CN101654485B - Spreading hedvotis herb polysaccharide for curing malignant tumor and preparation method thereof - Google Patents
Spreading hedvotis herb polysaccharide for curing malignant tumor and preparation method thereof Download PDFInfo
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- CN101654485B CN101654485B CN200910017798XA CN200910017798A CN101654485B CN 101654485 B CN101654485 B CN 101654485B CN 200910017798X A CN200910017798X A CN 200910017798XA CN 200910017798 A CN200910017798 A CN 200910017798A CN 101654485 B CN101654485 B CN 101654485B
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Abstract
The invention discloses a spreading hedvotis herb polysaccharide for curing malignant tumor, belonging to the traditional Chinese medicine field. The preparation method of the spreading hedvotis herb polysaccharide comprises the following steps: adopting spreading hedvotis herb as raw material, extracting with water, sedimenting with alcohol, adding tannin to remove protein, decoloring with activated carbon, ultrafiltrating, performing column chromatography separation and the like. The spreading hedvotis herb polysaccharide has better antitumor activity.
Description
Technical field
The present invention relates to a kind of Herba Hedyotidis Diffusae polysaccharide method for preparing, particularly a kind of Herba Hedyotidis Diffusae polysaccharide method for preparing and application thereof that is used to treat malignant tumor belongs to the field of Chinese medicines.
Background technology
Malignant tumor is one of disease of a kind of serious harm human health; According to the up-to-date statistical result showed of World Health Organization (WHO); Annual neopathy 1,000 ten thousand people of whole world cancer; Dead 7,000,000 people, China die from the patient of cancer every year more than 2,000,000 people, are No. second killers of the mankind who is only second to cardiovascular and cerebrovascular disease.At present, medical circle does not still have good method for malignant tumor.The targeted therapy of the operation of present routine clinical application, radiotherapy, chemotherapy and rising in recent years is all failed effectively to control tumor and is prevented its relapse and metastasis, and all has serious toxic and side effects, has a strong impact on patients ' life quality; Cost an arm and a leg simultaneously, increase the weight of country and patient's household economy burden.
Herba Hedyotidis Diffusae Oldenlandia diffusa (Willd.) Roxb. has another name called Herba Hedyotidis Diffusae, tang Huang, Serpentis needle grass, Radix Picriae felterrae, Clerodendrum thomsonae Balf., the young grass of pearl etc.; Cold in nature, sweet and slightly bitter taste, GUIXIN, liver, spleen channel; Beginning is stated from Shennong's Herbal; Tool heat clearing away, dampness removing, detoxifcation, effect such as anticancer are kindly controlled cough due to lung-heat, various cancer, are the antitumor drug of wide clinical application.A large amount of pharmacodynamic experiments and clinical practice experiment show that Herba Hedyotidis Diffusae has excellent curative to tumor, and its remarkable immunity of enhancing body, and great value of exploiting and utilizing is arranged.Document concentrates on polysaccharide basically to the research of Herba Hedyotidis Diffusae anti-tumor activity.But mostly the research for Herba Hedyotidis Diffusae polysaccharide is crude polysaccharides, not to the further refining purification research of Herba Hedyotidis Diffusae crude polysaccharides.For fundamentally eliminating the malignant tumor cause of disease; Improve symptom; With many side effect of avoiding in the present treatment means; Alleviate the patient suffering, necessary, Herba Hedyotidis Diffusae polysaccharide (POD) that anti-tumor activity stronger higher to the resulting purity of the further separation and purification of the polysaccharide in the Herba Hedyotidis Diffusae.
Summary of the invention
One object of the present invention is to provide a kind of Herba Hedyotidis Diffusae polysaccharide of treating malignant tumor;
Another object of the present invention is to provide the method for preparing of above-mentioned Herba Hedyotidis Diffusae polysaccharide.
The objective of the invention is to realize like this:
Get the Herba Hedyotidis Diffusae medical material, add 4～20 times of water gagings, extract 1～4 time, each 0.5～3h filters; Merging filtrate concentrates, and adds ethanol and makes and contain the alcohol amount and reach 40～90%, and cold preservation to deposition is separated out fully, filters; Deposition is dissolved in water, and adds 20% tannic acid alcoholic solution of 0.01～3% parts by volume, and cold preservation to deposition is separated out fully, filters, and filtrating adds 0.01～3% active carbon; Decolour 1～4 time, each 0.5～2h filters, and filtrate flow is the ultrafilter membrane of 10k through the relative molecular mass that dams, and collects relative molecular mass less than the 10k part; Drying, dry thing is dissolved in water, and through the DEAE-Sephrose ion exchange column, with 0.5mol/L sodium chloride solution eluting, UV-detector 210nm detects; Collect the eluent of the 4th detected peaks, behind the dialysis desalination,, use water elution through Superdex 30 gel columns; UV-detector 210nm detects, and collects the eluent of the 4th detected peaks, and drying promptly gets.
