CN102961424A - Panax notoginseng polysaccharide extract and preparation method, preparations and applications thereof - Google Patents
Panax notoginseng polysaccharide extract and preparation method, preparations and applications thereof Download PDFInfo
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Abstract
The invention discloses a panax notoginseng polysaccharide extract and a preparation method, preparations and applications thereof. The panax notoginseng polysaccharide extract is prepared by taking waste residues produced after panax notoginseng saponins are extracted as raw materials through the steps of extraction, concentration, purification, drying, crushing and detection, wherein the content of panax notoginseng polysaccharides is not less than 15%. The preparation method of the panax notoginseng polysaccharide extract comprises the steps of extraction, concentration, purification, drying, crushing and detection, and specifically comprises the steps of adding waste residues produced after panax notoginseng saponins are extracted into purified water for extracting, carrying out filtration and concentration on the obtained object, adding ethanol into the obtained object for precipitating, and carrying out filtration, purification and drying on the obtained product so as to obtain a target dried product. Pharmaceutical excipients are added into the panax notoginseng polysaccharide extract to be prepared into powder, capsules, granules, tablets or pills; and the panax notoginseng polysaccharide extract is applied to the preparation of antineoplastic drugs and immunity enhancing drugs. According to the invention, waste residues produced after panax notoginseng saponins are extracted are reused, thereby changing waste material into things of value, facilitating the comprehensive utilization of panax notoginseng resources, reducing the cost and improving the economic benefits.
Description
Technical field
The invention belongs to medicine Application and preparation technical field, be specifically related to a kind of Radix Notoginseng polysaccharide extract and preparation method thereof and preparation and application.
Background technology
The Chinese medicine Radix Notoginseng is Araliaceae Panax pseudoginseng Panax notoginseng(Burk.) dry root of F H.Chen, main product is in Yunnan, Guangxi and other places, wild or cultivation.Its sweet in the mouth, little hardship, warm in nature, main enter liver, stomach, large intestine channel, have nourishing, strong, enrich blood, the effects such as Repercusion analgesia, styptic powder addiction, enhancing immunity.In 20 end of the centurys, Chinese scholars has been carried out widely pharmacological research and clinical experiment to Radix Notoginseng, has obtained a large amount of new results, and the field of medical applications of Radix Notoginseng is expanded.
Up to now, Chinese scholar has been carried out comparatively systematic research to the chemical constituent in the Radix Notoginseng, and the Radix Notoginseng main component is Radix Notoginseng total arasaponins, polysaccharide, dencichine, flavone, volatile oil, aminoacid and various trace element etc.The for many years research of Radix Notoginseng mainly concentrates on the chemistry and pharmacological action of saponin, and research confirms that Radix Notoginseng total arasaponins has myocardium protecting action; Pre-preventing thrombosis and anti thrombotic action; Analgesic activity; The liver protecting; Anti-inflammatory effect; Radiation resistance; Increase superoxide dismutase activity etc., the research of other compositions is then less.Along with the reach of science, the bioactive ingredients polysaccharide of higher plant more and more causes people's interest, and the effect research of polysaccharide also gradually deeply.
At present, what application was more in the Radix Notoginseng chemical constituent is Radix Notoginseng total arasaponins, and in the process of extracting Radix Notoginseng total arasaponins, left residue usually all is taken as waste residue and processes.In fact, in these waste residues, also contain more other active substances except Radix Notoginseng total arasaponins, be used as waste residue and process, the Radix Notoginseng resource has been caused suitable waste, be unfavorable for the comprehensive utilization of Radix Notoginseng resource.Therefore, a kind of method that turns waste into wealth, can fully utilize the Radix Notoginseng raw material of exploitation is very important.
Summary of the invention
The first purpose of the present invention is to provide a kind of Radix Notoginseng polysaccharide extract; The second purpose is to provide the preparation method of described Radix Notoginseng polysaccharide extract; The preparation that provides described Radix Notoginseng polysaccharide extract to make is provided the 3rd purpose; The 4th purpose is to provide described Radix Notoginseng polysaccharide extract owing to the effect with enhancing immunity, and because the raising of immunity produces anticancer pharmacologically active, can be used for preparing the application in antitumor drug and the enhancing immunity medicine.
The present invention's the first purpose be achieved in that described Radix Notoginseng polysaccharide extract be that to extract waste residue behind the Radix Notoginseng total arasaponins be raw material through extract, concentrated, refining, drying, pulverizing, testing sequence obtain, wherein Radix Notoginseng polysaccharide content is not less than 15%.
