CN1524869A - Method for extracting anticarcinogenesis polysaccharide composite AVDS from A.valvataD. and its use as medicament - Google Patents
Method for extracting anticarcinogenesis polysaccharide composite AVDS from A.valvataD. and its use as medicament Download PDFInfo
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- CN1524869A CN1524869A CNA031155138A CN03115513A CN1524869A CN 1524869 A CN1524869 A CN 1524869A CN A031155138 A CNA031155138 A CN A031155138A CN 03115513 A CN03115513 A CN 03115513A CN 1524869 A CN1524869 A CN 1524869A
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Abstract
The invention relates to a the use of triterpenoid and polysaccharide composite (AVDS) extracted from traditional Chinese herbal cat gen-seng in preparing medicament with anticancer action, the polysaccharide composite has the actions of killing tumour cell and immunity regulation, thus can be used for the preparation with anticancer actions, including oral administration preparation, injection, fluid infusion agent, percutaneous absorption, and mucosa absorption dosage pattern.
Description
Technical field
The present invention discloses a kind of preparation method of cancer therapy drug, is specifically related to extract polysaccharide compounds (AVDS) such as triterpenoid, saccharide complex and has application in the medicine of antitumous effect in preparation from the Chinese medicine Root of Valvate Actinidia.
Background technology
Root of Valvate Actinidia's (another name: cat gas rattan, sand pear rattan, glutinous rice rattan), Actinidia Vaivata Dunn puts down in writing according to the Chinese medicine dictionary: have antipyretic and antidote functions, can control carbuncle furuncle, leukorrhea, leprosy.Also there is report can control pulmonary abscess, the report of treatment cancer is also arranged in recent years.Many reports that this other Chinese medicine of medicine compatibility are used for clinical treatment tumour are arranged.However, up to the present, yet there are no that relevant single Root of Valvate Actinidia extracts triterpene compound, saccharide complex (AVDS) also claims Root of Valvate Actinidia's polysaccharide compound, Root of Valvate Actinidia's glycopeptide to be prepared into the report of pharmaceutical preparation.
The pernicious place of cancer cells mainly is that propagation has broken away from normal control with apoptosis, and can attack healthy tissues and distant metastasis takes place, and cancer why constantly grow up rapidly by the fatal cancer cells that mainly do not lie in, and be its aggressive and transitivity.The whole process of cancer metastasis comes down to cancer cells and cancer cells, cancer cells and host cell (for example blood vessel and lymph endotheliocyte, thrombocyte and immunocyte etc.), and cancer cells and the interactional a series of complex processes of extracellular matrix molecule (ln in the basilar membrane).Interaction between the cell is selectively, rather than at random.Cell is distinguished the cell of having an effect and not having an effect by its recognition capability.This recognition function of cell is mediated by cell surface glycoprotein, and mainly is to be carried out by the sugar chain of cell surface.The free oligosaccharides of structural similitude or glycoconjugate can be blocked the glycoprotein of cell surface or the biological action that sugar chain participated on the glycolipid.Therefore develop polysaccharide compound, killing tumor cell and raising human immunity regulatory function are had great importance.
Summary of the invention
The object of the present invention is to provide a kind of method of from the Root of Valvate Actinidia, extracting polysaccharide compound AVDS, and propose its new purposes in the treatment cancer with antitumous effect.According to the bioactive ingredients-triterpene compound, the saccharide complex (AVDS) that extract is polysaccharide compound, be used to prepare the preparation (comprising formulations such as oral preparations, injection, infusion solution, Transdermal absorption, mucosal absorption) of antitumous effect, in clinical use, can dwindle dosage, improve curative effect, the convenient use.
The objective of the invention is to realize by following technological method: get Root of Valvate Actinidia's pharmaceutical decocting piece, be ground into coarse particles (crossing 20 mesh sieves), add 8-10 and doubly measure 70% ethanol, use alcohol heat reflux 2 times, 3-4h/ time, united extraction liquid; High speed centrifugation, 5000-16000r/min, impurity elimination in 30 minutes is reclaimed ethanol, concentrated extracting solution; Macroporous resin on the concentrated solution with distilled water 45%-55% ethanol wash-out successively, reclaims ethanol and concentrates; Concentrated solution 50%-70% ethanol gradient elution, elutriant reclaims ethanol, vacuum concentration, drying gets Root of Valvate Actinidia's triterpene compound.