Optimizing technology is: get the Herba Hedyotidis Diffusae medical material, add 8～15 times of water gagings, extract 1～3 time, each 1～2h filters; Merging filtrate concentrates, and adds ethanol and makes and contain the alcohol amount and reach 50～80%, and cold preservation is separated out fully to deposition, and deposition is dissolved in water; The 20% tannic acid alcoholic solution that adds 0.1～1% parts by volume, cold preservation to deposition is separated out fully, filters, and filtrating adds 0.2～1% active carbon, decolours 1～3 time; Each 0.5～1h filters, and filtrate flow is the ultrafilter membrane of 10k through the relative molecular mass that dams, and collects relative molecular mass less than 10k part, drying; Dry thing is dissolved in water, and through the DEAE-Sephrose ion exchange column, with 0.5mol/L sodium chloride solution eluting, UV-detector 210nm detects; Collect the eluent of the 4th detected peaks, behind the dialysis desalination,, use water elution through Superdex 30 gel columns; UV-detector 210nm detects, and collects the eluent of the 4th detected peaks, and drying promptly gets.
Further optimizing technology is: get the Herba Hedyotidis Diffusae medical material, add 75～85 ℃ of hot water of 12 times of amounts, insulation is extracted 3 times, and each 1h filters; Merging filtrate concentrates, and adds ethanol and makes and contain the alcohol amount and reach 80%, and cold preservation is separated out fully to deposition, and deposition is dissolved in water; The 20% tannic acid alcoholic solution that adds 0.5% parts by volume, cold preservation to deposition is separated out fully, filters, and filtrating adds 0.5% active carbon, decolours 3 times; Each 1h filters, and filtrate flow is the ultrafilter membrane of 10k through the molecular weight that dams, and collects relative molecular mass less than 10k part, lyophilization; Be dissolved in water, through the DEAE-Sephrose ion exchange column, with 0.5mol/L sodium chloride solution eluting, flow velocity 1ml/min, UV-detector 210nm detects; Collect the eluent of the 4th detected peaks, behind the dialysis desalination, through Superdex 30 gel columns, use water elution, flow velocity 0.5ml/min; UV-detector 210nm detects, and collects the eluent of the 4th detected peaks, and lyophilizing promptly gets.
Purity for the Herba Hedyotidis Diffusae polysaccharide of proving conclusively above-mentioned technology gained guarantees that it plays the material base of physiologically active, and the inventor has carried out molecular weight determination, and other purity testings to the Herba Hedyotidis Diffusae polysaccharide of above-mentioned technology gained.
(1) relative molecular mass is measured: adopt gel permeation chromatography that the molecular weight of the refining polysaccharide HPS of Herba Hedyotidis Diffusae is measured.Select polysaccharide standard substance (Dextran T5, T10, T40, T70, T500, the T2000 of 6 known molecular amounts for use; Molecular weight is respectively 0.5 ten thousand, 10,000,40,000,70,000,500,000,2,000,000), process the standard solution of 5mg/ml respectively with distilled water, get 100 μ l respectively and inject AKTA column chromatography system; Sodium chloride solution eluting with 0.15mol/L; Flow velocity is 0.5ml/min, detects polysaccharide in the 210nm place, tries to achieve elution volume Ve respectively.With T2000 as post void volume V
0, with Ve/V
0Be abscissa, 1gM is a vertical coordinate, obtains the molecular weight determination standard curve.
Assay method: get polysaccharide 5mg to be measured, be dissolved in 1ml water, sample size is 100 μ l, and elution requirement is tried to achieve corresponding elution volume with Dextran standard substance series, calculates Ve/V
0Value according to gained standard molecular weight standard curve, is tried to achieve its molecular weight.Adopt gel permeation chromatography (GPC) that the molecular weight of POD is measured, obtain the elution volume of different molecular weight standard polysaccharide and POD, result of the test is seen table 1.According to the mensuration result of standard polysaccharide in the table, according to lgM-Ve/V
0The relation mapping obtains molecular weight-elution volume canonical plotting, must return the standard curve equation and be: Y=-1.5340X+7.8137 R
2=0.9996, try to achieve its relative molecular mass according to the elution volume of Herba Hedyotidis Diffusae polysaccharide and be about 6310.
The elution volume of table 1 molecular weight standard polysaccharide and Herba Hedyotidis Diffusae polysaccharide and molecular weight
(2) monosaccharide composition analysis
Adopt the HPLC-ELSD method that the monosaccharide after the complete acid hydrolysis of Herba Hedyotidis Diffusae polysaccharide component is analyzed.Chromatographic condition is a chromatographic column: Zorbax Carbohydrate Analysis Column (4.6 * 250mm, 5 μ m); Mobile phase: acetonitrile: water=75: 25; Flow velocity: 1ml/min; Alltech ELSD2000 working condition: drift tube temperature: 81.3 ℃, flow rate of carrier gas: 2.1L/min, column temperature: 40 ℃.
