CN101112393B - Surface-active substance composition moireuphe and method for preparing the same and use thereof - Google Patents
Surface-active substance composition moireuphe and method for preparing the same and use thereof Download PDFInfo
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- CN101112393B CN101112393B CN200710015055XA CN200710015055A CN101112393B CN 101112393 B CN101112393 B CN 101112393B CN 200710015055X A CN200710015055X A CN 200710015055XA CN 200710015055 A CN200710015055 A CN 200710015055A CN 101112393 B CN101112393 B CN 101112393B
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Abstract
The present invention relates to a surfactant moryoupe and the preparation method and the application of the surfactant, which pertains to the technical field of pharmaceutical industry. The composition is made by extraction, purification and other steps of the tissues of bivalves mollusks, the composition contains rich phospholipid complex of the seafood which is composed of polyunsaturated fatty acids, wherein, the composition includes plasmalogen, active short-chain peptides and amino acids etc. The composition of the present invention has the surface activity of the surfactant, which can be used for preparing the drugs for replacement, therapy during the lack of pulmonary surfactants; furthermore, the invention has no immunogenicity.
Description
Technical field
The present invention relates to a kind of surface-active substance composition-" moireuphe " that from the sea mollusk organ, obtains and preparation method thereof, and the application in preparation treatment pulmonary surfactant disappearance disease drug, the pharmaceuticals industry field belonged to.
Background technology
The excretory pulmonary surfactant deficiency of alveolar epithelial cells will cause serious pulmonary's pathological phenomenon, cause pneumonia, cardiopulmonary pulmonic insufficiency etc. as disordered breathing, pulmonary atelectasis, edema etc.Have a kind of viewpoint to think that the low reason of Eskimos's pulmonary disease prevalence is the dietary habit that they are rich in omega-3 unsaturated fatty acid, for example eicosapentaenoic acid wherein (EPA) and docosahexenoic acid (DHA) have good health care and prophylactic function.If EPA and DHA are easy to oxidized rotten under free state, if but vitamin E exists simultaneously, and it is stable then can to become, referring to Song J.H., Jhoul Y., Miyazawa T., Biosci.Biotechnol.Biochem.1997,01, N 12.
Existing result of study shows that exogenous additional EPA can increase the quantity of phospholipid composition and reduce leukotriene level in the neutrophilic granulocyte.Epidemiological study has also disclosed omega-3 polyunsaturated fatty acids (PUFA) and has had prevention and therapeutical effect for pulmonary disease such as infantile asthma and bronchitis, referring to Nagakura T., and Natsuda S.et al.Eur, Respir.J, 2000; 16:861-865..Well-known maternity dress uses the medicament that is rich in iodine can reverse the intellectual deficiency of fetus, just contains fish oil fatty acid in this medicament.Also comprising high-load ω-3PUFA esters in " Tekom " preparation that uses during treatment chronic pulmonary cardiac, referring to UA 36137A, 16.04.2001, its shortcoming is very easy oxidation deterioration.
There is report from Mollusca-mussel tissue, to be separated to the biomacromolecule compositions; this compositions comprises compositions such as nitrogen-containing compound, saccharide, lipid; have biological activitys such as antioxidation, liver protection, class thyrotropin, referring to UA 60504A, 15.10.2003.Also have radioprotector " Bipolan ", by preparing in the shellfish Mollusca, composition wherein has free amino acid, amino sugar and derivant thereof, glycopeptide class and para-insulin material etc.Report in addition to comprise multiple phospholipid, fatty acid, metallic element and flavones ingredient in the marine organism extract, but the use of said extracted thing does not appear in the newspapers as yet.
Clinical atomizing sucks streptomycin merging hydrocortisone and glucose might be corrected the pulmonary surfactant disorders in 5 days, yet antibiotic and hormone do not advise using at present.
