CN100486983C - Medicine compound for preventing diabets mellitus and preparing method thereof - Google Patents
Medicine compound for preventing diabets mellitus and preparing method thereof Download PDFInfo
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Abstract
A medical compound for preventing and treating diabetes contains chitinous oligose sulfate (CSA) and/or chitosan sulfate (CSB). Its advantages are high activity in reducing blood sugar, regulating blood fat, improving immunity and resisting coagulation, low cost, less pollution and high output rate.
Description
(1). technical field
The present invention relates to the research and development of marine drug, specifically is a kind of lowering blood glucose marine drug---prevent and treat medical compounds of diabetes and preparation method thereof, it belongs to the marine biotechnology field.
(2). background technology
As everyone knows, hyperglycemia is a key character of diabetes.Diabetes are a kind of common endocrine and metabolic disorders diseases, and it is because insulin secretion is absolute or relative deficiency causes sugar, fat, protein metabolism disorder, the hyperglycemia state of persistence occurs.The patients on long-term of the diabetes chronic complicating diseases such as arteriosclerosis, cardiovascular, kidney, illness in eye and neural system that often occur together, the severe patient life-threatening, its sickness rate height, complication is many, has a strong impact on the healthy of people.There are diabetic subject 1.25 hundred million people in the whole world, and the diabetic subject of China has reached 4,000 ten thousand people, and also on the rise, and the World Health Organization classifies diabetes one of as the world's three big difficult disease.
The current ofhypoglycemic medicine that is usually used in treating diabetes clinically mainly contains two kinds, and a kind of is sulfourea, and a kind of is biguanides.Wherein, sulfonylurea drugs mainly is by stimulating insulin secretion, thus the lowering blood glucose level.A kind of diabetes pill medicine is arranged, and is exactly the medicine of treatment diabetes commonly used in the market, and its main ingredient composition glyburide belongs to the sulfourea hypoglycemic agents.Than the s-generation medicine diamicron that French Les Laboratoires servier is produced, its blood sugar reducing function height, advantage such as have that dosage is little, effect is fast, curative effect is high, longer duration, side effect are light.But this medicine has cumulative effect in human body, and this has just increased the load of internal organs such as liver, kidney, and is especially to the elderly and liver, renal insufficiency person, unconformable especially, and hypoglycemia easily takes place in addition.
In view of diabetes very harmful to HUMAN HEALTH, and still do not have the medicine of very good treatment diabetes in the market, people are making great efforts the optimal path with the diabetes that seek treatment always.Through discovering that chitosan has certain hypoglycemic activity, infer that its mechanism of action is that chitosan can stimulate beta Cell of islet synthetic, secretion and uelralante, and reduce alpha Cell of islet and secrete hyperglycemic-glycogenolytic factor, act on liver simultaneously, suppress the absorption of sugar in liver glycogen heteroplasia and the body, reduce sugar output, and strengthen surrounding tissue to the utilization of sugar and lowering blood glucose also may increase insulin receptor and glucoreceptor, improve insulin sensitivity, add strong biological activity, opposing behind the acceptor is weakened, suppress the endo-oxidase system and cause histanoxia, strengthen glucose metabolism, quicken its utilization, thus lowering blood glucose.As everyone knows, chitosan is a large amount of class natural biological polysaccharide that exist of nature, and is nontoxic, has a lot of biological activitys, and as antitumor, strengthening immunity etc., but the water-fast character of chitosan has limited its application.
Main contents to sulfated chitosan (CSB) research in the prior art have: sulfated chitosan (CSB) sulphur content is lower, and sulfate group content is less than 33%.Because of chitosan water insoluble, a lot of human inhomogeneous reactions, this just causes the chitosan utilization ratio very low.Now the someone studies again, uses homogeneous reaction, but its employed sour solvent is a dichloro acetic acid.This sour solvent price height (94 yuan/500ml), though can increase the utilization ratio of chitosan, total cost has no sign of improvement.The somebody is at N
2React 2～8h under the atmosphere and prepare sulfated chitosan (CSB), it can make the sulfur acid ester group amount of sulfated chitosan (CSB) only reach 32.7%; The somebody mainly studies the anticoagulant active of sulfated chitosan (CSB), and the anti-AIDS aspect is used, and to its hypoglycemic aspect effect, yet there are no report.
