CN100417390C - Effective part of sweet wormwood, its preparation method and application thereof in pharmacy - Google Patents
Effective part of sweet wormwood, its preparation method and application thereof in pharmacy Download PDFInfo
- Publication number
- CN100417390C CN100417390C CNB2006100965310A CN200610096531A CN100417390C CN 100417390 C CN100417390 C CN 100417390C CN B2006100965310 A CNB2006100965310 A CN B2006100965310A CN 200610096531 A CN200610096531 A CN 200610096531A CN 100417390 C CN100417390 C CN 100417390C
- Authority
- CN
- China
- Prior art keywords
- sweet wormwood
- effective part
- water
- herba artemisiae
- artemisiae annuae
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a method for using the effective part of sweet wormwood polysaccharide to prepare drug. Wherein, said effective part is extracted from sweet wormwood, whose polysaccharide content is higher than 50% at 5000-50000 molecule weight, as well asisodulcitol, arabinose, xylose, mannose, cerebrose and amylaceum. The invention can be used to prepare the anti-cancer drug or immunity drug.
Description
Technical field
The invention belongs to field of traditional Chinese medicine pharmacy, relate to effective part of sweet wormwood and preparation method thereof and the application in pharmacy.
Background technology
Herba Artemisiae Annuae is the dry aerial parts of feverfew Herba Artemisiae annuae Artemisia annua L..Be conventional Chinese medicine simply, have clearing away summer-heat, remove and steam, the function of preventing the attack (or recurrence) of malaria.Research to Herba Artemisiae Annuae at present mainly concentrates on ter penoids and volatile oil thereof such as arteannuin, and its contained polysaccharide composition is not seen that the research report is arranged.
Summary of the invention
The purpose of this invention is to provide a kind of effective part of sweet wormwood with antitumor action and immunoregulation effect.
Another object of the present invention provides the preparation method of above-mentioned effective part of sweet wormwood.
A further object of the invention provides above-mentioned effective part of sweet wormwood and uses in pharmacy.
The objective of the invention is to realize by following technical measures:
A kind of effective part of sweet wormwood (called after sweet wormwood HQG), this effective part of sweet wormwood obtains by extracting in the Herba Artemisiae Annuae, wherein polyoses content is not less than 50% by weight percentage, polysaccharide molecular weight is 5000-50000, and contains rhamnose, arabinose, xylose, mannose, galactose and six kinds of monosaccharide residues of glucose.
Described effective part of sweet wormwood, this effective part of sweet wormwood prepares by following method:
1) extraction of Herba Artemisiae Annuae crude polysaccharides: get Herba Artemisiae Annuae and decoct with water 2～3 times, each amount of water is 10～15 times of Herba Artemisiae Annuae weight, and each 1～2 hour, extracting solution merged, concentrating under reduced pressure adds ethanol precipitation to containing the alcohol amount more than 85%, leaves standstill, the leaching precipitation is used ethanol Xian Di, the dry Herba Artemisiae Annuae crude polysaccharides that gets;
2) effective part of sweet wormwood is refining: get the Herba Artemisiae Annuae crude polysaccharides, water fully dissolves, concentration is 0 7～1%, the employing cutoff value is that the dialyzer of 3500～5000D was dialysed 48～72 hours with pure water, remove molecular weight 5000 or 3500 following micromolecule, liquid glucose concentrates, and is centrifugal, the supernatant lyophilization gets powder, promptly gets effective part of sweet wormwood;
Perhaps
Get the Herba Artemisiae Annuae crude polysaccharides, water fully dissolves, and adopting cutoff value is dialyzer or ultrafilter membrane removal molecular weight 5000 or the 3500 following micromolecule of 3500～5000D, and liquid glucose concentrates, centrifugal, the weak anionic exchange is twisted on the supernatant, and water, 01～1mol/LNaCl gradient elution are collected Xian and taken off liquid, adopting cutoff value is dialyzer or ultrafilter membrane removal molecular weight 5000 or the 3500 following micromolecule of 3500～5000D, concentrate, drying promptly gets effective part of sweet wormwood.
The preparation method of described effective part of sweet wormwood, this method comprises the following steps:
1) extraction of Herba Artemisiae Annuae crude polysaccharides: get Herba Artemisiae Annuae and decoct with water 2～3 times, each amount of water is 10～15 times of Herba Artemisiae Annuae weight, and each 1～2 hour, extracting solution merged, concentrating under reduced pressure adds ethanol precipitation to containing the alcohol amount more than 85%, leaves standstill, the leaching precipitation is used washing with alcohol, the dry Herba Artemisiae Annuae crude polysaccharides that gets;
2) effective part of sweet wormwood is refining: get the Herba Artemisiae Annuae crude polysaccharides, water fully dissolves, concentration is 0.7～1%, the employing cutoff value is that the dialyzer of 3500～5000D was dialysed 48～72 hours with pure water, remove molecular weight 5000 or 3500 following micromolecule, liquid glucose concentrates, and is centrifugal, the supernatant lyophilization gets powder, is effective part of sweet wormwood;
Perhaps
Get the Herba Artemisiae Annuae crude polysaccharides, water fully dissolves, and adopting cutoff value is dialyzer or ultrafilter membrane removal molecular weight 5000 or the 3500 following micromolecule of 3500～5000D, and liquid glucose concentrates, centrifugal, weak anionic exchange column on the supernatant, water, 01～1mol/L NaCl gradient elution are collected eluent, adopting cutoff value is dialyzer or ultrafilter membrane removal molecular weight 5000 or the 3500 following micromolecule of 3500～5000D, concentrate, drying promptly gets effective part of sweet wormwood.
The preparation method of described effective part of sweet wormwood, this method also comprises the following steps:
Get effective part of sweet wormwood, suitable quantity of water dissolving, last weak anionic exchange column, water, 0.1～1mol/LNaCl gradient Xian take off, the phenolsulfuric acid method detects collects liquid, merges the eluent that the polysaccharide reaction is positive, and concentrates, the employing cutoff value is that the dialyzer of 3500～5000D was dialysed 48～72 hours with pure water, remove molecular weight 5000 or 3500 following micromolecule, 40～50 ℃ of concentrating under reduced pressure of liquid glucose, lyophilization, get powder, be the effective part of sweet wormwood of purification.
Perhaps
Getting effective part of sweet wormwood is that the molecular sieve chromatography of 1000～150000D separates with the exclusion scope respectively after with water dissolution, obtain a series of molecular weight different but each series is respectively the effective part of sweet wormwood of the purification of homogeneous molecular weight.
Described preparation method, wherein the weak anionic exchange column is a DEAE weak anionic exchange column.
Described preparation method, wherein DEAE weak anionic exchange column is DEAE Sepharose FF post or DEAE cellulose column.
Described preparation method, wherein molecular sieve chromatography is SephadexG or Sephacryl gel column.
The application of described effective part of sweet wormwood in the medicine of preparation antineoplastic agent or smelting treatment immune disease.
Beneficial effect of the present invention:
1, the chemical research of effective part of sweet wormwood
Adopting sulfuric acid-phynol method to measure sugared content to the sweet wormwood HQG (pressing embodiment 1 preparation, down together) that adopts extracting method of the present invention to obtain, is reference substance with the glucose, and measurement result is as follows:
| Sweet wormwood HQG lot number | Sugar content (%) |
| 050811 | 61 33 |
| 050912 | 62 17 |
| 051005 | 63.02 |
| 060307 | 58.64 |
| 060311 | 62.09 |
| 060411 | 61 84 |
The result shows that the sugared content of most of sample can reach 60%, and small part sample sugar content can reach 50%.
Adopt high-efficient liquid phase technique to carry out molecular weight determination to the sweet wormwood HQG that obtains with extracting method of the present invention:
Highly effective liquid phase chromatographic system: waters515 type high-pressure pump, 717 type automatic samplers, Tianjin, island 6A differential detector, chromatographic column is TSK G4000Pwxl Lot E3313 (7.5 * 300mm, 10 μ m) with TSK G5000Pwxl Lot G3367 columns in series, Millineum
32Data software.Sample size: 100 μ L; Column temperature: 30 ℃; Mobile phase: 0 2mol/LNaCl aqueous solution; Flow velocity: 0 4mL/min.Molecular weight becomes multiplet to distribute between 5000-50000 as a result.