Reference substance solution: D-glucose, D-fructose, D-mannose, D-xylose, D-arabinose, D-galactose, L-rhamnose standard substance are 100 μ g/ml, and solvent is 50% acetonitrile; Hybrid standard monosaccharide: the Concentraton gradient of D-glucose, D-fructose, D-mannose, D-xylose, D-arabinose, D-galactose, L-rhamnose hybrid standard monosaccharide is: 15.63 μ g/ml, 31.25 μ g/ml, 62.5 μ g/ml, 125 μ g/ml, 200 μ g/ml, 250 μ g/ml, and solvent is 50% acetonitrile;
Need testing solution: get Herba Hedyotidis Diffusae polysaccharide 10mg, put in the 10ml tool plug test tube, add 2mol/L trifluoroacetic acid solution 5ml, tube sealing, 100 ℃ of hydrolysis 8h.Behind the hydrolyzed solution vacuum drying, the trifluoroacetic acid that adds 5ml 2mol/L again repeats hydrolysis once, cooling, and it is clean that the adding distil water repeating vacuum is dried to the trifluoroacetic acid volatilization, dissolves with 0.5ml 50% acetonitrile at last.
Assay method: at first D-glucose, D-fructose, D-mannose, D-xylose, D-arabinose, D-galactose, 7 kinds of standard monosaccharide of L-rhamnose are distinguished sample introduction; Confirm each monosaccharide retention time; Application mix standard monosaccharide sample introduction is distinguished the production standard curve according to each monosaccharide peak area and standard monosaccharide concentration then.According to the ratio between each monosaccharide of calculated by peak area of need testing solution.Result of the test shows: Herba Hedyotidis Diffusae polysaccharide mainly contains rhamnose, xylose, galactose and glucose.With corresponding standard curve in the peak area substitution table of each monosaccharide among the HPS; The mass ratio that calculates each monosaccharide in the Herba Hedyotidis Diffusae polysaccharide is: rhamnose: xylose: galactose: glucose=36.77: 17.28: 64.00: 122.26, and convert mol ratio into and be: rhamnose: xylose: galactose: glucose=1.75: 1: 3.09: 5.90.Concrete experimental result is seen table 2,3.
Table 2 monosaccharide peak area and standard monosaccharide concentration relation
The standard curve of each standard monosaccharide of table 3
(3) purity is identified
The purity that adopts the gel filtration chromatography method to carry out the gained polysaccharide is identified.
Assay method: take by weighing Herba Hedyotidis Diffusae polysaccharide 6mg, be dissolved in the 0.75ml distilled water, applied sample amount is 0.5ml, and gel column is Superdex 30 posts, adopts 0.15mol/L sodium chloride to carry out eluting, and flow velocity is 0.5ml/min, carries out online ultraviolet detection in the 210nm place.Measure through going up appearance repeatedly, eluting peak is single symmetrical peak all, explains that the purity of gained polysaccharide is higher.
Beneficial effect
Herba Hedyotidis Diffusae polysaccharide of the present invention has very strong anti-tumor activity.
Be further checking pharmacological action of the present invention, carried out animal contrast pharmacodynamic experiment with Herba Hedyotidis Diffusae polysaccharide of the present invention.
Experimental example 1: the present invention is to the influence of S180 tumor-bearing mice
1 reagent, sample and animal
1.1 reagent
Cyclophosphamide for injection, 200mg/ props up, Hengrui Medicine Co., Ltd., Jiangsu Prov., lot number: 08051354
1.2 confession test agent:
Add 80 ℃ of hot water of 12 times of amounts, insulation is extracted 3 times, and each 1h filters merging filtrate; Concentrate, add ethanol and make and contain the alcohol amount and reach 80%, cold preservation is separated out fully to deposition, and deposition is dissolved in water, and adds 20% tannic acid alcoholic solution of 0.5% parts by volume; Cold preservation to deposition is separated out fully, filters, and filtrating adds 0.5% active carbon, decolours 3 times, each 1h; Filter, filtrate flow is the ultrafilter membrane of 10k through the molecular weight that dams, and collects relative molecular mass less than the 10k part, and lyophilization is dissolved in water; Through DEAE-Sephrose ion exchange column (blade diameter length ratio is 1: 15), with 0.5mol/L sodium chloride solution eluting, flow velocity 1ml/min, UV-detector 210nm detects, and collects the eluent of the 4th detected peaks; Behind the dialysis desalination,, use water elution, flow velocity 0.5ml/min through Superdex 30 gel columns (blade diameter length ratio is 1: 20); UV-detector 210nm detects, and collects the eluent of the 4th detected peaks, and lyophilizing is mixed with desired concn with preceding with distilled water.