The second purpose of the present invention is achieved in that and comprises extraction, concentrated, refining, drying, pulverizing, testing sequence, specifically comprises:
A, extraction: get the waste residue that extracts behind the Radix Notoginseng total arasaponins and insert in the extraction pot, the purified water that adds 8 ~ 24 times of amounts of volume ratio is 50 ~ 60 ℃ of lower lixiviate 4 ~ 8h in temperature, and circulation lixiviate 2 ~ 6 times is filtered, and merging filtrate is for subsequent use;
B, concentrated: at pressure≤0.8MPa, temperature≤90 ℃ are concentrated into relative density and are 1.16 ~ 1.24 extractum under concentrated steam pressure≤0.3MPa with the filtrate after the second extraction;
C, refining: extractum is left standstill 8 ~ 24h, behind the ethanol precipitation 8 ~ 24h of 3 ~ 8 times of amounts of adding volume ratio, filter crude polysaccharides is separated with mother solution, crude polysaccharides cleans 2 ~ 6 times to get object with ethanol again;
D, drying: Radix Notoginseng polysaccharide extract drying to the water content after refining is 1 ~ 9% to obtain Radix Notoginseng polysaccharide extract dry product;
E, be pulverized and mixed: Radix Notoginseng polysaccharide extract dry product was pulverized 80 mesh sieves, obtain Radix Notoginseng polysaccharide extract fine powder, with the quantity-produced Product mix;
F, check: with the Radix Notoginseng polysaccharide extract fine powder packing that mixes, pick test is provided inspection report.
The 3rd purpose of the present invention is achieved in that described Radix Notoginseng polysaccharide extract adds adjuvant suitable on medical science or the food and makes powder, capsule, granule, tablet or pill.
The 4th purpose of the present invention is achieved in that the application of described Radix Notoginseng polysaccharide extract in preparation antitumor drug and/or enhancing immunity medicine.
The present invention utilizes again with the waste residue that extracts behind the Radix Notoginseng total arasaponins, turns waste into wealth, and helps the comprehensive utilization of Radix Notoginseng resource, reduces cost, increases economic efficiency.
Description of drawings
Fig. 1 is process chart of the present invention;
Fig. 2 is that Radix Notoginseng polysaccharide is to the survival curve figure of lotus tumor H22 mice.
The specific embodiment
The present invention is further illustrated below in conjunction with drawings and Examples, but never in any form the present invention is limited, and any conversion or replacement based on training centre of the present invention is done all belong to protection scope of the present invention.
Radix Notoginseng polysaccharide extract of the present invention be that to extract waste residue behind the Radix Notoginseng total arasaponins be raw material through extract, concentrated, refining, drying, pulverizing, testing sequence obtain, wherein Radix Notoginseng polysaccharide content is not less than 15%.
Described Radix Notoginseng polysaccharide content is 15 ~ 45%.
Described Radix Notoginseng polysaccharide content is 45 ~ 70%.
The preparation method of Radix Notoginseng polysaccharide extract of the present invention comprises extraction, concentrated, refining, drying, pulverizing, testing sequence, specifically comprises:
Extraction is that the waste residue behind the extraction Radix Notoginseng total arasaponins is inserted in the extraction pot, and the purified water that adds 8 ~ 24 times of amounts of volume ratio is 50 ~ 60 ℃ of lower lixiviate 4 ~ 8h in temperature, and circulation lixiviate 2 ~ 6 times is filtered, and merging filtrate is for subsequent use;
Concentrated be with the filtrate after the second extraction at pressure≤0.8MPa, temperature≤90 ℃ are concentrated into relative density and are 1.16 ~ 1.24 extractum under concentrated steam pressure≤0.3MPa;
Refining is that extractum is left standstill 8 ~ 24h, behind the ethanol precipitation 8 ~ 24h of 3 ~ 8 times of amounts of adding volume ratio, filters crude polysaccharides is separated with mother solution, and crude polysaccharides cleans 2 ~ 6 times to get object with ethanol again;
Drying is that Radix Notoginseng polysaccharide extract drying to the water content after refining is 1 ~ 9% to obtain Radix Notoginseng polysaccharide extract dry product;
Being pulverized and mixed is that Radix Notoginseng polysaccharide extract dry product was pulverized 80 mesh sieves, obtains Radix Notoginseng polysaccharide extract fine powder, with the quantity-produced Product mix;
Check is the Radix Notoginseng polysaccharide extract fine powder packing that will mix, and pick test is provided inspection report.
Be filtered in the described extraction step and use 100 order stainless steel meshs to realize.
Ethanol is concentration greater than 90% food-grade alcohol in the described purification step; Be filtered into through the centrifuge of filter cloth aperture 50 ~ 100mm and realize.The preferred cotton filter cloth of described filter cloth; The preferred link-suspended basket centrifuge of described centrifuge.
Drying is one or more in vacuum drying, lyophilization, constant pressure and dry, infrared drying, microwave drying, the hygroscopic desiccation in the described drying steps.Preferred vacuum drying, baking temperature≤110 ℃, vacuum≤0.08MPa, steam pressure≤0.3MPa.
Described being pulverized and mixed pulverized in the step as using rustless steel pulverizing machine to finish; Be mixed into and use two dimension or three-dimensional motion mixer to realize incorporation time＞1h.
The preparation that Radix Notoginseng polysaccharide extract of the present invention is made is that described Radix Notoginseng polysaccharide extract adding pharmaceutic adjuvant is made powder, capsule, granule, tablet or pill.