Circulation of qi promoting phase method for measuring analysis is sugared to be formed to the preparation alditol acetate is gone forward side by side behind the sweet wormwood HQG employing acid hydrolysis that obtains with extracting method of the present invention:
Gas chromatography system: the GC-14B gas chromatograph, hydrogen flame ionization detector (SHIMADZU, Japan), chromatographic column: the OV-225 capillary chromatographic column (
0 32mm * 30m, 5 μ m) (the safe and sound analysis of section Science and Technology Ltd. in the Lanzhou), N2000 chromatographic work station (it is limited that Zhejiang University's intelligence reaches information engineering).
Take by weighing the about 10mg of sample, add the 2mol/L trifluoroacetic acid, the ampoule encapsulation was put 110 ℃ of convection oven internal reactions 45 minutes, took out, and it is an amount of to add methanol, evaporate to dryness; Adding distil water 20mL dissolving adds NaBH
430mg, room temperature reaction 25 hours, jolting frequently adds acetic acid to there not being bubble, adds the methanol evaporate to dryness, and totally 4 times, each 20mL adds 1 of acetic acid preceding twice, dry 15 minutes of 100 ℃ of convection oven; Add acetic anhydride 15mL, 100 ℃ of convection oven were reacted 1 hour, and jolting in per 20 minutes once adds toluene 15mL azeotropic distillation except that desolvating; Add chloroform 10mL, distilled water extraction 5 times discards water layer, and chloroform layer adds anhydrous Na SO
4An amount of dehydration is filtered, and filtrate decompression boils off solvent, adds chloroform 05mL dissolving, is the alditol acetate derivant.Sample introduction 2 μ l.
The result shows among the sweet wormwood HQG and contains rhamnose, arabinose, xylose, mannose, six kinds of monosaccharide residues of galactose and glucose.
2, sweet wormwood HQG provided by the invention is little to the mouse tail vein injection acute toxicity, mice transplanted tumor EAC, Heps, S180 there is obvious inhibitory action, external immunology studies show that, sweet wormwood HQG has significant immunologic enhancement, can be used for preparing antineoplastic agent or enhance immunity medicine.
3, preparation method provided by the invention is simple, is suitable for the industrialization utilization.
Below be that sweet wormwood HQG (being called for short HQG, by embodiment 1 preparation) and the sweet wormwood HQG injection (by embodiment 3 preparations) that adopts the inventive method to make carries out relevant pharmacodynamic study:
One, the acute toxicity testing of sweet wormwood HQG
1 experiment material, reagent and instrument
Animal: ICR kind white mice, 18-22g, male and female half and half; The quality certification number: SCXK (Soviet Union) 2002-0011;
Feedstuff: pellet;
Raising condition: air-conditioned room, temperature 18-24 ℃, relative humidity 70%.
Supply test agent: sweet wormwood HQG.
2 experimental techniques
Acute toxicity testing is divided into trial test and formal test, and trial test is formally tested after finding roughly dosage range.If six dosage groups, 10 mices of every dosage group, male and female half and half, drug dose is arranged by geometric ratio, ratio 08, be respectively 190 054,237.568,296 96,371 2,464,580mg/kg, tail vein injection, administration volume 0 4ml/, after the shot, after 24 hours, repeatedly observe, observed continuously 7 days.The record toxic reaction situation of animal and the distribution of dead animal, dead animal in time performs an autopsy on sb, and record pathological changes situation, if when the visible pathological tissues of naked eyes is arranged, need carry out the pathomorphology inspection of this tissue.The result calculates LD with the Bliss statistical method
50Value.
3. experimental result
Poisoning symptom is that the fine hair volume crouches after the mouse mainline administration, haltingly, the movable minimizing, drowsiness, breathing is slowed down.Dead animal is dissected, and each internal organs shows no obvious abnormalities.Dead distribution and statistical result see Table 1.
Table 1 sweet wormwood mouse tail vein injection LD
50Measurement result
Statistical analysis, regression equation are Y=-9.9322+5.8758X (r=0.9832) (Y is that probability unit adds the effect value after 5, and X is the logarithm of dosage); LD
50=423.8894mg/kg, the confidence interval during confidence coefficient a=0 05 is: 299 4602mg/kg≤LD
50≤ 403.8729mg/kg.The result shows that sweet wormwood intravenous injection toxicity is less, and safety range is big.
Two, sweet wormwood HQG is to the inhibitory action of transplanted tumor EAC, Heps, S180
(1) sweet wormwood HQG is to the inhibitory action of animal transplanting tumor EAC
1 test material:
11 are subjected to the reagent thing: sweet wormwood HQG injection.
1.2 animal: ICR kind white mice, 18-22g, male and female half and half are provided by animal housing of China Medicine University.The quality certification number: SCXK (Soviet Union) 2002-0011;
Feedstuff: pellet, supply with by animal housing of China Medicine University;
Raising condition: air-conditioned room, temperature 18-24 ℃, relative humidity 70%.
13 positive drug: cyclophosphamide (CTX), Hualian Pharmaceutical Co., Ltd., Shanghai.Specification 200mg/ bottle, lot number 030915
The main contents of 2 experimentatioies
The medicine tail vein injection is to the inhibitory action of mice-transplanted tumor EAC
3 experimental techniques and step
31 route of administration: tail vein injection (iv)
32 administration cycles: press transplanted tumor organon inoculation EAC solid type, inoculate administration after 24 hours, the iv administration, every other day once, administration is 4 times altogether, and mice is dissected in execution in the 2nd day after the drug withdrawal.
33 dosage settings: establish 5 groups altogether
34 administration volumes: 0 4ml/20g
35 experimental techniques: get 50 of above-mentioned specification mices and inoculate back 24 hours and claim Mus heavy, and be divided into 5 groups at random by transplanted tumor organon inoculation EAC solid type, 10 every group, male and female half and half.Inoculate administration after 24 hours, the iv administration, every other day once, administration is 4 times altogether, and the 2nd day mice weighed after drug withdrawal, puts to death tumor-bearing mice and separates the tumor piece, claims tumor heavy, and the gained data are carried out statistical procedures (t check).
36 experimental results:
The result shows, compare with the blank group, each dosage group of HQG all can significantly suppress the growth (P＜001) of mice-transplanted tumor EAC, but 100,50mg/kg dosage group influences big (P＜001) to the weight of animals, and HQG25mg/kg dosage group is to the weight of animals influence less relatively (P＜0.05).Positive drug CTX is to the inhibitory action of transplanted tumor also clearly (P＜001), and is also bigger to the influence of mice body weight.See Table 2.
Table 2 sweet wormwood HQG intravenous injection is to the inhibitory action of mice-transplanted tumor EAC
*P＜005
*Compare with the blank group P＜0.01.
(2) sweet wormwood HQG is to the inhibitory action of animal transplanting tumor Heps
1 test material
11 are subjected to the reagent thing:
Sweet wormwood HQG injection.
12 animals:
ICR kind white mice, 18-22g, male and female half and half are provided by animal housing of China Medicine University.
The quality certification number: SCXK (Soviet Union) 2002-0011;
Feedstuff: pellet, supply with by animal housing of China Medicine University;
Raising condition: air-conditioned room, temperature 18-24 ℃, relative humidity 70%.
13 positive drug: cyclophosphamide (CTX), Hualian Pharmaceutical Co., Ltd., Shanghai.Specification 200mg/ bottle, lot number 030915.
The main contents of 2 experimentatioies
The HQG intravenous injection is to the inhibitory action of mice-transplanted tumor Heps
3 experimental techniques and step
31 route of administration: tail vein injection (iv)
32 administration cycles: press the transplanted tumor organon, inoculate administration after 24 hours, the iv administration, every other day once, administration is 4 times altogether, and mice is dissected in execution in the 2nd day after the drug withdrawal.
33 dosage settings:
Establish 5 groups altogether
3.4 administration volume: 0.4ml/20g
35 experimental techniques: get 50 of above-mentioned specification mices and inoculate back 24 hours and claim Mus heavy, and be divided into 5 groups at random by transplanted tumor organon inoculation Heps solid type, 10 every group, male and female half and half.Inoculate administration after 24 hours, the iv administration, every other day once, administration is 4 times altogether, and the 2nd day mice weighed after drug withdrawal, puts to death tumor-bearing mice and separates the tumor piece, claims tumor heavy, and the gained data are carried out statistical procedures (t check).
3.6 experimental result:
The result shows, compare with the blank group, each dosage group of HQG all can significantly suppress the growth (P＜001) of mice-transplanted tumor Heps, but 100,50mg/kg dosage group influences big (P＜001) to the weight of animals, and HQG25mg/kg dosage group is to the weight of animals influence less relatively (P＜0.05).Positive drug CTX is to the inhibitory action of transplanted tumor also clearly (P＜0.01), and is also bigger to the influence of mice body weight.See Table 3.
