CN103131736A - Preparation and application of high-purity lysophosphatidylcholine - Google Patents
Preparation and application of high-purity lysophosphatidylcholine Download PDFInfo
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Abstract
The invention provides a novel effective method for preparing high-purity lysophosphatidylcholine, and a purpose for applying the high-purity lysophosphatidylcholine for preventing and/or treating metabolic bone diseases. According to the novel preparation method, phosphatidylcholine is converted into lysophosphatidylcholine under the catalysis of phospholipase A2. During the conversion process, a novel phospholipase A2 activity enhancer is added. Therefore, phospholipase A2 dose is reduced, lysophosphatidylcholine yield is improved, and high-purity lysophosphatidylcholine can be prepared. The prepared high-purity lysophosphatidylcholine can be used in medicine compositions used for preventing and/or treating metabolic bone diseases.
Description
Technical field
The present invention relates to a kind of preparation method and application thereof of high purity lyso-phosphatidylcholine.
Technical background
Lyso-phosphatidylcholine removes a fatty acyl group by phosphatidylcholine and the product that obtains.Phosphatidylcholine (also claiming Yelkin TTS) extensively is formed in the bodies of aminal and plant, and at the brain of animal, in suprarenal gland and cell, content is more.Than phosphatidylcholine, the HLB value of lyso-phosphatidylcholine is large, has not only kept lipophilicity, and also the minimizing because of non-polar group increases its hydrophilicity, and its wetting ability, emulsifying property, emulsifying capacity all are better than phosphatidylcholine.Lyso-phosphatidylcholine has important effect improving on the weave construction of grilled product.Lyso-phosphatidylcholine also has biological activity, and atherosclerotic prevention, diagnosis and treatment are had very important effect.At present, lyso-phosphatidylcholine is in an increasingly wide range of applications at aspects such as food, makeup, medicine, just seems most important so effectively prepare highly purified lyso-phosphatidylcholine.
Just know that phosphatidylcholine can change into lyso-phosphatidylcholine by enzymatically hydrolyse as far back as the earlier 1900s people.Make the early stage result of study of phosphatidylcholine degraded prove that the hemolytic action of snake venom occurs in the Yelkin TTS part of cytolemma by snake venom extract.Nineteen thirty-five, Hughes is with experimental results show that Yelkin TTS changes into the unimolecular film hydrolysis of lysolecithin (lyso-phosphatidylcholine) with relevant such as the factors such as surface concn of pH value, temperature and lecithin molecules.The lecithin molecules weighting material greatly reduces hydrolysis rate in unimolecular layer.The ether solubleness complex of Hanahan proof between egg lecithin and Phospholipase A2 causes discharging unsaturated fatty acids and lyso-phosphatidylcholine.When ethanol, chloroform or the sherwood oil of employing 95% made solvent, can not discover Phospholipase A2 to the hydrolytic action of phosphatidylcholine.In the report of 1963, the experiment that Dawson completes has found that also Phospholipase A2 is hydrolyzed into lyso-phosphatidylcholine and fatty acid molecule with phosphatidylcholine.It is relevant whether the activity that Dawson once determined enzyme and calcium ion exist, and interpolation ether or butanols will excite the activity of Phospholipid hydrolase.The English Patent that licenses to Unilever Ltd. has been introduced further scheme improvement for the 1st, 215, No. 868, and the hydrolysis reaction of the phosphatide of Phospholipase A2 participation is namely arranged under the condition that is having fat to exist.