1.3 laboratory animal
Male Kunming strain mice, the SPF level, body weight 20 ± 2g, available from Shandong Traditional Chinese Medicine University's Experimental Animal Center, the animal credit number is: SCXK (Shandong) 20050015.S180 ascites mice is available from Shandong Academy of Medical Sciences.
2 experimental techniques
The go down to posterity S180 ascites mouse peritoneal tumor cell of 8d of aseptic extraction adds normal saline adjustment concentration to 4X10
9/ L in the right oxter injection of every mice 0.2ml, inoculates 60.Behind the inoculation 24h, mice is divided into 5 groups at random, 10 every group, is divided into model group, the high, medium and low dose groups of the present invention and matched group.Model group is irritated stomach normal saline 10mLKg
-1, continuous 10d; The high, medium and low dose groups of Herba Hedyotidis Diffusae polysaccharide is irritated stomach 20,10,5mgKg respectively
-1Medicinal liquid, 10d continuously; The matched group intraperitoneal injection of cyclophosphamide, dosage 30mgKg
-1, continuous 2d; Dosage is 10mLKg
-1Behind the last administration 24h, the cervical vertebra dislocation is put to death, and peels off tumor, thymus, spleen, weighs, and calculates tumour inhibiting rate, thymus and index and spleen index, and computing formula is following:
Tumour inhibiting rate %=(blank control group tumor weight-average value-sample sets tumor weight-average value)/blank control group tumor weight-average value * 100%
Thymus (spleen) index=weight (mg)/body weight (g)
3 experimental results
The tumour inhibiting rate of 3 dose groups of the present invention is respectively 34.4%, 26.2%, 17.1%.Compare with model group, cyclophosphamide group and Herba Hedyotidis Diffusae polysaccharide are high, middle dose groups tumour inhibiting rate all has significant difference (P＜0.05), though low dose group has certain tumor killing effect, compare there was no significant difference (seeing table 4) with model group.The spleen of each dose groups of the present invention, thymus index and model group be there was no significant difference (P＞0.05) relatively, and cyclophosphamide group and model group relatively have utmost point significant difference (P＜0.01) (seeing table 5).
Table 4 the present invention is to tumor-inhibiting action in the body of S180 tumor-bearing mice
Annotate: compare * P＜0.05, * * P＜0.01 with model group.
Table 5 the present invention is to tumor-inhibiting action in the body of S180 tumor-bearing mice
Annotate: compare * P＜0.05, * * P＜0.01 with model group.
Experimental example 2: the present invention is to the influence of H22 tumor-bearing mice
1 reagent, sample and animal
1.1 reagent
Fluorouracil Injection (5-Fu), Shanghai Xudong Hipu Medicine Co., Ltd, lot number: 080302
1.2 supply test agent (with experimental example 1)
1.3 laboratory animal
Male Kunming strain mice, the SPF level, body weight 20 ± 2g, available from Shandong Traditional Chinese Medicine University's Experimental Animal Center, the animal credit number is: SCXK (Shandong) 20050015.The H22 liver cancer mouse is available from Shandong Academy of Medical Sciences.
2 experimental techniques
The go down to posterity H22 liver cancer mouse belly cavity tumor cell of 8d of aseptic extraction adds normal saline adjustment concentration to 4X10
9/ L in the right oxter injection of every mice 0.2ml, inoculates 60.Behind the inoculation 24h, mice is divided into 5 groups at random, 10 every group, is divided into model group, the high, medium and low dose groups of the present invention and matched group.Model group is irritated stomach normal saline 10mLKg
-1, continuous 10d; The high, medium and low dose groups of Herba Hedyotidis Diffusae polysaccharide is irritated stomach 20,10,5mgKg respectively
-1Medicinal liquid, 10d continuously; Matched group lumbar injection 5-Fu, dosage 25mgKg
-1, continuous 2d; Dosage is 10mLKg
-1Behind the last administration 24h, the cervical vertebra dislocation is put to death, and peels off tumor, thymus, spleen, weighs, and calculates tumour inhibiting rate, thymus and index and spleen index, and computing formula is following:
Tumour inhibiting rate %=(blank control group tumor weight-average value-sample sets tumor weight-average value)/blank control group tumor weight-average value * 100%
Thymus (spleen) index=weight (mg)/body weight (g)
3 experimental results
The tumour inhibiting rate of 3 dose groups of the present invention is respectively 43.3%, 33.5%, 23.6%.Compare with model group, the 5-Fu group all has significant difference (P＜0.05) (table 6) with each dose groups tumour inhibiting rate of Herba Hedyotidis Diffusae polysaccharide.The index and spleen index of each dose groups of the present invention and model group be there was no significant difference (P＞0.05) relatively; Among the present invention, the thymus index of low dose group and model group there was no significant difference (P＞0.05) relatively; But the thymus index of high dose group and model group relatively have significant difference (P＜0.05), and spleen, thymus index and the model group of 5-Fu group more all have utmost point significant difference (P＜0.01) (table 7).