Root of Valvate Actinidia's residue behind alcohol reflux, the chopping back is boiled with boiling water and is carried 2 times, each 3-4h, united extraction liquid, extracting solution adds 0.3-0.5% amylase, 0.01-0.02 proteolytic enzyme, 40 ℃ of insulations of 0.01-0.02% polygalacturonase 4-5h, high speed centrifugation (5000-16000r/nim) 30-45 minute discards insolubles; Supernatant liquor is crossed macroporous resin column, and effluent liquid carries out ultrafiltration and concentration with polysulfone membrane, adds 95% ethanol after concentrating and makes pure content reach 40-50%, and high speed centrifugation (5000-16000r/min) 0.5-1h gets and washes the shallow lake; The throw out water dissolution makes pure content reach 80% with 95% ethanol again, high speed centrifugation (5000-16000r/min) 0.5-1h; Get precipitation, throw out is used 95% ethanol respectively, dehydrated alcohol, acetone wash-out, and drying gets Root of Valvate Actinidia's saccharide complex.Together constitute Root of Valvate Actinidia's complex polysaccharide (AVDS) with Root of Valvate Actinidia's triterpene compound again, also claim Root of Valvate Actinidia's polysaccharide compound, Root of Valvate Actinidia's glycopeptide.
The Chinese medicine Root of Valvate Actinidia extract the polysaccharide compound comprise triterpene compound, saccharide complex (hereinafter to be referred as, AVDS), have killing tumor cell and immunoregulation effect, can prepare preparation (comprising formulations such as oral preparations, injection, infusion solution, Transdermal absorption, mucosal absorption) with antitumous effect.Its mechanism of action is:
1. the glycosylation that AVDS can anticancer suppresses it and shifts synthesizing of relevant sugar chain.Damage cyto-chromatin, influenced duplicating, transcribing of DNA gene, reduced rna content in the endochylema, the cancer cells vigor has been reduced, thereby suppressed cancer cell multiplication, finally caused cancer cell death.
2. AVDS can strengthen splenocyte T, B cell mitogen inductive propagation, and promptly the immunne response ability to T, B cell has promoter action.
3. the phagocytic function that AVDS can activating macrophage.
Disclosed in this invention from the Root of Valvate Actinidia technology of extracting effective components be different from extraction process described in the prior art.Its advantage shows:
1. adopt Amberlyst process and film (ultrafiltration) isolation technique, reduced the usage quantity of organic solvent, not only kept effective constituent to greatest extent, constant, the constant product quality of yield, easy and simple to handle, the effective constituent crystallization degree is good, and effective constituent reclaims fully, and can remove conventional extraction process and be difficult to isolating impurity, bacterium and bacterial endotoxin (pyrogen);
2. adopt separation " enzyme " technology, improved separating effect;
3. adopt high-speed centrifugation technology, replaced traditional ethanol freezing precipitation, make technical process (time) shorten;
4. reduced cost, improved the security of producing, for the safety in production of medicine provides reliable assurance.
Description of drawings
Fig. 1 is a process flow sheet of the present invention
Embodiment
Below in conjunction with embodiment the present invention is further described.
Embodiment 1 gets Root of Valvate Actinidia's pharmaceutical decocting piece, is ground into coarse particles (crossing 20 mesh sieves), adds 8 times of amount 70% ethanol, uses alcohol heat reflux 2 times, and 3h/ time, united extraction liquid; High speed centrifugation, 16000r/min, impurity elimination in 30 minutes is reclaimed ethanol, concentrated extracting solution; Macroporous resin on the concentrated solution with distilled water 45%, 55% ethanol wash-out successively, reclaims ethanol and concentrates; Concentrated solution reclaims ethanol with 50%, 70% ethanol gradient elution, elutriant, vacuum concentration, and drying gets Root of Valvate Actinidia's triterpene compound.
Detect: carry out the wine with dregs column chromatography, with chloroform one methyl alcohol gradient elution, the thin-layer chromatography with preparation separates (CHCI then
3-MeOH=9: 1) get two kinds of whitening compounds, be positive, be shown triterpene through the Liebermann-Burchard reaction.
Root of Valvate Actinidia's residue behind alcohol reflux, the chopping back is boiled with boiling water and is carried 2 times, each 3h, united extraction liquid, extracting solution add 0.5% amylase, 0.01 proteolytic enzyme, 40 ℃ of insulations of 0.01% polygalacturonase 4h, high speed centrifugation (16000r/nim) 30 minutes discards insolubles; Supernatant liquor is crossed macroporous resin column, and effluent liquid carries out ultrafiltration and concentration with polysulfone membrane, adds 95% ethanol after concentrating and makes pure content reach 50%, and high speed centrifugation (16000r/min) 0.75h gets and washes the shallow lake; The throw out water dissolution makes pure content reach 80% with 95% ethanol again, high speed centrifugation (16000r/min) 0.75h; Get precipitation, throw out is used 95% ethanol respectively, dehydrated alcohol, acetone wash-out, and drying gets Root of Valvate Actinidia's complex polysaccharide (AVDS).
