CN101654485A - 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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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 herbpolysaccharide 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 preparation method, particularly a kind of Herba Hedyotidis Diffusae polysaccharide preparation method and application thereof that is used for the treatment of malignant tumour belongs to the field of Chinese medicines.
Background technology
Malignant tumour is one of disease of a kind of serious harm human health, according to the up-to-date statistical result showed of the World Health Organization, annual neopathy 1,000 ten thousand people of whole world cancer, dead 7,000,000 people, China dies from the patient of cancer every year more than 2,000,000 people, is No. second killer of the mankind who is only second to cardiovascular and cerebrovascular disease.At present, medical circle does not still have good method for malignant tumour.The targeted therapy of the operation of present routine clinical application, radiotherapy, chemotherapy and rising in recent years is all failed effectively to control tumour and is prevented its relapse and metastasis, and all has serious toxic side effect, 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.
Spreading Hedyotis Herb Oldenlandia diffusa (Willd.) Roxb. has another name called Herba Hedyotidis Diffusae, tang Huang, snake needle grass, Radix Picriae felterrae, dragon and tells pearl, the young grass of pearl etc., cold in nature, sweet and slightly bitter taste, the thoughts of returning home, liver, the spleen channel, beginning is stated from Shennong's Herbal, tool heat-clearing, dampness removing, detoxifcation, effect such as anticancer are kindly controlled cough due to lung-heat, various cancer, are the antitumor drugs of wide clinical application.A large amount of pharmacodynamic experiments and clinical application experiment show that Spreading Hedyotis Herb has good therapeutic action to tumour, and its remarkable immunizing power of enhancing body, and great value of exploiting and utilizing is arranged.Document concentrates on polysaccharide basically to the research of Spreading Hedyotis Herb anti-tumor activity.But mostly the research for Herba Hedyotidis Diffusae polysaccharide is Crude polysaccharides, not to the further refining purifying research of Spreading Hedyotis Herb Crude polysaccharides.For fundamentally eliminating the malignant tumour cause of disease, improve symptom, with many side effects 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 Spreading Hedyotis Herb.
Summary of the invention
One object of the present invention is to provide a kind of Herba Hedyotidis Diffusae polysaccharide for the treatment of malignant tumour;
Another object of the present invention is to provide the preparation method of above-mentioned Herba Hedyotidis Diffusae polysaccharide.
The object of the present invention is achieved like this:
Get the Spreading Hedyotis Herb medicinal material, add 4～20 times of water gagings, extract 1～4 time, each 0.5～3h filters merging filtrate, concentrate, add ethanol and make and contain the alcohol amount and reach 40～90%, refrigeration to precipitation is separated out fully, filter, precipitation is dissolved in water, and adds 20% tannic acid ethanolic soln of 0.01～3% parts by volume, refrigeration to precipitation is separated out fully, filters, and filtrate adds 0.01～3% gac, decolour 1～4 time, each 0.5～2h filters, and filtrate flow is the ultra-filtration 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, by the DEAE-Sephrose ion exchange column, with 0.5mol/L sodium chloride solution wash-out, UV-detector 210nm detects, and collects the elutriant of the 4th detected peaks, behind the dialysis method desalination, by Superdex 30 gel columns, the water wash-out, UV-detector 210nm detects, and collects the elutriant of the 4th detected peaks, drying, promptly.
Optimizing technology is: get the Spreading Hedyotis Herb medicinal material, add 8～15 times of water gagings, extract 1～3 time, each 1～2h filters merging filtrate, concentrate, add ethanol and make and contain the alcohol amount and reach 50～80%, refrigeration to precipitation is separated out fully, precipitation is dissolved in water, and adds 20% tannic acid ethanolic soln of 0.1～1% parts by volume, and refrigeration to precipitation is separated out fully, filter, filtrate adds 0.2～1% gac, decolours each 0.5～1h 1～3 time, filter, filtrate flow is the ultra-filtration 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, by the DEAE-Sephrose ion exchange column, with 0.5mol/L sodium chloride solution wash-out, UV-detector 210nm detects, collect the elutriant of the 4th detected peaks, behind the dialysis method desalination, by Superdex 30 gel columns, water wash-out, UV-detector 210nm detects, collect the elutriant of the 4th detected peaks, drying, promptly.