In prior art the common method for preparing lyso-phosphatidylcholine have multiple, commonly used method be all with phosphatidylcholine as reaction substrate, add suitable enzyme to react the acquisition lyso-phosphatidylcholine under certain condition.For example allow the lytic enzyme that is formed by phospholipase A1 or A2 act on hydration Yelkin TTS production lysolecithin in CN1197115A, then acetone is stirred and add gained lysolecithin solution, the ratio of acetone is 1 to 4 or 5 or more times of described lysolecithin solution water yield volume, make this lysolecithin mutually floating or the precipitation, separate described lysolecithin phase, then repeat the acetone extraction process, produce the high-purity vegetable lysolecithin of having removed the free fatty acids byproduct in described lysolecithin production and having derived from the oil soluble impurity of raw material; For example in CN102277393A, phosphatidylcholine is added in low-carbon alcohol solution again, after mixing at a certain temperature, add lipase, stir under constant temperature lipase-catalyzed phosphatidylcholine alcoholysis is generated lyso-phosphatidylcholine.In above-mentioned these preparation methods, remain in some shortcomings, for example the price of Phospholipase A2 is more expensive, so cost is higher; The transformation efficiency of phosphatidylcholine is not high, therefore exists the ratio of unreacted hyle and byproduct higher in the end product of reaction; Or used lipase, although reduced cost, the byproduct that brings increases, and purifying technique has been had higher requirement.
Metabolic osteopathy is the destroyed and disease that occurs of a kind of osteoclast in vivo and osteoblastic balance.Most representative osteopathia is osteoporosis.Osteoporosis is to cause the symptom of bone amount due to reducing because of osteoblastic active the increasing of the specific activity of osteoclast.In case the generation osteoporosis, cortex bone attenuation, pulp cavity increase, bone trabecula attenuates, bone becomes porous structure gradually.Along with the progress of osteoporosis, bone strength descends, and brings out pain in the back and arthrodynia, and slight impact is also easily fractured.The inflammatory frontal resorption disease that occurs after the destroyed periodontal disease of alveolar bone, transplanting dental graft after metabolic osteopathy also has transfers of cancer bone, primary osteocarcinoma (such as multiple myeloma), rheumatic or the degenerative osteoarthritis of mammary cancer or prostate cancer, the bacterium that is caused periodontal disease to infect in addition, in field of orthopedic surgery, the struvite bone resorption disease that causes after the transplanting thing for bone fixation, the Paget`s disease that is occured by multiple inherited genetic factors etc.
In recent years, very active to the molecular biology research of the metabolic osteopathys such as treatment osteoporosis, develop bone forming and promoted the factor and osteoclast supressor.Bone forming promotes that the factor has fluorochemical, parathyroid hormone, transforming growth factor (TGF-β), bone morphogenetic protein, oestrogenic hormon, calcitonin, vitamins D and its derivative, Diphosphonate (bisphosphonate) (Jardine et al., Annual Reports in Medicinal Chemistry, 31,211 (1996)) etc.
Now existing a lot of materials are developed to the medicine of osteoporosis, wherein the most frequently used medicine is oestrogenic hormon, but also do not identify its actual usefulness fully, the shortcoming that all will take in all one's life is arranged, and can increase the sickness rate of mammary cancer and uterus carcinoma during long-term taking.It is indefinite that Alendronate (alrendronate) also has its usefulness, and the absorption in digestive tube is slow, causes the problems such as stomach and esophageal mucosa membrane inflammation.Although calcium preparation is considered to few side effects and excellent, be that medicine it would be better to say that it is preventing preparation.Also have in addition the Vitamin D preparations such as calcitonin, but its usefulness and side effect aspect are also lacked sufficient research.
Summary of the invention
Purpose of the present invention provides a kind of new method that effectively prepares the high purity lyso-phosphatidylcholine just for existing problem in above-mentioned prior art, and uses the high purity lyso-phosphatidylcholine to prevent and/or treat the purposes of metabolic osteopathy.This novel preparation method uses phosphatidylcholine to be converted into lyso-phosphatidylcholine under Phospholipase A2 catalysis, in conversion process, adds a kind of novel Phospholipase A2 active reinforcing agent.Reduce the consumption of Phospholipase A2, improved the yield of lyso-phosphatidylcholine, can prepare the high purity lyso-phosphatidylcholine.