The intravenous injection of table 3 sweet wormwood HQG medicine is to the inhibitory action of mice-transplanted tumor Heps
*P＜005
*Compare with the blank group P＜001
(3) sweet wormwood HQG is to the inhibitory action of animal transplanting tumor S180
1 test material
11 are subjected to the reagent thing:
Sweet wormwood HQG injection.
12 animals:
ICR kind white mice, 18-22g, male and female half and half are provided by animal housing of China Medicine University.
The quality certification number: SCXK (Soviet Union) 2002-0011;
Feedstuff: pellet, supply with by animal housing of China Medicine University;
Raising condition: air-conditioned room, temperature 18-24 ℃, relative humidity 70%.
13 positive drug
Cyclophosphamide (CTX), Hualian Pharmaceutical Co., Ltd., Shanghai.Specification 200mg/ bottle, lot number 030915
The main contents of 2 experimentatioies
The intravenous injection of sweet wormwood HQG injection is to the inhibitory action of mice-transplanted tumor S180
3 experimental techniques and step
31 route of administration: tail vein injection (iv)
The 32 administration cycles: press the transplanted tumor organon
[1]Inoculation S180 solid type is inoculated administration after 24 hours, the iv administration, and every other day once, administration is 4 times altogether, and mice is dissected in execution in the 2nd day after the drug withdrawal.
33 dosage settings: establish 7 groups altogether
34 administration volumes: 0 4ml/20g
35 experimental techniques: get 50 of above-mentioned specification mices and inoculate back 24 hours and claim Mus heavy, and be divided into 5 groups at random by transplanted tumor organon inoculation S180 solid type, 10 every group, male and female half and half.Inoculate administration after 24 hours, the iv administration, every other day once, administration is 4 times altogether, and the 2nd day mice weighed after drug withdrawal, puts to death tumor-bearing mice and separates the tumor piece, claims tumor heavy, and the gained data are carried out statistical procedures (t check).
36 experimental results:
The result shows, compares with the blank group, and each dosage group of HQG all can significantly suppress the growth (P＜001) of mice-transplanted tumor S180, but 100mg/kg dosage group influences big (P＜005) to the weight of animals.Positive drug CTX is to the inhibitory action of transplanted tumor also clearly (P＜001), and is also bigger to the influence of mice body weight.See Table 4.
Table 4HQG intravenous injection is to the inhibitory action of mice-transplanted tumor S180
*P＜005
*Compare with the blank group P＜0.01
Repeatedly experimental result shows, this product is to transplanted solid tumor EAC, and Heps and S180 all have certain curative effect, its inhibitory rate of basic, normal, high this product is more than 30～50%, and antitumor action has a certain amount of effect relationship, though effect is less than positive drug CTX, but toxicity is little, and is safe.
Three, the Study immune regulation of sweet wormwood HQG
(1) lymphocytotoxicity test (LCT) of sweet wormwood HQG
1 experiment material, reagent and instrument
Reagent and material: RPMI-1640 basal medium dry powder (GBICO), calf serum (HyClone), MTT (sigma), DMSO (Shanghai Ling Feng chemical reagent company limited), other biochemical reagents are analytical pure.
96 porocyte culture plates (costar).
Laboratory animal: pure lines Kunming white mice: in 7 ages in week, 20 ± 2g is provided by China Medicine University's Experimental Animal Center.
Instrument: superclean bench (Sujing Group Co., Suzhou City), cell culture incubator (Thermo), enzyme-linked immunosorbent assay instrument (Thermo), LXJ-II centrifuge (Shanghai medical analytical instrument factory).
2 experimental techniques
21 experimental principles
Adopt tetramethyl azo azoles salt (MTT) method.
The succinate dehydrogenase that produces in the living cells center line La physical ability metabolic process makes faint yellow MTT be reduced into hepatic crystallization (formazan, the first a ceremonial jade-ladle, used in libation) be deposited in the cell and cell peripheral, and the succinate dehydrogenase that the living cells interior lines La physical ability metabolism of propagation produces is more than the living cells of non-propagation, so, darker than the living cells of non-propagation in the proliferative cell with bluish violet that cell peripheral is reduced into, but dead cell loses the function that the metabolism of line La physical ability produces succinate dehydrogenase, so dead cell is not painted.Therefore, according to the painted depth of cell, can distinguish the living cells and the dead cell (the first a ceremonial jade-ladle, used in libation can be dissolved fully) of the living cells of propagation, non-propagation with enzyme mark tintometer under the situation that specific solvent exists.The external no any environmental stimuli of splenocyte is not bred, but can survive about 1 week in the short time.Test group illustrates that than negative group data height medicine can promote the propagation of splenocyte.Otherwise, toxic effect is described.
2.2 the preparation of solution
2 21 PBS (0.01M, pH7.4): take by weighing 8.0g NaCl, 3.84g Na respectively
2HPO
412H
2O, 0 2g KCl, 0 24gKH
2PO
4, be settled to 1000mL with deionized water, through 121 ℃ * 15min autoclaving.
2.22 Hank ' s liquid: take by weighing 80g NaCl, 0.06g Na respectively
2HPO
412H
2O, 0 4gKCl, 0 06gKH
2PO
4, 0 098gMgSO
47H
2O, 1.0g glucose, 0.14gCaCl
2, be dissolved in appropriate amount of deionized water, use 7%NaHCO
3Solution is transferred PH to 74, is settled to 1000mL, and 0.22 μ m membrane filtration degerming is in 4 ℃ of preservations.
2 23 Tris-NH
4Cl (erythrocyte cracked liquid): take by weighing 0 747g NH respectively
4Cl and 0 26g Tris (Tris) are settled to 100mL with deionized water, 0 22 μ m membrane filtration degerming, 4 ℃ of preservations.
2 24RPMI-1640 complete mediums: get the RPMI1640 culture medium dry powder, the by specification preparation, 0 22 μ m membrane filtration degerming add 10% calf serum, are sub-packed in 4 ℃ of preservations.
2.25 MTT: be made into 5mg/mL solution with 0.01MPBS, 0 22 μ m membrane filtration degerming are kept in Dark Place in-20 ℃.
2.26 the preparation of sweet wormwood HQG sample: it is an amount of that precision takes by weighing sweet wormwood HQG sample, is dissolved among the sterilization PBS 0.22 μ m membrane filtration degerming, 4 ℃ of preservations; Face with preceding and be diluted to desired concn with the RPMI-1640 complete medium.
The preparation of 23 splenocyte suspensions
Mice is taken off neck put to death, be dipped in 5min in 75% the ethanol; The sterile working takes out spleen, grinds 200 order steel sieve, collects isolating splenocyte suspension, and 2000rpm * 5min abandons supernatant; Add erythrocyte cracked liquid 10mL, re-suspended cell leaves standstill behind the 5min centrifugally, uses Hank ' s liquid to wash centrifugal 3 times; Re-suspended cell is in the RPMI-1640 complete medium, and the trypan blue dyeing counting is transferred cell concentration to 10
7Individual/mL.
The lymphocytotoxicity test (LCT) of 24 sweet wormwood HQG
At first the splenocyte suspension for preparing is added 96 porocyte culture plates, 100 μ L/ holes.Add sample then, 100 μ L/ holes, multiple 3 holes of each concentration of every duplicate samples (final concentration is respectively 100 μ g/mL, 10 μ g/mL and 1 μ g/mL); Simultaneously, set up the negative control that only adds the RPMI-1640 complete medium.The cell plates that will add sample are put 5%CO
2In the cell culture incubator, cultivate 44h for 37 ℃.Take out cell plates, add MTT, 4h is continued to cultivate in 20 μ L/ holes.Take out cell plates, 3000rpm * 5mm abandons most supernatant; Add DMSO, 100 μ L/ holes, 37 ℃ of vibration cell lysis 10mm; Measure OD with enzyme-linked immunosorbent assay instrument
570Value.
3 experimental results: the lymphocytotoxicity test (LCT) of sweet wormwood HQG the results are shown in Table 5.
The lymphocytotoxicity test (LCT) result of table 5 sweet wormwood HQG
aMeansigma methods ± SD.
Experimental result shows, the OD of sweet wormwood HQG
570Value illustrates that sweet wormwood HQG can significantly promote splenocyte to transform propagation, and does not have obvious cytotoxicity apparently higher than negative control group.