Table 6 the present invention is to tumor-inhibiting action in the body of H22 tumor-bearing mice
Annotate: compare * P＜0.05, * * P＜0.01 with model group.
Table 7 the present invention is to tumor-inhibiting action in the body of H22 tumor-bearing mice
Annotate: compare * P＜0.05, * * P＜0.01 with model group.
Experimental example 3: the present invention is to the influence of human cervical carcinoma Hela cell and the growth of people's hepatocarcinoma Bel-7402 cell
1 reagent, sample
1.1 reagent
People's hepatocarcinoma Bel-7402 cell strain, human cervical carcinoma Hela cell's strain (U.S. Sciencell company) are available from Beijing North great achievement Development Co., Ltd; Herba Hedyotidis Diffusae polysaccharide, self-control; 5-fluorouracil (5-Fu) is available from Hefei Huo Shi Pharmaceutical Technology Co., Ltd; RPMI-1640 culture fluid (U.S. GIBCO company) is available from Shanghai Zhuo Kang bio tech ltd; Hyclone is available from Beijing Baeyer enlightening Bioisystech Co., Ltd; Trypsin, ribonuclease (RNase) and propidium iodide (PI) are available from Chemical Reagent Co., Ltd., Sinopharm Group; Tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) are available from Sigma company.
1.2 supply test agent (with experimental example 1)
2 experimental techniques
2.1 cell culture
Get people's hepatocarcinoma Bel-7402 cell strain, human cervical carcinoma Hela cell's strain tumor cell is inoculated in the culture bottle with an amount of concentration, add to contain 10
6The RPMI-1640 culture fluid of U/L gentamycin, 0.1g/L streptomycin and 10% hyclone places to contain 5%CO
2, 37 ℃ of constant temperature and saturated humidity incubator in cultivate, the cell attachment growth was gone down to posterity once in per 2～3 days; Topple over original culture medium, add the digestion of pancreatin solution, treat cell from the bottle wall come off be suspended in the liquid after; Add a small amount of hyclone and stop digestion, the stupid supernatant liquid that contains cell in the former bottle is moved in the centrifuge tube, to discard liquid behind the centrifugal 5min of 1000rpm; Add fresh culture piping and druming evenly, move in the new culture bottle, add complete culture solution to an amount of by required cell concentration.The continuous freeze-stored cell of experimental session, all cell exponential phase of growth is all used in experiment, and each is organized cell and all cultivates 72h.
2.2 test is divided into groups
Be divided into 5 groups at random, the blank group: in containing 10
6The conventional cultivation in the RPMI-1640 culture fluid of U/L gentamycin, 0.1g/L streptomycin and 10% hyclone; Supply the examination drug group: it is an amount of to get test liquid of the present invention, adds suitable RPMI1640 culture fluid dilution, and final concentration is respectively 10g/L, 50g/L and 100g/L (in raw medicinal herbs); Positive controls: 5-fluorouracil adds the dilution of RPMI1640 culture fluid through 0.22 μ m microporous filter membrane EK, and final concentration is 0.1g/L.
2.3 adopt the metamorphosis of cell in the inverted microscope direct observation incubation, the itemized record observe phenomena.
2.4 mtt assay is measured inhibitory rate of cell growth
With each tumor cell inoculation in 96 well culture plates, every porocyte several 10
4Individual (it is the same to divide into groups, and the blank group adds the equivalent complete medium); After the cell synchronization, change fresh culture, add a corresponding drug solution respectively, cultivate 72h.The careful suction of liquid-transfering gun goes all supernatant, every hole to add 160 μ l serum-free mediums and 40 μ lMTT (the MTT final concentration is 1g/L); Aluminium foil is wrapped culture plate, continues to cultivate 4h; Inhale when stopping cultivating and remove all supernatant in the hole, every hole adds 200 μ lDMSO, and the 10min that on microwell plate agitator under the room temperature, vibrates adopts ELIASA to measure corresponding optical density (D value) in the 570nm wavelength.
2.5 cells were tested by flow cytometry apoptosis rate
Collect above-mentioned Bel-7402 and Hela cell after treatment respectively, using PBS to transfer cell concentration is 10
9/ L gets the 1ml single cell suspension, and centrifugal, rinsing with 70% ethanol 1ml fixed cell, is used the PBS rinsing then.In unicellular sample, add RNase; Adding final concentration again is the PI dyeing of 50mg/L; Adopt cells were tested by flow cytometry apoptosis (PBLC with the normal person is the normal diploid standard, with the apoptosis rate of CELLQUEST analysis software Bel-7402 and Hela cell) after the filtration immediately.