Further optimizing technology is: get the Spreading Hedyotis Herb medicinal material, add 75～85 ℃ of hot water of 12 times of amounts, insulation is extracted 3 times, each 1h filters merging filtrate, concentrate, add ethanol and make and contain the alcohol amount and reach 80%, refrigeration to precipitation is separated out fully, precipitation is dissolved in water, and adds 20% tannic acid ethanolic soln of 0.5% parts by volume, and refrigeration to precipitation is separated out fully, filter, filtrate adds 0.5% gac, decolours 3 times, each 1h filters, and filtrate flow is the ultra-filtration membrane of 10k through the molecular weight that dams, collect relative molecular mass less than the 10k part, lyophilize is dissolved in water, by the DEAE-Sephrose ion exchange column, with 0.5mol/L sodium chloride solution wash-out, flow velocity 1ml/min, UV-detector 210nm detects, and collects the elutriant of the 4th detected peaks, behind the dialysis method desalination, by Superdex 30 gel columns, water wash-out, flow velocity 0.5ml/min, UV-detector 210nm detects, collect the elutriant of the 4th detected peaks, freeze-drying, promptly.
Purity for the Herba Hedyotidis Diffusae polysaccharide of proving conclusively above-mentioned technology gained guarantees that it plays the basic substance of physiologically active, and the contriver 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 Spreading Hedyotis Herb 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), make the standardized solution of 5mg/ml respectively with distilled water, get 100 μ l respectively and inject AKTA column chromatography system, sodium chloride solution wash-out 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 X-coordinate, lgM is an ordinate zou, obtains the molecular weight determination typical curve.
Measuring 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 typical 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, test-results sees Table 1.According to the measurement 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 typical 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) monose compositional analysis
Adopt the HPLC-ELSD method that the monose after the complete acid hydrolysis of Spreading Hedyotis Herb polysaccharide fraction is analyzed.Chromatographic condition is a chromatographic column: Zorbax Carbohydrate Analysis Column (4.6 * 250mm, 5 μ m); Moving phase: acetonitrile: water=75: 25; Flow velocity: 1ml/min; Alltech ELSD2000 working conditions: drift tube temperature: 81.3 ℃, flow rate of carrier gas: 2.1L/min, column temperature: 40 ℃.
Reference substance solution: D-glucose, D-fructose, D-seminose, D-wood sugar, D-pectinose, D-semi-lactosi, L-rhamnosyl standard substance are 100 μ g/ml, and solvent is 50% acetonitrile; Hybrid standard monose: the concentration gradient of D-glucose, D-fructose, D-seminose, D-wood sugar, D-pectinose, D-semi-lactosi, L-rhamnosyl hybrid standard monose 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.After 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, at last with the dissolving of 0.5ml 50% acetonitrile.
Measuring method: at first D-glucose, D-fructose, D-seminose, D-wood sugar, D-pectinose, D-semi-lactosi, 7 kinds of standard monose of L-rhamnosyl are distinguished sample introduction, determine each monose retention time, application mix standard monose sample introduction is distinguished the production standard curve according to each monose peak area and standard monosaccharide concentration then.According to the ratio between each monose of calculated by peak area of need testing solution.Test-results shows: Herba Hedyotidis Diffusae polysaccharide mainly contains rhamnosyl, wood sugar, semi-lactosi and glucose.With corresponding standard curve in the peak area substitution table of each monose among the HPS, the mass ratio that calculates each monose in the Herba Hedyotidis Diffusae polysaccharide is: rhamnosyl: wood sugar: semi-lactosi: glucose=36.77: 17.28: 64.00: 122.26, and be converted to mol ratio and be: rhamnosyl: wood sugar: semi-lactosi: glucose=1.75: 1: 3.09: 5.90.Concrete experimental result sees Table 2,3.