(3) technology contents
The objective of the invention is to provide a kind of hypoglycemic ocean medicinal material, promptly a kind of medical compounds of preventing and treating diabetes and preparation method thereof.Owing to discover that chitosan is gained sulfated chitosan (CSB) derivative after sulfonation, have and the similar structure of heparin, and have strong polyanion character, thereby caused numerous scholars' concern.Therefore chitosan is carried out chemical modification and modification,, become current hot topic to enlarge its range of application.The medical compounds of preventing and treating diabetes of the present invention has and heparin similar structures and strong polyanion character, studied it except having anti-freezing, anti-bolt and effect such as antiviral, and human body is had no side effect, be expected to be exploited, for the mankind benefit at deep structure research.It is high that the active sulphur content of two kinds of Sulfate of polysaccharide of the medical compounds of preventing and treating diabetes of the present invention is wanted, chitosan raw material availability height; Used preparation material and used the comparing of forefathers are wanted ratio of performance to price height; Two kinds of Sulfate of polysaccharide are first as the application of hypoglycemic marine drug aspect.Since the influence of receptor 1 activity group sulfate group and molecular weight thereof, the activity that this medical compounds of preventing and treating diabetes demonstrates at aspects such as anti-blood sugar, accent blood fat, strengthening immunity, anticoagulations.This preparation method who prevents and treats the medical compounds of diabetes will adopt homogeneous reaction, and simple, running cost is low, pollutes and lacks, the raw material availability height.
Task of the present invention has following technical scheme to finish, developed a kind of medical compounds of preventing and treating diabetes, it comprises: CSA (CSA) and/or sulfated chitosan (CSB) is characterized in that: the typical structure formula of two these compounds is:
Wherein n is the polymerization degree: 3～600, and R
1Be SO
3Na or H, R
2Be SO
3Na or H or Ac, R
1With R
2Be all except H or the Ac person; The active group of this compound---sulfate group (OSO
3 -) content be 40.73～42.21%; Wherein, the CSA of faint yellow cotton shape (CSA) molecular weight is 1～6KD, and the sulfated chitosan of faint yellow cotton shape (CSB) molecular weight is 30～100KD.
Described CSA (CSA), its polymerization degree: 3～15; Described sulfated chitosan (CSB), its polymerization degree: 100～600.
The preparation process of the CSA of described control diabetes medicament (CSA) is as follows:
(1) preparation of esterifying reagent: a certain amount of N, N-dimethyl formamide (DMF) put with there-necked flask in, after cryosel is bathed cooling, stir and slowly drip an amount of chlorsulfonic acid down, remain temperature of reaction system less than 5 ℃;
(2) take by weighing quantitative chitosan raw material;
(3) add 2% an amount of acetic acid solution, raw material is fully dissolved;
(4), be put in the microwave oven stoichiometric number minute with above-mentioned abundant dissolved raw material;
(5) with the raw material of above-mentioned reacting by heating, be neutralized to neutrality with the NaOH solution of 2N, place for some time again;
(6) will be neutralized to the above-mentioned feed liquid of neutral and carry out suction filtration;
(7) get filter cake and dry, the faint yellow solid material of gained; Pulverize this material of oven dry again, survey its molecular weight, this material is the chitin oligosaccharide of lower molecular weight;
(8) get the product that an amount of above-mentioned pulverizing is dried, add an amount of formamide solvent;
(9), after stirring, add an amount of formic acid or dichloro acetic acid, after mixing with said mixture;
(10) add a certain amount of sulphonating agent DMFSO again
3, after stirring;
(11) again under 40～60 ℃ of temperature, stirring reaction 1～2h;
(12) above-mentioned reaction solution is added 95% ethanol of 3 times of volumes again, when a large amount of white flocks occurring, place heavyization 30min in 4 ℃ of refrigerators, the throw out that obtains promptly is the thick product of CSA;
(13) above-mentioned throw out is carried out suction filtration, get filter cake;
(14) above-mentioned filter cake is used water dissolution again, the NaOH solution that adds 2N then is neutralized to neutrality;
(15) above-mentioned solution is dialysed with dialysis tubing, get dialyzate behind the desalination; Or use the hyperfiltration process desalination, get and hold back solution;
(16) again with above-mentioned solution, be concentrated into 1/10 volume;
(17) above-mentioned concentrated solution is put in the refrigerator-freezer freezes, lyophilize gets faint yellow cotton shape material then;
(18) pulverize this cotton shape material, measure its sulphur content and molecular weight, the CSA (CSA) of getting lower molecular weight 1～6KD up to specification is as preventing and treating the medicine of diabetes.