Radix Notoginseng polysaccharide extract of the present invention be applied as described Radix Notoginseng polysaccharide extract for the preparation of the application in antitumor drug and/or the enhancing immunity medicine.
The present invention is raw materials used---and the waste residue that extracts behind the Radix Notoginseng total arasaponins is unrestricted, all can realize the present invention, and only the present invention will be further described with residue obtained in the part extraction process for notoginseng saponin for the below:
Embodiment 1
Get Radix Notoginseng 750 ㎏, clean, being dried to moisture is 10%, and the coarse powder that is crushed to particle diameter and is 6 * 6 ~ 9 * 9mm is for subsequent use; Coarse powder is inserted in the extraction pot, and 60% the ethanol that adds 10 times of amounts of volume ratio is 50 ℃ of lower lixiviate 12h in temperature, circulation lixiviate 5 times, and through 100 order stainless steel sift net filtrations, merging filtrate uses as producing Radix Notoginseng total arasaponins, and it is for subsequent use to merge residue.
Embodiment 2
Get Radix Notoginseng 750 ㎏, clean, being dried to moisture is 20%, and the coarse powder that is crushed to particle diameter and is 8 * 8mm is for subsequent use; Coarse powder is inserted in the extraction pot, and 90% the ethanol that adds 14 times of amounts of volume ratio is 60 ℃ of lower lixiviate 8h in temperature, circulation lixiviate 3 times, and through 100 order stainless steel sift net filtrations, merging filtrate uses as producing Radix Notoginseng total arasaponins, and it is for subsequent use to merge residue.
Embodiment 3
Get Radix Notoginseng 750 ㎏, clean, being dried to moisture is 10%, and the coarse powder that is crushed to particle diameter and is 9 * 9mm is for subsequent use; Coarse powder is inserted in the extraction pot, and 60% the ethanol that adds 10 times of amounts of volume ratio is 55 ℃ of lower lixiviate 10h in temperature, circulation lixiviate 4 times, and through 100 order stainless steel sift net filtrations, merging filtrate uses as producing Radix Notoginseng total arasaponins, and it is for subsequent use to merge residue.
Embodiment 4
Get Radix Notoginseng 750 ㎏, clean, being dried to moisture is 15%, and the coarse powder that is crushed to particle diameter and is 10mm is for subsequent use; Coarse powder is inserted in the extraction pot, and 70% the ethanol that adds 13 times of amounts of volume ratio is 58 ℃ of lower lixiviate 10h in temperature, circulation lixiviate 5 times, and through 100 order stainless steel sift net filtrations, merging filtrate uses as producing Radix Notoginseng total arasaponins, and it is for subsequent use to merge residue.
Embodiment 5
Get Radix Notoginseng 750 ㎏, clean, being dried to moisture is 11%, and the coarse powder that is crushed to particle diameter and is 8mm is for subsequent use; Coarse powder is inserted in the extraction pot, and 75% the ethanol that adds 12 times of amounts of volume ratio is 55 ℃ of lower lixiviate 10h in temperature, circulation lixiviate 4 times, and through 100 order stainless steel sift net filtrations, merging filtrate uses as producing Radix Notoginseng total arasaponins, and it is for subsequent use to merge residue.
Embodiment 6
Get embodiment 1 residue obtained inserting in the extraction pot, the purified water that adds 8 times of amounts of volume ratio is 50 ℃ of lower lixiviate 8h in temperature, circulation lixiviate 6 times, and through 100 order stainless steel sift net filtrations, merging filtrate is for subsequent use; Be 0.8MPa with the filtrate after the second extraction at pressure, temperature is 90 ℃, and concentrated steam pressure is that relative density is 1.16 fluid extract when being concentrated into 60 ℃ under the 0.3MPa; Fluid extract is left standstill 24h, after adding 90% ethanol precipitation 24h of 3 times of amounts of volume ratio, be filtered into to filter through the link-suspended basket centrifuge of cotton filter cloth aperture 50mm crude polysaccharides is separated with mother solution, crude polysaccharides cleans 2 times with 90% ethanol again must object Radix Notoginseng polysaccharide extract; Radix Notoginseng polysaccharide extract after refining is through vacuum drying, and baking temperature is 110 ℃, and vacuum is 0.08MPa, and steam pressure is 0.3MPa, when water content is 5% to obtain Radix Notoginseng polysaccharide extract dry product; Radix Notoginseng polysaccharide extract dry product was pulverized 80 mesh sieves with the stainless steel type pulverizer obtain Radix Notoginseng polysaccharide extract fine powder, quantity-produced is mixed with a collection of product two-dimensional motion mixer, incorporation time is 1h; With the Radix Notoginseng polysaccharide extract fine powder packing that mixes, pick test is provided inspection report, and wherein Radix Notoginseng polysaccharide content is 54.05%.