A first aspect of the present invention provides the preparation method of high purity lyso-phosphatidylcholine:
The mixture of phosphatidylcholine and Phospholipase A2 active reinforcing agent is formed dispersion in the solution of water-rudimentary ether, allow this dispersion contact with Phospholipase A2, to form reaction mixture.After reaction is completed, reclaim lyso-phosphatidylcholine.
Wherein said Phospholipase A2 active reinforcing agent can be selected from triglyceride level and Semen Ricini extract.
The molecule of wherein said triglyceride level is comprised of the glyceryl that the acyl group with three lipid acid is connected, and the carbochain of the acyl group of triglyceride level has 8 to 20 carbon atoms and 1 to 4 unsaturated link(age).
Wherein said Semen Ricini extract be first with Semen Ricini through pulverizing degreasing, re-using 95% extraction using alcohol and obtain.Be specially: Semen Ricini is shelled, smashes, then carry out degreasing: add the ratio of 4mL ether and sherwood oil mixed solution to add the mixed solution of ether and sherwood oil evenly mixed in every gram Semen Ricini, use again the Büchner funnel suction filtration, material on filter paper is used above-mentioned degreasing method degreasing 3 times again, then composition on filter paper is carried out drying, obtain the loose milk look Semen Ricini cake dry powder after fuel-displaced, standby; The mixed solution of wherein said ether and sherwood oil, wherein the volume ratio of ether and sherwood oil is 1: 1;
the Semen Ricini cake dry powder that makes is wrapped up with filter paper, put into cable type extractor according, adding the ratio of 10mL ethanol to add mass concentration according to every gram Semen Ricini cake dry powder is 95% ethanol, extract 8h, obtain tawny oily matter extracting solution, be distilled to 1/5 of original volume with Rotary Evaporators again, obtain paste, then according to the ratio of every gram paste 20mL distilled water, the paste that obtains is dissolved in the distilled water of 80 ℃, be cooled to ether continuous extraction 3-4 time of using again 4 ℃ after 25 ℃, to remove a small amount of remaining grease, after extraction finishes, take off layer brown clear solution dry, use again the acetonitrile repetitive scrubbing, the merging washings makes the paste Semen Ricini extract after sending into and revolving steaming in Rotary Evaporators, standby.
Wherein the mass ratio of phosphatidylcholine and Phospholipase A2 active reinforcing agent is between 10: 1 to 1: 1, preferred 5: 1-7: 1.
Wherein said rudimentary ether is C1-C4 ether, preferably ether.
In the solution of wherein said water-rudimentary ether, the volume ratio of water and rudimentary ether is about 5: 1-10: 1, preferred 7: 1-9: 1, and more preferably 8: 1.
Wherein the consumption of Phospholipase A2 is the 1/3-1/2 of conventional amount used, is about the every kg phosphatidylcholine of 0.5ml-1ml Phospholipase A2.
The step of wherein said recovery lyso-phosphatidylcholine comprises the use acetone extract.
Wherein the transformation efficiency of phosphatidylcholine can reach more than 95%, and preferred 99%.
Take preparation method of the present invention, reduced the consumption of Phospholipase A2, need the above Phospholipase A2 of 2ml by every kg phosphatidylcholine in prior art, being reduced to only needs 0.5ml-1ml.Improve the yield of lyso-phosphatidylcholine and the transformation efficiency of phosphatidylcholine, by the transformation efficiency of 60-70% in prior art, risen to more than 95%, even can reach 99%.On the other hand, greatly shorten the time of reacting required, only needed 24-36 hour by generally needing 48h to foreshorten in prior art.Can effectively reduce reaction cost, Reaction time shorten, raising reaction efficiency by preparation method of the present invention, obtain more highly purified lyso-phosphatidylcholine.
A second aspect of the present invention provides the purposes of high purity lyso-phosphatidylcholine in the pharmaceutical composition of preparation treatment metabolic osteopathy of using preparation method of the present invention to obtain.The present invention also provides a kind of pharmaceutical composition be used to preventing and/or treating metabolic osteopathy, wherein comprises high purity lyso-phosphatidylcholine and the pharmaceutically acceptable carrier by preparation method's acquisition of the present invention of effective dose.