The DMF in (1) described in the preparation process of control diabetes medicament preparation method's of the present invention CSA (CSA) and the volume ratio of chlorsulfonic acid are 10～5:1; The amount of 2% acetic acid solution in described (3) with the envelope-bulk to weight ratio of (2) middle chitosan is: 6～30:1 (V/W); Described (4) are reacted to 100 ℃ in microwave oven, the reaction times: 3～6min; The molecular weight of the faint yellow solid material in described (7) is 9.21 * 10
3～3.86 * 10
4Dalton; The envelope-bulk to weight ratio of getting an amount of oven dry product and an amount of formamide solvent in described (8) is: 15～50:1 (V/W); The volume ratio of the dense amount of formic acid of adding 88% and the mixture in (8) is in described (9): 1:3～10 (V/V); Add sulphonating agent DMFSO in described (10)
3Amount be 50～100ml; Stirring reaction in described (11), synthesis under normal pressure under air atmosphere; Dialysis tubing in described (15) is that the dialyzate that intercepts is a product of holding back 1～6KD molecular weight size with 1 and two kinds of film bags of 8KD.
The preparation process of the preparation method's of control diabetes medicament of the present invention described sulfated chitosan (CSB) is as follows:
(1) preparation of esterifying reagent: a certain amount of DMF is inserted in the there-necked flask, after cryosel is bathed cooling, stir and slowly drip an amount of chlorsulfonic acid down, remain temperature of reaction system less than 5 ℃;
(2) get an amount of chitosan raw material, add an amount of formamide solvent;
(3), after stirring, add an amount of formic acid or dichloro acetic acid, after mixing with said mixture;
(4) add a certain amount of sulphonating agent DMFSO again
3, after stirring; Again under 40～60 ℃ of temperature, stirring reaction 1～2h;
(5) above-mentioned reaction solution is added 95% ethanol of 3 times of volumes again, when a large amount of white flocks occurring, place heavyization 30min in 4 ℃ of refrigerators, the throw out that obtains promptly is the thick product of sulfated chitosan;
(6) above-mentioned throw out is carried out suction filtration, get filter cake;
(7) above-mentioned filter cake is used water dissolution again, the NaOH solution that adds 2N then is neutralized to neutrality;
(8) above-mentioned solution is dialysed with dialysis tubing, get dialyzate behind the desalination; Or use the hyperfiltration process desalination to get and hold back solution;
(9) again with above-mentioned solution, be concentrated into 1/10 volume;
(10) above-mentioned concentrated solution is put in the refrigerator-freezer freezes, lyophilize gets faint yellow cotton shape material---sulfated chitosan then;
(11) pulverize this cotton shape material, measure its sulphur content and molecular weight, the sulfated chitosan (CSB) of getting molecular weight 30～100KD up to specification is as preventing and treating the medicine of diabetes.
In the preparation process of the sulfated chitosan among the preparation method of control diabetes medicament of the present invention (CSB), the volume ratio of DMF and chlorsulfonic acid is 10～5:1 in described (1); The amount of getting an amount of chitosan raw material in described (2) is 15～50:1 (V/W) with the ratio of an amount of formamide solvent; Adding 88% dense amount of formic acid in described (3) is 3～10ml; Add sulphonating agent DMFSO in described (4)
3Amount be 50～100ml stirring reaction wherein synthesis under normal pressure under air atmosphere; Used dialysis tubing is the dialysis tubing with the minimum 1.2KD of being limited to of molecular weight cut-off in described (8), carries out dialysis desalting.
CSA of the present invention (CSA) and/or sulfated chitosan (CSB) are another important protection domains of the present invention as the application of the hypoglycemic marine drug of preparation.
The medical compounds of preventing and treating diabetes of the present invention, it comprises: CSA (CSA) and/or sulfated chitosan (CSB), the test positively effect of these two kinds control diabetes medicaments is as follows:
(A) toxicology test of CSA (CSA):
1. laboratory animal
Laboratory animal: Kunming mouse, body weight 18-22 gram, male and female half and half, laboratory animal room provides by new drug pharmacological research center, Shandong Province, conformity certification number: 970102
The raising condition: 18-22 ℃ of room temperatures, 5 in the every cage of mouse is freely taken the photograph water and is ingested, and feed is particle mouse material, and laboratory animal room provides by new drug pharmacological research center, Shandong Province.
Test method: trial test shows that this product toxicity is lower, is difficult to measure LD50, so carry out maximum tolerance determination in its mouse one day.
2. administering mode
2.1 i.g administration: sample CSA (CSA) solution preparation 25g/100ml, dosage 0.4ml/10g, get 20 of healthy Kunming mouses, male and female half and half, test fasting in preceding 12 hours and can't help water, pressed 0.4ml/10g body weight i.g CSA suspension on the 1st, in 8:00am ig once, observe the reaction of animals situation behind the medicine immediately, and the activity of record animal behavior, breathing, the mental status, stool and urine proterties and color, quilt hair, nose, eye, oral secretion and death condition, observed continuously 7 days.