Embodiment 7
Get embodiment 2 residue obtained inserting in the extraction pot, the purified water that adds 24 times of amounts of volume ratio is 60 ℃ of lower lixiviate 5h in temperature, circulation lixiviate 2 times, and through 100 order stainless steel sift net filtrations, merging filtrate is for subsequent use; Be 0.6MPa with the filtrate after the second extraction at pressure, temperature is 85 ℃, and concentrated steam pressure is that relative density is 1.24 fluid extract when being concentrated into 60 ℃ under the 0.2MPa; Fluid extract is left standstill 8h, after adding 95% ethanol precipitation 14h of 4 times of amounts of volume ratio, be filtered into to filter through the link-suspended basket centrifuge of cotton filter cloth aperture 100mm crude polysaccharides is separated with mother solution, crude polysaccharides cleans 6 times with 95% ethanol again must object Radix Notoginseng polysaccharide extract; Radix Notoginseng polysaccharide extract after refining is through vacuum drying, and baking temperature is 100 ℃, and vacuum is 0.06MPa, and steam pressure is 0.2MPa, when water content is 9% to obtain Radix Notoginseng polysaccharide extract dry product; Radix Notoginseng polysaccharide extract dry product was pulverized 80 mesh sieves with the stainless steel type pulverizer obtain Radix Notoginseng polysaccharide extract fine powder, quantity-produced is mixed with three-dimensional motion mixer with a collection of product, incorporation time is 2h; With the Radix Notoginseng polysaccharide extract fine powder packing that mixes, pick test is provided inspection report, and wherein Radix Notoginseng polysaccharide content is 54.36%.
Embodiment 8
Get embodiment 3 residue obtained inserting in the extraction pot, the purified water that adds 15 times of amounts of volume ratio is 55 ℃ of lower lixiviate 6h in temperature, circulation lixiviate 3 times, and through 100 order stainless steel sift net filtrations, merging filtrate is for subsequent use; Be 0.7MPa with the filtrate after the second extraction at pressure, temperature is 80 ℃, and concentrated steam pressure is that relative density is 1.21 fluid extract when being concentrated into 60 ℃ under the 0.1MPa; Fluid extract is left standstill 12h, after adding 93% ethanol precipitation 11h of 4 times of amounts of volume ratio, be filtered into to filter through the link-suspended basket centrifuge of cotton filter cloth aperture 50mm crude polysaccharides is separated with mother solution, crude polysaccharides cleans 4 times with 93% ethanol again must object Radix Notoginseng polysaccharide extract; Radix Notoginseng polysaccharide extract after refining is through vacuum drying, and baking temperature is 98 ℃, and vacuum is 0.06MPa, and steam pressure is 0.3MPa, when water content is 2% to obtain Radix Notoginseng polysaccharide extract dry product; Radix Notoginseng polysaccharide extract dry product was pulverized 80 mesh sieves with the stainless steel type pulverizer obtained Radix Notoginseng polysaccharide extract fine powder, quantity-produced was mixed incorporation time 3h with a collection of product with two-dimensional motion mixer; With the Radix Notoginseng polysaccharide extract fine powder packing that mixes, pick test is provided inspection report, and wherein Radix Notoginseng polysaccharide content is 62.43%.
Embodiment 9
Get embodiment 4 residue obtained inserting in the extraction pot, the purified water that adds 16 times of amounts of volume ratio is 58 ℃ of lower lixiviate 7h in temperature, circulation lixiviate 3 times, and through 100 order stainless steel sift net filtrations, merging filtrate is for subsequent use; With the filtrate after the second extraction at pressure 0.8MPa, 80 ℃ of temperature, relative density is 1.19 fluid extract when being concentrated into 60 ℃ under the concentrated steam pressure 0.2MPa; Fluid extract is left standstill 12h, after adding 94% ethanol precipitation 12h of 5 times of amounts of volume ratio, being filtered into through cotton filter cloth aperture 100mm is that the link-suspended basket centrifuge of 3mm filters crude polysaccharides is separated with mother solution through the filter cloth aperture, and crude polysaccharides cleans 4 times with 94% ethanol again must object Radix Notoginseng polysaccharide extract; Radix Notoginseng polysaccharide extract after refining is through vacuum drying, and baking temperature is 98 ℃, and vacuum is 0.05MPa, and steam pressure is 0.1MPa, when water content is 7% to obtain Radix Notoginseng polysaccharide extract dry product; Radix Notoginseng polysaccharide extract dry product was pulverized 80 mesh sieves with the stainless steel type pulverizer, obtain Radix Notoginseng polysaccharide extract fine powder, quantity-produced is mixed with two-dimensional motion mixer with a collection of product, incorporation time is 2h; With the Radix Notoginseng polysaccharide extract fine powder packing that mixes, pick test is provided inspection report, and wherein Radix Notoginseng polysaccharide content is 65.89%.