Modern analysis shows that the human body pulmonary surfactant is lipid and proteic mixture,, is covered and reduces surface tension on the alveolar in alveolar fluid by alveolar type II epithelial cell synthesis secretion.Unsaturated lipids as main component is very important for surface active function.Neonatal pulmonary surfactant comprises two Palmic acid phosphatidylcholines (DPPC), PHOSPHATIDYL ETHANOLAMINE (PEA), Phosphatidylserine (PS), phosphatidyl glycerol ester (PG), sphingomyelins (SM), phosphinositides (PI), phosphatidic acid (PA).In fatty acid phospholipid, Palmic acid and DHA are two kinds of most important fatty acids.Pulmonary surfactant is a mixture, and wherein DPPC is solvent, accounts for 41%, and cholesterol is about 9%, also has protein-9%, PEA-5%.PG and PS-4%, PI-2%, SM-1%, FA-1% in addition.The main effect of pulmonary surfactant is the liquid-vapor interface tension force that reduces the alveolar skin covering of the surface, has a kind of lipoid monolayer in this film, wherein contains the DPPC of the 40-50% that has an appointment.It is two saturated acyl chains that DPPC has an end, and the unsaturated fatty acid ester dense arrangement of the other end is on monolayer, for keeping the stable extremely important of film.DPPC can reduce the capillary topmost factor of lung, and in addition, PC (phosphatidylcholine), PEA, PG and PI have then strengthened the distribution of surfactant and adhered to, referring to Berezovsky V.A., Vrachebnoe delo, 1984, N 41, P.107-111.
Mentioned component can be used for being grown up and child's respiratory distress syndrome, and the characteristics of this disease are the pulmonary surfactant disappearances, as hyaline membrane disease or the synthetic Function of Pulmonary Surfactant obstacle of alveolar type II epithelial cell.Pulmonary surfactant loss may cause by histanoxia, also may by tissue oxygen too much or respiratory tract infection cause.This disease may occur in any age bracket, particularly relies on the patient who uses the respirator rehabilitation, referring to Carrie I, and Clement M, de Javel D etal.J Lipid Res., 2000, Mar., 41 (3): 473-480.
At present, alternative medicine is verified can correct respiratory distress syndrome.Can be used as alternative medicine with exogenous material like the human body pulmonary surfactant constituent class uses.From pulmonis Bovis seu Bubali (referring to RU, 2198670, Cl, 20.02.2003) and amniotic fluid referring to (RU, 2066197, Cl, 10.09.1996) in the exogenous surfactant that extracts in the treatment of child's respiratory distress syndrome, shown gratifying effect, and do not find any toxicity as yet.
Present class surfactant medicine all is to be main component with DPPC or phospholipid, also has aminoacid, alcohols, fatty acid and saccharide.Existing medicine has synthetic as " Ecxosurf " (U.S.) (referring to USA 5,110,866 1992), " Alek " (Britain); Semisynthetic as " Survanta " (U.S.); Natural extract as " Curosurf " (U.S.).But be not that all above medicines all show good biological effectiveness.Have research to report that some medicines that contain aquation soybean lecithin (phospholipon-90H) have surface activity, but in fact, this class medicine reduce capillary ability and does not have the so good of people's imagination.Therefore, for natural origin, effectively, with the research of the similar phospholipid composite of pulmonary surfactant be quite urgent.
Have the generation that the disappearance that studies show that some essential fatty acid (EFA) can cause multiple disease now, the decline of the level of some crucial phospholipid, ω-6 and omega-3 unsaturated fatty acid-arachidonic acid (AA), DGA caused during these diseases can be organized, referring to Ukr.Biochem.Journ., 2004, v.76, N 3, P.91-97.Clinical research discloses, as some diseases such as pulmonary disease, pulmonary surfactant disappearance diseases, and may be relevant with the disappearance of omega-3 unsaturated fatty acid in the tissue and some crucial phospholipid.Have and studies confirm that the have significant impact of the disappearance of the crucial fatty acid of ω in the tissue-3 to multiple disease, the disappearance of pulmonary surfactant is the clinical manifestation of these pulmonary disease.
Under normal circumstances, omega-3 unsaturated fatty acid phospholipid in the food can appear in the alveolar immobilized artificial membrane fast, omega-3 unsaturated fatty acid content of phospholipid in the film significantly increases, and can't cause the decline of arachidonic acid (AA) level, AA is most important ω-6 a fatty acid phospholipid in the biomembrane.
Up to the present, the inventor has createed a kind of surface-active material that has that obtains that extracts from squid, be used for the treatment of adult and child's respiratory distress syndrome.Zoopery shows that this material can reduce blistered severe degree, regulates the reaction of cell to pulmonary surfactant, and can resist the toxic damages of pulmonary, and does not cause any untoward reaction, referring to UA, and 76074, C2,15.06.2006.
Summary of the invention
At the deficiencies in the prior art, the invention provides a kind of surface-active substance composition moireuphe (Mollyufil) and preparation method, the present invention also provides the application of this surface-active substance composition moireuphe at field of medicaments.