Table 2 monose peak area and standard monosaccharide concentration relation
The typical curve of each standard monose of table 3
(3) purity is identified
The purity that adopts the gel filtration chromatography method to carry out the gained polysaccharide is identified.
Measuring 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-chlor to carry out wash-out, and flow velocity is 0.5ml/min, carries out online ultraviolet detection in the 210nm place.Measure through going up sample repeatedly, elution peak is single symmetrical peak all, illustrates 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 supply test agent:
Add 80 ℃ of hot water of 12 times of amounts, insulation is extracted 3 times, each 1h, filter, merging filtrate concentrates, add ethanol and make and contain alcohol amount and reach 80%, refrigeration is separated out fully to precipitation, and precipitation is dissolved in water, the 20% tannic acid ethanolic soln that adds 0.5% parts by volume, refrigeration to precipitation is separated out fully, filters, filtrate adds 0.5% gac, decolours 3 times, each 1h, filter, filtrate flow is the ultra-filtration membrane of 10k through the molecular weight that dams, and collects relative molecular mass less than 10k part, lyophilize, be dissolved in water, by DEAE-Sephrose ion exchange column (blade diameter length ratio is 1: 15), with 0.5mol/L sodium chloride solution wash-out, flow velocity 1ml/min, UV-detector 210nm detects, collect the elutriant of the 4th detected peaks, behind the dialysis method desalination, by Superdex 30 gel columns (blade diameter length ratio is 1: 20), the water wash-out, flow velocity 0.5ml/min, UV-detector 210nm detects, and collects the elutriant of the 4th detected peaks, freeze-drying 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 mouse is available from Shandong Academy of Medical Sciences.
2 experimental techniques
The go down to posterity S180 ascites mouse peritoneal tumour cell of 8d of aseptic extraction adds physiological saline and adjusts concentration to 4X10
9/ L in the right oxter injection of every mouse 0.2ml, inoculates 60.Behind the inoculation 24h, mouse is divided into 5 groups at random, 10 every group, is divided into model group, the high, medium and low dosage group of the present invention and control group.Model group is irritated stomach physiological saline 10mLKg
-1, continuous 10d; The high, medium and low dosage group of Herba Hedyotidis Diffusae polysaccharide is irritated stomach 20,10,5mgKg respectively
-1Soup, 10d continuously; The control 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 tumour, thymus gland, spleen, weighs, and calculates tumour inhibiting rate, thymus gland and index and spleen index, and calculation formula is as follows:
Tumour inhibiting rate %=(blank group knurl weight-average value-sample sets knurl weight-average value)/blank group knurl weight-average value * 100%
Thymus gland (spleen) index=weight (mg)/body weight (g)
3 experimental results
The tumour inhibiting rate of 3 dosage groups of the present invention is respectively 34.4%, 26.2%, 17.1%.Compare with model group, endoxan group and Herba Hedyotidis Diffusae polysaccharide height, middle dosage group tumour inhibiting rate all have 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 dosage group of the present invention, thymus index and model group be there was no significant difference (P＞0.05) relatively, and endoxan group and model group relatively have utmost point significant difference (P＜0.01) (seeing Table 5).
Table 4 the present invention to tumor-inhibiting action in the body of S180 tumor-bearing mice (
N=10)
Annotate: compare * P＜0.05, * * P＜0.01 with model group.