(2) sweet wormwood HQG induces the spleen lymphocyte proliferation test
1 experiment material, reagent and instrument
Reagent and material: RPMI-1640 basal medium dry powder (GBICO), calf serum (HyClone), ConA (sigma), LPS (sigma), MTT (sigma), DMSO (Shanghai Ling Feng chemical reagent company limited), other biochemical reagents are analytical pure.Lentinus edodes polysaccharide injecta (good fortune continent Mei Feng pharmaceutical factory).
96 porocyte culture plates (costar).
Laboratory animal: pure lines Kunming white mice: in 7 ages in week, 20 ± 2g is provided by China Medicine University's Experimental Animal Center.
Instrument: super-clean bench (Sujing Group Co., Suzhou City), cell culture incubator (Thermo), enzyme-linked immunosorbent assay instrument (Thermo), centrifugal precipitation mechanism LXJ-II (Shanghai medical analytical instrument factory).
2 experimental techniques
Adopt mtt assay.
The preparation of 21 solution
2 11 PBS (001M, pH74): take by weighing 8 0g NaCl, 3 84g Na respectively
2HPO
412H
2O, 0 2g KCl, 0 24g KH
2PO
4, be settled to 1000mL with deionized water, through 121 ℃ * 15min autoclaving.
2 12 Hank ' s liquid: take by weighing 8 0g NaCl, 0 06g Na respectively
2HPO
412H
2O, 0 4gKCl, 0 06gKH
2PO
4, 0 098g MgSO
47H
2O, 1 0g glucose, 0.14g CaCl are dissolved in appropriate amount of deionized water, use 7%NaHCO
3Transfer PH to 74, be settled to 1000mL, 0 22 μ m membrane filtration degerming are sub-packed in 4 ℃ of preservations.
2 13 Tris-NH
4Cl (erythrocyte cracked liquid): take by weighing 0 747gNH respectively
4Cl and 0 26g Tris (Tris) are settled to 100mL with deionized water, 0 22 μ m membrane filtration degerming, 4 ℃ of preservations.
2 14 RPMI-1640 complete mediums: get the RPMI1640 culture medium dry powder, the by specification preparation, 0 22 μ m membrane filtration degerming add 10% calf serum, are sub-packed in 4 ℃ of preservations.
2 15 ConA: be made into 1mg/mL solution with 1640 basal mediums, 0 22 μ m membrane filtration degerming are sub-packed in-20 ℃ of preservations.
2 16 LPS: be made into 1mg/mL solution with 1640 basal mediums, 0.22 μ m membrane filtration degerming is sub-packed in-20 ℃ of preservations.
2 17 MTT: be made into 5mg/mL solution with 0.01M PBS, 0 22 μ m membrane filtration degerming are sub-packed in-20 ℃ and keep in Dark Place.
The preparation of 2 18 polysaccharide samples: precision takes by weighing sweet wormwood HQG and the lentinan sample is an amount of, is dissolved among the sterilization PBS 0.22 μ m membrane filtration degerming, 4 ℃ of preservations; Face with preceding and be diluted to desired concn with the RPMI-1640 complete medium.
2.2 the preparation of splenocyte suspension
Mice is taken off neck put to death, be dipped in 5min in 75% the ethanol; The sterile working takes out spleen, grinds 200 order steel sieve, collects isolating splenocyte suspension, and 2000rpm * 5min abandons supernatant; Add erythrocyte cracked liquid 10mL, re-suspended cell leaves standstill behind the 5min centrifugally, uses Hank ' s liquid to wash centrifugal 3 times; Re-suspended cell is in the RPMI-1640 complete medium, and the trypan blue dyeing counting is transferred cell concentration to 10
7Individual/mL.
The inductive lymphocyte proliferation assay of 23 sweet wormwood HQG
At first the splenocyte suspension for preparing is added 96 porocyte culture plates, 100 μ L/ holes.Add sample then, 100 μ L/ holes, multiple 3 holes of each concentration of every duplicate samples (final concentration is respectively 100 μ g/mL, 10 μ g/mL and 1 μ g/mL); Simultaneously, set up positive control and negative control, wherein positive control lentinan final concentration is 50 μ g/mL, and the ConA final concentration is 10 μ g/mL, and negative control only adds the RPMI-1640 complete medium.The cell plates that will add sample are put 5%CO
2In the cell culture incubator, cultivate 44h for 37 ℃.Take out cell plates, add MTT, 4h is continued to cultivate in 20 μ L/ holes.Take out cell plates, 3000rpm * 5mm abandons most supernatant; Add DMSO, 100 μ L/ holes, 37 ℃ of vibration cell lysis 10mm; Measure OD with enzyme-linked immunosorbent assay instrument
570Value.
The lymphocyte proliferation assay of 24 sweet wormwood HQG and ConA combined induction
At first the splenocyte suspension for preparing is added 96 porocyte culture plates, 100 μ L/ holes.Add ConA and sample then respectively, 50 μ L/ holes, wherein the ConA final concentration is 5 μ g/mL, multiple 3 holes of each concentration of every duplicate samples (final concentration is respectively 100 μ g/mL, 10 μ g/mL and 1 μ g/mL); Simultaneously, set up positive control, sample blank contrast and negative control, wherein positive control lentinan final concentration is 50 μ g/mL, and the sample blank contrast only adds ConA, and negative control only adds the RPMI-1640 complete medium.The cell plates that will add sample are put 5%CO
2In the cell culture incubator, cultivate 44h for 37 ℃.Take out cell plates, add MTT, 4h is continued to cultivate in 20 μ L/ holes.Take out cell plates, 3000rpm * 5mm abandons most supernatant; Add DMSO, 100 μ L/ holes, 37 ℃ of vibration cell lysis 10min; Measure OD with enzyme-linked immunosorbent assay instrument
570Value.
The lymphocyte proliferation assay of 25 sweet wormwood HQG and LPS combined induction
At first the splenocyte suspension for preparing is added 96 porocyte culture plates, 100 μ L/ holes.Add LPS and sample then respectively, 50 μ L/ holes, wherein the LPS final concentration is 10 μ g/mL, multiple 3 holes of each concentration of every duplicate samples (final concentration is respectively 100 μ g/mL, 10 μ g/mL and 1 μ g/mL); Simultaneously, set up positive control, sample blank contrast and negative control, wherein positive control lentinan final concentration is 50 μ g/mL, and the sample blank contrast only adds LPS, and negative control only adds the RPMI-1640 complete medium.The cell plates that will add sample are put 5%CO
2In the cell culture incubator, cultivate 44h for 37 ℃.Take out cell plates, add MTT, 4h is continued to cultivate in 20 μ L/ holes.Take out cell plates, 3000rpm * 5mm abandons most supernatant; Add DMSO, 100 μ L/ holes, 37 ℃ of vibration cell lysis 10min; Measure OD with enzyme-linked immunosorbent assay instrument
570Value.
3 experimental results
The inductive lymphocyte proliferation assay of 31 sweet wormwood HQG
The inductive lymphocyte proliferation assay of sweet wormwood HQG the results are shown in Table 6.
The inductive lymphocyte proliferation assay result of table 6 sweet wormwood HQG
aMeansigma methods ± SD.
Experimental result shows, the OD of sweet wormwood HQG experimental group
570Value all is higher than negative control group, illustrates mouse spleen lymphocyte had significantly to induce proliferation function, and obvious dose dependent is arranged.Because lentinan is present clinical practice antitumor polyose medicament the most widely, so determined curative effect is with its medicine in contrast, the OD of sweet wormwood HQG experimental group
570Value and lentinan are approaching, show that each position of sweet wormwood HQG induces proliferation function suitable with lentinan to mouse spleen lymphocyte, greater than ConA in the set dosage range of experiment.
The lymphocyte proliferation assay of 32 sweet wormwood HQG and ConA combined induction
The lymphocyte proliferation assay of sweet wormwood HQG and ConA combined induction the results are shown in Table 7
The lymphocyte proliferation assay result of table 7 sweet wormwood HQG and ConA combined induction
aMeansigma methods ± SD.
Experimental result shows, the OD of sweet wormwood HQG experimental group
570Value all is higher than the sample blank group, illustrates that sweet wormwood HQG transforms propagation to the inductive mouse spleen lymphocyte of ConA obvious facilitation is arranged, and obvious dose dependent is arranged.The OD of sweet wormwood HQG experimental group
570Value and lentinan are approaching, illustrate that in the set dosage range of experiment, sweet wormwood HQG is suitable to facilitation and the lentinan that the inductive mouse spleen lymphocyte of ConA transforms propagation.