Summary of the invention
Characteristics of the present invention are to use new method to obtain a kind of natural surface-active substance composition moireuphe from seashells biological tissue, described moireuphe feature be a kind of be main component with omega-3 polyunsaturated fatty acids phospholipid, also comprise components such as plasmalogen, small peptide and aminoacid.The feature of described compositions also is to have the surface of good activity, can be used for the replacement therapy of the disease of surface activity system disorders.Preparation of compositions method of the present invention can be guaranteed in the said composition preparation process to be to the enrichment of surfactant and to reject antigenic substance as much as possible.
Detailed Description Of The Invention
A kind of surface-active substance composition moireuphe; this surfactant extracts from seashells biological tissue and makes; its composition comprises omega-3 polyunsaturated fatty acids composite phospholipid 60～85%; T-CHOL 0.5～2.5%; small peptide 0.01～1.5%; saccharide 0.1～2.5%; free fatty 0.1～2.8%; vitamin E 0.01～0.5%; heparin 0.1～0.5%; by aminoacid (I) 0.045-0.08% that taurine, arginine, glutamic acid, glycine are formed, surplus materials is other aminoacid (II) except that aminoacid (I).Below all be weight percentage.
Phosphatidylcholine (PC) that described omega-3 polyunsaturated fatty acids composite phospholipid is 30-50% and the PHOSPHATIDYL ETHANOLAMINE (PEA) of 7.0-12.8%, 30-45% plasmalogen (ethanolamine plasmalogens and choline plasmalogen), all the other are sphingomyelins, phosphatidyl glycerol fat, phosphatidylinositols, Phosphatidylserine.
In the above-mentioned aminoacid of being made up of taurine, arginine, glutamic acid, glycine (I), the weight ratio of each component is a taurine: arginine: glutamic acid: glycine=20～30: 2.5～5.0: 11.0～25.0: 12.0～20.0.
Preferably, also contain heparin 0.1～0.5% in the above-mentioned composition moireuphe.Percentage by weight.
Preferably, also contain content of vitamin E 0.01～0.5% in the above-mentioned composition moireuphe.Percentage by weight.
A kind of preparation method of surface-active substance composition moireuphe, step is as follows:
The biological tissue of frozen seashells biology is smashed to pieces, carry out solvent extraction under the room temperature, extracting liquid filtering concentrates filtrate decompression and boils off organic solvent, adds cleanser again and extracts processing, collect upper strata liquid and be concentrated into certain volume, decolouring is handled, and concentrating under reduced pressure obtains surfactant (SAS) then, add distilled water and heparin again, last low-pressure refrigeration drying obtains lyophilized powder.
Preferably, in the above-mentioned preparation method, after decolouring is handled, add vitamin E earlier, making the percentage by weight of vitamin E in the finished product compositions is 0.01～0.5%, and then concentrating under reduced pressure, can protect compositions not oxidated destruction in preparation process like this.
Preferably, described shellfish biology is the lamellibranchiate seashells biology of Mollusca; Specifically be selected from mussel, Concha Ostreae, Margarita or Conch Meretricis seu Cyclinae etc.; Described biological tissue all organizes except that shell.
Preferably, the extraction solvent that uses in the described solvent extraction can be the mixed solvent of lower alcohols such as acetone, ethyl acetate, ethanol, methanol and/or these solvents and water, and preferred solvent is an ethanol.
Above-mentioned solvent use amount is biological tissue's raw material: solvent=1: (5～20), w/v.The preferred biological tissue's raw material that uses: solvent=1: 10 w/v.
Preferably, solvent extraction is no less than 2 times, and extracting solvent adjustment is that acid back effect is better, can remove impurity parts such as saccharide, pigment, cholesterol oxidation product and ester thereof; Preferably, extract solvent organic acid adjustment of acidity, preferred, can be acetic acid, formic acid, hydrochloric acid; Preferably, regulate extraction solvent acidity and be not less than 4 for pH value.
Preferably, the cleanser that uses can be the mixed liquor of ethanol, normal hexane and ethyl acetate; Preferably, the volume ratio of this mixed liquor is 10: (15～25): (0.7～1.4).
Preferably, add heparin, the percentage by weight that makes heparin in the compositions is 0.1～0.5%.Such combination and ratio are preferably to get through overtesting, make the present composition be convenient to clinic trial, and have taken into full account its stability and curative effect.