2.2 i.p administration: select sample CSA, obtain solution concentration 12.5g/100ml, dosage 0.2ml/10g gets 20 of healthy Kunming mouses, male and female half and half, test fasting in preceding 12 hours and can't help water, press 0.2ml/10g body weight i.p suspension on the 1st, in 8:00am i.p once, observe the reaction of animals situation behind the medicine immediately, and the activity of record animal behavior, breathing, the mental status, stool and urine proterties and color, quilt hair, nose, eye, oral secretion and death condition, observed continuously 7 days.
3 experimental results
3.1. mouse behavioral activity, breathing, the mental status, stool and urine proterties and color, quilt hair, nose, eye, oral cavity etc. show no obvious abnormalities after the CSA.ig administration, observe continuously and do not see considerable change in 7 days, put to death and cutd open inspection part mouse on the 7th day, it is as seen unusual that main organs such as the visual inspection heart, liver, spleen, lung, kidney all do not have naked eyes.The maximum tolerated dose on the one that shows CSA mouse ig administration is greater than 10g/kg, and this product a drug does not have the overt toxicity effect.
3.2.CSA the mouse activity increases immediately after the i.p administration, animal engenders and turns round body after ten minutes, the movable minimizing, turn round the Signs shape after one hour and disappear, animal begins to occur prostrate, and it is motionless to close order, still moved less in eight hours behind medicine, second day animal recovers normal behind the medicine.Observe continuously and do not see considerable change in 7 days, put to death and cutd open inspection part mouse on the 7th day, it is as seen unusual that main organs such as the visual inspection heart, liver, spleen, lung, kidney all do not have naked eyes.The maximum tolerated dose on the one that shows CSA mouse i.p administration is greater than 2.5g/kg, and this product a drug does not have the overt toxicity effect.
The raw materials of chitosan Nantural non-toxic of this sample has biocompatibility and degradation in vivo, introduces SO through sulfonation
4 2-, the similar heparin contains SO with other
4 2-Natural polysaccharide the same, its toxic side effect is little.That passes through us experimental results show that the CSA toxic side effect is little, and visible CSA (CSA) is feasible as new drug development.
(B) sulfated chitosan (CSB) hypoglycemic test 1 (CSB):
1. laboratory animal: Kunming mouse, body weight 24-28 grams, male and female half and half, laboratory animal room provides by new drug pharmacological research center, Shandong Province, conformity certification number: 970102
2. main medicine and reagent and equipment
Pale yellow powder CSA is made into the desired concn suspension with 0.5% carboxymethylcellulose sodium solution.Lab-500 type full-automatic biochemical analytical method is produced by peach garden, Nanjing Applied Biotechnology company limited.Blood sugar test kit (glucose oxidase method) is produced lot number 981129 by Beijing Zhongsheng Biological Engineering High Technology Company.
3. test grouping and hyperglycemia moulding
Get 6 groups of mouse, each is organized mouse and causes hyperglycemia model through tail vein injection tetraoxypyrimidine 70mg/kg, measures fasting blood glucose level behind the 72h.The result shows that tetraoxypyrimidine can cause mouse blood sugar significantly to raise.Distinguish administration igCSA, CSB 300mg/kg and 1000mg/kg then, positive controls diabetes pill group 1250mg/kg, negative control group is given isopyknic 0.5% carboxymethylcellulose sodium solution.Respectively with administration after 2,4,6,12 and 24h measure glucose level, CSA (CSA) can obviously suppress tetraoxypyrimidine induced mice blood sugar increasing, sees Table 1:
Table 1: the chitooligosaccharide-sulfuric ester is to the influence of tetraoxypyrimidine induced mice hyperglycemia:
Annotate: x ± s, n=10, each control group compares,
* 1P＜0.001,
* 2P＜0.01
As can be seen from the above table, the low high dose group of CSA (CSA) has the obvious suppression effect to the mouse hyperglycemia due to the tetraoxypyrimidine, and high dose group is than low dose group inhibiting rate height, and illustrating has tangible dose-effect relationship between the high low dose group.The effect that they suppress blood sugar is better than control group diabetes pill, and inhibiting rate is than diabetes pill height.High molecular weight chitosan sulfuric ester (CSB) by the table in as can be seen, its blood sugar decreasing effect is more effective than low-molecular weight chitoglycan sulfuric ester (CSB), also shows tangible dose-effect relationship.Illustrate that molecular weight is an important factor that influences hypoglycemic activity.
Tetraoxypyrimidine can optionally destroy beta Cell of islet, and sulfated chitosan (CSB) has the obvious suppression effect to the blood sugar increasing due to the tetraoxypyrimidine, and this shows that sulfated chitosan (CSB) may weaken tetraoxypyrimidine to the damage of beta Cell of islet or improve the function of the β cell of damaged.Thereby effect with blood sugar increasing due to the anti-tetraoxypyrimidine.