Table 5 the present invention to tumor-inhibiting action in the body of S180 tumor-bearing mice (
N=10)
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 for 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 physiological saline and adjusts concentration to 4X10
9/ L in the right oxter injection of every mouse 0.2ml, inoculates 60.Behind the inoculation 24h, mouse is divided into 5 groups at random, 10 every group, is divided into model group, the high, medium and low dosage group of the present invention and control group.Model group is irritated stomach physiological saline 10mLKg
-1, continuous 10d; The high, medium and low dosage group of Herba Hedyotidis Diffusae polysaccharide is irritated stomach 20,10,5mgKg respectively
-1Soup, 10d continuously; Control group abdominal 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 tumour, thymus gland, spleen, weighs, and calculates tumour inhibiting rate, thymus gland and index and spleen index, and calculation formula is as follows:
Tumour inhibiting rate %=(blank group knurl weight-average value-sample sets knurl weight-average value)/blank group knurl weight-average value * 100%
Thymus gland (spleen) index=weight (mg)/body weight (g)
3 experimental results
The tumour inhibiting rate of 3 dosage 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 dosage group tumour inhibiting rate of Herba Hedyotidis Diffusae polysaccharide.The index and spleen index of each dosage group 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 to tumor-inhibiting action in the body of H22 tumor-bearing mice (
N=10)
Annotate: compare * P＜0.05, * * P＜0.01 with model group.
Table 7 the present invention to tumor-inhibiting action in the body of H22 tumor-bearing mice (
N=10)
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 liver cancer Bel-7402 cell
1 reagent, sample
1.1 reagent
People's liver cancer 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 FU 5 fluorouracil (5-Fu) is available from Hefei Huo Shi Pharmaceutical Technology Co., Ltd; RPMI-1640 nutrient solution (U.S. GIBCO company) is available from Shanghai Zhuo Kang bio tech ltd; Foetal calf serum is available from Beijing Baeyer enlightening Bioisystech Co., Ltd; Trypsinase, rnase (RNase) and propidium iodide (PI) are available from Chemical Reagent Co., Ltd., Sinopharm Group; Tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) (DMSO) are available from Sigma company.
1.2 for test agent (with experimental example 1)
2 experimental techniques
2.1 cell cultures
Get people's liver cancer Bel-7402 cell strain, human cervical carcinoma Hela cell's strain tumour cell is inoculated in the culturing bottle with an amount of concentration, add to contain 10
6The RPMI-1640 nutrient solution of U/L gentamicin, 0.1g/L Streptomycin sulphate and 10% foetal calf serum 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, and was toppled over original substratum in per 2～3 days, 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 foetal calf serum 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 culturing 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 grouping
Be divided into 5 groups at random, the blank group: in containing 10
6The conventional cultivation in the RPMI-1640 nutrient solution of U/L gentamicin, 0.1g/L Streptomycin sulphate and 10% foetal calf serum; Supply reagent thing group: it is an amount of to get test liquid of the present invention, adds suitable RPMI1640 nutrient solution dilution, and final concentration is respectively 10g/L, 50g/L and 100g/L (in raw medicinal herbs); Positive controls: 5 FU 5 fluorouracil adds the dilution of RPMI1640 nutrient solution through 0.22 μ m millipore filtration Entkeimung, and final concentration is 0.1g/L.
2.3 adopt the metamorphosis of cell in the inverted microscope direct viewing culturing process, the detail record observe phenomena.
2.4MTT method 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 perfect 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 liquors, 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 liquors in the hole, every hole adds 200 μ lDMSO, and the 10min that vibrates on microwell plate vibrator under the room temperature adopts microplate reader to measure corresponding optical density(OD) (D value) in 570nm wavelength place.
2.5 cells were tested by flow cytometry apoptosis rate
Collect above-mentioned Bel-7402 and Hela cell after treatment respectively, transferring cell concn with PBS 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 (peripheral blood lymphocyte with the normal people 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 form changes
Can be observed by 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 nutrient solution, and cell transparency and adhesive power descend, cell rounding, and volume dwindles gradually; The growth of blank group cell is more normal, is fusiformis, adherent growth, and nuclear-cytoplasmic ratio is normal, intact nuclear membrane; Positive controls is to heavy dose of more similar for reagent thing group.