Detect: anthrone one sulfuric acid process.Collect single sugared peak, the chromatographic peak retention value of sample and standard monose is contrasted to determine the kind of monose in the sample.Adopt the relative percentage composition of chromatographic peak area normalization method working sample monose.
The effective constituent that Root of Valvate Actinidia of the present invention extracts, triterpene compound, saccharide complex be preparation separately, also can be with the effective constituent of Root of Valvate Actinidia's extraction, the activeconstituents compatibility of triterpene compound, saccharide complex and other traditional Chinese medicine extraction is made compound preparation.As, biologically active polypeptides-r-hirudin that Root of Valvate Actinidia's polysaccharide compound and Chinese traditional medicine leech are extracted, the compound preparation with antitumous effect of the compatible preparation of Unidasa (comprising formulations such as oral preparations, injection, infusion solution, Transdermal absorption, mucosal absorption).Wherein said leech is biologically active polypeptides-r-hirudin, and molecular weight is about 7000, and the hyaluronic acid enzyme molecular weight is about 28500.
Embodiment 2 gets Root of Valvate Actinidia's pharmaceutical decocting piece, is ground into coarse particles (crossing 20 mesh sieves), adds 9 times of amount 70% ethanol, uses alcohol heat reflux 2 times, and 4h/ time, united extraction liquid; High speed centrifugation, 8000r/min, impurity elimination in 40 minutes is reclaimed ethanol, concentrated extracting solution; Macroporous resin on the concentrated solution with distilled water 45%, 55% ethanol wash-out successively, reclaims ethanol and concentrates; Concentrated solution reclaims ethanol with 50%, 70% ethanol gradient elution, elutriant, vacuum concentration, and drying gets Root of Valvate Actinidia's triterpene compound.
Detect: carry out the wine with dregs column chromatography, with chloroform one methyl alcohol gradient elution, the thin-layer chromatography with preparation separates (CHCI then
3-MeOH=9: 1) get two kinds of whitening compounds, be positive, be shown triterpene through the Liebermann-Burchard reaction.
Root of Valvate Actinidia's residue behind alcohol reflux, the chopping back is boiled with boiling water and is carried 2 times, each 3h, united extraction liquid, extracting solution add 0.5% amylase, 0.015 proteolytic enzyme, 40 ℃ of insulations of 0.015% polygalacturonase 5h, high speed centrifugation (8000r/nim) 30 minutes discards insolubles; Supernatant liquor is crossed macroporous resin column, and effluent liquid carries out ultrafiltration and concentration with polysulfone membrane, adds 95% ethanol after concentrating and makes pure content reach 50%, and high speed centrifugation (8000r/min) 0.5h gets and washes the shallow lake; The throw out water dissolution makes pure content reach 80% with 95% ethanol again, high speed centrifugation (8000r/min) 0.5h; Get precipitation, throw out is used 95% ethanol respectively, dehydrated alcohol, acetone wash-out, and drying gets Root of Valvate Actinidia's complex polysaccharide (AVDS).
Detect: anthrone one sulfuric acid process.Collect single sugared peak, the chromatographic peak retention value of sample and standard monose is contrasted to determine the kind of monose in the sample.Adopt the relative percentage composition of chromatographic peak area normalization method working sample monose.
The AVDS that the present invention extracts is to rat liver cancer and S
180The tumor-inhibiting action of solid tumor
1. rat liver cancer, S
180The preparation of oncocyte suspension
Choose and inoculate after the liver cancer 8～10 days, tumor-bearing mice that tumor growth is good, on Bechtop, operate, the cervical vertebra dislocation is put to death, after the skin of abdomen sterilization, extract seroperitoneum (the dense magma of oyster white) and insert in the disinfectant beaker, dilute by 1: 1 with physiological saline and make liver cancer seroperitoneum suspension and S
180The seroperitoneum suspension.
2. inhibition test
2.1 AVDS is to the inhibition test of rat liver cancer
Get 60 of Kunming mouses, male and female half and half, body weight (20 ± 2) g, with the liver cancer seroperitoneum suspension that has prepared, every mouse armpit subcutaneous vaccination 0.2ml is divided into 5 group: AVDS100mg/ (kgd) at random after the inoculation, 50mg/ (kgd), 25mg/ (kgd) group, promptly high, in, low 3 dosage groups, other establishes endoxan positive controls 50mg/ (kgd) and liver cancer model control group, every group 12, behind the inoculation 24h, administered intramuscular, every day 1 time, successive administration 10 days, the liver cancer control group such as awards at the physiological saline of capacity, behind last administration 24h mouse is weighed, and puts to death animal, claim knurl, calculate the tumour inhibiting rate of each group.The average knurl of tumour inhibiting rate=(the average ratio of outflow of the average knurl weight-test group of control group)/control group is heavy by * 100%, takes statistics to learn with the t check and handles.
