JP3884488B2 - Oligoglycolipid - Google Patents

Description

[0001]
[Industrial application fields]
The present invention can synthesize oligosaccharide chains stably on phospholipid endoplasmic reticulum (hereinafter referred to as endoplasmic reticulum), oil droplets or micelles composed of a known surfactant, can be easily synthesized, and has a clear structure. Related to oligoglycolipids. That is, the present invention relates to an oligoglycolipid that is intended to stabilize the dispersion of vesicles comprising surfactants such as phospholipids, or oil droplets and micelles that are O / W emulsions.
[0002]
That is, the vesicle modified with the oligosaccharide chain of the present invention and the oil droplets and micelles of the O / W emulsion have excellent dispersion stability and can prevent fusion between the vesicles. Emulsion type cosmetics can be prepared.
The endoplasmic reticulum can be used as a drug delivery system or sustained-release material in which a physiologically active substance such as adriamycin or prostaglandin is encapsulated in the inner aqueous phase. It can also be red blood cells. Similarly, a lipophilic physiologically active substance such as tocopherol can be contained in the oil droplets of the O / W emulsion and the oil phase of the micelle.
[0003]
[Prior art]
It is normal for oil droplets or micelles of endoplasmic reticulum and O / W emulsion to have instability such as aggregation, fusion, disintegration or morphological change accompanying the above phenomenon or leakage of inclusions. A number of proposals have been made as means for this.
First, stabilization of an aggregate of unsaturated phospholipids has been attempted (for example, review: H. Ringsdorf et al., Angew. Chem., Int. Engl., 27, 113 (1988)). Due to the film polymerization, the characteristics of the original lipid, such as the gel-liquid crystal phase transition phenomenon, phase separation and fluidity of the membrane, are lost. The metabolic mechanism in the body is also unclear.
[0004]
Next, the endoplasmic reticulum surface coating with carboxymethyl chitin (H. Izawa et al., Biochem. Biophys. Acta, 855, 243 (1986)) and polysaccharides such as amylopectin and pullulan were added with hydrophobic groups such as cholesterol. A technique for stabilizing the endoplasmic reticulum structure by coating the surface of the endoplasmic reticulum with a bound compound (Japanese Patent Application Laid-Open No. 3-292301) is disclosed. In addition, a derivative obtained by introducing a fatty acid into a polysaccharide such as hyaluronic acid as a coating agent for microcapsules such as phospholipid vesicles (Japanese Patent Laid-Open No. 3-47801), or a glucosamine derivative as a compounding agent for forming cationic liposomes (JP No. 3-218389) is disclosed, but none of these inventions has a drawback of easily inducing aggregation of the endoplasmic reticulum, although it does not greatly change the membrane fluidity of the endoplasmic reticulum.
[0005]
It has been reported that introduction of an amphiphilic molecule in which polyethylene glycol is bonded to the hydrophilic part of cholesterol or phosphatidylethanolamine into the endoplasmic reticulum suppresses the aggregation of the endoplasmic reticulum and extends the retention time of the endoplasmic reticulum in blood. (TM Allen et al., Biochem. Biophys. Acta, 1066, 29 (1991)). Further, a technique for stabilizing a phospholipid by incorporating a polyoxyethylene surfactant or the like as a liposome for an immunoassay reagent (Japanese Patent Laid-Open No. 3-61859) has been disclosed. There is concern about behavior.
[0006]
In addition, the present inventors have already introduced oligosaccharide fatty acid ester into the endoplasmic reticulum bilayer membrane, and can effectively stabilize the dispersion of the endoplasmic reticulum, and the longer the sugar chain of the oligosaccharide becomes, It has been disclosed that an excellent effect is exhibited (Japanese Patent Laid-Open No. 1-294701). However, although the oligosaccharide fatty acid ester offers many solutions to the stability of the endoplasmic reticulum, long-chain fatty acids are introduced non-selectively into oligosaccharides having a large number of reactive groups (hydroxyl groups). The number and site of introduction vary. Therefore, the separation and purification of the oligosaccharide fatty acid ester is complicated and the yield is low, and the aggregation inhibition effect, fusion inhibition effect, and degree of fixation on the surface of the endoplasmic reticulum membrane of the endoplasmic reticulum itself into which the oligosaccharide fatty acid ester is introduced are The problem is that it differs depending on the isomer of the oligosaccharide. Furthermore, the relationship between the structure and effect of the isomer is unclear.