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 restraining effect to Hela and the growth of Bel-7402 cell
The present invention all has certain restraining effect to the proliferate of Hela and Bel-7402 cell, and along with the increase of concentration, inhibiting rate has obvious rising, and test-results sees Table 8.
Table 8 the present invention is to the influence (n=5) of Hela and Bel-7402 cell proliferation growth
Annotate: compare * P＜0.05, * * P＜0.01, * * * P＜0.001 with control 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 test-results sees Table 9.
Table 9 the present invention to Hela and the apoptotic influence of Bel-7402 (
N=5)
Annotate: compare * P＜0.05, * * P＜0.01, * * * P＜0.001 with control group.
Above-mentioned experiment shows that the present invention has better antitumor activity.
Below further set forth technical scheme of the present invention with embodiment, but the claimed content of the present patent application and not only in these preparation methods.The consumption of bulk drug can be a unit with ton, kilogram, gram etc. also not only in the embodiment consumption in the actual production.
Embodiment
Embodiment 1:
Get Spreading Hedyotis Herb medicinal material 1kg, add 100 ℃ of hot water of 20 times of amounts, boil and extract 4 times, each 3h filters merging filtrate, concentrate, add ethanol and make and contain the alcohol amount and reach 90%, refrigeration to precipitation is separated out fully, filter, precipitation is dissolved in water, and adds 20% tannic acid ethanolic soln of 3% parts by volume, refrigeration to precipitation is separated out fully, filters, and filtrate adds 3% gac, decolour 4 times, each 2h filters, filtrate flow is the ultra-filtration 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, by DEAE-Sephrose ion exchange column (blade diameter length ratio is 1: 15), with 0.5mol/L sodium chloride solution wash-out, flow velocity 1ml/min, UV-detector 210nm detects, collect the elutriant of the 4th detected peaks, behind the dialysis method desalination, by Superdex 30 gel columns (blade diameter length ratio is 1: 20), the water wash-out, flow velocity 0.5ml/min, UV-detector 210nm detects, and collects the elutriant of the 4th detected peaks, drying gets polysaccharide 1.2g.
Embodiment 2:
Get Spreading Hedyotis Herb medicinal material 2kg, add 60 ℃ of hot water of 4 times of amounts, 0.5h is extracted in insulation, filter, filtrate concentrates, and adds ethanol and makes and contain the alcohol amount and reach 40%, refrigeration to precipitation is separated out fully, filters, and precipitation is dissolved in water, the 20% tannic acid ethanolic soln that adds 0.01% parts by volume, refrigeration to precipitation is separated out fully, filters, filtrate adds 0.01% gac, decolouring 0.5h filters, and filtrate flow is the ultra-filtration 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, by DEAE-Sephrose ion exchange column (blade diameter length ratio is 1: 12), with 0.5mol/L sodium chloride solution wash-out, flow velocity 2ml/min, UV-detector 210nm detects, and collects the elutriant of the 4th detected peaks, behind the dialysis method desalination, by Superdex 30 gel columns (blade diameter length ratio is 1: 15), water wash-out, flow velocity 1ml/min, UV-detector 210nm detects, collect the elutriant of the 4th detected peaks, drying under reduced pressure gets polysaccharide 1.8g.
Embodiment 3:
Get Spreading Hedyotis Herb medicinal material 1.5kg, add 70 ℃ of hot water of 8 times of amounts, 1h is extracted in insulation, filter, filtrate concentrates, and adds ethanol and makes and contain the alcohol amount and reach 50%, refrigeration is separated out fully to precipitation, and precipitation is dissolved in water, and adds 20% tannic acid ethanolic soln of 0.1% parts by volume, refrigeration to precipitation is separated out fully, filter, filtrate adds 0.2% gac, decolouring 0.5h, filter, filtrate flow is the ultra-filtration 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, by DEAE-Sephrose ion exchange column (blade diameter length ratio is 1: 15), with 0.5mol/L sodium chloride solution wash-out, flow velocity 2ml/min, UV-detector 210nm detects, collect the elutriant of the 4th detected peaks, behind the dialysis method desalination, by Superdex 30 gel columns (blade diameter length ratio is 1: 20), the water wash-out, flow velocity 1ml/min, UV-detector 210nm detects, and collects the elutriant of the 4th detected peaks, freeze-drying gets polysaccharide 1.5g.
