccharide according to claim 6, wherein the method comprises the following steps: the molar ratio of the phosphatidylcholine to the agar oligosaccharide is 1: 50; the volume ratio of the organic phase to the aqueous phase is 1: 1; the enzyme adding amount of the phospholipase D is 1.4U.
8. The method for preparing phosphatidyl-agaropectin oligosaccharide according to claim 1, wherein the method comprises the following steps: the specific reaction conditions for catalyzing the transphosphatidylation are as follows: the reaction system is subjected to water bath reaction at 37-42 ℃ for 8-12 h.
9. The phosphatidyl-agar oligosaccharide prepared by the preparation method of any one of claims 1 to 8.
10. Use of the phosphatidyl-agaropectide of claim 9 as an active substance in the preparation of liposomes, drug delivery.
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CN111514152A (en) * 2020-05-18 2020-08-11 中国海洋大学 Application of n-3PUFA phosphatidyl glycoside in preparation for improving blood brain barrier damage
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JPH0656856A (en) * 1993-04-16 1994-03-01 Meito Sangyo Kk Phospholipid derivative
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