Embodiment 10
Get embodiment 5 residue obtained inserting in the extraction pot, the purified water that adds 16 times of amounts of volume ratio is 55 ℃ of lower lixiviate 6h in temperature, circulation lixiviate 3 times, and through 100 order stainless steel sift net filtrations, merging filtrate is for subsequent use; With the filtrate after the second extraction at pressure 0.8MPa, 90 ℃ of temperature, relative density is 1.24 fluid extract when being concentrated into 60 ℃ under the concentrated steam pressure 0.3MPa; Fluid extract is left standstill 14h, after adding 96% ethanol precipitation 14h of 5 times of amounts of volume ratio, be filtered into to filter through the link-suspended basket centrifuge of filter cloth aperture 5mm through cotton filter cloth aperture 50mm crude polysaccharides is separated with mother solution, crude polysaccharides cleans 4 times with 96% ethanol again must object Radix Notoginseng polysaccharide extract; Radix Notoginseng polysaccharide extract after refining is through vacuum drying, and baking temperature is 110 ℃, and vacuum is 0.08MPa, and steam pressure is 0.3MPa, when water content is 6% to obtain Radix Notoginseng polysaccharide extract dry product; Radix Notoginseng polysaccharide extract dry product was pulverized 80 mesh sieves with the stainless steel type pulverizer, obtain Radix Notoginseng polysaccharide extract fine powder, quantity-produced is mixed with two-dimensional motion mixer with a collection of product, incorporation time is 1.5h; With the Radix Notoginseng polysaccharide extract fine powder packing that mixes, pick test is provided inspection report, and wherein Radix Notoginseng polysaccharide content is 70.99%.
Embodiment 11
Get any one Radix Notoginseng polysaccharide among the embodiment 5 ~ 10, add pharmaceutic adjuvant, packing is powder.
Get any one Radix Notoginseng polysaccharide among the embodiment 5 ~ 10, add pharmaceutic adjuvant, through granulating, drying, be filled in the capsule, namely get capsule.
Embodiment 13
Get any one Radix Notoginseng polysaccharide among the embodiment 5 ~ 10, add 1% magnesium stearate, mixing is filled in the capsule, namely gets capsule.
Embodiment 14
Get any one Radix Notoginseng polysaccharide among the embodiment 5 ~ 10, add pharmaceutic adjuvant, through granulating, drying, namely get granule.
Embodiment 15
Get any one Radix Notoginseng polysaccharide among the embodiment 5 ~ 10, add pharmaceutic adjuvant, through granulation, tabletting, namely get tablet.
Embodiment 16
Get any one Radix Notoginseng polysaccharide among the embodiment 5 ~ 10, add pharmaceutic adjuvant, pill, oven dry namely get pill.
Test example 1 Radix Notoginseng polysaccharide capsule is to the Regulating study of mouse immunity
1 materials and methods
1.1 sample: cloud section board Radix Notoginseng polysaccharide capsule, specification: the 0.4g/ grain, batch number: 20101018, cloud
Pharmaceutcal corporation, Ltd of Nan Yun section.Putting shady and cool dry place preserves.Human oral recommends consumption to be everyone (adult) every day 2 times, and each 2, become body weight for humans to press 60kg calculating, amounting to dosage is 26.7mg/kg BW.
1.2 laboratory animal and grouping: 240 of the healthy Male Kunming strain mice of SPF level, body weight are 18～22 grams,
By Guangdong Medical Lab Animal Center breeding, laboratory animal production licence number: SCXK (Guangdong) 2008-0002, the Quality of Experimental Animals quality certification number: 0087715.Per 40 mices are one group, totally 6 groups.Immunity I group is carried out the mouse spleen lymphocyte transformation experiment that ConA induces; Immunity II group is carried out the delayed allergy experiment; Immunity III group is carried out dirty/body ratio measurement, and serum hemolysin is measured and the antibody-producting cell number is measured; Immunity IV～VI group carries out respectively that carbon is cleaned up experiment, peritoneal macrophage is engulfed chicken red blood cell and tested and the NK cytoactive detection.
1.3 experimental animal room environmental condition: SPF level laboratory animal occupancy permit number: SYXK (osmanthus) 2007-0003.
The laboratory animal room temperature: 22～25 ℃, relative humidity: 55～70%.
1.4 dosage is selected to give mode with tested material: recommend consumption according to human oral, it is basic, normal, high to establish sample
The dosage group is respectively 133.5,267.0,534.0 mg/kg BW(and is equivalent to respectively human body and recommends 5,10,20 times of consumption), establish a negative control group, every group of 10 animals.Take by weighing respectively cloud section board Radix Notoginseng polysaccharide capsule 's content 667.5,1335.0,2670.0mg, respectively add pure water to 100mL, mixing, be made into 6.675,13.350,26.700mg/mL concentration solution, volume by 0.2mL/10g BW gives corresponding dosage treated animal gavage, negative control group gives isopyknic pure water, every day gavage once, continuously gavage is 30 days.
1.5 key instrument and reagent: animal platform balance, electronic analytical balance, centrifuge, clean bench, CO2 gas incubator, constant water bath box, microscope, semi-automatic biochemical analyzer, microplate reader etc.