Embodiment 4:
Get Spreading Hedyotis Herb medicinal material 1kg, add 90 ℃ of hot water of 15 times of amounts, insulation is extracted 3 times, each 2h filters merging filtrate, concentrate, add ethanol and make and contain the alcohol amount and reach 80%, refrigeration to precipitation is separated out fully, precipitation is dissolved in water, and adds 20% tannic acid ethanolic soln of 1% parts by volume, and refrigeration to precipitation is separated out fully, filter, filtrate adds 1% gac, decolours 3 times, each 1h filters, and filtrate flow is the ultra-filtration 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, by DEAE-Sephrose ion exchange column (blade diameter length ratio is 1: 20), with 0.5mol/L sodium chloride solution wash-out, flow velocity 1ml/min, UV-detector 210nm detects, and collects the elutriant of the 4th detected peaks, behind the dialysis method desalination, by Superdex 30 gel columns (blade diameter length ratio is 1: 15), water wash-out, flow velocity 0.5ml/min, UV-detector 210nm detects, collect the elutriant of the 4th detected peaks, freeze-drying gets polysaccharide 1.1g.
Embodiment 5:
Get Spreading Hedyotis Herb medicinal material 2kg, add 80 ℃ of hot water of 12 times of amounts, insulation is extracted 3 times, each 1h filters merging filtrate, concentrate, add ethanol and make and contain the alcohol amount and reach 80%, refrigeration to precipitation is separated out fully, precipitation is dissolved in water, and adds 20% tannic acid ethanolic soln of 0.5% parts by volume, and refrigeration to precipitation is separated out fully, filter, filtrate adds 0.5% gac, decolours 3 times, each 1h filters, and filtrate flow is the ultra-filtration membrane of 10k through the molecular weight that dams, collect relative molecular mass less than the 10k part, lyophilize is dissolved in water, by DEAE-Sephrose ion exchange column (blade diameter length ratio is 1: 15), with 0.5mol/L sodium chloride solution wash-out, flow velocity 1ml/min, UV-detector 210nm detects, and collects the elutriant of the 4th detected peaks, behind the dialysis method desalination, by Superdex 30 gel columns (blade diameter length ratio is 1: 20), water wash-out, flow velocity 0.5ml/min, UV-detector 210nm detects, collect the elutriant of the 4th detected peaks, lyophilize gets polysaccharide 2g.
Claims (7)
1, a kind of Herba Hedyotidis Diffusae polysaccharide that is used for the treatment of malignant tumour, it is characterized in that this Herba Hedyotidis Diffusae polysaccharide is prepared by following method: get the Spreading Hedyotis Herb medicinal material, add 4～20 times of water gagings, extract 1～4 time, each 0.5～3h, filter, merging filtrate, concentrate, add ethanol and make and contain the alcohol amount and reach 40～90%, refrigeration to precipitation is separated out fully, filter, precipitation is dissolved in water, and adds 20% tannic acid ethanolic soln of 0.01～3% parts by volume, and refrigeration to precipitation is separated out fully, filter, filtrate adds 0.01～3% gac, decolours each 0.5～2h 1～4 time, filter, filtrate flow is the ultra-filtration 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, by the DEAE-Sephrose ion exchange column, with 0.5mol/L sodium chloride solution wash-out, UV-detector 210nm detects, collect the elutriant of the 4th detected peaks, behind the dialysis method desalination, by Superdex 30 gel columns, water wash-out, UV-detector 210nm detects, collect the elutriant of the 4th detected peaks, drying, promptly.