The lymphocyte proliferation assay of 33 sweet wormwood HQG and LPS combined induction
The lymphocyte proliferation assay of sweet wormwood HQG and LPS combined induction the results are shown in Table 8.
The lymphocyte proliferation assay result of table 8 sweet wormwood HQG and LPS combined induction
aMeansigma methods ± SD.
Experimental result shows, the OD of sweet wormwood HQG experimental group
570Value all is higher than the sample blank group, illustrates that sweet wormwood HQG transforms propagation to the inductive mouse spleen lymphocyte of LPS obvious facilitation is arranged, and obvious dose dependent is arranged.The OD of sweet wormwood HQG experimental group
570Value and lentinan are approaching, illustrate that in the set dosage range of experiment, sweet wormwood HQG is suitable to facilitation and the lentinan that the inductive mouse spleen lymphocyte of LPS transforms propagation.
(3) sweet wormwood HQG induces IFN-γ, IL-2 and the excretory influence of TNF-α to ConA, LPS
1 experiment material, reagent and instrument
Reagent and material: RPMI-1640 basal medium dry powder (GBICO), calf serum (HyClone), ConA (sigma), LPS (sigma), MTT (sigma), DMSO (Shanghai Ling Feng chemical reagent company limited).IL-2, IFN-γ and TNF-α ELISA detection kit (every gloomy male Scientific and Technical Industry Co., Ltd is gone up in the import packing).Other biochemical reagents are analytical pure.Lentinus edodes polysaccharide injecta (good fortune continent Mei Feng pharmaceutical factory).
96 porocyte culture plates (costar).
Laboratory animal: pure lines Kunming white mice: in 7 ages in week, 20 ± 2g is provided by China Medicine University's Experimental Animal Center.
Instrument: superclean bench (Sujing Group Co., Suzhou City), cell culture incubator (Thermo), enzyme-linked immunosorbent assay instrument (Thermo), centrifugal precipitation mechanism LXJ-II (Shanghai medical analytical instrument factory).
2 experimental techniques
21 experimental principles
Cytokine is a group small-molecular weight (about 8-80kDa) albumen, plays a role with autocrine (acting on the cell of these cytokines of secretion) or paracrine (acting on contiguous cell) form usually.Cytokine active high can play a role in picomole level even femtomole level.Cytokine is the ingredient of intercellular signal network.Various functional incidents in this network regulation natural immunity and the specific immune response are as the propagation of inflammatory reaction, defend against computer virus infection, specificity T, B cell clone and the adjusting of function thereof.Cytokine comprises interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), somatomedin (GF), colony stimulating factor (CSF) and tendency cytokine [4].This experiment adopts Enzyme Linked Immunoadsorbent Assay (ELISA) test kit pair cell factor content to measure, the effect that induces of the research sweet wormwood HQG pair cell factor.The ELISA test kit is the specificity interaction according to antigen, antibody, and design by enzyme connection expansion reaction signal.IFN-γ, IL-2 and TNF-α ELISA test kit are specifically designed to the changes of contents of measuring IFN-γ, IL-2 and TNF-α.
2.2 the preparation of solution
2.21 PBS (0.01M, pH7.4): take by weighing 8 0g NaCl, 3.84g Na respectively
2HPO
412H
2O, 0 2g KCl, 0 24g KH
2PO
4, be settled to 1000mL with deionized water, through 121 ℃ * 15mm autoclaving.
2 22 Hank ' s liquid: take by weighing 8 0g NaCl, 0 06g Na respectively
2HPO
412H
2O, 0 4gKCl, 0 06gKH
2PO
4, 0 098g MgSO
47H
2O, 1.0g glucose, 0 14g CaCl are dissolved in appropriate amount of deionized water, use 7%NaHCO
3Transfer PH to 7.4, be settled to 1000mL, 0.22 μ m membrane filtration degerming is sub-packed in 4 ℃ of preservations.
2 23 Tris-NH
4Cl (erythrocyte cracked liquid): take by weighing 0.747gNH respectively
4Cl and 0.26gTris (Tris) are settled to 100mL with deionized water, 0 22 μ m membrane filtration degerming, 4 ℃ of preservations.
2 24 RPMI-1640 complete mediums: get the RPMI1640 culture medium dry powder, the by specification preparation, 0 22 μ m membrane filtration degerming add 10% calf serum, are sub-packed in 4 ℃ of preservations.
2 25 ConA: be made into 1mg/mL solution with 1640 basal mediums, 0 22 μ m membrane filtration degerming are sub-packed in-20 ℃ of preservations.
2 26 LPS: be made into 1mg/mL solution with 1640 basal mediums, 0 22 μ m membrane filtration degerming are sub-packed in-20 ℃ of preservations.
2 27 MTT: be made into 5mg/mL solution with 0 01M PBS, 0 22 μ m membrane filtration degerming are sub-packed in-20 ℃ and keep in Dark Place.
The preparation of 2 28 polysaccharide samples: it is an amount of that precision takes by weighing sweet wormwood HQG sample, is dissolved among the sterilization PBS 0 22 μ m membrane filtration degerming, 4 ℃ of preservations; Face with preceding and be diluted to desired concn with the RPMI-1640 complete medium.
The preparation of 23 splenocyte suspensions
Mice is taken off neck put to death, be dipped in 5mm in 75% the ethanol; The sterile working takes out spleen, grinds 200 order steel sieve, collects isolating splenocyte suspension, and centrifugal 2000rpm * 5min abandons supernatant; Add erythrocyte cracked liquid 10mL, re-suspended cell leaves standstill behind the 5min centrifugally, washs centrifugal 3 times with Hank ' s liquid; Re-suspended cell is in the RPMI-1640 complete medium, and the trypan blue dyeing counting is transferred cell concentration to 10
7Individual/mL.
The preparation of 24 peritoneal macrophage suspensions
Mice is taken off neck put to death, be dipped in 5min in 75% the ethanol, collect intraperitoneal liquid.Centrifugal 1000rpm * 5min abandons supernatant, washs centrifugal 3 times with Hank ' s liquid.Re-suspended cell is in the RPMI-1640 complete medium, and the trypan blue dyeing counting is transferred cell concentration to 10
6Individual/mL.
2.5 the cell culture supernatant preparation of sweet wormwood HQG associating ConA induction of lymphocyte secretion of gamma-IFN and IL-2
At first the splenocyte suspension for preparing is added 96 porocyte culture plates, 100 μ L/ holes.Add ConA and sample then respectively, 50 μ L/ holes, wherein the ConA final concentration is 5 μ g/mL, multiple 3 holes of each concentration of every duplicate samples (final concentration is respectively 100,10 and 1 μ g/mL); Simultaneously, set up positive control, sample blank contrast and negative control, wherein positive control lentinan final concentration is 50 μ g/mL, and the sample blank contrast only adds ConA, and negative control only adds the RPMI-1640 complete medium.The cell plates that will add sample are put 5%CO
2In the cell culture incubator, cultivate 48h for 37 ℃.Sucking-off and the culture fluid that merges identical well, 3000rpm * 5min abandons precipitation, and supernatant is to be measured in-20 ℃ of preservations.
26 sweet wormwood HQG associating ConA induces the cell culture supernatant preparation of macrophage TNF secretion-α
At first the peritoneal macrophage suspension for preparing is added 96 porocyte culture plates, 1mL/ hole, 37 ℃, 5%CO
2Cultivated 2 hours in the incubator, abandon supernatant, Hank ' s liquid flush away non-adherent cell is required macrophage.Add ConA and sample respectively, 50 μ L/ holes, wherein the ConA final concentration is 5 μ g/mL, multiple 3 holes of each concentration of every duplicate samples (final concentration is respectively 100 μ g/mL, 10 μ g/mL and 1 μ g/mL); Simultaneously, set up positive control, sample blank contrast and negative control, wherein positive control lentinan final concentration is 50 μ g/mL, and the sample blank contrast only adds ConA, and negative control only adds the RPMI-1640 complete medium.The cell plates that will add sample are put 5%CO
2In the cell culture incubator, cultivate 24h for 37 ℃.Sucking-off and the culture fluid that merges identical well, 3000rpm * 5min abandons precipitation, and supernatant is to be measured in-20 ℃ of preservations.