Surface-active substance composition moireuphe of the present invention, its medical usage are, are used to prepare the medicine that treatment can reverse pulmonary surfactant disappearance disease.
The medicine made from composition moireuphe of the present invention, can be used for the treatment of pulmonary surfactant disappearance disease, during use the moireuphe medicine be dissolved with the 10ml sterile saline, pour into 1-2 days, every day 1-2 time, continue operating position and decide according to clinical needs.
Surface-active substance composition moireuphe that obtains in the seashells biology and the surfactant that extracts from the mammal lung are analyzed, be the results are shown in Table 1
Table 1 moireuphe (Mollyufil) main component qualitative and quantitative analysis table (%: weight ratio)
Table 2 has been listed in the experimental V-E deficiency disease moireuphe effect to essential fatty acid (EFA) disappearance associated diseases, and to CCl
4Due to the influence of oxidation resistance of Hepar Mus microsomal enzyme.
Table 2 moireuphe is for the MC influence of EFA disappearance each internal organs of Mus
*-have significant difference (p＜0.05, variance analysis) compared with the control
DPG: diphosphoglyceric acid; LPC: hemolytic phosphatidylcholine; LPE: hemolytic PHOSPHATIDYL ETHANOLAMINE
Table 3 moireuphe is to lipid peroxidation product in the Mus lung microsome of V-E deficiency disease (n=7, M ± m)
*-compare with the normal control group and to have significant difference (p＜0.05);
*-compare with V-E deficiency disease group with the normal control group and all to have significant difference (p＜0.05);
* *-compare with V-E deficiency disease group and to have significant difference (p＜0.05)
Content of phospholipid (%) in the table 4 Mus pneumonocyte microsome
| Phospholipid | The normal control group | V-E deficiency disease group | V-E deficiency disease group+V-E | V-E deficiency disease group+Mollyufil |
| PC | 59.8±2.0 | 47.8±0.49 * | 50.0±0.96 | 55.8±1.72 ** |
| PEA | 20.3±0.5 | 17.4±1.06 | 19.5±1.15 | 19.4±1.5 |
| SM | 13.4±0.64 | 17.2±0.48 * | 15.6±1.13 | 16.8±1.15 |
| PS | 7.2±0.1 | 8.6±1.45 | 7.8±0.20 | 7.3±0.63 |
| DPG | 1.38±0.2 | 3.±0.74 | 2.40.9 | 1.6±0.42 |
| PG | 1.2±0.02 | 2.6±0.42 * | 2.1±0.4 | 1.8±0.14 |
| LPC | 1.92±0.27 | 3.2±0.38 | 2.6±1.0 | 1.5±0.35 ** |
| LPE | 2.13±0.4 | 3.1±0.07 * | 2.4±0.05 ** | 0.9±0.31 ** |
*-compared significant difference (p＜0.05) with the normal control group,
*-compared significant difference (p＜0.05) with V-E deficiency disease group
Table 5 moireuphe is to CCl
4The influence of malonaldehyde in the Hepar Mus homogenate under the effect
| The experiment group | Fe 2+The POL of-dependence, (nmol/mg albumen) | The POL that NADPH-relies on, (nmol/mg albumen) |
| The normal control group | 1.80±0.03 | 2.10±0.01 |
| CCl 4Handle 4d | 3.50±0.02 * | 3.3±0.03 * |
| CCl 4Handle 4d+ Mollyufil and handle 12d | 2.80±0.04 ** | 2.30±0.02 ** |
*-compared significant difference (p＜0.05) with the normal control group;
*-with CCl
4Processed group has been compared significant difference (p＜0.05) POL: superoxide dismutase
Table 6 moireuphe is to the influence of the lung microsome oxidation resistance of V-E deficiency disease rat (n=7, M ± m)
*-compared significant difference (p＜0.05) with the normal control group;
*-with CCl
4Processed group has been compared significant difference (p＜0.05)
The property list of table 7 moireuphe
| Medicine | Surface tension (din/cm) | The air flue peak value is pressed |
| Moireuphe Mollyufil | Min.-21.0 Max.-47.5 | |
| The mammal surfactant | Min.-10.3 Max.-42.3 | - |
| Ka Mofei Calmofil | Min.-11.7 Max.-48.5 | 24.91.1±1.1 |
| Contrast medicine " Survanta " | Min.-4.1 Max.-37.5 | 21.2±1.1 |
| Contrast medicine " Exosurf " | Min.-21.0 Max.-67.5 | 21.2±1.1 |
The influence of table 8 mussel surface-active substance confrontation rat immunity system (n=7, M ± m)