Aseptic operation apparatus, microsyringe, cell counter, the flat Tissue Culture Plate in 24 holes and 96 holes, the U-shaped Tissue Culture Plate in 96 holes, glass dish, gauze, microscope slide, test tube, 200 eye mesh screens, timer, hemoglobin pipet etc.
The RPMI1640 culture fluid, calf serum, 2-ME, penicillin, streptomycin, ConA, the HCL solution of 1mol/L, isopropyl alcohol, MTT, Hank ' s liquid (pH 7.2～7.4), PBS buffer (Ph7.2～7.4), platform phenol is blue, DNFB, acetone, barium sulfide, complement (guinea pig serum), the SA buffer, agarose, sheep red blood cell (SRBC) (SRBC), normal saline, india ink, 0.1% sodium carbonate, chicken red blood cell, methanol, the Gimsa dye liquor, the YAC-1 cell, EINECS 212-761-8, the nitro tetrazolium chloride, PMS, oxidized form of nicotinamide-adenine dinucleotide, 0.2mol/L the Tris-HCL buffer, 1% glacial acetic acid, 1% NP40 etc.
1.6 experimental technique: according to Ministry of Public Health " health food check and assessment technique standard " (2003 editions)
[10], carry out internal organs/weight ratio pH-value determination pH; The mouse spleen lymphocyte transformation experiment (mtt assay) that ConA induces; The mouse DTH experiment (ear swelling method) that dinitrofluorobenzene is induced; Antibody-producting cell detects (Jerne improves slide method); The mensuration of serum hemolysin (Hemagglutination Method); Mice carbon is cleaned up experiment; Turnover of Mouse Peritoneal Macrophages is engulfed chicken red blood cell experiment (half intracorporal method); NK cytoactive detection (LDH algoscopy).
1.7 experimental data statistics: use the SPSS statistical software and carry out the variance analysis statistical disposition.
1.8 the result judges: any two aspects result is positive aspect cellular immune function, humoral immune function, monocytes/macrophages function, NK cytoactive four, can judge that this given the test agent has the enhancing immunity function.
2 results
2.1 sample is on the impact of Mouse Weight
Test between first, as to test mid-term, the Mouse Weight of testing each dosage group of sample in latter stage and experimental session mice body weight gain and negative control group and compare, there are no significant for difference (P〉0.05), show that this sample has no significant effect the body weight gain of mice.
2.2 sample is on the impact of mouse immune organ internal organs/body weight ratio
Per os gives the cloud section board Radix Notoginseng polysaccharide capsule 30 days of mice various dose, thymus/body weight of mice and spleen/body weight ratio and negative control group are relatively, difference that there are no significant (P〉0.05), show that this sample has no significant effect the immune organ weight of mice.
2.3 sample is on the impact of the cellular immunization of mice
2.3.1 the impact of the mouse spleen lymphocyte conversion capability that sample is induced ConA
The mouse spleen lymphocyte transformation experiment result of table 1 cloud section board Radix Notoginseng polysaccharide capsule (
± s)
As seen from Table 1, per os gives the cloud section board Radix Notoginseng polysaccharide capsule 30 days of mice various dose, the lymphocyte transformation ability of each dosage group all is higher than negative control group, and the difference of high dose group and negative control group has significance (P＜0.01), shows that this sample has the lymphopoiesis that promotes mice, the effect of conversion capability.
2.3.2 sample is on the impact of mice delayed allergy (DTH)
Mice delayed allergy (DTH) experimental result of table 2 cloud section board Radix Notoginseng polysaccharide capsule (
± s)
As seen from Table 2, per os gives the cloud section board Radix Notoginseng polysaccharide capsule 30 days of mice various dose, the left and right sides auricle weight difference of each dosage group all is higher than negative control group, and the difference of height, middle dosage group and negative control group has significance (respectively P＜0.01 and P＜0.05), shows that this sample has the effect of the delayed allergy that promotes mice.
2.4 sample is on the impact of the humoral immunization of mice
2.4.1 sample is on the impact of the antibody-producting cell number of mice
As seen from Table 3, per os gives the cloud section board Radix Notoginseng polysaccharide capsule 30 days of mice various dose, the antibody-producting cell number average of each dosage group is higher than negative control group, and the difference of height, middle dosage group and negative control group has significance (respectively P＜0.01 and P＜0.05), shows that this sample has the effect of the antibody-producting cell propagation that promotes mice.
The antibody-producting cell test experience result of table 3 cloud section board Radix Notoginseng polysaccharide capsule (
± s)
2.4.2 sample is on the impact of mice serum hemolysin
As seen from Table 4, per os gives the cloud section board Radix Notoginseng polysaccharide capsule 30 days of mice various dose, the antibody product of each dosage group all is higher than negative control group, and the difference of high dose group and negative control group has significance (P＜0.05), shows that this sample has the effect of the serum hemolysin that improves mice.