Term " bone metabolic disease " is that destruction and the absorption of phalanges is increased, and causes the disease of physiological pathological state.Metabolic osteopathy comprise the transfer of cancer bone, primary osteocarcinoma, rheumatic or the degenerative osteoarthritis of osteoporosis, mammary cancer and prostate cancer etc., the bacterium that is caused periodontal disease infect after the destroyed periodontal disease of alveolar bone, transplant the inflammatory frontal resorption disease that occurs after the dental graft, in field of orthopedic surgery, the struvite bone resorption disease that causes after the transplanting thing for bone fixation, the Paget`s disease that is occured by multiple inherited genetic factors etc.
Pharmaceutical preparation of the present invention can come administration by oral, local, injection or non-oral mode.These formulations can comprise the high purity lyso-phosphatidylcholine of effective treatment bone metabolic disease of 0.5～20% weight percent.Oral preparations of the present invention comprises the formulations such as pill, tablet, coating tablet, powder, granule, hard capsule, soft capsule, solution, coated tablet, emulsion, suspensoid, aerosol.Non-oral formulation can comprise injection, microcapsule, through formulations such as skin agent.
Can add pharmaceutically acceptable carrier in pharmaceutical preparation of the present invention.For example, in order to prepare pill, tablet, coating tablet, hard capsule, can use the excipient such as lactose, W-Gum, pregelatinized Starch, stearic acid and its salt.Soft capsule and suppository can use fat, wax, semisolid or liquid shape polyalcohols, the natural or wet goods excipient that solidifies.Solution and syrup can make the excipient such as water, sucrose, starch, glucose, polyalcohols.Injection can use the excipient such as preservatives, pain killer, solvating agent, stablizer.Microcapsule can use the excipient such as multipolymer, oxyacetic acid, lactic acid.Except comprising active compound and excipient, can also comprise other additives, such as fill out agent, extender, disintegrating agent, wedding agent, lubricant, wetting agent, stabilization agent, emulsifying agent, sanitas, sweeting agent, tinting material, flavour agent or perfume compound, enriching agent, thinner, buffer reagent, other solvent or solvating agent, obtain long-acting effect material, regulate osmotic pressure and the material that adds, Drug coating, antioxidant etc.
Embodiment
Following instance is only to further illustrate the present invention, is not the restriction the scope of protection of the invention.
Preparation Example 1:
Phosphatidylcholine and triglyceride level are mixed, and wherein the mass ratio of phosphatidylcholine and triglyceride level is 1: 1.With mixture heating up to 85 ℃, obtain uniform melt.Add the water of capacity and the solution that ether forms, wherein the volume ratio of water and ether is 10: 1, is heated to 80 ℃ reactant is mixed.Regulating the pH value is 8-9.Add the zymin 0.5ml of a LecitaseTM10L Phospholipase A2 according to the per kilogram phosphatidylcholine, keep reacting 36 hours, until till the phosphatidylcholine complete hydrolysis under the condition of stirring and pH value.After complete hydrolysis, add heat extraction water and ether, obtain mashed prod.
In order to obtain highly purified lyso-phosphatidylcholine, with the end product of acetone precipitation reaction, wherein acetone will extract triglyceride level and two kinds of compositions of lipid acid from the above-mentioned hydrolysate that comprises lyso-phosphatidylcholine, triglyceride level and lipid acid.When adding acetone, lyso-phosphatidylcholine will be precipitated out from solution, and can reclaim with standard method.The lyso-phosphatidylcholine purity that obtains is 95.4%.
Preparation Example 2:
Phosphatidylcholine and triglyceride level are mixed, and wherein the mass ratio of phosphatidylcholine and triglyceride level is 7: 1.With mixture heating up to 85 ℃, obtain uniform melt.Add the water of capacity and the solution that ether forms, wherein the volume ratio of water and ether is 8: 1, is heated to 80 ℃ reactant is mixed.Regulating the pH value is 8-9.Add the zymin 1ml of a LecitaseTM10L Phospholipase A2 according to the per kilogram phosphatidylcholine, keep reacting 36 hours, until till the phosphatidylcholine complete hydrolysis under the condition of stirring and pH value.After complete hydrolysis, add heat extraction water and ether, obtain mashed prod.