Table 9 moireuphe is to the influence of experimental PUFA deficiency disease rat acetal fatty acids in phospholipids content
| Acid | Normal diet | PUFA shortage group | PUFA shortage group+Mollyufil |
| C 16:0 | 12.6 | 46.5 | 18.7 |
| C 18:0 | 27.2 | 15.1 | 33.3 |
| C 18:1 | 14.5 | 7.04 | 4.89 |
| C 18:2 | 2.63 | 1.82 | 1.23 |
| C 18:3ω-3 | 0.64 | 0.47 | 1.27 |
| C 20:3 | 1.48 | 0.33 | 0.56 |
| C 20:4 | 16.14 | 6.8 | 13.7 |
| C 22:3 | 1.14 | 0.64 | 0.87 |
| C 22:4 | 2.4 | 0.54 | 1.81 |
| C 22:5 | 8.7 | 3.35 | 9.84 |
| n-6 | 33.13 | 13.95 | 29.28 |
| C 22:6ω-3 | 0.97 | 0.31 | 4.4 |
| n-3 | 0.97 | 0.31 | 5.67 |
| n-6/n-3 | 34.15 | 45.0 | 5.16 |
| AA/DGA | 16.6 | 21.93 | 3.11 |
[H in the table 10 lung microsome TL
3]-arachidonic acid content (n=3, p＜0.05)
The invention provides a kind of preparation method of from the sea mollusk tissue, obtaining surface-active substance composition, and a kind of novel seashells GLP compositions that acquires.
Characteristics of the present invention are:
1, utilized marine shellfish as raw material;
2, obtained to be rich in the new compositions of omega-fatty acid phospholipid, phospholipid mainly is two Palmic acid phosphatidylcholines, also have plasmalogen (ethanolamine plasmalogens and choline plasmalogen), be rich in arginine, glycine, taurine, glutamic acid, free amino acid, can strengthen surface-active adjusting peptide and short-chain peptide in addition, and the compositions of a considerable amount of cholesteryl ester, free fatty, carbohydrate content;
3, preparation method of the present invention relates to extraction acquisition surface-active substance composition from the seashells tissue, and the extraction that relates in this method, separation, purge process help to strengthen the surface activity of this product;
4, in preparation method of the present invention, adopt normal hexane, ethyl acetate mixed liquor to carry out purified treatment, helped to strengthen the surface activity of this product;
5, extract the SAS yield from the mammal lungs and be up to 1.5%, extract yield then is 1% from the marine products sepiellae seu sepiae, and the SAS yield that extraction obtains from the seashells tissue that the present invention relates to can reach 5%;
6, the superiority of the compositions of preparation method acquisition of the present invention also is embodied in and contains a series of omega-fatty acid phospholipid, and main is DPPC (containing Palmic acid 35-50%, DHA20%, EPA10-15%);
7, also contain adjusting small peptide, aminoacid in the compositions that preparation method of the present invention obtains, zoopery proves that this product does not have toxicity, also non-immunogenicity, macrophage is compared with matched group and there was no significant difference in 1h, 1d, 3d, the 7d lung after giving this product, and Comparatively speaking this and other like product is the characteristics of a superiority.
The novel surface active compound that preparation method of the present invention obtains has following characteristics:
1, the moireuphe of the present invention's acquisition is rich in omega-fatty acid phospholipid, can be used to correct pulmonary surfactant disappearance disease, also have film protection and immunoregulation effect, can replenish EPA and DHA, the synthetic closely-related arachidonic content of prostaglandin in adjusting and the tissue;
But 2, the heparin microcirculation improvement in the composition moireuphe of the present invention's acquisition, vitamin E can play Stabilization for the low dispersive structure of this product;
3, higher phospholipid and the plasmalogen of content strengthened surface activity in the moireuphe of the present invention's acquisition;
4, do not add pigment or albumen in addition in the composition moireuphe that the present invention obtains;
5, the moireuphe short-term of the present invention's acquisition is used and can be improved phospholipid amount in the alveolar membrane, pulmonary alveolar macrophage and pulmonary surfactant quantity fast.