[0007]
Furthermore, the present inventors have disclosed that the endoplasmic reticulum can be excellently stabilized by an oligosaccharide derivative in which a hydrophobic group is efficiently introduced into the anomeric position of the reducing end group of the oligosaccharide by an ether bond and an amide bond. (Japanese Patent Application No. 4-126716 invention, Japanese Patent Application No. 4-148922 invention, Japanese Patent Application No. 4-191364 invention, Japanese Patent Application No. 4-206136 invention). However, the production method is complicated by introducing a protective group into the reactive group (hydroxyl group), or by attaching the hydrophobic group after dehydration and ring closure after oxidative ring opening of the reducing end group, and the production process becomes complicated. Is also low.
[0008]
[Problems to be solved by the invention]
In order to solve the above problems, the present invention has a technical problem to develop an oligoglycolipid having a clear structure in which a hydrophobic group is easily introduced into a specific group of the oligoglycolipid in a high yield.
[0009]
[Means for Solving the Problems]
In order to solve the above-described problems, the present invention provides an integer having a sugar polymerization degree of 2 to 30 and a reducing end group composed of aldose, and an ester of a hydrophobic group at the anomeric position of the reducing end group. The present invention relates to an oligoglycolipid introduced by binding, and further relates to the use of the oligoglycolipid as a stabilizer for phospholipid endoplasmic reticulum.
[0010]
The present invention is described in detail below.
Since the oligosaccharide lipid of the present invention is a compound that needs to adjust the hydrophilicity / hydrophobicity balance, the oligosaccharide used in the present invention is an oligosaccharide having an integer of 2 to 30 saccharide units, and Oligosaccharides in which the reducing end group is composed of aldose are preferred.
[0011]
The oligosaccharides represented by an integer of 2 to 30 saccharide units can be obtained with high purity by using a gel filtration column that has been kept warm, for fractionation by degree of polymerization and by type. A differential refractometer is used for the detection. Confirmation of qualitative and purity of the fractionated oligosaccharides can be carried out by multiple development with a mixed solvent (1-butanol / pyridine / water) using a silica gel thin layer plate, or sulfuric acid coloration, or a stationary phase having an amino group, Liquid chromatography using a mobile phase of a mixed solvent (acetonitrile / water), or liquid chromatography using a stationary phase having an octadecyl group and a mobile phase of pure water can be used. The oligosaccharide used in the present invention may be a commercially pure product as long as the structure is clear.
[0012]
Since the oligoglycolipid of the present invention needs to emphasize the adjustment of the hydrophilicity / hydrophobicity balance, the hydrophobic group used here has, for example, an alkyl chain having 12 to 22 carbon atoms and an unsaturated bond. In some cases, the number of unsaturations is preferably 1 to 4. In addition, since the carboxylate obtained by oxidizing the anomeric position of the reducing end group of the oligosaccharide chain and the hydrophobic group are ester-bonded, the hydrophobic group-introducing agent used in the present invention has a halogen.
[0013]
Examples of the halogen-containing hydrophobic group-introducing agent include primary and secondary alkyl halides having 12 to 22 alkyl carbon atoms. That is, examples of the halogen having a hydrophobic group include chlorine, bromine and iodine. Examples of the long-chain alkyl having a halogen include lauryl chloride, cetyl bromide, stearyl bromide, and behenyl iodide as the linear primary halogenated alkyl. In addition, linear primary unsaturated alkyl halides such as 1-chloro-1-dodecene, oleyl bromide, linolenyl iodide, branched primary saturated alkyl halides, branched primary unsaturated alkyl halides, secondary saturated Alkyl halides or secondary unsaturated alkyl halides can also be used.
Further, a hydrophobic group of a halide having two or more long-chain alkyl groups, for example, a hydrophobic group-introducing agent of a halide having long-chain alkyl groups at the 1,2-position and 1,3-position of the glycerol skeleton is also effective. is there.
Furthermore, substances such as sterols such as cholesteryl bromide are also included in the scope of the hydrophobic group-introducing agent having halogen.
[0014]
Introduction of a hydrophobic group into the sugar chain terminal anomeric position by an ester bond is performed as follows.
That is, the reducing end of the oligosaccharide is oxidized using hypoiodous acid or bromine water to open the anomeric position as a carboxylate, and then converted into a carboxylic acid by a cation exchange column.