3 experimental results
3.1 cellular morphology changes
Can be observed through inverted microscope, the growth of of the present invention group Bel-7402 cell obviously is suppressed, and cell is by adherent and come off gradually, and floating being suspended from the culture fluid, cell transparency and adhesion decline, and cell rounding, and volume dwindles gradually; The growth of blank group cell is more normal, is fusiformis, adherent growth, and nucleocytoplasmic ratio is normal, intact nuclear membrane; Positive controls is more similar with heavy dose of examination drug group that supplies.
Of the present invention group Hela cell prolongation in time, cell volume increase, and a large amount of suspension cells engender, division stage cell rare, and various dose is not obvious to the influence of Hela cell; The growth of the Hela cell of blank group is more active, behind the inoculation 24h promptly adherent fully, visible division stage cell, suspension cell is few.
3.2 inhibitory action to Hela and the growth of Bel-7402 cell
The present invention all has certain inhibitory action to the proliferate of Hela and Bel-7402 cell, and along with the increase of concentration, suppression ratio has obvious rising, and result of the test is seen table 8.
Table 8 the present invention is to the influence (n=5) of Hela and the growth of Bel-7402 cell proliferation
Annotate: compare * P＜0.05, * * P＜0.01, * * * P＜0.001 with matched group.
3.3 to Hela and the apoptotic influence of Bel-7402
Discover that along with dosage of the present invention increases and prolongation action time, the apoptosis rate of Hela and Bel-7402 cell obviously raises, the heavy dose of group of the present invention is better than positive controls, has shown better antitumor activity, and result of the test is seen table 9.
Table 9 the present invention is to Hela and the apoptotic influence of Bel-7402
Annotate: compare * P＜0.05, * * P＜0.01, * * * P＜0.001 with matched group.
Above-mentioned experiment shows that the present invention has better antitumor activity.
Below come further to set forth technical scheme of the present invention with the specific embodiment, but the claimed content of application of the present invention and not only in these method for preparinies.The consumption of crude drug can be a unit with ton, kilogram, gram etc. also not only in the embodiment consumption in the actual production.
The specific embodiment
Embodiment 1:
Get Herba Hedyotidis Diffusae medical material 1kg, add 100 ℃ of hot water of 20 times of amounts, boil and extract 4 times, each 3h filters; Merging filtrate concentrates, and adds ethanol and makes and contain the alcohol amount and reach 90%, and cold preservation to deposition is separated out fully, filters; Deposition is dissolved in water, and adds 20% tannic acid alcoholic solution of 3% parts by volume, and cold preservation to deposition is separated out fully, filters, and filtrating adds 3% active carbon; Decolour 4 times, each 2h filters, and filtrate flow is the ultrafilter membrane of 10k through the relative molecular mass that dams, and collects relative molecular mass less than the 10k part; Drying, dry thing is dissolved in water, through DEAE-Sephrose ion exchange column (blade diameter length ratio is 1: 15), with 0.5mol/L sodium chloride solution eluting, flow velocity 1ml/min; UV-detector 210nm detects, and collects the eluent of the 4th detected peaks, behind the dialysis desalination, through Superdex 30 gel columns (blade diameter length ratio is 1: 20), uses water elution; Flow velocity 0.5ml/min, UV-detector 210nm detects, and collects the eluent of the 4th detected peaks, and drying gets polysaccharide 1.2g.
Embodiment 2:
Get Herba Hedyotidis Diffusae medical material 2kg, add 60 ℃ of hot water of 4 times of amounts, 0.5h is extracted in insulation, filters, and filtrating concentrates; Add ethanol and make and contain alcohol amount and reach 40%, cold preservation to deposition is separated out fully, filters, and deposition is dissolved in water, and adds 20% tannic acid alcoholic solution of 0.01% parts by volume; Cold preservation to deposition is separated out fully, filters, and filtrating adds 0.01% active carbon, and decolouring 0.5h filters; Filtrate flow is the ultrafilter membrane of 10k through the relative molecular mass that dams, and collects relative molecular mass less than the 10k part, drying, and dry thing is dissolved in water, through DEAE-Sephrose ion exchange column (blade diameter length ratio is 1: 12); With 0.5mol/L sodium chloride solution eluting, flow velocity 2ml/min, UV-detector 210nm detects, and collects the eluent of the 4th detected peaks; Behind the dialysis desalination,, use water elution, flow velocity 1ml/min through Superdex 30 gel columns (blade diameter length ratio is 1: 15); UV-detector 210nm detects, and collects the eluent of the 4th detected peaks, and drying under reduced pressure gets polysaccharide 1.8g.