The cell culture supernatant preparation of 27 sweet wormwood HQG associating LPS induction of lymphocyte secretion of gamma-IFN, IL-2
At first the splenocyte suspension for preparing is added 96 porocyte culture plates, 100 μ L/ holes.Add LPS and sample then respectively, 50 μ L/ holes, wherein the LPS final concentration is 10 μ g/mL, multiple 3 holes of each concentration of every duplicate samples (final concentration is respectively 100,10 and 1 μ g/mL); Simultaneously, set up positive control, sample blank contrast and negative control, wherein positive control lentinan final concentration is 50 μ g/mL, and the sample blank contrast only adds LPS, and negative control only adds the RPMI-1640 complete medium.The cell plates that will add sample are put 5%CO
2In the cell culture incubator, cultivate 48h for 37 ℃.Sucking-off and the culture fluid that merges identical well, 3000rpm * 5mm abandons precipitation, and supernatant is to be measured in-20 ℃ of preservations.
28 sweet wormwood HQG associating LPS induces the cell culture supernatant preparation of macrophage TNF secretion-α
At first the peritoneal macrophage suspension for preparing is added 96 porocyte culture plates, 1mL/ hole, 37 ℃, 5%CO
2Cultivated 2 hours in the incubator, abandon supernatant, Hank ' s liquid flush away non-adherent cell is required macrophage.Add LPS and sample then respectively, 50 μ L/ holes, wherein the LPS final concentration is 10 μ g/mL, multiple 3 holes of each concentration of every duplicate samples (final concentration is respectively 100,10 and 1 μ g/mL); Simultaneously, set up positive control, sample blank contrast and negative control, wherein positive control lentinan final concentration is 50 μ g/mL, and the sample blank contrast only adds LPS, and negative control only adds the RPMI-1640 complete medium.The cell plates that will add sample are put 5%CO
2In the cell culture incubator, cultivate 48h for 37 ℃.Sucking-off and the culture fluid that merges identical well, 3000rpm * 5min abandons precipitation, and supernatant is to be measured in-20 ℃ of preservations.
The mensuration of IFN-γ, IL-2 and TNF-α in 29 cell culture supernatants
Press the explanation of ELISA test kit and detect, wherein the cells and supernatant of sample 0,1,2,5,10 and lentinan is answered 2 holes, and standard curve is done multiple 2 holes, all the other single holes.Measure OD
492Value is obtained the concentration of IFN-γ, IL-2 and TNF-α according to standard curve, and analytic sample is to the effect that induces of different cytokines.
3 experimental results
The mensuration of IFN-γ in 31 cell culture supernatants
The drafting of 3 11 standard curves
Table 9IFN-γ content measuring standard curve data
The IFN-γ content measuring standard curve of drawing as shown in Figure 1, visible IFN-γ content measuring standard curve is y=1 6877x-2 0257 (r=09943).
3 12 sample determinations
Measurement result sees Table 10.
Table 10 sweet wormwood HQG is to the result that influences of ConA, LPS induction of lymphocyte secretion of gamma-IFN
aUnit is pg/mL.
Experimental result shows, the IFN-gamma cells factor concentration numerical value of sweet wormwood HQG experimental group is apparently higher than sample blank, illustrate that sweet wormwood HQG has obvious facilitation to the inductive lymphocytic emiocytosis IFN-of ConA, LPS γ, and tangible dose dependent is arranged; The IFN-gamma cells factor concentration numerical value of sweet wormwood HQG experimental group is greater than lentinan, illustrate in the set dosage range of experiment, sweet wormwood HQG to the facilitation of the inductive lymphocytic emiocytosis IFN-of ConA, LPS γ greater than lentinan.
3.2 the mensuration of IL-2 in the cell culture supernatant
3.21 the drafting of standard curve
Table 11 IL-2 content measuring standard curve data
The IL-2 content measuring standard curve of drawing as shown in Figure 2, visible IL-2 content measuring standard curve is y=1 6658x-1 9869 (r=0 9911).
3 22 sample determinations
Measurement result sees Table 6-8.
Table 12 sweet wormwood HQG is to the result that influences of ConA, LPS induction of lymphocyte secretion IL-2
aUnit is pg/mL.
Experimental result shows that the IL-2 cytokine concentrations numerical value of sweet wormwood HQG experimental group illustrates that sweet wormwood HQG has obvious facilitation to the inductive lymphocytic emiocytosis IL-2 of ConA, LPS, and tangible dose dependent is arranged apparently higher than sample blank.To the facilitation aspect of the inductive lymphocytic emiocytosis IL-2 of ConA, in the set dosage range of experiment, effect is greater than lentinan; To the facilitation aspect of the inductive lymphocytic emiocytosis IL-2 of LPS, act on suitable with lentinan.
The mensuration of TNF-α in 33 cell culture supernatants
The drafting of 3 31 standard curves
Table 13 TNF-alpha content bioassay standard curve data
The TNF-alpha content bioassay standard curve of drawing as shown in Figure 3, visible TNF-alpha content bioassay standard curve is y=1 6164x-1 9884 (r=09959)
3 32 sample determinations
Measurement result sees Table 14.
Table 14 sweet wormwood HQG induces the result that influences of macrophage TNF secretion-α to ConA, LPS
aUnit is pg/mL.
Experimental result shows that the TNF-α cytokine concentrations numerical value of sweet wormwood HQG experimental group is higher than sample blank, illustrates that sweet wormwood HQG has obvious facilitation to the inductive macrophage TNF secretion-α of ConA, LPS, and tangible dose dependent is arranged.To the facilitation aspect of the inductive macrophage TNF secretion-α of ConA, effect is greater than lentinan; To the facilitation aspect of the inductive macrophage TNF secretion-α of LPS, effect is less than lentinan.
(4) brief summary:
Result of study shows that sweet wormwood HQG does not have obvious cytotoxicity, can transform propagation by induction of lymphocyte, promote lymphocytic emiocytosis IFN-γ, IL-2, promote macrophage TNF secretion-α, dose dependent is arranged in its effect and the lentinan effect of the general use of factory is suitable clinically.Sweet wormwood HQG is safe, and is little to body damage, and tangible immunologic enhancement is arranged, and is a kind of good immunopotentiating agent, aspect the immunization therapy of diseases such as tumor, boundless application prospect arranged.
Sweet wormwood HQG disclosed by the invention, it is little that acute toxicity is penetrated in tail vein river, obvious to the mice transplanted tumor inhibitory action, experiment in vitro shows that it has significant immunologic enhancement, the cytokine relevant to immunotherapy of tumors has growth-promoting functions, and a kind of active plant polysaccharide with clinical antitumor medical value is provided.
Description of drawings
Fig. 1 is an IFN-γ content measuring standard curve.
Fig. 2 is an IL-2 content measuring standard curve.
Fig. 3 is a TNF-alpha content bioassay standard curve.
The specific embodiment
The invention will be further elaborated by the following examples, but do not limit the present invention.
Embodiment 1: the preparation of effective part of sweet wormwood
The extraction of 1 Herba Artemisiae Annuae crude polysaccharides
Get Herba Artemisiae Annuae and decoct with water 3 times, each amount of water is 15 times of Herba Artemisiae Annuae weight, and each 1.5 hours, extracting solution merged, and concentrating under reduced pressure adds ethanol precipitation to containing alcohol amount 85%, leaves standstill, and the leaching precipitation use ethanol Xian Di, the dry Herba Artemisiae Annuae crude polysaccharides that gets,
Making with extra care of 2 effective part of sweet wormwood
Get the Herba Artemisiae Annuae crude polysaccharides, water fully dissolves, concentration is 07～1%, adopt the dialyzer of cutoff value 5000 to dialyse 72 hours with pure water, to remove the small molecular weight impurity of molecular weight 5000 below, liquid glucose is concentrated, 15000 rev/mins of high speed centrifugations 18 minutes, lyophilization gets powder, is effective part of sweet wormwood.Adopting sulfuric acid-phynol method to measure sugared content, is reference substance with the glucose, and the result shows that sugared content is 61 3%.This polysaccharide molecular weight is distributed as 5000-50000 after measured, comprises rhamnose, arabinose, xylose, mannose, six kinds of monosaccharide residues of galactose and glucose.