Next, neutralization is performed with a 10% methanol solution or a 10% aqueous solution of tetra-n-butylammonium hydroxide or the like to obtain a quaternary ammonium salt of carboxylic acid. Or it neutralizes with 0.5N aqueous solution, such as sodium hydroxide, potassium hydroxide, or lithium hydroxide, to make an alkali metal salt. Or ammonia basic AgNO _Three It is set as silver carboxylate using aqueous solution. These are dissolved in dimethylformamide, dimethylsulfoxide, or formamide, and then mixed with a hydrophobic halide to form a uniform solution, which is reacted at a temperature of 20 to 90 ° C, preferably 40 to 70 ° C. Obtain a solution. The oligoglycolipid obtained here is purified by precipitation with a poor solvent such as acetone, and the precipitate is dissolved in water, water-methanol or water-ethanol and applied to a reverse phase silica gel column.
[0015]
Confirmation of the structure of the oligoglycolipid by the ester bond obtained by the above method is as follows: ¹ H-NMR and ¹³ Performed by C-NMR. For purity confirmation, a thin layer chromatography based on an infrared spectrophotometer (hereinafter referred to as IR) and silica gel, or a reversed-phase or normal-phase silica gel column is applied and a differential refraction detector is used as the detector. Using high performance liquid chromatography.
[0016]
The stability of the endoplasmic reticulum modified with the oligoglycolipid of the present invention can be confirmed as follows.
Phospholipids alone, or mixed lipids made by adding membrane stabilizers (sterols such as cholesterol and cholestanol) to phospholipids or negatively charged lipids (stearic acid, diacylphosphatidic acid, etc.) to phospholipids In addition, 0.01 to 50 mol%, preferably 0.05 to 20 mol%, of the oligoglycolipid of the present invention is added as a stabilizer for the endoplasmic reticulum to prepare a hydrate. The hydrate dispersion of 50 to 200 nm is prepared from the hydrate by a known method such as an ultrasonic stirrer, a microfluidizer or an extruder. The particle size of the vesicle dispersion obtained here can be measured by a particle size distribution measuring device or a negative-stained transmission electron microscope.
[0017]
After gel filtration of the dispersion of the endoplasmic reticulum, or after removing oligoglycolipids that have not been introduced into the endoplasmic reticulum as a supernatant by ultracentrifugation, the sugars of each fraction are phenol-sulfuric acid. By quantifying using the method, it is possible to determine the rate of introduction of oligoglycolipids into the endoplasmic reticulum. More than 70% of the added amount of the oligoglycolipid of the present invention is introduced into the endoplasmic reticulum.
The dispersion stability of the endoplasmic reticulum should be evaluated and confirmed by measuring the time-dependent change in absorbance (turbidity) at 500 to 800 nm in a dispersion liquid excluding free oligoglycolipids at room temperature. Can do.
[0018]
In addition, as a method for evaluating the dispersion stability of the endoplasmic reticulum, there are a method of measuring the viscosity of the endoplasmic reticulum dispersion using a cone plate type rotational viscometer, a spectroscopic method using an electron microscope, and the like.
[0019]
Next, oil droplets will be exemplified as an O / W emulsion modified with the oligoglycolipid of the present invention, and the confirmation of its stability will be described.
Oils and fats, for example triglycerides such as soybean oil (oil / oligoglycolipid molar ratio = 0.1 to 100, preferably 1 to 10), aqueous media such as water, phosphate buffer (pH 5 to 9), physiological saline , Krebs-Ringers solution, etc. are added, followed by shaking or stirring with a mixer.
The O / W-type oligoglycolipid-modified microsphere thus prepared is a hydrophilic emulsion.
Furthermore, by carrying out an emulsification process using a high-pressure homogenizer such as an ultrasonic process or a microfluidizer, a hydrophilic emulsion containing small droplets of oil droplets can be obtained. Oil droplet globules can be prepared in the range of several μm to several tens of nm depending on the conditions of the preparation method. These particle sizes are stable without change after storage for several months at room temperature or 5 ° C.
[0020]
[Action]
The oligoglycolipid of the present invention is a compound with a clear structure obtained in a high yield by a very simple method, and has a very good affinity with an amphiphilic molecule. Suitable as a modifier for aggregates (endoplasmic reticulum, oil droplets, micelles, etc.). Furthermore, the oligoglycolipid introduced into the aggregate can maintain a state in which the sugar structure is exposed to the aqueous phase.