2.2 AVDS is to mouse S
180Inhibition test
Get 60 of Kunming mouses, male and female half and half, body weight (20 ± 2) g is with the S that has prepared
180The seroperitoneum suspension, every mouse armpit subcutaneous vaccination 0.2ml is divided into 5 groups at random after the inoculation: AVDS dosage is 100mg/ (kgd), 50mg/ (kgd), 25mg/ (kgd), promptly high, medium and low 3 dosage groups, endoxan positive controls 50mg/ (kgd) and S
180Model control group.All the other working method are with the inhibition test of rat liver cancer.
Respectively organize mouse down 2.3 get 2.2 tests, cut open and get thymus gland, spleen, calculate thymus index and spleen index.Heavy (the mg)/body weight (g) of thymus gland (spleen) index=thymus gland (spleen).
3. result
3.1 AVDS the results are shown in Table 1 to the inhibition test of rat liver cancer, high, medium and low three the dosage groups of AVDS are respectively 55.49%, 53.54% and 40.17% to the tumour inhibiting rate of rat liver cancer, and endoxan group tumour inhibiting rate is 65.30%.AVDS height, middle dosage group and endoxan group and model control group (physiological saline) relatively have significant difference (P＜0.01); AVDS small dose group and model control group (physiological saline) relatively have notable difference (P＜0.05).The result shows that AVDS has tumor-inhibiting action to rat liver cancer.
Table 1 AVDS is to the tumor-inhibiting action of rat liver cancer
The heavy tumour inhibiting rate of the average knurl of experiment grouping dosage number of mice
(mg/kgd) (only) (x ± S, g) (%)
Model control group 12 2.049 ± 0.612
Endoxan group 50 12 0.711 ± 0.158 65.30
The heavy dose of group 100 12 0.912 ± 0.365 55.49 of AVDS
Dosage group 50 12 0.952 ± 0.354 53.54 among the AVDS
AVDS small dose group 25 12 1.226 ± 0.189 40.17
Annotate: compare with model control group, be P＜0.05, be P＜0.01
3.2 AVDS is to mouse S
180Inhibition test the results are shown in Table 2, high, medium and low three the dosage groups of AVDS are to mouse S
180Tumour inhibiting rate be respectively 51.13%, 50.74% and 41.61%, endoxan group tumour inhibiting rate is 60.44%.AVDS height, middle dosage group and endoxan group and model control group (physiological saline) relatively have significant difference (P＜0.01); AVDS small dose group and model control group (physiological saline) relatively have notable difference (P＜0.05).The result shows that AVDS is to mouse S
180Tumor-inhibiting action is arranged.
Table 2 AVDS is to mouse S
180Tumor-inhibiting action
The heavy tumour inhibiting rate of the average knurl of experiment grouping dosage number of mice
(mg/kgd) (only) (x ± S, g) (%)
Model control group 12 1.764 ± 0.412
Endoxan group 50 12 0.698 ± 0.147 60.44
The heavy dose of group 100 12 0.862 ± 0.268 51.13 of AVDS
Dosage group 50 12 0.869 ± 0.255 50.74 among the AVDS
AVDS small dose group 25 12 1.030 ± 0.189 41.61
Annotate: compare with model control group, be P＜0.05, be P＜0.01
3.3 AVDS is to S
180The result that influences of tumor-bearing mice immune organ weight shows that endoxan causes tumor-bearing mice thymus gland, and the obvious atrophy of spleen relatively has notable difference (P＜0.05) with model control group; AVDS can increase tumor-bearing mice thymus gland, and the weight of spleen relatively has notable difference (P＜0.05) with model control group, sees Table 3.
Table 3 AVDS is to S
180The influence of tumor-bearing mice immune organ weight
Dosage thymus index (mgg
-1) spleen index (mgg
-1)
Group
(mg·kg
-1)
Model control group-1.54 ± 0.36 4.28 ± 0.35
The heavy dose of group 100 1.77 ± 0.25 5.64 ± 0.34 of AVDS
Dosage group 50 1.68 among the AVDS ± 0.27 5.26 ± 0.41
Endoxan group 50 0.92 ± 0.23
Δ3.20 ± 0.28
Δ
AVDS and model control group be P＜0.05 relatively; Endoxan group and model control group are relatively
ΔP＜0.05.