2, Herba Hedyotidis Diffusae polysaccharide as claimed in claim 1 is characterized in that preparation process is: get the Spreading Hedyotis Herb medicinal 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%, refrigeration is separated out fully to precipitation, and precipitation is dissolved in water, and adds 20% tannic acid ethanolic soln of 0.1～1% parts by volume, refrigeration to precipitation is separated out fully, filters, and filtrate adds 0.2～1% gac, decolour 1～3 time, each 0.5～1h filters, and filtrate flow is the ultra-filtration 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, by the DEAE-Sephrose ion exchange column, with 0.5mol/L sodium chloride solution wash-out, UV-detector 210nm detects, and collects the elutriant of the 4th detected peaks, behind the dialysis method desalination, by Superdex 30 gel columns, the water wash-out, UV-detector 210nm detects, and collects the elutriant of the 4th detected peaks, drying, promptly.
3, Herba Hedyotidis Diffusae polysaccharide as claimed in claim 2 is characterized in that preparation process is: get the Spreading Hedyotis Herb medicinal 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%, refrigeration is separated out fully to precipitation, and precipitation is dissolved in water, and adds 20% tannic acid ethanolic soln of 0.5% parts by volume, refrigeration to precipitation is separated out fully, filters, and filtrate adds 0.5% gac, decolour 3 times, each 1h filters, filtrate flow is the ultra-filtration membrane of 10k through the molecular weight that dams, and collects relative molecular mass less than 10k part, lyophilize, be dissolved in water, by the DEAE-Sephrose ion exchange column, with 0.5mol/L sodium chloride solution wash-out, flow velocity 1ml/min, UV-detector 210nm detects, collect the elutriant of the 4th detected peaks, behind the dialysis method desalination, by Superdex 30 gel columns, the water wash-out, flow velocity 0.5ml/min, UV-detector 210nm detects, and collects the elutriant of the 4th detected peaks, freeze-drying, promptly.
4, as each described Herba Hedyotidis Diffusae polysaccharide in the claim 1 to 3, it is characterized in that the relative molecular mass of this Herba Hedyotidis Diffusae polysaccharide is about 6310.
5,, it is characterized in that containing rhamnosyl, wood sugar, semi-lactosi and glucose in this Herba Hedyotidis Diffusae polysaccharide, and its mol ratio is 1.75: 1: 3.09: 5.90 as each described Herba Hedyotidis Diffusae polysaccharide in the claim 1 to 3.
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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| CN103868840A (en) * | 2014-03-26 | 2014-06-18 | 南京中医药大学 | Determination method of molecular weight cutoff of ultrafiltration membrane |
| CN103893325A (en) * | 2014-04-18 | 2014-07-02 | 李晓明 | Application of oldenlandia diffusa |
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| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103868840A (en) * | 2014-03-26 | 2014-06-18 | 南京中医药大学 | Determination method of molecular weight cutoff of ultrafiltration membrane |
| CN103893325A (en) * | 2014-04-18 | 2014-07-02 | 李晓明 | Application of oldenlandia diffusa |
| CN103893325B (en) * | 2014-04-18 | 2015-11-04 | 李晓明 | The application of Herba Hedyotidis Diffusae |
| CN104059159A (en) * | 2014-06-17 | 2014-09-24 | 湖南农业大学 | Cellulase-catalyzed method for extracting spreading hedyotis herb polysaccharides |
| 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 |
| CN104292353A (en) * | 2014-09-30 | 2015-01-21 | 河南科技大学 | Anti-oxidation natural plant polysaccharide and preparation method thereof |
| 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 |
| CN111484564A (en) * | 2019-01-27 | 2020-08-04 | 复旦大学 | Spreading hedyotis herb polysaccharide, preparation method thereof and application thereof in preparing anticomplement medicines |
| 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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