[0021]
The oligoglycolipid as an aggregate modifier of the present invention is used, for example, as a dispersion stabilizer for endoplasmic reticulum or a leakage inhibitor for inclusions, as well as for dispersion stabilization, parentalization, and specification for solutes such as proteins. It can be used in accordance with the purpose of use, such as recognition labeling for antibodies and suppression of phagocytosis in vivo.
[0022]
When a carrier containing a functional molecule in the inner aqueous phase of the endoplasmic reticulum is administered in vivo, the endoplasmic reticulum is usually excluded from the blood in a short time by incorporation into the reticuloendothelial system or phagocytic cells. By the surface modification of the endoplasmic reticulum with the oligoglycolipid of the present invention, the residence time in the blood of the endoplasmic reticulum can be controlled.
Furthermore, the oligosaccharide lipid of the present invention in which an oligosaccharide chain and a structure capable of being metabolized together with a hydrophobic group are ester-bonded can be expected not to cause any obstacle in in vivo metabolism.
[0023]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1.
After dissolving 1.83 g of iodine in 50 ml of 20% potassium iodide aqueous solution and adding an aqueous solution in which 5.0 g of maltopentaose was dissolved in 5 ml of pure water, 10% tetra-n-butyl was added at room temperature in the dark. Ammonium hydroxide ((n-Bu) _Four The reaction was stirred for 3 hours while dropping the NOH) aqueous solution until the iodine color disappeared. The reaction was washed with acetone and ethanol to remove free salts. As a result, the anomeric position of the reducing end group of the oligosaccharide is changed to carboxylic acid (n-Bu). _Four N ⁺ 4.51 g of an oligosaccharide derivative that was opened as a salt was obtained. The yield was 68.9%. The confirmation of the reaction is 1620 cm based on carboxylate ions in the IR spectrum (KBr). ^-1 This was confirmed by the appearance of a peak in the vicinity.
[0024]
0.5 g of the resulting oligosaccharide ammonium salt was dissolved in 40 ml of distilled N, N-dimethylformamide (hereinafter referred to as DMF), mixed with 307 mg of 2-fold molar stearyl bromide, and stirred at 50 ° C. for 40 hours. did. This was concentrated and reprecipitated in acetone to obtain a crude sample of maltopentaose monostearyl ester (hereinafter referred to as MPMS) of the oligoglycolipid of the present invention. That is, this is one in which an ester group is introduced into maltopentaose by an ester bond using stearyl bromide.
The obtained MPMS was dissolved in methanol, and using liquid chromatography, a capsule pack C18 column (trade name, manufactured by Shiseido Co., Ltd.) (may be an octadecyl-modified silica gel column) as a stationary phase and methanol / As a result of removing unreacted substances using a pure water (95/5) mixed solvent, 0.37 g of purified MPMS was obtained, and the yield was 76.0% based on the ammonium salt.
[0025]
As a result of IR measurement, purified MPMS has peaks based on alkyl chains of 2800 to 2950 cm. ^-1 Further, the peak based on the C═O stretching vibration of the ester bond is confirmed at 1740 cm. ^-1 It was confirmed in the vicinity. Also, ¹ From the 1 H-NMR measurement, peaks of α-position and β-position methylene protons based on ester bonds were observed at 4.04 and 1.58 ppm, respectively. Therefore, the MPMS can be an oligoglycolipid with a clear structure in which a stearyl group is introduced into the anomeric position of the reducing end group of maltopentaose by an ester bond.
[0026]
Evaluation of ER stabilization of MPMS was performed as follows.
3 mg of MPMS and 100 mg of dipalmitoylphosphatidylcholine (hereinafter referred to as DPPC) as phospholipid were dissolved in methanol and mixed, and then a thin film was formed on the inner wall of the pear-shaped flask by a rotary evaporator. 10 ml of pure water and several glass beads were added and stirred with heating at 50 ° C. by vortexing to obtain a cloudy liquid. This white turbid liquid was treated with an extruder. By changing the pore size of the polycarbonate filter at the time of treatment to 0.4 μm, 0.2 μm, 0.1 μm and 0.05 μm in sequence, A transparent DPPC vesicle dispersion was obtained. The particle size of the endoplasmic reticulum obtained here was measured with a particle size distribution measuring device and a negative-stained transmission electron microscope. The average particle size was 56 ± 11 nm and 53 ± 9 nm, respectively.