Embodiment 2: the separation and purification of effective part of sweet wormwood
Get effective part of sweet wormwood by embodiment 1 preparation, the suitable quantity of water dissolving, last DEAE Sepharose FF. post, water, 0 1～1mol/LNaCl gradient elution, the phenolsulfuric acid method detects collects liquid, merge the eluent that the reaction of each several part polysaccharide is positive, concentrate, the dialyzer that adopts cutoff value 5000 is with pure water dialysis 72 hours, to remove the small molecular weight impurity of molecular weight below 5000,48 ℃ of concentrating under reduced pressure of liquid glucose, lyophilization gets powder, is the effective part of sweet wormwood of purification, have 6 positions, water, 01,02,04,0.6,1 0mol/L NaCl eluting position.Adopting sulfuric acid-phynol method to measure sugared content, is reference substance with the glucose, and the result shows that the sugared content at each eluting position is respectively 958%, 85 0%, 78 5%, 70 1%, 40 1%, 20 3%.The each several part polysaccharide molecular weight is distributed as 5000-50000 after measured, includes rhamnose, arabinose, xylose, mannose, six kinds of monosaccharide residues of galactose and glucose.
Embodiment 3: the preparation of sweet wormwood injection
Get the sweet wormwood HQG that makes by embodiment 1 method and add the conventional adjuvant of injection, be configured to 1000ml by the injection common process with sterilized water for injection, aseptic filtration, fill.
Embodiment 4: the preparation of sweet wormwood lyophilized injectable powder
Get the sweet wormwood HQG that makes by embodiment 1 method, add the conventional adjuvant of lyophilized powder, be configured to 1000ml with sterilized water for injection by the lyophilized powder common process, aseptic filtration, quantitative perfusion to the glass bottle, the plug plug half, lyophilization, the plug total head is rolled lid.
Embodiment 5: the preparation of sweet wormwood injection
The effective part of sweet wormwood of getting the purification that makes by embodiment 2 methods adds the conventional adjuvant of injection, is configured to 1000ml by the injection common process with sterilized water for injection, aseptic filtration, fill.
Embodiment 6: the preparation of sweet wormwood lyophilized injectable powder
Get the effective part of sweet wormwood that makes by embodiment 2 methods, add the conventional adjuvant of lyophilized powder, be configured to 1000ml with sterilized water for injection by the lyophilized powder common process, aseptic filtration, quantitative perfusion to the glass bottle, the plug plug half, lyophilization, the plug total head is rolled lid.
Embodiment 7: the preparation of effective part of sweet wormwood
The extraction of 1 Herba Artemisiae Annuae crude polysaccharides
Get Herba Artemisiae Annuae and decoct with water 3 times, each amount of water is 10 times of Herba Artemisiae Annuae weight, and each 2 hours, extracting solution merged, and concentrating under reduced pressure adds ethanol precipitation to containing alcohol amount 90%, leaves standstill, and the leaching precipitation use washing with alcohol, the dry Herba Artemisiae Annuae crude polysaccharides that gets;
Making with extra care of 2 effective part of sweet wormwood
Get the Herba Artemisiae Annuae crude polysaccharides, water fully dissolves, adopt the ultrafilter membrane of cutoff value 5000 to remove the micromolecule of molecular weight below 5000, parameter during ultrafiltration: temperature is that room temperature, operating pressure are 0 05MPa, circular flow 40L/hr, mean residence time 3hr, liquid glucose concentrates, last DEAE Sepharose FF post, water, 0 1～1mol NaCl gradient elution, collect eluent, the dialyzer that adopts cutoff value 5000, concentrates to remove the small molecular weight impurity of molecular weight below 5000 with pure water dialysis 72 hours, drying gets effective part of sweet wormwood.Adopting sulfuric acid-phynol method to measure sugared content, is reference substance with the glucose, and the result shows that sugared content is 62 1%.This polysaccharide molecular weight is distributed as 5000-50000 after measured, and rhamnose, arabinose, xylose, mannose, six kinds of monosaccharide residues of galactose and glucose.
Embodiment 8: the purification of effective part of sweet wormwood
Get effective part of sweet wormwood by embodiment 7 preparations, the suitable quantity of water dissolving, last DEAE Sepharose FF post, water, 0 1～1mol/LNaCl gradient elution, the phenolsulfuric acid method detects collects liquid, merge the eluent that the polysaccharide reaction is positive, concentrate, the dialyzer that adopts cutoff value 3500 is removed the micromolecule of molecular weight below 3500 with pure water dialysis 48 hours, 45 ℃ of concentrating under reduced pressure of liquid glucose, lyophilization gets powder, is the effective part of sweet wormwood of purification, have 6 positions, water, 0.1,02,0.4,06,1 0mol/L NaCl eluting position.Adopting sulfuric acid-phynol method to measure sugared content, is reference substance with the glucose, and the result shows that the sugared content of each several part is respectively 95 1%, 84 3%, 74 1%, 71 8%, 37 9%, 21 5%.The each several part polysaccharide molecular weight is distributed as 5000-50000 after measured, includes rhamnose, arabinose, xylose, mannose, six kinds of monosaccharide residues of galactose and glucose.
Embodiment 9: the preparation of effective part of sweet wormwood
The extraction of 1 Herba Artemisiae Annuae crude polysaccharides
Get Herba Artemisiae Annuae and decoct with water 3 times, each amount of water is 12 times of Herba Artemisiae Annuae weight, and each 2 hours, extracting solution merged, and concentrating under reduced pressure adds ethanol precipitation to containing alcohol amount 95%, leaves standstill, and the leaching precipitation use washing with alcohol, the dry Herba Artemisiae Annuae crude polysaccharides that gets;
Making with extra care of 2 effective part of sweet wormwood
Get the Herba Artemisiae Annuae crude polysaccharides, water fully dissolves, concentration is 0 7～1%, adopt the dialyzer of cutoff value 3500 to dialyse 72 hours with pure water, to remove the small molecular weight impurity of molecular weight 3500 below, liquid glucose is concentrated, 15000 rev/mins of high speed centrifugations 15 minutes, lyophilization gets powder, is effective part of sweet wormwood.Adopting sulfuric acid-phynol method to measure sugared content, is reference substance with the glucose, and the result shows that sugared content is 61 8%.This polysaccharide molecular weight is distributed as 5000-50000 after measured, comprises rhamnose, arabinose, xylose, mannose, six kinds of monosaccharide residues of galactose and glucose.
Embodiment 10: the purification of effective part of sweet wormwood
Get by water, 01,02,0 4mol/LNaCl eluting position in the effective part of sweet wormwood of embodiment 2 preparations, respectively by the purgation operation.The suitable quantity of water dissolving, with the exclusion scope is molecular sieve chromatography (SephadexG or the Sephacryl gel column) separation of 1000～150000D, is in charge of collection, and sulfuric acid-phynol method or differential refractometer detect, describe Xian and take off curve, the collection liquid that merges peak position, concentrating under reduced pressure adopts the dialyzer of cutoff value 3500 to dialyse 48 hours with pure water, remove the micromolecule of molecular weight below 3500,45 ℃ of concentrating under reduced pressure of liquid glucose, lyophilization gets powder.It is 13000,11000,20000,42000 that water, 01,02,0 Xian 4mol/LNaCl take off the polysaccharide molecular weight that obtains after separate through molecular sieve chromatography at the position; Adopting sulfuric acid-phynol method to measure sugared content, is reference substance with the glucose, and the result shows that the sugared content of each several part is respectively 95 8%, 95 2%, 93.7%, 94 2%; Include rhamnose after measured, arabinose, xylose, mannose, six kinds of monosaccharide residues of galactose and glucose.
Claims (7)
1. effective part of sweet wormwood, it is characterized in that this effective part of sweet wormwood obtains by extracting in the Herba Artemisiae Annuae, wherein polyoses content is not less than 50% by weight percentage, polysaccharide molecular weight is 5000-50000, and contains rhamnose, arabinose, xylose, mannose, galactose and six kinds of monosaccharide residues of glucose;
This effective part of sweet wormwood prepares by following method:
1) extraction of Herba Artemisiae Annuae crude polysaccharides: get Herba Artemisiae Annuae and decoct with water 2～3 times, each amount of water is 10～15 times of Herba Artemisiae Annuae weight, and each 1～2 hour, extracting solution merged, concentrating under reduced pressure adds ethanol precipitation to containing the alcohol amount more than 85%, leaves standstill, the leaching precipitation is used washing with alcohol, the dry Herba Artemisiae Annuae crude polysaccharides that gets;
2) effective part of sweet wormwood is refining: get the Herba Artemisiae Annuae crude polysaccharides, water fully dissolves, concentration is 0.7～1%, the employing cutoff value is that the dialyzer of 3500～5000D was dialysed 48～72 hours with pure water, remove micromolecule, liquid glucose concentrates, and is centrifugal, the supernatant lyophilization gets powder, promptly gets effective part of sweet wormwood;
Perhaps
Get the Herba Artemisiae Annuae crude polysaccharides, water fully dissolves, and adopting cutoff value is dialyzer or the ultrafilter membrane removal micromolecule of 3500～5000D, and liquid glucose concentrates, centrifugal, weak anionic exchange column on the supernatant, water, 0.1～1mol/L NaCl gradient elution are collected eluent, adopting cutoff value is dialyzer or the ultrafilter membrane removal micromolecule of 3500～5000D, concentrate, drying promptly gets effective part of sweet wormwood.