(C) test of the blood sugar regulation of CSA (CSA) 2:
1. laboratory animal: Wistar rat, body weight 200-300 grams, male and female half and half.Laboratory animal room provides by new drug pharmacology center, Shandong Province, conformity certification numbers 970102
2. test grouping and hyperglycemia moulding
Mouse is divided into 6 groups at random, 10 every group.Positive controls ig glyburide 25mg/kg, negative control group is given isopyknic 0.5% carboxymethylcellulose sodium solution, distinguishes ig CSA, CSB300mg/kg and 1000mg/kg for all the other four groups.With 4h after the administration, each organizes ip in mice suprarenin 0.02mg/kg, gets blood in injection back 1h broken end and surveys blood sugar.The result shows that CSA (CSA) 300mg/kg and 1000mg/kg can obviously suppress the effect of adrenergic rising blood sugar, see Table 2.
Table 2: sulfated chitosan (CSB) causes the influence of blood sugar increasing to suprarenin
| Group | Dosage (mg/kg) | Blood sugar (mmol/L) |
| The suprarenin group | 0.02 | 13.67±3.25 |
| Add the glyburide group | 25 | 8.60±3.73 * |
| CSA | 300 1000 | 7.03±2.60 ** 5.41±3.47 ** |
| CSB | |300 1000 | 6.79±2.69 ** 4.97±2.99 ** |
As can be seen from the above table, the low high dose group of CSA (CSA) has the obvious suppression effect to the mouse hyperglycemia due to the suprarenin, and high dose group is than low dose group inhibiting rate height, and illustrating has tangible dose-effect relationship between the high low dose group.The effect that they suppress blood sugar is better than control group glyburide, and inhibiting rate is than glyburide height.By it can also be seen that high molecular weight chitosan sulfuric ester (CSB) blood sugar decreasing effect is more effective than low-molecular weight crust oligosaccharide sulfate (CSA) in the table, also show tangible dose-effect relationship.Illustrate that molecular weight is an important factor that influences hypoglycemic activity.Adrenergic promotes the decomposition of liver starch and muscle glycogen and causes blood sugar increasing, and the adrenergic blood glucose increasing effect of CSA (CSA) energy antagonism may be relevant with the inhibition glycogenolysis.
In the preparation process (9) of CSA of the present invention (CSA) with the preparation process (3) of described sulfated chitosan (CSB) in the sour solvent that added all be 88% dense formic acid (preferred 88% the dense formic acid 5ml that uses in the embodiments of the invention).Need not dewater and other impurity when using this acid solvent, simplify processing sequence, save drying cost.Select formic acid also because the price of formic acid (10 yuan/500ml) well below dichloro acetic acid (94 yuan/500ml), with the homogeneous reaction that this sour solvent carried out, the sulphur content of products therefrom and productive rate are all with suitable with dichloro acetic acid, and its personality valency can be than having improved greatly.In art technology, use formic acid to yet there are no report as sour solvent.
In the preparation process (11) of CSA of the present invention (CSA) with the preparation process (4) of described sulfated chitosan (CSB) in stirring reaction, normal pressure reacts under air atmosphere.This improvement and forefathers are " at N
2React 2～8h under the atmosphere " technology compare, its method is more simple, and has saved the energy.
(4) accompanying drawing and embodiment, protection scope of the present invention not only is confined in following examples.
Embodiments of the invention further specify as follows in conjunction with the accompanying drawings:
Fig. 1 is the infrared spectrogram of chitosan raw material.
Fig. 2 is the infrared spectrogram of sulfated chitosan.
Fig. 3 is the chitooligosaccharide-sulfuric ester
13C-NMR collection of illustrative plates.
It is as follows that the present invention prevents and treats the preparation method embodiment of marine drug of diabetes:
The preparation embodiment of I, CSA:
1.. the preparation of esterifying reagent
Adopt N, N-dimethyl formamide (DMF) prepares sulfonated reagent for reaction medium---DMFSO
3(dimethyl formamide-chlorsulfonic acid) sulphonating agent.Add 50mlN in the there-necked flask that whipping appts is housed, N-dimethyl formamide places the cryosel water-bath.Under whipped state, drip the certain volume chlorsulfonic acid, the speed that control drips remains on below 5 ℃ the temperature of reaction system.At room temperature continue after dropwising to stir certain hour, make sulfonated reagent safer effectively.Chlorsulfonic acid add-on such as table 3:
The ratio of table 3:DMF and chlorsulfonic acid is to the influence of sulphonating agent state
| DMF(ml) | Chlorsulfonic | State | |
| 50 | 50 | The reaction solution thickness stirs motionless | |
| 50 | 40 | The reaction solution thickness, more slightly smaller than above-mentioned |
|
| 50 | 30 | The reaction solution thickness can stir | |
| 50 | 20 | Still has |
|
| 50 | 10 | It is liquid that mixture is, and mixes | |
| 60 | 10 | It is liquid that mixture is, and mixes | |
| 100 | 10 | It is liquid that mixture is, and mixes |
As seen from the above table, the ratio of this experimental selection DMF and chlorsulfonic acid be 10～5:1 all can, but 5:1 best results.