[0027]
The dispersion stability of the endoplasmic reticulum was evaluated by measuring the change in absorbance (turbidity) at 500 nm after standing at 25 ° C. for 3 days with an ultraviolet-visible spectrophotometer (MPS-2000, manufactured by Shimadzu Corporation).
In the unmodified DPPC vesicle, when the sample immediately after preparation was left at room temperature, the turbidity of the solution immediately started to increase and aggregation of the vesicle occurred. Further, in the sugar ester-modified vesicle, the increase in turbidity reached about 70% when the unmodified system was taken as 100%, and aggregation was not sufficiently suppressed.
On the other hand, in the system modified by introducing MPMS, an increase in turbidity of about 7% of that of the unmodified system was only observed immediately after the preparation was allowed to stand, and the aggregation was suppressed with little increase over time.
Therefore, the effect of imparting dispersion stability to the endoplasmic reticulum of the MPMS of the present invention was extremely large.
[0028]
Example 2
5.20 g of iodine was dissolved in 50 ml of a 40 ° C. heated methanol solution, and an aqueous solution in which 5.0 g of maltopentaose was dissolved in 5 ml of water was added thereto, followed by stirring at 40 ° C. for 60 minutes. Then 4% KOH / methanol solution was added and added dropwise until the iodine color disappeared. As a result, the anomeric position of the oligosaccharide was converted to a carboxylic acid potassium salt, resulting in a precipitated substance. The precipitated material was then filtered and washed with ethanol and acetone.
After the washing, the precipitated substance is dissolved in water, and this aqueous solution is further dissolved in the cation exchange resin Amberlite-IR-120B (H ⁺ Type) (manufactured by Organo Co., Ltd.) was used as an oligosaccharide having a carboxylic acid, and free salts were removed with an electrodialysis desalting apparatus (manufactured by Asahi Kasei Co., Ltd.).
[0029]
To the oligosaccharide having the carboxylic acid, 3.1 ml of a 10% tetra-n-butylammonium hydroxide-methanol solution was added dropwise in distilled water to adjust the pH of the aqueous solution to 7.5, followed by lyophilization. The free salt was washed with acetone and dried to obtain 4.3 g of ammonium carboxylate. The reaction is 1620 cm in IR spectrum (KBr). ^-1 This was confirmed by the appearance of a peak based on carboxylate ions in the vicinity.
[0030]
0.5 g of the resulting oligoglycolipid ammonium salt was dissolved in 40 ml of distilled DMF, mixed with 324 mg of 2-fold molar cetyl iodide, and stirred at 70 ° C. for 12 hours to be reacted. Further, the reaction solution was concentrated and then reprecipitated in acetone to obtain a crude sample of maltopentaose monocetyl ester (hereinafter referred to as MPMC).
The obtained MPMC was dissolved in methanol, and using liquid chromatography, the stationary phase was a recloprep RP-18 column (trade name, manufactured by Merck) (may be an octadecyl-modified silica gel column) and the mobile phase was methanol / As a result of removing unreacted substances using a pure water (95/5) mixed solvent, 0.39 g of purified MPMC was obtained, and the yield was 79.3% based on the ammonium salt of the oligoglycolipid. .
[0031]
As a result of IR measurement, purified MPMC has peaks based on alkyl chains of 2800 to 2950 cm. ^-1 Further, the peak based on the C═O stretching vibration of the ester bond is confirmed at 1740 cm. ^-1 It was confirmed in the vicinity. Also, ¹ From the 1 H-NMR measurement, peaks of α-position and β-position methylene protons based on ester bonds were observed at 4.04 and 1.58 ppm, respectively. Accordingly, the MPMC can be an oligoglycolipid with a clear structure in which a cetyl group is introduced by an ester bond at the anomeric position of the reducing end group of maltopentaose.
[0032]
Mixed lipid powder containing the oligosaccharide lipid of the present invention (molar ratio: DPPC / cholesterol / palmitic acid / MPMC = 7/7/2/1) 2 g of 5 (6) -carboxyfluorescein (hereinafter referred to as CF) 40 mM aqueous solution ( pH 7.4) Add 40 ml and stir at 4 ° C. for 24 hours. The obtained cloudy liquid was passed through a polycarbonate filter having a pore diameter of 0.2 μm five times by an extrusion method to obtain a CF-encapsulated vesicle dispersion with an average particle diameter of 217 ± 39 nm. Gel filtration was performed using a 50 mM Hepes buffer solution as a developing solvent to remove unencapsulated CF and unintroduced MPMC. Further, centrifugation (50,000 × g, 60 minutes) was performed to obtain a CF-encapsulated endoplasmic reticulum layer having a lipid concentration of 5 wt%.