Embodiment 3:
Get Herba Hedyotidis Diffusae medical material 1.5kg, add 70 ℃ of hot water of 8 times of amounts, 1h is extracted in insulation, filters, and filtrating concentrates; Add ethanol and make and contain alcohol amount and reach 50%, cold preservation is separated out fully to deposition, and deposition is dissolved in water, and adds 20% tannic acid alcoholic solution of 0.1% parts by volume, and cold preservation to deposition is separated out fully; Filter, filtrating adds 0.2% active carbon, and decolouring 0.5h filters, and filtrate flow is the ultrafilter membrane of 10k through the relative molecular mass that dams; Collect relative molecular mass less than the 10k part, drying, dry thing is dissolved in water, through DEAE-Sephrose ion exchange column (blade diameter length ratio is 1: 15); With 0.5mol/L sodium chloride solution eluting, flow velocity 2ml/min, UV-detector 210nm detects, and collects the eluent of the 4th detected peaks; Behind the dialysis desalination,, use water elution, flow velocity 1ml/min through Superdex 30 gel columns (blade diameter length ratio is 1: 20); UV-detector 210nm detects, and collects the eluent of the 4th detected peaks, and lyophilizing gets polysaccharide 1.5g.
Embodiment 4:
Get Herba Hedyotidis Diffusae medical material 1kg, add 90 ℃ of hot water of 15 times of amounts, insulation is extracted 3 times, and each 2h filters; Merging filtrate concentrates, and adds ethanol and makes and contain the alcohol amount and reach 80%, and cold preservation is separated out fully to deposition, and deposition is dissolved in water; The 20% tannic acid alcoholic solution that adds 1% parts by volume, cold preservation to deposition is separated out fully, filters, and filtrating adds 1% active carbon, decolours 3 times; Each 1h filters, and filtrate flow is the ultrafilter membrane of 10k through the relative molecular mass that dams, and collects relative molecular mass less than 10k part, drying; Dry thing is dissolved in water, through DEAE-Sephrose ion exchange column (blade diameter length ratio is 1: 20), and with 0.5mol/L sodium chloride solution eluting, flow velocity 1ml/min, UV-detector 210nm detects; Collect the eluent of the 4th detected peaks, behind the dialysis desalination,, use water elution, flow velocity 0.5ml/min through Superdex 30 gel columns (blade diameter length ratio is 1: 15); UV-detector 210nm detects, and collects the eluent of the 4th detected peaks, and lyophilizing gets polysaccharide 1.1g.
Embodiment 5:
Get Herba Hedyotidis Diffusae medical material 2kg, add 80 ℃ of hot water of 12 times of amounts, insulation is extracted 3 times, and each 1h filters; Merging filtrate concentrates, and adds ethanol and makes and contain the alcohol amount and reach 80%, and cold preservation is separated out fully to deposition, and deposition is dissolved in water; The 20% tannic acid alcoholic solution that adds 0.5% parts by volume, cold preservation to deposition is separated out fully, filters, and filtrating adds 0.5% active carbon, decolours 3 times; Each 1h filters, and filtrate flow is the ultrafilter membrane of 10k through the molecular weight that dams, and collects relative molecular mass less than 10k part, lyophilization; Be dissolved in water, through DEAE-Sephrose ion exchange column (blade diameter length ratio is 1: 15), with 0.5mol/L sodium chloride solution eluting, flow velocity 1ml/min, UV-detector 210nm detects; Collect the eluent of the 4th detected peaks, behind the dialysis desalination,, use water elution, flow velocity 0.5ml/min through Superdex 30 gel columns (blade diameter length ratio is 1: 20); UV-detector 210nm detects, and collects the eluent of the 4th detected peaks, and lyophilization gets polysaccharide 2g.
Claims (7)
1. a Herba Hedyotidis Diffusae polysaccharide that is used to treat malignant tumor is characterized in that this Herba Hedyotidis Diffusae polysaccharide is prepared by following method: get the Herba Hedyotidis Diffusae medical material, add 4～20 times of water gagings, extract each 0.5～3h 1～4 time; Filter, merging filtrate concentrates, and adds ethanol and makes and contain the alcohol amount and reach 40～90%, and cold preservation to deposition is separated out fully; Filter, deposition is dissolved in water, and adds 20% tannic acid alcoholic solution of 0.01～3% parts by volume, and cold preservation to deposition is separated out fully; Filter, filtrating adds 0.01～3% active carbon, decolours each 0.5～2h 1～4 time; Filter, filtrate flow is the ultrafilter membrane of 10k through the relative molecular mass that dams, and collects relative molecular mass less than 10k part, drying; Dry thing is dissolved in water, and through the DEAE-Sephrose ion exchange column, with 0.5mol/L sodium chloride solution eluting, UV-detector 210nm detects; Collect the eluent of the 4th detected peaks, behind the dialysis desalination,, use water elution through Superdex 30 gel columns; UV-detector 210nm detects, and collects the eluent of the 4th detected peaks, and drying promptly gets.