2.. the degraded of chitosan under the microwave action is prepared as follows:
(1) gets the product that the above-mentioned pulverizing of 2～5g is dried, add 30～150ml formamide solvent;
(2), after stirring, add 3～10ml formic acid or dichloro acetic acid, after mixing with said mixture;
(3) add 50～100ml sulphonating agent DMFSO again
3, after stirring;
(4) again under 40～60 ℃ of temperature, stirring reaction 1～2h;
(5) above-mentioned reaction solution is added 95% ethanol of 3 times of volumes again, when a large amount of white flocks occurring, place heavyization 30min in 4 ℃ of refrigerators;
(6) above-mentioned throw out is carried out suction filtration, get filter cake;
(7) above-mentioned filter cake is used water dissolution again, the NaOH solution that adds 2N then is neutralized to neutrality;
(8) above-mentioned solution is dialysed with dialysis tubing, get dialyzate behind the desalination; Or use the hyperfiltration process desalination, get and hold back solution;
(9) again with above-mentioned solution, be concentrated into 1/10 volume;
(10) above-mentioned concentrated solution is put in the refrigerator-freezer freezes, lyophilize gets faint yellow cotton shape material then;
(11) pulverize this cotton shape material, measure its sulphur content and molecular weight, the CSA (CSA) of getting lower molecular weight 1～6KD up to specification is as preventing and treating the medicine of diabetes.
4. produce two kinds of glycan sulfuric esters of the present invention (CSA, condition CSB) sees Table 5,6:
Table 5: (CSA) preparation condition of CSA under the dichloro acetic acid effect and result
| Instance number number | Solvent | Solvent load ml | Chitosan dosage g | Sulphonating agent consumption ml | Reaction times h | Temperature of reaction ℃ | Productive rate % | Sulfate radical content % | The |
| 1 | |
50 | 2 | 50 | 2 | 40 | 95 | 40.15 | 7509 |
| 2 | |
50 | 2 | 50 | 2 | 45 | 91 | 41.70 | 7089 |
| 3 | |
50 | 2 | 50 | 1 | 50 | 90 | 40.33 | 6562 |
| 4 | |
50 | 2 | 50 | 1 | 55 | 87 | 40.17 | 5869 |
| 5 | |
50 | 2 | 50 | 2 | 50 | 84 | 40.39 | 3809 |
| 6 | |
50 | 2 | 50 | 2 | 55 | 79 | 41.27 | 2763 |
Table 6: (CSA) preparation condition of CSA under the formic acid effect and result:
| Instance number | Solvent | Solvent load ml | Chitosan dosage g | Sulphonating agent consumption ml | Reaction times h | Temperature of reaction ℃ | Productive rate % | Sulfate radical content % | The |
| 1 | |
50 | 2 | 50 | 2 | 40 | 95 | 40.73 | 7209 |
| 2 | |
50 | 2 | 50 | 2 | 45 | 93 | 42.21 | 6889 |
| 3 | |
50 | 2 | 50 | 1 | 50 | 93 | 40.62 | 6362 |
| 4 | |
50 | 2 | 50 | 1 | 55 | 89 | 41.15 | 5469 |
| 5 | |
50 | 2 | 50 | 2 | 50 | 86 | 40.59 | 3789 |
| 6 | |
50 | 2 | 50 | 2 | 55 | 83 | 41.25 | 2564 |
By last two tables as can be known, formic acid is suitable as sour solvent effect as solvent effect and dichloro acetic acid, and is quite a lot of a little to a certain degree, and the formic acid price is well below dichloro acetic acid, so recently weigh with sexual valence, and formic acid is better than dichloro acetic acid.
The preparation embodiment of II, sulfated chitosan (CSB):
The preparation embodiment of II, sulfated chitosan (CSB):
1.. the preparation of esterifying reagent is identical with the preparation of esterifying reagent in the CSA.