[0033]
2 ml of human plasma obtained by centrifuging human blood (1900 × g) was added to 2 ml of the endoplasmic reticulum layer, and allowed to stand at 37 ° C., and the leakage rate and turbidity of CF were measured.
As a result, 19.9% of CF leaked in the unmodified system, whereas in the MPMC modified system, the CF leakage rate was suppressed to 4.3%, and a very large stabilizing effect of the endoplasmic reticulum was observed in plasma. It was.
[0034]
Example 3
100.0 mg of a lactone derived from di-N-acetylchitobiose (hereinafter referred to as chitobiose lactone) in an aqueous solution was added with 0.1N ammonia basic AgNO. _Three The mixture was mixed with 2.4 ml of an aqueous solution to obtain silver chitobiose acid salt.
The product and 2 moles of oleyl iodide were stirred in dimethyl sulfoxide (hereinafter referred to as DMSO) at 80 ° C. for 75 hours to obtain di-N-acetylchitobiose monooleyl ester (hereinafter referred to as DACMO). ) This was concentrated and then precipitated in acetone, and unreacted substances were removed using liquid chromatography in the same manner as in Example 1 to obtain 111 mg of purified DACMO. The total yield at this time was 68%.
[0035]
As a result of IR measurement of the purified DACMO, 2700 to 3000 cm ^-1 A peak based on an alkyl chain can be confirmed in the vicinity, 700 cm ^-1 A peak based on CH-out-of-plane bending vibration of the oleyl group was observed in the vicinity. Moreover, the peak based on the C = O stretching vibration of the ester bond is 1735 cm. ^-1 In the vicinity, ¹ From the 1 H-NMR, peaks of methylene protons at the α-position and β-position based on the ester bond were confirmed at 4.05 and 1.59 ppm, respectively. Therefore, the DACMO obtained here could have an oleyl group introduced by an ester bond at the anomeric position of the reducing end group of chitobiose.
[0036]
Next, 250 mg of mixed lipid powder containing DACMO (molar ratio: DPPC / cholesterol / distearyl phosphatidic acid / DACMO = 10/9 / 0.9 / 5) was obtained by lyophilization from a mixed solvent of dioxane / methanol / pure water. It was. After adding 5 ml of physiological saline to this freeze-dried sample, the white turbid solution obtained by stirring for 12 hours was pulverized at room temperature for 30 minutes with Hiscotron (manufactured by Nissin Medical Science Co., Ltd.). This was sequentially passed through a sterilizing filter having a pore size of 1.0 μm, 0.4 μm, and 0.2 μm to obtain a white transparent DACMO-modified vesicle dispersion (average particle size 200 nm). After adding 5 mM calcium chloride (as calcium ions) to this dispersion, the viscosity of the dispersion was measured with a rotational viscometer (Bismetron VS-AK) (1.2 sec. ^-1 ) And 5 cp, and the stability of the endoplasmic reticulum was evaluated to be extremely high.
[0037]
On the other hand, when an endoplasmic reticulum dispersion was similarly prepared from a mixed lipid powder not containing DACMO (molar ratio: DPPC / cholesterol / distearyl phosphatidic acid = 10/9 / 0.9) and calcium ions were added, Because of the negative charge, the endoplasmic reticulum immediately aggregated and the viscosity increased to 11 cp.
[0038]
Example 4
An ammonium salt was prepared from 7.0 g of cellotriose in the same manner as in Example 1, dissolved in distilled DMF, and twice as much cholesteryl bromide previously dissolved in chloroform. Cellotriose monocholesteryl ester (hereinafter referred to as S-Chol.) Was synthesized, further purified according to the same procedure as in Example 1, and purified S-Chol. As a result, 9.9 g of a raw sugar, that is, a glycolipid produced with respect to the number of moles used of cellotriose, that is, S-Chol. When the number of moles of sugar in the product was compared, the yield was 80%. In the IR spectrum of this one, 1740 cm ^-1 A peak based on the C═O stretching vibration of the ester bond was observed in the vicinity, and it was confirmed that the esterification reaction proceeded.