In order to obtain highly purified lyso-phosphatidylcholine, with the end product of acetone precipitation reaction, wherein acetone will extract triglyceride level and two kinds of compositions of lipid acid from the above-mentioned hydrolysate that comprises lyso-phosphatidylcholine, triglyceride level and lipid acid.When adding acetone, lyso-phosphatidylcholine will be precipitated out from solution, and can reclaim with standard method.The lyso-phosphatidylcholine purity that obtains is 96.2%.
Preparation Example 3:
Semen Ricini is shelled, smashes, then carry out degreasing: add 4mL ether and sherwood oil mixed solution to mix evenly by every gram Semen Ricini, use again the Büchner funnel suction filtration, material on filter paper is used above-mentioned degreasing method degreasing 3 times again, then composition on filter paper is carried out drying, obtain the loose Semen Ricini cake dry powder after fuel-displaced, standby; The mixed solution of wherein said ether and sherwood oil, wherein the volume ratio of ether and sherwood oil is 1: 1;
the Semen Ricini cake dry powder that makes is wrapped up with filter paper, put into cable type extractor according, adding the ratio of 10mL ethanol to add mass concentration according to every gram Semen Ricini cake dry powder is 95% ethanol, extract 8h, obtain tawny oily matter extracting solution, be distilled to 1/5 of original volume with Rotary Evaporators again, obtain paste, then according to the ratio of every gram paste 20mL distilled water, the paste that obtains is dissolved in the distilled water of 80 ℃, be cooled to ether continuous extraction 3-4 time of using again 4 ℃ after 25 ℃, to remove a small amount of remaining grease, after extraction finishes, take off layer brown clear solution dry, use again the acetonitrile repetitive scrubbing, the merging washings makes the paste Semen Ricini extract after sending into and revolving steaming in Rotary Evaporators, standby,
Phosphatidylcholine and above-mentioned Semen Ricini extract are mixed, and wherein the mass ratio of phosphatidylcholine and Semen Ricini extract is 7: 1.With mixture heating up to 75 ℃, obtain uniform melt.Add the water of capacity and the solution that ether forms, wherein the volume ratio of water and ether is 6: 1, is heated to 80 ℃ reactant is mixed.Regulating the pH value is 8-9.
Reduce temperature to 35 ℃, add the zymin 0.5ml of a LecitaseTM10L Phospholipase A2 according to the per kilogram phosphatidylcholine, keep stirring and the condition of pH value under, reacted 34 hours, until till the phosphatidylcholine complete hydrolysis.After complete hydrolysis, add heat extraction water and ether, obtain mashed prod.
In order to obtain highly purified lyso-phosphatidylcholine, use the acetone extract mashed prod.When adding acetone, lyso-phosphatidylcholine will be precipitated out from solution, and can reclaim with standard method.The lyso-phosphatidylcholine purity that obtains is 98.7%.
Preparation Example 4:
The Semen Ricini extract that obtains in phosphatidylcholine and Preparation Example 3 is mixed, and wherein the mass ratio of phosphatidylcholine and Semen Ricini extract is 5: 1.With mixture heating up to 75 ℃, obtain uniform melt.Add the water of capacity and the solution that ether forms, wherein the volume ratio of water and ether is 9: 1, is heated to 80 ℃ reactant is mixed.Regulating the pH value is 8-9.
Reduce temperature to 35 ℃, add the zymin 0.5ml of a LecitaseTM10L Phospholipase A2 according to the per kilogram phosphatidylcholine, keep stirring and the condition of pH value under, reacted 36 hours, until till the phosphatidylcholine complete hydrolysis.After complete hydrolysis, add heat extraction water and ether, obtain mashed prod.