2.. the preparation of high-molecular weight sulfated chitosan (CSB), preparation process is as follows:
(1) gets 2～5g chitosan raw material, add 30～150ml formamide solvent; With said mixture, after stirring, add 3～10ml formic acid or dichloro acetic acid, after mixing;
(2) add 50～100ml sulphonating agent DMF again
, after stirring;
(3) again under 40～60 ℃ of temperature, stirring reaction 1～2h;
(4) above-mentioned reaction solution is added 95% ethanol of 3 times of volumes again, when a large amount of white flocks occurring, place heavyization 30min in 4 ℃ of refrigerators, the throw out that obtains promptly is the thick product of sulfated chitosan;
(5) above-mentioned throw out is carried out suction filtration, get filter cake;
(6) above-mentioned filter cake is used water dissolution again, the NaOH solution that adds 2N then is neutralized to neutrality;
(7) above-mentioned solution is dialysed with dialysis tubing, get dialyzate behind the desalination; Or use the hyperfiltration process desalination to get and hold back solution;
(8) again with above-mentioned solution, be concentrated into 1/10 volume;
(9) above-mentioned concentrated solution is put in the refrigerator-freezer freezes, lyophilize gets faint yellow cotton shape material then---sulfated chitosan (CSB);
(10) pulverize this cotton shape material, measure its sulphur content and molecular weight, the sulfated chitosan (CSB) of getting molecular weight 30～100KD up to specification is as preventing and treating the medicine of diabetes.
The CSA of producing (CSA) condition sees Table 7,8:
Table 7: (CSA) preparation condition of CSA under the dichloro acetic acid effect and result
| Instance number number | Solvent | Solvent load (ml) | Chitosan dosage (g) | Sulphonating agent consumption (ml) | Reaction times (h) | Temperature of reaction (℃) | Productive rate (%) | Sulfate radical content amount (%) | In the molecular weight (* 10 4) |
| 1 | |
50 | 2 | 50 | 2 | 40 | 95 | 40.10 | 10.71 |
| 2 | |
50 | 2 | 50 | 2 | 45 | 93 | 41.37 | 6.63 |
| 3 | |
50 | 2 | 50 | 1 | 50 | 95 | 40.24 | 9.62 |
| 4 | |
50 | 2 | 50 | 1 | 55 | 95 | 40.05 | 8.75 |
| 5 | |
50 | 2 | 50 | 2 | 50 | 92 | 40.27 | 5.16 |
| 6 | |
50 | 2 | 50 | 2 | 55 | 90 | 41.03 | 3.05 |
Table 8: (CSA) preparation condition of CSA under the formic acid effect and result
| Instance number | Solvent | Solvent load ml | Chitosan dosage g | Sulphonating agent consumption ml | Reaction times h | Temperature of reaction ℃ | Productive rate % | Sulfate radical content % | The |
| 1 | |
50 | 2 | 50 | 2 | 40 | 95 | 40.23 | 8.91 |
| 2 | |
50 | 2 | 50 | 2 | 45 | 95 | 42.01 | 7.01 |
| 3 | |
50 | 2 | 50 | 1 | 50 | 95 | 40.22 | 8.59 |
| 4 | |
50 | 2 | 50 | 1 | 55 | 95 | 41.25 | 7.89 |
| 5 | |
50 | 2 | 50 | 2 | 50 | 93 | 40.19 | 4.46 |
| 6 | |
50 | 2 | 50 | 2 | 55 | 91 | 41.35 | 3.16 |
By last two tables as can be known, formic acid is suitable as sour solvent effect as solvent effect and dichloro acetic acid, and is quite a lot of a little to a certain degree, and the formic acid price is well below dichloro acetic acid, so recently weigh with performance and price, and formic acid is better than dichloro acetic acid.
4.. the atlas analysis of sulfated chitosan (CSB)
(1). the infrared spectrogram analytical results of sulfated chitosan (CSB):
Infrared spectra by Fig. 1 chitosan raw material is found out 3435cm
-1Be the imido grpup stretching vibration, 2917cm
-1And 2874cm
-1Be respectively-CH
3,-CH
2Stretching vibration, 1659,1597cm
-1Be acid amides I band, acid amides II band, 1379cm
-1Be-CH
2Out-of-plane deformation vibration, 1076cm
-1Be the stretching vibration of ehter bond (c-o-c), in addition, 897cm
-1, 930cm
-1, 750cm
-1The place illustrates that chitosan is with β-1.4 glycosidic link banded polymer, The results of FT-IR consistent (Li Jihang, 1997 with reported in literature a little less than absorbing; It is colored that the king changes, and 1997; Week is red, and 1996).
Sulfonated product IR sees Fig. 2, compares with Fig. 1, at 3429cm
-1, 2923cm
-1, 1629cm
-1, 1535cm
-1, 1062cm
-1The characteristic peak that the chitosan skeleton occurred, and at 1257cm
-1, 803cm
-1, 1383cm
-1Three peaks appear in the place, are respectively Vs (s=0), V (s-o-c) and Vas (s=0), and infrared spectra consistent with the reference collection of illustrative plates (A.M.Naggi, 1985) has been introduced sulfate group on chitosan molecule behind the proved response.
(2). sulfated chitosan (CSB)
13C-NMR spectroscopic analysis result:
As can be seen from Figure 3, at the C of chitosan
6Sulfate group (-OSO is introduced in the position
3 -) after, because the effect of deshielding of sulfate group makes C
6Chemical shift obviously drift to 69.3ppm, C by 62.85ppm to low field
3Chemical shift obviously drift to 78.1ppm, C by 75.3ppm to low field
2Chemical shift obviously drift to 58.2ppm to low by 56.7ppm, show successful sulfate group is incorporated on the chitosan of this reaction, at 100.45ppm, 77.78ppm 75.40ppm locates to occur respectively C-1, C-4, the C-5 signal, this three sites chemical shift is almost constant.
Claims (2)
1, a kind of preparation method who prevents and treats the medical compounds of diabetes, it is characterized in that: the preparation process of described CSA is as follows:
(1) preparation of esterifying reagent: with N, N-dimethyl formamide is inserted in the there-necked flask, after cryosel is bathed cooling, stirs and slowly drips chlorsulfonic acid down, N, the volume ratio of N-dimethyl formamide and chlorsulfonic acid remains temperature of reaction system less than 5 ℃ for (5:1)-(10:1);
(2) take by weighing quantitative chitosan raw material;
(3) add 2% acetic acid solution, 2% acetic acid solution with the volume/weight ratio of (2) middle chitosan is: (6:1)-(30:1), this raw material is fully dissolved;
(4), be put in the microwave oven reacting by heating number minute with above-mentioned abundant dissolved raw material;
(5) with the raw material of above-mentioned reacting by heating, be neutralized to neutrality with the NaOH solution of 2N, place for some time again;
(6) will be neutralized to the above-mentioned feed liquid of neutral and carry out suction filtration;
(7) get filter cake and dry, the faint yellow solid material of gained; Pulverize this material of oven dry again, survey its molecular weight, this material is low-molecular-weight chitin oligosaccharide;
(8) get the product that above-mentioned pulverizing is dried, add formamide solvent, formamide solvent with the volume/weight ratio of oven dry product is: (15:1)-(50:1);
(9) with said mixture, after stirring, add formic acid or dichloro acetic acid, the dense amount of formic acid of adding 88% and the volume ratio of the mixture in (8) are: (1:3)-(1:10), after mixing;
(10) add 50～100ml sulphonating agent DMFSO again
3, after stirring;
(11) again under 40～60 ℃ of temperature, stirring reaction 1～2h;
(12) above-mentioned reaction solution is added 95% ethanol of 3 times of volumes again, when a large amount of white flocks occurring, place heavyization 30min in 4 ℃ of refrigerators, the throw out that obtains promptly is the thick product of CSA;
(13) above-mentioned throw out is carried out suction filtration, get filter cake;
(14) above-mentioned filter cake is used water dissolution again, the NaOH solution that adds 2N then is neutralized to neutrality;
(15) above-mentioned solution is dialysed with dialysis tubing, get dialyzate behind the desalination; Or use the hyperfiltration process desalination, get and hold back solution;
(16) again with above-mentioned solution, be concentrated into 1/10 volume;
(17) above-mentioned concentrated solution is put in the refrigerator-freezer freezes, lyophilize gets faint yellow cotton shape material then;
(18) pulverize this cotton shape material, measure its sulphur content and molecular weight, the CSA of getting lower molecular weight up to specification 1～6 KD is as preventing and treating the medicine of diabetes.
2, according to the preparation method of the described control diabetes medicament of claim 1, it is characterized in that: described (4) are reacted to 100 ℃ in microwave oven, the reaction times: 3～6min; The molecular weight of the faint yellow solid material in described (7) is 9.21 * 10
3～3.86 * 10
4Dalton; Stirring reaction in described (11), synthesis under normal pressure under air atmosphere; Dialysis tubing in described (15) is that the dialyzate that intercepts is a product of holding back 1～6KD molecular weight size with 1 and two kinds of film bags of 8KD.
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| CN105968231B (en) * | 2016-07-21 | 2019-11-12 | 中国科学院海洋研究所 | A kind of different molecular weight β-C-2,3,6- sulfated chitosan and preparation method thereof |
| CN106188339A (en) * | 2016-07-21 | 2016-12-07 | 中国科学院海洋研究所 | A kind of immunostimulant and preparation method thereof |
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Non-Patent Citations (1)
| Title |
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| Effect of sulphated derivatives of chitosan on lipoproteinlipaseactivity of rabbit plasma after their intravenous injection. Shigehiro Hirano, Junko Kinugawa.Carbahydrate Research,Vol.150 No.1. 1986 * |
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