[0039]
The purified S-Chol. Mixed lipid powder (molar ratio: hydrogenated soybean lecithin / cholesterol / stearic acid / S-Chol. = 7/4/2/3) was dispersed in physiological saline and stirred at 4 ° C. for 24 hours. . This was treated using a microfluidizer at 2500 psi for 15 minutes at 4 ° C., and S-Chol. A modified endoplasmic reticulum dispersion was obtained, and further centrifuged (100,000 × g, 30 minutes) to obtain an endoplasmic reticulum layer as a lower layer. In order to observe the agglutination effect of dextran, dextran (molecular weight, 40000) was added to the ER layer, and turbidity measurement at 800 nm was performed.
As a result, in the unmodified endoplasmic reticulum, turbidity was increased and aggregation was observed even in the 1 wt% hydrate, but S-Chol. In the modified endoplasmic reticulum, there is almost no increase in turbidity, and S-Chol. It was possible to confirm the inhibitory effect.
[0040]
Example 5 FIG.
A mixed lipid powder containing MPMS (molar ratio: DPPC / cholesterol / palmitic acid / MPMS = 7/7/2 / 0.5) 10.0 g was purified from a stromal-removed hemoglobin solution (40 wt%, 200 ml [pH 7.4). , NaCl 100 mM, pyridoxal-5 ^, -Phosphate 19.2 mM]) and stirred at 4 ° C. for 2 hours, and a vesicle dispersion having an average particle size of 300 ± 99 nm was obtained using a microfluidizer. Further, the filter was passed through a filter having a pore size of 0.2 μm by the particle size extrusion method, and the outer aqueous phase hemoglobin and unintroduced MPMS were removed by gel filtration using physiological saline as a solvent, and the hemoglobin-encapsulated particles having an average particle size of 215 ± 58 nm A dispersion of vesicles was obtained.
[0041]
The hemoglobin-containing endoplasmic reticulum dispersion was adjusted to a lipid concentration of 5 wt%, a hemoglobin concentration of 10 wt%, and a pH of 7.4, and then 2000 mg / kg was administered into a group of rats via the tail vein. After blood was collected over time, erythrocytes and hemoglobin-containing endoplasmic reticulum were separated by centrifugation, and the time course of the disappearance amount of hemoglobin-containing endoplasmic reticulum from the blood was observed. As a result, the average half-life in blood in the system in which the endoplasmic reticulum was modified with MPMS, which is the oligoglycolipid of the present invention, and in the unmodified system was 39 hours and 23 hours, respectively. It was recognized that there was.
[0042]
Example 6
After 25 g of corn starch was treated with pullulanase, 18 g of 95 ° C. heat-dissolved fraction was obtained. The obtained fraction was subjected to liquid chromatography using an octadecyl-modified silica gel column (ODS-AQ, manufactured by YMC) as a stationary phase and pure water as a mobile phase under the condition of being kept at 60 ° C. 650 mg of maltoeicosapentaose with 25 units was obtained.
[0043]
After dissolving 500 mg of the maltoeicosapentaose in hot water, 1.0 ml of 0.5N sodium hydroxide was added while keeping the temperature at 60 ° C., and the liquid temperature was immediately cooled to 40 ° C. Here, 175 mg of potassium iodide and 37 mg of iodine were added and dissolved, and the mixture was stirred and reacted at 40 ° C. for 18 hours under sealed light shielding. After the reaction, the product was washed with hot ethanol and acetone to obtain a desalted precipitate. This was dissolved in hot water, cooled to 5 ° C., and the resulting precipitate was collected and washed with dilute hydrochloric acid to convert the sugar-terminated carboxylic acid potassium salt into a free carboxylic acid. This was freeze-dried to obtain 450 mg of white powder.
[0044]
After 450 mg of the white powder was dissolved in 50 ml of pure water at 95 ° C., 10% (W / V) tetra-n-butylammonium hydroxide aqueous solution was added to a pH of 7.5, and then dried to dryness on a rotary evaporator. This was dissolved in 50 ml of 5% (W / V) lithium chloride added formamide. Furthermore, 0.14 g of 2-fold mole of 1-bromo-2,3-O-octadecylpropane was added and stirred at 70 ° C. for 48 hours. 1,2-di-O-octadecyl-3-α in which a hydrophobic group having two alkyl groups is ester-bonded to the anomeric position of the sugar reducing end by reprecipitation in acetone after the reaction yield. A crude sample of (β) -eicopentaonyl-rac-glyceride (hereinafter referred to as DODEPG) was obtained.