In order to obtain highly purified lyso-phosphatidylcholine, use the acetone extract mashed prod.When adding acetone, lyso-phosphatidylcholine will be precipitated out from solution, and can reclaim with standard method.The lyso-phosphatidylcholine purity that obtains is 97.7%.
Preparation Example 5
The Semen Ricini extract that obtains in phosphatidylcholine and Preparation Example 3 is mixed, and wherein the mass ratio of phosphatidylcholine and Semen Ricini extract is 6: 1.With mixture heating up to 70 ℃, obtain uniform melt.Add the water of capacity and the solution that ether forms, wherein the volume ratio of water and ether is 8: 1, is heated to 80 ℃ reactant is mixed.Regulating the pH value is 8-9.
Reduce temperature to 35 ℃, add the zymin 0.5ml of a Lec i taseTM10L Phospholipase A2 according to the per kilogram phosphatidylcholine, keep stirring and the condition of pH value under, reacted 30 hours, until till the phosphatidylcholine complete hydrolysis.After complete hydrolysis, add heat extraction water and ether, obtain mashed prod.
In order to obtain highly purified lyso-phosphatidylcholine, use the acetone extract mashed prod.When adding acetone, lyso-phosphatidylcholine will be precipitated out from solution, and can reclaim with standard method.The lyso-phosphatidylcholine purity that obtains is 99.1%.
EXPERIMENTAL EXAMPLE
1, to the inhibition of differentiation of osteoclast: medullary cell
The separation of medullary cell
The ICR female mice in birth 6-7 week is taken off cervical vertegra put to death after, with 70% ethanol disinfection back leg position, isolate shin bone under aseptic technique, be placed in 3HBSS (Gibco BRL), peel off the removal soft tissue.Cut off the two ends of shin bone, be injected into marrow, get medullary cell with 1cc syringe holder 1 * α-MEM.With the abundant cell dispersion of pipette, at 1600rpm centrifugal 5 minutes, obtain medullary cell composition (medullary cell and red blood corpuscle).Add 15-20ml ACK damping fluid (155mM NH4Cl in cell, 11mM KHCO3,0.01mM EDTA) processing added phosphoric acid buffer after 2 minutes, the damage of medullary cell is reduced to inferior limit, lyse red blood cells, after 1600rpm is centrifugal 5 minutes cell suspension in the α-MEM that contains 10%FBS.
The cultivation of medullary cell
Separate according to the method described above the medullary cell obtain, be inoculated in 48 well culture plates co-cultivation in α-MEM of 10%FBS with the cell density in 4 * 105/ holes.After adding ODF (50ng/ml) and M-CSF (30ng/ml), then the lyso-phosphatidylcholine that adds the present invention of different concns to make, its concentration is respectively 0.825 μ m, 1.65 μ m, 3.3 μ m, 6.6 μ m.Cultivate after 3 days, exchange fresh α-MEM nutrient solution, at this moment catch up with and state the lyso-phosphatidylcholine that equally adds ODF (50ng/ml) and M-CSF (30ng/ml) and different concns.The osteoclast that finishes differentiation is to confirm with the TRAP staining.TRAP dyeing is (Sigma, the Cat.No.387-A) that carries out with the Tartrate resistant acid phosphatase test kit.Mature osteoclast is fixed 5 minutes with 10% formalin.Remove formalin, add 0.1% Tryptones-100, processed for 10 seconds.After discarding Tryptones-100, carry out TRAP dyeing 5 minutes.After removing the TRAP dyeing solution, clean 2 times with distilled water, drying is counted the osteoclast of the TRAP positive at microscopically.
In experimental result, as seen, the TRAP of control group is positive, and the osteoclast number is 240 ± 44.But the positive osteoclast number of TRAP of pressing the experimental group of different concns processing lyso-phosphatidylcholine (0.825 μ m, 1.65 μ m, 3.3 μ m, 6.6 μ m) is respectively 12.9,185 soil, 172 ± 9.3,87 ± 3.5,36 ± 3.6.Experimental result shows, in the medullary cell culture systems, lyso-phosphatidylcholine can suppress the differentiation of osteoclast, and its restraining effect and concentration are the line style relation.