The activity of PUFA scalable transcription factor, can influence the signal transcription and the expression of lymphocyte and phagocyte series of genes, it is the substrate that makes up a kind of important lipid regulating agent-eicosanoid, they comprise prostaglandin, prostacyclin, leukotriene, thromboxane etc., have participated in processes such as immunomodulating, inflammation, blood coagulation, spasm and vascular leakage.
The difference of three prostanoids and thromboxane is that the double key number order in the molecular side chain is different.By the synthetic eicosanoid of linoleic acid 3 two keys are arranged, 4 two keys are arranged, 5 two keys are arranged by α-linoleic acid is synthetic by arachidonic acid is synthetic.Omega-fatty acid phospholipid in the shellfish surfactant of the present invention can influence the biosynthesis of eicosanoid in tissue.In the body development growth course, EFA lacks can cause multiple disease: broncho-pulmonary dysplasia, child's brain and heart disease etc.Treating in the research of EFA deficiency disease with omega-fatty acid phospholipid medicine, the arachidonic acid of usage flag has disclosed the mechanism of action of medicine, be promptly as an arachidonic competitive antagonist, the antagonism arachidonic acid is to the conversion of prostaglandin (PGE2), so reduced the biosynthesis of eicosanoid.
Shellfish surface-active substance composition of the present invention can be used to prepare medicine.DPPC in the omega-fatty acid phospholipid, plasmalogen are topmost compositions.They can effectively reduce surface tension, stablize the hexagonal structure of structure of phospholipid, and strengthen the surface activity of surfactant-based phosphatide complexes.These components comprise the peptide class, are rich in arginine, free amino acid, vitamin E and the heparin etc. of glycine, taurine, glutamic acid.
The characteristics of preparation method of the present invention also are to remove parts such as unwanted impurity (pigment), protein simultaneously, and the shellfish surfactant that obtains through purifying the back can be used for recovering the function of lung surface active system, the treatment respiratory distress syndrome.
Inventive point of the present invention is the separation of surfactant in the marine organisms mussel (based on the phospholipid-surface activity sample complex of omega-3-polyunsaturated fatty acid) and the preparation that is used for the treatment of the pharmaceutical formulation of the pneumonopathy (especially neonatal relevant disease) that lacks surfactant thereof.Compared with the omega-3-polyunsaturated fatty acid medicine that DHA forms by free fatty acid EPA in the fish oil with well-known, this component has many advantages, below in conjunction with specific embodiment and experimental result this is proved absolutely.
The specific embodiment
Below by specific embodiment the present invention is set forth, but do not limit the scope of the invention in any form.
Embodiment 1: refrigerated marine products mussel is organized 100g, and meat grinder rubs, the extraction vessel that places band to stir, add 10 times of volume of ethanol, extracted 2 hours, filter, the filtrate solvent recovered under vacuum, the raw material that leaches extracted 1 hour with acidic ethanol (acetic acid is transferred pH 〉=4) again.Merge extractive liquid,, filter, vacuum evaporating solvent, dissolve again with ethanol, and the mixture of adding normal hexane and ethyl acetate, ethanol: normal hexane: the volume ratio of ethyl acetate is 10: 20: 1, tells normal hexane-ethyl acetate layer, vacuum concentration to 1/10 volume, add activated carbon, remove residual protein component, in ethyl acetate filtrate, add vitamin E with the filtering with microporous membrane of 0.2um, making the percentage by weight of vitamin E in the finished product compositions is 0.2%, concentrating under reduced pressure wherein adds 0.1% heparin, lyophilization, obtain pharmaceutical formulation, output 5g (5%).
Embodiment 2:100 restrains freezing sepiellae seu sepiae, meat grinder rubs, the extraction vessel that places band to stir, add 10 times of volume of ethanol, extracted 2 hours, and filtered the filtrate solvent recovered under vacuum, the raw material that leaches extracted 1 hour with acidic ethanol (acetic acid is transferred pH 〉=4) again, merge extractive liquid, filters vacuum evaporating solvent, dissolve again with ethanol, and the mixture of adding acetoneand ethyl acetate, ethanol: normal hexane: the volume ratio of ethyl acetate is 10: 20: 1, tells acetone-ethyl acetate layer, vacuum concentration to 1/10 volume, add activated carbon, remove residual protein component, in acetone-ethyl acetate filtrate, add vitamin E with the filtering with microporous membrane of 0.2um, making the percentage by weight of vitamin E in the finished product compositions is 0.5%, concentrating under reduced pressure wherein adds 0.1% heparin, lyophilization, obtain pharmaceutical formulation, output 1g (1%).