2. Herba Hedyotidis Diffusae polysaccharide as claimed in claim 1 is characterized in that the preparation process is: get the Herba Hedyotidis Diffusae medical material, add 8～15 times of water gagings, extract each 1～2h 1～3 time; Filter, merging filtrate concentrates, and adds ethanol and makes and contain the alcohol amount and reach 50～80%, and cold preservation to deposition is separated out fully; Deposition is dissolved in water, and adds 20% tannic acid alcoholic solution of 0.1～1% parts by volume, and cold preservation to deposition is separated out fully, filters, and filtrating adds 0.2～1% active carbon; Decolour 1～3 time, each 0.5～1h filters, and filtrate flow is the ultrafilter membrane of 10k through the relative molecular mass that dams, and collects relative molecular mass less than the 10k part; Drying, dry thing is dissolved in water, and through the DEAE-Sephrose ion exchange column, with 0.5mol/L sodium chloride solution eluting, UV-detector 210nm detects; Collect the eluent of the 4th detected peaks, behind the dialysis desalination,, use water elution through Superdex 30 gel columns; UV-detector 210nm detects, and collects the eluent of the 4th detected peaks, and drying promptly gets.
3. Herba Hedyotidis Diffusae polysaccharide as claimed in claim 2 is characterized in that the preparation process is: get the Herba Hedyotidis Diffusae medical material, add 75～85 ℃ of hot water of 12 times of amounts, insulation is extracted 3 times, each 1h; Filter, merging filtrate concentrates, and adds ethanol and makes and contain the alcohol amount and reach 80%, and cold preservation to deposition is separated out fully; Deposition is dissolved in water, and adds 20% tannic acid alcoholic solution of 0.5% parts by volume, and cold preservation to deposition is separated out fully, filters, and filtrating adds 0.5% active carbon; Decolour 3 times, each 1h filters, and filtrate flow is the ultrafilter membrane of 10k through the molecular weight that dams, and collects relative molecular mass less than the 10k part; Lyophilization is dissolved in water, through the DEAE-Sephrose ion exchange column, with 0.5mol/L sodium chloride solution eluting, flow velocity 1ml/min; UV-detector 210nm detects, and collects the eluent of the 4th detected peaks, behind the dialysis desalination, through Superdex 30 gel columns, uses water elution; Flow velocity 0.5ml/min, UV-detector 210nm detects, and collects the eluent of the 4th detected peaks, and lyophilizing promptly gets.
4. like each described Herba Hedyotidis Diffusae polysaccharide in the claim 1 to 3, the relative molecular mass that it is characterized in that this Herba Hedyotidis Diffusae polysaccharide is 6310.
5. like each described Herba Hedyotidis Diffusae polysaccharide in the claim 1 to 3, it is characterized in that containing rhamnose, xylose, galactose and glucose in this Herba Hedyotidis Diffusae polysaccharide, and its mol ratio is 1.75: 1: 3.09: 5.90.
6. the preparation technology of each described Herba Hedyotidis Diffusae polysaccharide in the claim 1 to 5.
7. each described Herba Hedyotidis Diffusae polysaccharide is used for the application of anti-tumor drug in the claim 1 to 5 in preparation.
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| CN103868840B (en) * | 2014-03-26 | 2017-02-15 | 南京中医药大学 | Determination method of molecular weight cutoff of ultrafiltration membrane |
| CN103893325B (en) * | 2014-04-18 | 2015-11-04 | 李晓明 | The application of Herba Hedyotidis Diffusae |
| CN104059159B (en) * | 2014-06-17 | 2016-04-20 | 湖南农业大学 | The processing method of Mierocrystalline cellulose Enzymatic Extraction Herba Hedyotidis Diffusae polysaccharide |
| CN104292353B (en) * | 2014-09-30 | 2016-06-29 | 河南科技大学 | A kind of antioxidation natural plant polyose and preparation method thereof |
| CN109096410A (en) * | 2018-09-04 | 2018-12-28 | 山东中医药大学附属医院 | Spreading hedvotis herb polysaccharide is preparing the application in intestinal flora adjusting drug |
| CN111484564B (en) * | 2019-01-27 | 2022-07-08 | 复旦大学 | Spreading hedyotis herb polysaccharide, preparation method thereof and application thereof in preparing anticomplement medicines |
| CN109932446A (en) * | 2019-03-21 | 2019-06-25 | 苏州大学 | A kind of detection method of Lycium barbarum polysaccharide extract |
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