2, to the inhibition of differentiation of osteoclast: the osteoclast precursor cell
The separation of osteoclast precursor cell
The ICR female mice in birth 5-6 week is taken off neck put to death after, with 70% ethanol disinfection back leg position, isolate shin bone and Thigh bone under aseptic technique.The shin bone that separates and Thigh bone are peeled off with the identical method of above-described embodiment 2 and are removed after soft tissue several and inject 3X HBSS (Gibco BRL) and remove medullary cell.Be cut into small pieces with surgical scissors, put in the enzyme solution (1mg/ml collagenase typeII, 0.05% trypsinase, 4mM EDTA, Gibco BRL) that contains collagenase, 37 ℃ of digestion 15 minutes, repeatedly digest 5 times.After the 3rd digestion, bone shears is become more fritter., placed 15 minutes in ice cube in α-MEM through 5 cell suspensions of obtaining of digestion.Then mixed 1 minute with vortex, add the cold α-MEM with amount, placed 15 minutes in ice cube.Bone suspension after processing like this just can obtain medullary cell by the sterilization net, cultivates in containing α-MEM of 10%FBS.
The cultivation of osteoclast precursor cell
The osteoclast precursor cell that obtains is inoculated in 48 well culture plates co-cultivation in α-MEM of 10%FBS with the cell density in 0.5 * 106/ hole.After adding ODF (100ng/ml) and M-CSF (30ng/ml), then the lyso-phosphatidylcholine that adds the present invention of different concns to make, its concentration is respectively 0.825 μ m, 1.65 μ m, 3.3 μ m, 6.6 μ m.Control group is not for adding the co-cultivation cell of lyso-phosphatidylcholine.Cultivate after 3 days, exchange fresh α-MEM nutrient solution, at this moment catch up with and state the lyso-phosphatidylcholine that equally adds ODF and M-CSF and different concns.Cultivate after 6 days, with the fresh α of identical method exchange-MEM nutrient solution.Cultivate after 9 days, carry out TRAP dyeing, count the osteoclast of the TRAP positive with microscope.
In experimental result as seen, several 344 ± 43.7 of the positive osteoclast of the TRAP of control group.But the positive osteoclast number of TRAP of pressing the experimental group of different concns processing lyso-phosphatidylcholine (0.825 μ m, 1.65 μ m, 3.3 μ m, 6.6 μ m) is respectively 318 ± 19.3,318 ± 50.4, and 267 ± 24,152 ± 29.1.Experimental result shows, separates in the medullary cell precursor cell culture systems that obtains from bone, and lyso-phosphatidylcholine can suppress the differentiation of osteoclast, and its restraining effect and concentration are the line style relation.
3, conclusion
Lyso-phosphatidylcholine of the present invention can suppress the differentiation of osteoclast in the cultivation of medullary cell is, its restraining effect presents dose-dependence.And lyso-phosphatidylcholine of the present invention also suppresses the differentiation of osteoclast in the cultivation of the osteoclast precursor that separates system from bone, and its restraining effect presents dose-dependence.Lyso-phosphatidylcholine of the present invention is to scleroblast, medullary cell, equal no cytotoxicity in peritoneal macrophage and kidney cell.Thus, lyso-phosphatidylcholine of the present invention and pharmaceutical preparation thereof can be used for prevention and the treatment of bone metabolic disease.
FORMULATION EXAMPLE 1: tablet
Will
Be pressed into tablet according to ordinary method.
FORMULATION EXAMPLE 2: dripping pill
Will
Lyso-phosphatidylcholine 2.5g
Polyethylene glycol 6000 13.4g
Polysorbate 80 0.2g
Make dripping pill according to ordinary method, make altogether 1000.
FORMULATION EXAMPLE 3: ointment
Will
Make according to a conventional method ointment.