The mice hemolysin test result of table 4 cloud section board Radix Notoginseng polysaccharide capsule (
± s)
2.5 sample is on the impact of the monocytes/macrophages phagocytic function of mice
2.5.1 the impact that sample is cleaned up the monocytes/macrophages carbon of mice
Per os gives the cloud section board Radix Notoginseng polysaccharide capsule 30 days of mice various dose, the phagocytic index of each dosage group all is higher than negative control group, but there are no significant for each dosage group and negative control group comparing difference (P〉0.05), shows that this sample cleans up function without obvious facilitation to the monocytes/macrophages carbon of mice.
2.5.2 sample is engulfed the impact of chicken red blood cell ability on the peritoneal macrophage of mice
Per os gives the cloud section board Radix Notoginseng polysaccharide capsule 30 days of mice various dose, the peritoneal macrophage of each dosage group all is higher than negative control group to phagocytic rate and the phagocytic index of chicken red blood cell, but except phagocytic rate and the negative control group difference of high dose group has the significance (P＜0.05), there are no significant for each dosage group and negative control group comparing difference (P〉0.05), show this sample to the phagocytic activity of the peritoneal macrophage of mice without obvious facilitation.
2.6 sample is on the impact of the NK cytoactive of mice
Per os gives the cloud section board Radix Notoginseng polysaccharide capsule 30 days of mice various dose, the NK cytoactive of each dosage group is higher than negative control group, but there are no significant for the difference of each dosage group and negative control group (P〉0.05), show this sample to the NK cytoactive of mice without obvious facilitation.
3 results
Per os gives the cloud section board Radix Notoginseng polysaccharide capsule 30 days of mice 133.5,267.0,534.0 mg/kg BW (be equivalent to human body and recommend consumption 5,10,20 times) dosage, can promote spleen lymphocyte proliferation, the transformation of mice, promote the delayed allergy of mice, promote the antibody-producting cell propagation of mice, improve the serum hemolysin of mice, body weight gain, thymus/body weight ratio, spleen/body weight ratio to mice, the phagocytic function of monocytes/macrophages and NK cytoactive have no significant effect, and point out this sample to have the enhancing immunity function.
Experimental example 2 cloud section board Radix Notoginseng polysaccharide capsules are to the effect of lotus H22 hepatoma mice
1 material
1.1 medicine and main agents
Cloud section board Radix Notoginseng polysaccharide capsule, lot number: 20101018, specification: the 0.4g/ grain is provided by Yunnan cloud section Pharmaceutical.Normal saline (N.S), Kuming Nanjiang Pharmacy Co., Ltd.
1.2 mice-transplanted tumor and laboratory animal
Mouse bearing liver cancer H22, key lab provides by unming Medical College Yunnan Province natural drug pharmacology.Kunming mouse, the SPF level, female, 18～22g is provided by unming Medical College's Experimental Animal Center, the laboratory animal production licence number: SCXK(Yunnan) 2005-0008.
1.3 key instrument
Electronic analytical balance (prunus mume (sieb.) sieb.et zucc. Teller-Tuo benefit Instr Ltd.); 1.0R type; Inverted phase contrast microscope, Japanese Olympus company produces, the BH-2 type; Ordinary electronic claims.
2 methods
2.1 the foundation of mice with tumor model
Select the well-grown lotus H22 of intraperitoneal inoculation 7 d tumor mice, the skin of abdomen sterilization is thrust the abdominal cavity with disposable sterilized hemostix through stomach wall, extract ascites, put into aseptic beaker, become the cancerous cell suspension of 1:3 with normal saline dilution, the Microscopic observation counting, oncocyte concentration is 3.3 * 10
7Individual/ml, the right axillary fossa of every Mus 0.2 ml Mice Inoculated is subcutaneous, inoculates to begin administration after 24 hours.
2.2 the experiment grouping, dosage and route of administration
Random packet behind the inoculation 24h is divided into 4 groups, and 10 every group, the beginning administration, be administered once every day.
Negative group (normal saline), 0.4ml/, gastric infusion;
Radix Notoginseng polysaccharide low dose group (133.5 mg/kg are equivalent to human body and recommend 5 times of consumptions), 0.4ml/, gastric infusion; Once a day.
Dosage group in the Radix Notoginseng polysaccharide (267.0 mg/kg are equivalent to human body and recommend 7.5 times of consumptions), 0.4ml/, gastric infusion; Once a day.
Radix Notoginseng polysaccharide high dose group (534.0 mg/kg are equivalent to human body and recommend 10 times of consumptions), 0.4ml/, gastric infusion; Once a day.
2.3 the life span of record tumor-bearing mice is survival curve figure.
2.4 immunological memory experiment
Select the well-grown lotus H22 of intraperitoneal inoculation 7 d tumor mice, the skin of abdomen sterilization is thrust the abdominal cavity with disposable sterilized hemostix through stomach wall, extract ascites, put into aseptic beaker, become the cancerous cell suspension of 1:3 with normal saline dilution, the Microscopic observation counting, oncocyte concentration is 3.3 * 10
7Individual/ml, every Mus gives 4 recoveries from illness about 6 months right axillary fossa subcutaneous vaccination of mice 0.3ml H22 cancerous cell suspensions again.Matched group: 2 mices, the right axillary fossa subcutaneous vaccination of mice 0.2ml H22 cancerous cell suspension.