[0045]
The DODEPG crude sample was purified by removing unreacted material using liquid chromatography using an octadecyl-modified silica gel column as a stationary phase and pure water as a mobile phase under the condition of keeping at 60 ° C. 280 mg of product was obtained.
As a result of IR measurement of purified DODEPG, 2800 to 3000 cm ^-1 A peak based on an alkyl chain can be confirmed in the vicinity, and a peak based on C═O stretching vibration of an ester bond is 1740 cm ^-1 It was recognized in the vicinity. Also, ¹³ From the C-NMR, it was confirmed that the dialkyl glyceride was selectively introduced at the terminal anomeric position because the chemical shift of the sugar terminal anomeric carbon moved from 95 ppm to 170 ppm.
[0046]
280 mg of trioctanoyl glyceride and 10 ml of 1/30 mM phosphate buffer solution (pH 7.4) were added to 280 mg of the above-mentioned DODEPG freeze-dried product, stirred with a magnetic stirrer, and then with a microfluidizer (7000 psi, 10 minutes). An aqueous dispersion containing oil droplets was prepared. When these oil droplet globules were observed with a scanning electron microscope, they were observed to be in the form of microspheres having an average particle size of 90 ± 45 nm. These particles were stable with no change in particle size even when stored at room temperature or 5 ° C. for 3 months.
[0047]
Example 7
20.0 g of fluidized barafine (Silicol P70: trade name, manufactured by Matsumura Oil Co., Ltd.) and 0.5 g of polyoxyethylene sorbitan monostearate were added to 0.2 g of MPMS obtained in Example 1, and heated to 80 ° C to dissolve. T. K. While stirring with an auto homomixer (manufactured by Tokushu Kika Kogyo Co., Ltd.), water at 80 ° C. was gradually added to cause phase inversion, and a 0 / W type emulsion having micelles with a small particle diameter was obtained with Hiscotron.
The MPMS-added system 0 / W type emulsion was observed for the micelle particle size immediately after its preparation and after 1 month of standing at 30 ° C. using an optical microscope. As a result, the average particle size was 5.7 ± 2. 1 μm and 5.8 ± 2.3 μm, the particle size was maintained and no separation of the 0 / W emulsion was observed, and it was stable.
On the other hand, in the simultaneous observation of the 0 / W type emulsion with no MPMS added, the average particle size immediately after preparation was 6.7 ± 3.8 μm, but it was not separated after standing at 30 ° C. for 1 month. However, the average particle size was 24 ± 13 μm, and an apparent increase in particle size was observed.
From this, it was shown that the MPMS contributes to stabilization of the micelle particle size in the 0 / W type emulsion.
[0048]
【The invention's effect】
The oligoglycolipid of the present invention is used as a vesicle composed of a surfactant such as phospholipid, and as a modifier for oil droplets or micelles which are 0 / W emulsions. In addition to suppressing aggregation of the 0 / W type emulsion itself, it is possible to stabilize the dispersion of the endoplasmic reticulum in an ionic solution, and it is possible to bring out the effect of suppressing phagocytosis in vivo.
In addition, when used as a stabilizer for oil droplets or micelles that are 0 / W type emulsions, excellent cosmetics, quasi drugs, pharmaceuticals, and the like can be prepared.
Furthermore, both the efficiency of synthesis and the effect of modification are superior to conventional oligosaccharide fatty acid esters.
Therefore, the oligoglycolipid of the present invention is extremely large as it contributes to the industry.

Claims (3)

Introduction of hydrophobic group as a carboxylate and a halide obtained by oxidizing the anomeric position of the reducing end group, wherein the sugar polymerization degree is an integer of 2 to 30 and the reducing end group is composed of aldose An oligoglycolipid in which a hydrophobic group is introduced into the anomeric position of the reducing end group by an ester bond by reacting with an agent, wherein the hydrophobic group is an alkyl group having 12 to 22 carbon atoms, 12 carbon atoms Selected from the group consisting of an alkenyl group having 1 to 4 unsaturated bonds of ˜22, a glyceryl group having an alkyl group of 12 to 22 carbon atoms in the 1,2-position or 1,3-position, and a steryl group. , Oligoglycolipids. A stabilizer for phospholipid vesicles comprising the oligoglycolipid according to claim 1. A stabilizer for an O / W emulsion comprising the oligoglycolipid according to claim 1.