Embodiment 3: the effect experiment that comes from the phospholipid composite moireuphe of marine products mussel.
The bait of feeding conventional bait respectively and being added with moireuphe to rat is to disclose the formation of eicosanoid in the organism.Eicosanoid derives from their precursor-ω-6 and omega-3 polyunsaturated fatty acids in vivo, and these precursors enter the metabolic conversion process after breaking away from membrane phospholipid.
Measuring follows these steps to carry out:
(1) the poisoning modeling destroys the surface activity system of animal lungs, makes it will cause death if do not carry out professional treatment;
(2) adopt suction to the laboratory animal administration;
(3) observe animal state (dead quantity, surface activity system mode index);
((body weight 23.4 ± 1.7g) is carried out inhalation to rat, at interval 1 hour, 1 day, 3 days and administration respectively in 7 days for body weight 195.6 ± 4.5g) and mice.Animal is divided 4 groups, and 10 every group, the toxicology data of investigation " moireuphe ".The concentration of component of aerosol is 33333.0mg/m3-666mg/m
3, rat continues to suck 4 hours, and white mice sucked 2 hours, and animal is in the common lab condition before and after sucking.Observe animal state and dead quantity after the administration in 14 days.The dead dosage of rat is 11200.0 ± 606.4mg/m as a result
3, white mice is 8000 ± 542.0mg/m
3Under the inhalation situation, " moireuphe " belongs to three grades of hazardous compounds, is a kind of medical substance of safety.
By inhalation " moireuphe " carried out pharmacological evaluation.The administration concentration of aerosol is 1/10 a multiple of rat fatal dose, and the body weight of rat is 135.6 ± 4.5g, dosing interval 1 hour, 1 day, 3 days and 7 days, and test divides four groups, every group of 10 animals.The aerosol concentration of each separation is 1120.0 ± 254.3mg/m3 during suction, keeps to suck 4 hours.
There is not a laboratory animal death under the above-mentioned experiment condition, laboratory animal in good condition after the administration and in the observation process, animal organ and system find no significantly unusual.
The animals survived number of using " moireuphe " is apparently higher than contrast.In vivo with experiment in vitro in, use CCl
4Acute intoxicating, the application of this medical substance has reduced the poisonous effect of poisonous substance, and animals survived is had facilitation effect, has reduced fat peroxidating (LP) intensity, has regulated CCl
4Poisonous effect.
" moireuphe " do not cause any variation to the functional activity and the form of pulmonary macrophage, show its with respect to the cell immune response of lungs without any antigenic characteristic, do not see significant change on the 26S Proteasome Structure and Function of pulmonary macrophage yet, this is that preparation method of the present invention is created the result who optimizes, make protein content negligible (0.1-0.5%) in the final products, this is a very favorable factor.
The preparation method that use the present invention relates to has following excellent results:
1, simplified the method that obtains target product;
2, the output that derives from the pulmonary surfactant " moireuphe " of mussel tissue is 5%, is higher than the output of the surface reactive material 1.0% in ox lung source far away;
3, effectively reduce surface tension, and its contained plasmalogen can suppress lipid peroxidation as antioxidant;
4, vitamin E can protect phospholipid composition and pulmonary surfactant protein oxidized;
5, the heparin that adds can improve blood microcirculation.
The shellfish surface-active substance composition that adopts preparation method of the present invention to obtain has following excellent results:
1, increased the surface-active of animal lungs by reducing surface tension;
2, whole duration of test, compared with the control, the quantity of pulmonary alveolar macrophage does not have any variation.
Experimental result provides evidence for the invention that it further is applied to the clinical patient treatment. " moireuphe " can be used for the treatment of the acute toxicity case of lung surface active systemic-function disorder. The intact animal application derives from halobiontic surface reactive material and does not show any variation, yet under the poisoning condition, PFT needs normalization; It has reduced the intensity of bubbling process, has replenished surface reactive material, makes the cell effect normalization of surface reactive material, slows down the process of poisoning process. The medical substance that obtains has been guaranteed the increase of lung surface active and the reduction of antigenic characteristic.