2. the preparation method of effective part of sweet wormwood as claimed in claim 1 is characterized in that this method comprises the following steps:
1) extraction of Herba Artemisiae Annuae crude polysaccharides: get Herba Artemisiae Annuae and decoct with water 2～3 times, each amount of water is 10～15 times of Herba Artemisiae Annuae weight, and each 1～2 hour, extracting solution merged, concentrating under reduced pressure adds ethanol precipitation to containing the alcohol amount more than 85%, leaves standstill, the leaching precipitation is used washing with alcohol, the dry Herba Artemisiae Annuae crude polysaccharides that gets;
2) effective part of sweet wormwood is refining: get the Herba Artemisiae Annuae crude polysaccharides, water fully dissolves, concentration is 0.7～1%, the employing cutoff value is that the dialyzer of 3500～5000D was dialysed 48～72 hours with pure water, remove micromolecule, liquid glucose concentrates, and is centrifugal, the supernatant lyophilization gets powder, is effective part of sweet wormwood;
Perhaps
Get the Herba Artemisiae Annuae crude polysaccharides, water fully dissolves, and adopting cutoff value is dialyzer or the ultrafilter membrane removal micromolecule of 3500～5000D, and liquid glucose concentrates, centrifugal, weak anionic exchange column on the supernatant, water, 0.1～1mol/L NaCl gradient elution are collected eluent, adopting cutoff value is dialyzer or the ultrafilter membrane removal micromolecule of 3500～5000D, concentrate, drying promptly gets effective part of sweet wormwood.
3. the preparation method of effective part of sweet wormwood according to claim 2 is characterized in that this method also comprises the following steps:
Get effective part of sweet wormwood, suitable quantity of water dissolving, last weak anionic exchange column, water, 0.1～1mol/LNaCl gradient elution, the phenolsulfuric acid method detects collects liquid, merges the eluent that the polysaccharide reaction is positive, and concentrates, the employing cutoff value is that the dialyzer of 3500～5000D was dialysed 48～72 hours with pure water, remove micromolecule, 40～50 ℃ of concentrating under reduced pressure of liquid glucose, lyophilization, get powder, be the effective part of sweet wormwood of purification.
Perhaps
Getting effective part of sweet wormwood is that the molecular sieve chromatography of 1000～150000D separates with the exclusion scope respectively after with water dissolution, obtain a series of molecular weight different but each series is respectively the effective part of sweet wormwood of the purification of homogeneous molecular weight.
4. according to claim 2 or 3 described methods, it is characterized in that the weak anionic exchange column is a DEAE weak anionic exchange column.
5. method according to claim 4 is characterized in that DEAE weak anionic exchange column is DEAESepharose F.F. post or DEAE cellulose column.
6. method according to claim 3 is characterized in that molecular sieve chromatography is SephadexG or Sephacryl gel column.
7. the application of effective part of sweet wormwood as claimed in claim 1 in the medicine of preparation antineoplastic agent or treatment immune disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100965310A CN100417390C (en) | 2006-09-30 | 2006-09-30 | Effective part of sweet wormwood, its preparation method and application thereof in pharmacy |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100965310A CN100417390C (en) | 2006-09-30 | 2006-09-30 | Effective part of sweet wormwood, its preparation method and application thereof in pharmacy |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1951396A CN1951396A (en) | 2007-04-25 |
| CN100417390C true CN100417390C (en) | 2008-09-10 |
Family
ID=38058079
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2006100965310A Active CN100417390C (en) | 2006-09-30 | 2006-09-30 | Effective part of sweet wormwood, its preparation method and application thereof in pharmacy |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100417390C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109674811B (en) * | 2018-12-26 | 2021-03-26 | 遵义医科大学 | Agastache rugosus polysaccharide composition and application and extraction method thereof |
| CN111484565B (en) * | 2019-01-27 | 2022-06-07 | 复旦大学 | Sweet wormwood polysaccharide, preparation method thereof and application thereof in preparation of anticomplement medicines |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020046488A (en) * | 2000-12-14 | 2002-06-21 | 채문식 | Agents for gastroenteric disorder by Helicobacter pylori and screening method thereof |
| KR20030092805A (en) * | 2002-05-31 | 2003-12-06 | 안병용 | Composition of crude polysaccharide of water extract of Artemisia iwayomogi Kitamura on melanin biosynthesis |
-
2006
- 2006-09-30 CN CNB2006100965310A patent/CN100417390C/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020046488A (en) * | 2000-12-14 | 2002-06-21 | 채문식 | Agents for gastroenteric disorder by Helicobacter pylori and screening method thereof |
| KR20030092805A (en) * | 2002-05-31 | 2003-12-06 | 안병용 | Composition of crude polysaccharide of water extract of Artemisia iwayomogi Kitamura on melanin biosynthesis |
Non-Patent Citations (2)
| Title |
|---|
| 青蒿提取物康单纯疱疹病毒活性研究. 张军峰等.天然产物研究与开发，第15卷第2期. 2003 |
| 青蒿提取物康单纯疱疹病毒活性研究. 张军峰等.天然产物研究与开发，第15卷第2期. 2003 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1951396A (en) | 2007-04-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102600219B (en) | Total flavone extract of abelmoschus manihot and preparing method of total flavone extract | |
| CN108546306B (en) | Cordyceps militaris culture medium polysaccharide, and separation and purification method and application thereof | |
| CN109400741B (en) | Separation and purification method of ganoderma lucidum spore polysaccharide | |
| CN102652792A (en) | Anti-cancer application of composition containing garlic oil, garlic total polysaccharide and garlic total saponin | |
| CN107722131B (en) | Total ganoderma lucidum spore powder refined polysaccharide with significant auxiliary antitumor activity and preparation method and application thereof | |
| CN102875689A (en) | Preparation method and application of ophiopogon japonicus polysaccharide | |
| CN106084087A (en) | A kind of preparation method of Fructus Trichosanthis polysaccharide | |
| CN106008729B (en) | A kind of sunset abelmoschus stem or bark leaf polyose and preparation method and application | |
| CN108503724B (en) | Cordyceps militaris culture medium polysaccharide, and separation and purification method and application thereof | |
| CN1965894B (en) | Prunella vulgaris polysaccharide having immune activity, preparation method and application thereof | |
| CN100417390C (en) | Effective part of sweet wormwood, its preparation method and application thereof in pharmacy | |
| CN104877037B (en) | Separation and purification method, products and application of Christia vespertilionis polysaccharides | |
| CN103275237B (en) | Preparation method and application of eggplant branch polysaccharide | |
| CN105796587B (en) | Caulis bambusae in taenian polysaccharide immunological regulation, it is antitumor in application | |
| CN110862463A (en) | Preparation of alfalfa root polysaccharide with bioactivity and selenizing modified polysaccharide thereof | |
| CN113717296B (en) | Eucommia acidic polysaccharide, extraction method and application of eucommia acidic polysaccharide in preparation of anti-colon cancer drugs | |
| CN1266164C (en) | Lucid ganoderma spore powder polysaccharide, production method and use | |
| CN110522776B (en) | Preparation method and application of anti-tumor effective part in hibiscus syriacus | |
| CN108752498B (en) | Senecio cannabifolius less polysaccharide with immunity enhancing activity and preparation method and application thereof | |
| CN1864725A (en) | Method for extracting active ingredient of starofbethlehem and use thereof | |
| CN107412730A (en) | A kind of glutinous rehmannia protein nano particle and preparation method thereof | |
| CN103374078A (en) | Homopolysaccharide in ganoderma sinensis submerged fermentation mycelium, as well as preparation method and applications thereof | |
| CN102114047B (en) | Woodlouse lyophilized powders 1 and 2 prepared by combined enzymolysis of pepsin and trypsinase and application thereof | |
| CN105061615A (en) | Chinese arborvitae twig polysaccharide with antiviral and immunity-enhancing activity, and preparation method and application thereof | |
| CN116731217B (en) | Ampelopsis grossedentata acidic polysaccharide AGP-2a, preparation method thereof and application thereof in preparing anti-inflammatory cosmetics |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |