CN1309731C - Poria cocos mycellium glucan sulphate derivative and its preparation method and use - Google Patents
Poria cocos mycellium glucan sulphate derivative and its preparation method and use Download PDFInfo
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- CN1309731C CN1309731C CNB2005100191813A CN200510019181A CN1309731C CN 1309731 C CN1309731 C CN 1309731C CN B2005100191813 A CNB2005100191813 A CN B2005100191813A CN 200510019181 A CN200510019181 A CN 200510019181A CN 1309731 C CN1309731 C CN 1309731C
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Abstract
The present invention relates to a poria cocos mycellium glucan sulphate esterification derivative. The present invention is prepared through the following steps: poria cocos mycellium is extracted, which leads water-insoluble alpha-glucan to be obtained; the alpha-glucan is suspended in anhydrous N, N-dimethylformamide, and pyridine and chlorosulfonic acid are orderly added; reaction solution reacts for 40-120 minutes in the temperature of 50 to 90 DEG C and is cooled to the room temperature; distilled water is added, and clear solution is formed; the pH value is adjusted to 9-10; precipitating agent alcohol is added, and the sulphate esterification derivative of the alpha-glucan is obtained. The sulphate esterification reaction of the alpha-glucan mainly happens at a C-6-position hydroxyl group, the substitution degree is from 0.86 to 1.38, the weight average molecular weight is from 2.36*10< 4 > to 14.5*10< 4 >, and good water solubility can be obtained. Through the proof of experimental results inside a body and outside a body, the sulphate esterification derivative of the alpha-glucan has the obvious braking effect on a sarcoma 180 implanted in the body of a BALB/c mouse. The present invention can be used for preparing anticancer drugs and health products for enhancing immunity of a human body.
Description
Technical field
The present invention relates to poria cocos mycellium glucan sulphate derivative that has anti-tumor activity and its production and use.It belongs to chemical field, also belongs to biology field.
Background technology
Recent study shows that fungus polysaccharide has pharmaceutical use widely, as antitumor, antiviral, antibiotic, anti-infective, anti-peptic ulcer, anti-oxidant, radioprotective, anti-aging, hypoglycemic blood fat etc.; As immunomodulator, panimmunity cells such as activating macrophage, T lymphocyte, LAK cell promote cytokine to generate complement activation, and toxic side effect is low, safe, so STUDY ON POLYSACHAROSE has been become the popular domain of contemporary biology, chemistry.Yet the main component of extracting from Poria mycelium is (1 → 3)-α-D-dextran, its water insoluble and lifeless matter activity almost, thereby do not have using value.
Summary of the invention
Problem to be solved by this invention provides a kind of poria cocos mycellium glucan sulphate derivative with anti-tumor activity and its production and use, the water-insoluble alpha-glucan that soon extracts from Poria mycelium obtains water-soluble polysaccharide by the sulfuric ester derivatize, this poria cocos mycellium glucan sulphate derivative has anti-tumor activity, and the preparation method is simple, with low cost, can be used for preparing the healthcare products of antitumor drug or enhance immunity function.
For achieving the above object, the technical solution adopted in the present invention is as follows:
A kind of poria cocos mycellium glucan sulphate derivative is made by laxative remedy: Poria cocos (Poria cocos) mycelium is carried out Soxhlet with ethyl acetate, acetone successively extract except that fat; Pass through physiological saline, hot water extraction then successively, centrifugal; Centrifugal back residuum 0.5-1.0M NaOH/0.05-0.1wt%NaBH
4Solution extracts down at 5-10 ℃ and obtains weight-average molecular weight M
WBe 7.75 * 10
4-57.3 * 10
4Water-insoluble alpha-glucan; Described alpha-glucan is suspended in anhydrous N, in the dinethylformamide (DMF), stirred 12-24 hour down, add pyridine again, be warming up to 50-90 ℃ and continue to stir 0.5-1 hour at 20-50 ℃; Then under 50-90 ℃ and lasting agitation condition, dropwise add chlorsulfonic acid with the speed of 0.4-0.6mL/min, wherein the mol ratio of dextran hydroxyl and chlorsulfonic acid, pyridine is 1: 5-7: 10-17.5; 40-120 minute postcooling of reaction is to room temperature down at 50-90 ℃ for reaction solution, and adding distilled water is regulated pH to 9-10 to form limpid solution, adds the thick product that precipitation agent ethanol obtains the sulphating derivative then; Should being dissolved in the distilled water by thick product again, injecting the Mierocrystalline cellulose dialysis tubing, is that pyridine is removed in dialysis in the NaOH solution of 9-11 at pH, uses distill water dialysis 4-10 days then, and lyophilize obtains poria cocos mycellium glucan sulphate derivative.
The sulfate group substitution value of above-mentioned poria cocos mycellium glucan sulphate derivative is 0.86-1.38, and weight-average molecular weight is 2.36 * 10
4-14.5 * 10
4
The application of above-mentioned poria cocos mycellium glucan sulphate derivative in the healthcare products of preparation antitumor drug or enhance immunity function.
Take into account laser light scattering instrument with infrared spectra, nuclear magnetic resonance spectrum, ultimate analysis, viscosity and determined its chemical constitution and secondary structure.The sulfuric acid esterification that the nuclear-magnetism result illustrates this alpha-glucan belongs to non-selective replacement, mainly occurs in C-6 position hydroxyl, and substitution value is 0.86-1.38, weight-average molecular weight (M
W) be 2.36 * 10
4-14.5 * 10
4
The poria cocos mycellium glucan sulphate derivative of different molecular weight the results are shown in subordinate list 1 to the in-vitro multiplication inhibiting rate of human hepatoma cell HepG2 and Sarcoma180 sarcoma.Obviously, all poria cocos mycellium glucan sulphate derivatives all have the remarkable vitro restraining effect, and inhibiting rate has tangible time, dose-dependence, illustrate that it has stable curative effect.Sulphating derivative that it should be noted that intermediate molecular weight shows higher anti tumor activity in vitro.In vivo test is the result also illustrate, and the sulphating derivative of all alpha-glucanes all has significant inhibition effect to implanting intravital Sarcoma 180 sarcomas of BALB/c mouse, the results are shown in subordinate list 2.The sulphating derivative (6 * 10 of intermediate molecular weight
4-10 * 10
4) show the highest anti-tumor in vivo activity, and compare body with five Fluracils and have no side effect.Therefore, this poria cocos mycellium glucan sulphate derivative is with a wide range of applications as the healthcare products of cancer therapy drug and enhancing body immunity.
Compare with existing technology, innovation of the present invention is as follows:
Poria cocos mycellium glucan sulphate derivative provided by the invention is a kind of brand-new material, has in the remarkable body, anti tumor activity in vitro, and body is had no side effect, and especially weight average molecular weight range is 6 * 10
4-10 * 10
4The sulfate derivative body in, extracorporeal inhibiting rate is near five Fluracils, but do not have its such toxic side effect.Therefore, this poria cocos mycellium glucan sulphate derivative can be made into the healthcare products of antitumor drug or enhancing body immunologic function.The main component alpha-glucan that employing is extracted from Poria mycelium is a raw material, and that water is insoluble by the method for sulphating, the active dextran of lifeless matter changes into is water miscible, notable biological activity, narrow dispersive dextran are arranged.This method is simple, and cost is low, therefore has broad application prospects.
Embodiment
Below in conjunction with concrete example technical scheme of the present invention is described further:
Embodiment 1
Poria mycelium (Hua Zhong Agriculture University provides, bacterium numbering P0) is carried out Soxhlet with ethyl acetate, acetone successively to be extracted except that fat; Pass through physiological saline, hot water extraction then successively, centrifugal; Centrifugal back residuum 0.5NaOH/0.05wt%NaBH
4Solution extracts down at 5 ℃ and obtains water-insoluble alpha-glucan.Adopt the reprecipitation stage division that this alpha-glucan is carried out classification: this alpha-glucan is dissolved in 0.25M LiCl/Me
2Among the SO, under 25 ℃ of conditions with 4: 1 acetone of volume ratio and 0.25M LiCl/Me
2The SO mixing solutions is that precipitation agent slowly is added drop-wise in the alpha-glucan solution up to solution appearance muddiness; Under agitation this turbid solution is warming up to 50 ℃, naturally be cooled to 25 ℃, and kept 12 hours, then this turbid solution centrifugation is gone out first fraction, supernatant liquor carries out classification next time by above-mentioned reprecipitation stage division, through 9 classifications, obtain 9 fraction F1-F9 of alpha-glucan successively.
With 200mg alpha-glucan fraction F2 (M
w=45.2 * 10
4) be suspended in the anhydrous N of 10mL, in the dinethylformamide (DMF), under 50 ℃, stir and spend the night, property adding pyridine 3mL is warming up to 60 ℃ and continues to stir 30 minutes again; Then under 60 ℃ and lasting agitation condition, dropwise slowly add chlorsulfonic acid 1.25mL with the speed of 0.6mL/min, wherein dextran hydroxyl and chlorsulfonic acid, the mol ratio of pyridine is 1: 5: 10; 70 minutes postcooling of reaction are to room temperature down at 60 ℃ for reaction solution, and adding distilled water is regulated pH to 10 to form limpid solution, adds the thick product that precipitation agent ethanol obtains the sulphating derivative then; Be dissolved in thick product in the distilled water again, injecting the Mierocrystalline cellulose dialysis tubing, is that pyridine is removed in dialysis in the NaOH solution of 9-10 at pH, uses distill water dialysis then 7 days, lyophilize obtains the cotton-shaped poria cocos mycellium glucan sulphate derivative of white, is numbered S2.Sample S2 has well water-soluble, and substitution value is 0.89, and weight-average molecular weight is 9.10 * 10
4To be 0.5mg/mL in concentration be respectively 36.6% and 44.9% to the in-vitro multiplication inhibiting rate of human hepatoma cell HepG2 and Sarcoma 180 sarcomas to sample S2.When sample S2 was 20mg/kg at dosage, it was 52.0% to the inhibiting rate of implanting intravital Sarcoma 180 sarcomas of BALB/c mouse, and body is had no side effect.Therefore, but this poria cocos mycellium glucan sulphate derivative S2 anticancer not only, but and enhancing body's immunological function.
Embodiment 2
200mg is pressed the foregoing description 1 fractionated sample F3 (M
w=37.5 * 10
4) be suspended in the anhydrous N of 10mL, in the dinethylformamide (DMF), under 40 ℃, stir and spend the night, property adding pyridine 3mL is warming up to 70 ℃ and continues to stir 30 minutes again; Then under 70 ℃ and lasting agitation condition, dropwise slowly add chlorsulfonic acid 1.25mL with the speed of 0.4mL/min, wherein dextran hydroxyl and chlorsulfonic acid, the mol ratio of pyridine is 1: 7: 10; 60 minutes postcooling of reaction are to room temperature down at 70 ℃ for reaction solution, and adding distilled water is regulated pH to 10 to form limpid solution, adds the thick product that precipitation agent ethanol obtains the sulphating derivative then; Be dissolved in thick product in the distilled water again, injecting the Mierocrystalline cellulose dialysis tubing, is that pyridine is removed in dialysis in the NaOH solution of 9-10 at pH, uses distill water dialysis then 6 days, lyophilize obtains the cotton-shaped poria cocos mycellium glucan sulphate derivative of white, is numbered S3.Sample S3 has well water-soluble, and substitution value is 0.98, and weight-average molecular weight is 6.88 * 10
4To be 0.5mg/mL in concentration be respectively 50.7% and 46.6% to the in-vitro multiplication inhibiting rate of human hepatoma cell HepG2 and Sarcoma 180 sarcomas to sample S3.When sample S3 was 20mg/kg at dosage, it was 50.0% to the inhibiting rate of implanting intravital Sarcoma 180 sarcomas of BALB/c mouse, and body is had no side effect.Therefore, but this poria cocos mycellium glucan sulphate derivative S3 anticancer not only, but and enhancing body's immunological function.
Can obtain the poria cocos mycellium glucan sulphate derivative S1-S9 of 9 fraction F1-F9 of corresponding alpha-glucan with reference to embodiment 2,3.The results of FT-IR illustrates, and sulphating derivative S1-S9 is 1260 and 820cm
-1S=O and C-O-S stretching vibration peak appear respectively in the place.Nuclear-magnetism is the result illustrate, and the chemical shift that replaces carbon is moved 6-10ppm relative to what do not replace carbon to low field; The sulfuric acid esterification of this alpha-glucan belongs to non-selective replacement, mainly occurs in C-6 position hydroxyl.Ultimate analysis and laser light scattering instrument result illustrate, and substitution value is 0.86-1.38, and weight-average molecular weight is 2.36 * 10
4-14.5 * 10
4
The poria cocos mycellium glucan sulphate derivative of subordinate list 1. different molecular weights is the in-vitro multiplication inhibiting rate of 0.5mg/mL to human hepatoma cell HepG2 and Sarcoma 180 sarcomas in concentration
| The sulphating derivative | Inhibiting rate (%) | |
| Human hepatoma cell HepG2 | Sarcoma 180 sarcomas | |
| S1 S2 S3 S4 S5 S6 | 32.8 36.6 50.7 50.6 50.1 44.1 | 41.7 44.9 46.6 49.9 43.2 46.7 |
The poria cocos mycellium glucan sulphate derivative of subordinate list 2. different molecular weights is to implanting the inhibiting rate of intravital Sarcoma 180 sarcomas of BALB/c mouse
| The sulphating derivative | Molecular weight (M w× 10 -4 | Dosage (mg/kg * days) | Inhibiting rate (%) | Body weight gain rate (%) |
| The negative positive group of group of S1 S2 S3 S4 S5 S6 (5-Fluorouracil) | 14.5 9.10 6.88 4.71 3.50 2.36 | 20×8 20×8 20×8 20×8 20×8 20×8 20×8 20×8 | 31.4 52.0 50.0 40.8 31.2 21.2 46.8 | 22.9 25.7 24.9 19.6 27.4 34.0 38.6 11.1 |
Claims (4)
1. a poria cocos mycellium glucan sulphate derivative is made by laxative remedy: Poria mycelium is carried out Soxhlet with ethyl acetate, acetone successively extract except that fat; Pass through physiological saline, hot water extraction then successively, centrifugal; Centrifugal back residuum 0.5-1.0M NaOH/0.05-0.1wt%NaBH
4Solution extracts under 5-10 ℃ and obtains weight-average molecular weight is 7.75 * 10
4-57.3 * 10
4Water-insoluble alpha-glucan; Described alpha-glucan is suspended in anhydrous N, in the dinethylformamide, stirred 12-24 hour down, add pyridine again, be warming up to 50-90 ℃ and continue to stir 0.5-1 hour at 20-50 ℃; Then under 50-90 ℃ and lasting agitation condition, dropwise add chlorsulfonic acid with the speed of 0.4-0.6mL/min, wherein the mol ratio of dextran hydroxyl and chlorsulfonic acid, pyridine is 1: 5-7: 10-17.5; 40-120 minute postcooling of reaction is to room temperature down at 50-90 ℃ for reaction solution, and adding distilled water is regulated pH to 9-10 to form limpid solution, adds the thick product that precipitation agent ethanol obtains the sulphating derivative then; Should being dissolved in the distilled water by thick product again, injecting the Mierocrystalline cellulose dialysis tubing, is that pyridine is removed in dialysis in the NaOH solution of 9-11 at pH, uses distill water dialysis 4-10 days then, and lyophilize obtains poria cocos mycellium glucan sulphate derivative.
2. by the described poria cocos mycellium glucan sulphate derivative of claim 1, it is characterized in that: the sulfate group substitution value of poria cocos mycellium glucan sulphate derivative is 0.86-1.38, and weight-average molecular weight is 2.36 * 10
4-14.5 * 10
4
3. the preparation method of claim 1 or 2 described poria cocos sclerotium dextran sulfates is characterized in that: Poria mycelium is carried out Soxhlet with ethyl acetate, acetone successively extract except that fat; Pass through physiological saline, hot water extraction then successively, centrifugal; Centrifugal back residuum 0.5-1.0M NaOH/0.05-0.1wt%NaBH
4Solution extracts under 5-10 ℃ and obtains weight-average molecular weight is 7.75 * 10
4-57.3 * 10
4Water-insoluble alpha-glucan; Described alpha-glucan is suspended in anhydrous N, in the dinethylformamide, stirred 12-24 hour down, add pyridine again, be warming up to 50-90 ℃ and continue to stir 0.5-1 hour at 20-50 ℃; Then under 50-90 ℃ and lasting agitation condition, dropwise add chlorsulfonic acid with the speed of 0.4-0.6mL/min, wherein the mol ratio of dextran hydroxyl and chlorsulfonic acid, pyridine is 1: 5-7: 10-17.5; 40-120 minute postcooling of reaction is to room temperature down at 50-90 ℃ for reaction solution, and adding distilled water is regulated pH to 9-10 to form limpid solution, adds the thick product that precipitation agent ethanol obtains the sulphating derivative then; Should being dissolved in the distilled water by thick product again, injecting the Mierocrystalline cellulose dialysis tubing, is that pyridine is removed in dialysis in the NaOH solution of 9-11 at pH, uses distill water dialysis 4-10 days then, and lyophilize obtains poria cocos mycellium glucan sulphate derivative.
4. claim 1 or the 2 described poria cocos mycellium glucan sulphate derivatives application in the healthcare products of preparation antitumor drug or enhance immunity function.
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| CNB2005100191813A CN1309731C (en) | 2005-07-29 | 2005-07-29 | Poria cocos mycellium glucan sulphate derivative and its preparation method and use |
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| CNB2005100191813A CN1309731C (en) | 2005-07-29 | 2005-07-29 | Poria cocos mycellium glucan sulphate derivative and its preparation method and use |
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| CN1309731C true CN1309731C (en) | 2007-04-11 |
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| CN101250236B (en) * | 2008-04-11 | 2010-09-29 | 武汉大学 | Tuckahoe dextran phosphate esterified derivative as well as preparation and use thereof |
| CN102603910B (en) * | 2012-02-29 | 2013-11-13 | 合肥学院 | Preparation method of pachyman sulfate |
| CN108484792B (en) * | 2018-05-24 | 2020-09-25 | 山东谷雨春生物科技有限公司 | Dextran sulfate and method for preparing dextran sulfate |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS58138392A (en) * | 1982-02-13 | 1983-08-17 | Japan Synthetic Rubber Co Ltd | Beta-d-gulcan |
| CN1124249A (en) * | 1994-12-09 | 1996-06-12 | 沈阳药科大学 | Antineoplastic new poria polysaccharide water soluble derivative |
| CN1583800A (en) * | 2004-06-01 | 2005-02-23 | 武汉大学 | Anti-tumor activating fuling scloerotium glucan sulfate and its preparation and use |
-
2005
- 2005-07-29 CN CNB2005100191813A patent/CN1309731C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS58138392A (en) * | 1982-02-13 | 1983-08-17 | Japan Synthetic Rubber Co Ltd | Beta-d-gulcan |
| CN1124249A (en) * | 1994-12-09 | 1996-06-12 | 沈阳药科大学 | Antineoplastic new poria polysaccharide water soluble derivative |
| CN1583800A (en) * | 2004-06-01 | 2005-02-23 | 武汉大学 | Anti-tumor activating fuling scloerotium glucan sulfate and its preparation and use |
Non-Patent Citations (1)
| Title |
|---|
| 磺酰化新茯苓多糖的制备及抗肿瘤作用 赵吉福等，沈阳药科大学学报，第13卷第2期 1996 * |
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