Claims (7)
1, a kind of method of extracting the polysaccharide compound AVDS with antitumous effect from the Root of Valvate Actinidia is characterized in that it comprises extraction triterpene compound and Root of Valvate Actinidia's saccharide complex two portions.
2, the method for claim 1 is characterized in that the method for described extraction triterpene compound is:
A gets Root of Valvate Actinidia's pharmaceutical decocting piece, is ground into coarse particles (crossing 20 mesh sieves), adds 8-10 and doubly measures 70% ethanol, uses alcohol heat reflux 2 times, and 3-4h/ time, united extraction liquid;
B, high speed centrifugation, 5000-16000r/min, impurity elimination in 30 minutes is reclaimed ethanol, concentrated extracting solution;
C, macroporous resin on the concentrated solution with distilled water 45%-55% ethanol wash-out successively, reclaims ethanol and concentrates;
D, concentrated solution 50%-70% ethanol gradient elution, elutriant reclaims ethanol, vacuum concentration, drying gets Root of Valvate Actinidia's triterpene compound.
3, the method for claim 1 is characterized in that the method for described extraction Root of Valvate Actinidia saccharide complex is:
A, the Root of Valvate Actinidia's residue behind alcohol reflux, the chopping back is boiled with boiling water and is carried 2 times, each 3-4h, united extraction liquid,
B, extracting solution add 0.3-0.5% amylase, 0.01-0.02 proteolytic enzyme, and 40 ℃ of insulations of 0.01-0.02% polygalacturonase 4-5h, high speed centrifugation (5000-16000r/nim) 30-45 minute discards insolubles;
C, supernatant liquor is crossed macroporous resin column, and effluent liquid carries out ultrafiltration and concentration with polysulfone membrane, adds 95% ethanol after concentrating and makes pure content reach 40-50%, and high speed centrifugation (5000-16000r/min) 0.5-1h gets and washes the shallow lake;
D, the throw out water dissolution makes pure content reach 80% with 95% ethanol again, high speed centrifugation (5000-16000r/min) 0.5-1h;
E gets precipitation, and throw out is used 95% ethanol respectively, dehydrated alcohol, acetone wash-out, and drying gets Root of Valvate Actinidia's saccharide complex.
F together constitutes Root of Valvate Actinidia's complex polysaccharide (AVDS) with Root of Valvate Actinidia's triterpene compound again, also claims Root of Valvate Actinidia's polysaccharide compound, Root of Valvate Actinidia's glycopeptide.
4, the preparation with antitumous effect that a kind of polysaccharide compound that extracts with the method for claim 1 prepares is characterized in that described preparation can be formulations such as oral preparations, injection, infusion solution, Transdermal absorption, mucosal absorption.
5, a kind of compound of the polysaccharide compound that extracts with the method for claim 1, it is characterized in that it is biologically active polypeptides-r-hirudin that Root of Valvate Actinidia's polysaccharide compound and Chinese traditional medicine leech are extracted, the compound preparation with antitumous effect of the compatible preparation of Unidasa.
6, compound as claimed in claim 5 is characterized in that described leech is biologically active polypeptides-r-hirudin, and molecular weight is about 7000, and the hyaluronic acid enzyme molecular weight is about 28500.
7, compound as claimed in claim 5 is characterized in that described preparation can be formulations such as oral preparations, injection, infusion solution, Transdermal absorption, mucosal absorption.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101323873B (en) * | 2008-07-16 | 2011-08-10 | 珠海市星远科技有限公司 | Ginseng polysaccharide extraction process |
| CN101584718B (en) * | 2008-05-19 | 2012-02-22 | 中国人民解放军第二军医大学 | Antisepalous kiwi fruit leaf extractive as well as preparation method and application thereof |
| CN107205461A (en) * | 2014-07-29 | 2017-09-26 | 韩国食品研究院 | The ginseng source polysaccharide component handled through ferment and preparation for improving immunity function |
-
2003
- 2003-02-24 CN CNA031155138A patent/CN1524869A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101584718B (en) * | 2008-05-19 | 2012-02-22 | 中国人民解放军第二军医大学 | Antisepalous kiwi fruit leaf extractive as well as preparation method and application thereof |
| CN101323873B (en) * | 2008-07-16 | 2011-08-10 | 珠海市星远科技有限公司 | Ginseng polysaccharide extraction process |
| CN107205461A (en) * | 2014-07-29 | 2017-09-26 | 韩国食品研究院 | The ginseng source polysaccharide component handled through ferment and preparation for improving immunity function |
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