3 results
3.1 the life span of tumor-bearing mice and survival curve figure (seeing accompanying drawing 2)
Table 5: tumor-bearing mice life span
3.2 immunological memory experimental result
2 control group mice began to find tumor in rear the 7th day in inoculation, and tumor and more long larger; Do not find tumor growth behind 4 recovery from illness mouse inoculations.
4 conclusions
Dosage group in the Radix Notoginseng polysaccharide, 10 lotus H22 hepatoma mices have 2 recoveries from illness; Every group of Radix Notoginseng polysaccharide low dose group and Radix Notoginseng polysaccharide high dose group all have a recovery from illness, and 10 of negative groups are all dead.Observe the state of mice, high dose group mice integrality was better during Radix Notoginseng polysaccharide was low, and hair color is smooth, and the mental status is negative the group.From the survival rate broken line graph of each group mice, the life cycle of dosage group is the longest in the Radix Notoginseng polysaccharide.
The immunological memory experimental result confirms: the Radix Notoginseng polysaccharide capsule has the effect of enhancing immunity, and the immunocyte kill cancer cell also produces memory.
Claims (10)
1. Radix Notoginseng polysaccharide extract, it is characterized in that extracting waste residue behind the Radix Notoginseng total arasaponins and be raw material through extract, concentrated, refining, drying, pulverizing, testing sequence obtain, wherein Radix Notoginseng polysaccharide content is not less than 15%.
2. Radix Notoginseng polysaccharide extract according to claim 1 is characterized in that described Radix Notoginseng polysaccharide content is 15 ~ 45%.
3. Radix Notoginseng polysaccharide extract according to claim 1 is characterized in that described Radix Notoginseng polysaccharide content is 45 ~ 70%.
4. the preparation method of a Radix Notoginseng polysaccharide extract claimed in claim 1 is characterized in that comprising extraction, concentrated, refining, drying, pulverizing, testing sequence, specifically comprises:
A, extraction: get the waste residue that extracts behind the Radix Notoginseng total arasaponins and insert in the extraction pot, the purified water that adds 8 ~ 24 times of amounts of volume ratio is 50 ~ 60 ℃ of lower lixiviate 4 ~ 8h in temperature, and circulation lixiviate 2 ~ 6 times is filtered, and merging filtrate is for subsequent use;
B, concentrated: at pressure≤0.8MPa, temperature≤90 ℃ are concentrated into relative density and are 1.16 ~ 1.24 extractum under concentrated steam pressure≤0.3MPa with the filtrate after the second extraction;
C, refining: extractum is left standstill 8 ~ 24h, behind the ethanol precipitation 8 ~ 24h of 3 ~ 8 times of amounts of adding volume ratio, filter crude polysaccharides is separated with mother solution, crude polysaccharides cleans 2 ~ 6 times to get object with ethanol again;
D, drying: Radix Notoginseng polysaccharide extract drying to the water content after refining is 1 ~ 9% to obtain Radix Notoginseng polysaccharide extract dry product;
E, be pulverized and mixed: Radix Notoginseng polysaccharide extract dry product was pulverized 80 mesh sieves, obtain Radix Notoginseng polysaccharide extract fine powder, with the quantity-produced Product mix;
F, check: with the Radix Notoginseng polysaccharide extract fine powder packing that mixes, pick test is provided inspection report.
5. the preparation method of Radix Notoginseng polysaccharide extract according to claim 4 is characterized in that being filtered in the described A step and uses 100 order stainless steel meshs to realize.
6. the preparation method of Radix Notoginseng polysaccharide extract according to claim 4 is characterized in that ethanol in the described C step is concentration greater than 90% food-grade alcohol; Be filtered into through the centrifuge of filter cloth aperture 50 ~ 100mm and realize.
7. the preparation method of Radix Notoginseng polysaccharide extract according to claim 4 is characterized in that dry one or more in vacuum drying, lyophilization, constant pressure and dry, infrared drying, microwave drying, the hygroscopic desiccation in the described D step.
8. the preparation method of Radix Notoginseng polysaccharide extract according to claim 1 is characterized in that pulverizing in the described E step for using rustless steel pulverizing machine to finish; Be mixed into and use two dimension or three-dimensional motion mixer to realize incorporation time＞1h.
9. the preparation that the described Radix Notoginseng polysaccharide extract of claim 1 ~ 3 is made is characterized in that described Radix Notoginseng polysaccharide extract adds adjuvant suitable on medical science or the food and makes powder, capsule, granule, tablet or pill.
10. described Radix Notoginseng polysaccharide extract is for the preparation of the application in antitumor drug and/or the enhancing immunity medicine according to claim 1 ~ 3.
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