Claims (10)
1. method for preparing the high purity lyso-phosphatidylcholine, it is characterized in that, comprise the following steps: the mixture of phosphatidylcholine and Phospholipase A2 active reinforcing agent is formed dispersion in the solution of water-rudimentary ether, allow this dispersion contact with Phospholipase A2, to form reaction mixture, after reaction is completed, reclaim lyso-phosphatidylcholine, obtain the high purity lyso-phosphatidylcholine.
2. preparation method claimed in claim 1, wherein said Phospholipase A2 active reinforcing agent can be selected from triglyceride level and Semen Ricini extract.
3. preparation method claimed in claim 2, wherein the mass ratio of phosphatidylcholine and Phospholipase A2 active reinforcing agent is between 1: 1 to 10: 1, preferred 5: 1-7: 1.
4. preparation method claimed in claim 2, the molecule of wherein said triglyceride level is comprised of the glyceryl that the acyl group with three lipid acid is connected, and the carbochain of the acyl group of triglyceride level has 8 to 20 carbon atoms and 1 to 4 unsaturated link(age).
5. preparation method claimed in claim 2, wherein said Semen Ricini extract be first with Semen Ricini through pulverizing degreasing, re-using 95% extraction using alcohol and obtain.
6. preparation method claimed in claim 1, wherein in the solution of water-rudimentary ether, the volume ratio of water and rudimentary ether is about 5: 1-10: 1, preferred 7: 1-9: 1, more preferably 8: 1, wherein said rudimentary ether was C1-C4 ether, preferably ether.
7. preparation method claimed in claim 1, the step of wherein said recovery lyso-phosphatidylcholine comprises the use acetone extract.
8. lyso-phosphatidylcholine prevents and/or treats purposes in the pharmaceutical composition of metabolic osteopathy in preparation, and wherein lyso-phosphatidylcholine is that the preparation method described in claim 1 makes.
9. purposes claimed in claim 8, wherein bone transfer, primary osteocarcinoma, rheumatic or degenerative osteoarthritis, the periodontal disease etc. of the cancers such as the optional autophya osteoporosis of metabolic osteopathy, mammary cancer and prostate cancer.
10. a pharmaceutical composition that is used for preventing and/or treating metabolic osteopathy, wherein comprise high purity lyso-phosphatidylcholine and pharmaceutically acceptable carrier that preparation method claimed in claim 1 makes.
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| CN103535526A (en) * | 2013-10-22 | 2014-01-29 | 广州智特奇生物科技股份有限公司 | Feed additive for improving fat utilization rate |
| CN103571614A (en) * | 2013-10-14 | 2014-02-12 | 盐城工学院 | Preparation method of castor oil and castor oil prepared through same |
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| US4849132A (en) * | 1986-05-16 | 1989-07-18 | Asahi Denka Kogyo Kabushiki Kaisha | Surfactant composition having improved functions |
| WO1997028270A1 (en) * | 1996-02-02 | 1997-08-07 | Biomolecular Products, Inc. | Methods for making lysophosphatidylcholine |
| CN1608073A (en) * | 2001-12-13 | 2005-04-20 | 钱吉子 | A process for preparing N-acylated lysophosphatidylcholine compounds and a pharmaceutical composition for treatment of metabolic bone disease comprising said compounds |
| CN102633832A (en) * | 2011-02-09 | 2012-08-15 | 北京绿色金可生物技术股份有限公司 | Method for preparing high-purity phosphatidylcholine |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103571614A (en) * | 2013-10-14 | 2014-02-12 | 盐城工学院 | Preparation method of castor oil and castor oil prepared through same |
| CN103535526A (en) * | 2013-10-22 | 2014-01-29 | 广州智特奇生物科技股份有限公司 | Feed additive for improving fat utilization rate |
| CN103535526B (en) * | 2013-10-22 | 2015-04-22 | 广州智特奇生物科技股份有限公司 | Feed additive for improving fat utilization rate |
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| CN103131736B (en) | 2015-08-05 |
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