The objective of the invention is to obtain surface-active medical substance " moireuphe " from the tissue of shellfish, this medical substance comprises omega-3 polyunsaturated fatty acids phosphatide, plasmalogen, adjusting peptide, amino acid, vitamin E and heparin. Prepared bioactive composition has the former and anti-oxidation characteristics of adaptation, and the surface reactive material " moireuphe " of producing with this method can by replenishing the surface reactive material that lacks, recover disorderly lung surface active systemic-function.
Claims (6)
1. surface-active substance composition moireuphe, it is characterized in that its composition comprises omega-3 polyunsaturated fatty acids composite phospholipid 60～85% by weight percentage, T-CHOL 0.5～2.5%, small peptide 0.01～1.5%, saccharide 0.1～2.5%, free fatty 0.1～2.8%, vitamin E 0.01～0.5%, heparin 0.1～0.5%, by aminoacid (I) 0.045-0.08% that taurine, arginine, glutamic acid, glycine are formed, surplus materials is other aminoacid (II) except that aminoacid (I);
Described omega-3 polyunsaturated fatty acids composite phospholipid is the PHOSPHATIDYL ETHANOLAMINE of phosphatidylcholine and the 7.0-12.8% of 30-50%, the 30-45% plasmalogen, and all the other are sphingomyelins, phosphatidyl glycerol fat, phosphatidylinositols, Phosphatidylserine;
The described aminoacid of being made up of taurine, arginine, glutamic acid, glycine (I), the weight ratio of each component are taurine: arginine: glutamic acid: glycine=(20～30): (2.5～5.0): (11.0～25.0): (12.0～20.0);
This surface-active substance composition moireuphe makes as follows:
The biological tissue of frozen seashells biology is smashed to pieces, carry out solvent extraction under the room temperature, extracting liquid filtering is with the concentrated organic solvent that boils off of filtrate decompression, add cleanser again and extract processing, collect upper strata liquid and be concentrated into certain volume, decolouring is handled, and adds vitamin E, concentrating under reduced pressure then, obtain surfactant, add distilled water and heparin again, last low-pressure refrigeration drying obtains lyophilized powder;
The extraction solvent that uses in the described solvent extraction is biological tissue's raw material as the mixed solvent of acetone, ethyl acetate, ethanol, methanol or acetone, ethyl acetate, ethanol, methanol and water, described solvent use amount: solvent=1: (5～20), w/v;
Described cleanser is the mixed liquor of ethanol, normal hexane and ethyl acetate.
2. surface-active substance composition moireuphe as claimed in claim 1 is characterized in that described shellfish biology is Mollusca lamellibranchiata seashells biology; Described biological tissue all organizes except that shell.
3. surface-active substance composition moireuphe as claimed in claim 1, the dosage that it is characterized in that the extraction solvent that uses in the described solvent extraction are by biological tissue's raw material: solvent=1: 10 w/v.
4. surface-active substance composition moireuphe as claimed in claim 1 is characterized in that described extraction solvent is adjusted to acidity with organic acid, and preferred pH value is not less than 4.
5. surface-active substance composition moireuphe as claimed in claim 1 is characterized in that described cleanser volume ratio is an ethanol: normal hexane: ethyl acetate=10: (15～25): (0.7～1.4).
6. the application of claim 1 surface-active substance composition moireuphe is used to prepare the medicine that treatment can reverse pulmonary surfactant disappearance disease.
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| CN101112393B true CN101112393B (en) | 2011-01-19 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| ES2347119B2 (en) * | 2009-04-22 | 2011-04-28 | Universidad De Santiago De Compostela | POLYARGININE NANOCAPSULES. |
| WO2012103685A1 (en) * | 2011-02-01 | 2012-08-09 | Nippon Suisan Kaisha, Ltd. | Sexual function improving agent |
| CN102564825B (en) * | 2012-01-05 | 2014-01-08 | 上海海洋大学 | Method for preparing mixed phospholipid standard substance by using aquatic products and developing agent thereof |
| CN115192528B (en) * | 2022-07-01 | 2023-12-01 | 国科温州研究院(温州生物材料与工程研究所) | Lung surface active composition containing plasmalogens and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1120907A (en) * | 1994-10-20 | 1996-04-24 | 国家海洋局第三海洋研究所 | Marine shellfish gonad and yolk extract product and its prodn process and uses |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1120907A (en) * | 1994-10-20 | 1996-04-24 | 国家海洋局第三海洋研究所 | Marine shellfish gonad and yolk extract product and its prodn process and uses |
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