CN102008494B - 20(R)-ginsenoside Rg3 medicinal water-solubility intermediate, freeze-dried powder injection and preparation method thereof - Google Patents
20(R)-ginsenoside Rg3 medicinal water-solubility intermediate, freeze-dried powder injection and preparation method thereof Download PDFInfo
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Abstract
The invention relates to 20(R)-ginsenoside Rg3 medicinal water-solubility intermediate, a freeze-dried powder injection and a preparation method thereof. The preparation method comprises the following steps of: dissolving 20(R)-ginsenoside Rg3 into organic solvent to form solution of ginsenoside, wherein the ratio of 20(R)-ginsenoside Rg3 to the 2-hydroxypropyl-beta-cyclodextrin is 1:220-400; dissolving the 2-hydroxypropyl-beta-cyclodextrin into water, and heating and keeping the temperature between 40 and 100 DEG C; dripping the solution of the ginsenoside with stirring, recovering the solvent under the reduced pressure until the solvent is almost dried, and adding water for injection, shaking uniformly and dissolving to form 0.5 to 2.0 milligrams/milliliter of 20(R)-ginsenoside Rg3-containing intermediate; and filtering the intermediate, heating for heat insulating, sterilizing and freeze-drying or drying the intermediate to form powder, dissolving the powder, filtering, heating for heat insulating, sterilizing and freeze-drying to form a powder injection, namely powder which is dissolved into the water and does not contain the organic solvent, wherein the absolute bioavailability of the powder is 100 percent, and the cost of the preparation is lower than that of clathrate compounds of the component.
Description
Technical field the present invention relates to a kind of ginsenoside's medicinal soluble intermediate and preparation method thereof.
Background technology ginsenoside Rg3 is a kind of tetracyclic triterpenes saponin compound that is present in Radix Ginseng, molecular weight is 784.13, it exists with 20 (R)-ginsenoside Rg3s and two kinds of optical isomer forms of 20 (s)-ginsenoside Rg3s again, 20 (R)-ginsenoside Rg3 stable chemical natures wherein, water insoluble, and 20 (s)-ginsenoside Rg3 unstable chemcial properties are soluble in water, and their structural formula is respectively:
It is found that, 20 (R)-ginsenoside Rg3s have stronger inhibition tumor and metastasis effect, but it is again a kind of water-fast material, so the bioavailability of its oral formulations is very low, greatly limit the performance of its clinical drug effect, also limited the parenterai administration approach simultaneously.
For making 20 (R)-ginsenoside Rg3s water-soluble, people are studied this, as on January 29th, 2003 Gazette of Patent for Invention disclosed a kind of＜Ginsenoside Rg3 hydroxypropyl-beta-cyclodextrin inclusion and preparation and preparation method (application number 01119929.6) its to the effect that: the hydroxypropyl-beta-cyclodextrin inclusion of Ginsenoside Rg3, it is comprised of by following weight ratio following raw material, i.e. the ginsenoside: HP-β-CD=1: 1～200; Its preparation method is that (1) is dissolved in Ginsenoside Rg3 in organic solvent, (2) HP-β-CD is soluble in water, (3) under strong agitation, Ginsenoside Rg3 solution slowly is added drop-wise in the aqueous solution that contains HP-β-CD, all adding rear continuation stirred 2～24 hours, use 0.45 μ m filtering with microporous membrane, filtrate is concentrated, eliminate organic solvent, add injection water again to dissolve, cross 0.22 μ m film, the filtrate lyophilization gets loose white powder, is ginsenoside's clathrate.
Its weak point is: (1) clathrate is in preparation process, its reactant liquor is recycled to 1/3 o'clock of original volume, still have after measured more organic solvent residual, and lyophilization is invalid to removing residual solvent, thereby this clathrate powder is difficult to meet the limit standard of injection residual solvent for the preparation of injection.(2) clathrate is in preparation process, existence due to residual organic solvent, help Ginsenoside Rg3 and HP-β-CD to form water-soluble clathrate with 1: 1～200 ratio, if residual organic solvent is really eliminated, can not form stable clathrate soluble in water in the aforementioned proportion scope, Ginsenoside Rg3 can be separated out precipitation very soon from its clathrate aqueous solution, make this clathrate can not be for the preparation of injection.(3) Ginsenoside Rg3 can not all form clathrate, and utilization rate is only 86%, and productive rate reduction and the production cost of injection are obviously increased.
Summary of the invention the object of the present invention is to provide low 20 (R)-ginseng sapoglycoside Rg 3 medicinal soluble intermediate and lyophilized injectable powder and preparation method and the medical usage that easily is absorbed by the body again of a kind of product cost.
Main material of the present invention (abbreviation major ingredient) is 20 (R)-ginsenoside Rg3s, and auxiliary material (abbreviation adjuvant) is 2-HP-BETA-CD (english abbreviation: 2-HP-β-CD); The weight ratio of above-mentioned principal agent and adjuvant is 1: 220～400.
The preparation method of 20 (R)-ginseng sapoglycoside Rg 3 medicinal soluble intermediate:
1. 20 (R)-ginsenoside Rg3s are dissolved in mixed organic solvents, make ginsenoside's solution of 0.1～2%.Mixed organic solvents used and their ratio are as follows: (1) chloroform: ethyl acetate: methanol: water=10～25: 30～45: 18～30: 5～15, and lower floor; (2) chloroform: methanol: water=70～60: 40～30: 10, lower floor; (3) ethanol: water=90～95: 10～5, (4) methanol: water=85～90: 15～10, (5) acetonitrile: water=40～60: 60～40, (6) dichloromethane: methanol: water=60～65: 40～35: 10, (7) acetonitrile: methanol=70～80: 30～20.
2-HP-BETA-CD is soluble in water 2., make 20～65% aqueous solution.
3. will contain the aqueous solution heating of 2-HP-BETA-CD, and make its temperature maintain 40～100 ℃, and at the uniform velocity drip ginsenoside Rg3's solution, and stir it, and namely stop stirring when ginsenoside Rg3's solution drips.
4. the reclaim under reduced pressure mentioned solution is closely dried, its vacuum 0.01～0.08MPa, 60～100 ℃ of temperature.Preferably add water for injection that it is dissolved in condensed cream, then carry out the reclaim under reduced pressure operation, repeat twice, in order to eliminate organic solvent.
5. add water for injection to original volume in concentrate, shake and make it dissolving, the aqueous solution that obtains is 20 (R)-ginseng sapoglycoside Rg 3 medicinal soluble intermediate of content 0.5～2 mg/ml, and 20 (R)-ginsenoside Rg3s all form the water solublity intermediate, and namely gained water solublity intermediate yield is 100%.
Make raw material with above-mentioned 20 (R)-ginseng sapoglycoside Rg 3 medicinal soluble intermediate, can prepare freezing-dried powder injection: (1) with above-mentioned aqueous solution with ultrafilter or add the injection-use activated carbon mix homogeneously of its weight 0.1%, in 80 ℃ the insulation 30 minutes after, remove thermal source with 0.45 μ m membrane filtration, under aseptic condition with 0.22 μ m filtering with microporous membrane degerming packing, lyophilization gets the white loose powder, is 20 (R)-ginsenoside Rg3 lyophilized injectable powders.(2) above-mentioned aqueous solution is carried out vacuum, spraying or lyophilization (seeing the following form), make the Rg3 water-soluble powder, then add water for injection to redissolve, and then repeat the operation of (1).
Different drying equipments, drying means and drying condition catalog
The pharmaceutical preparation made from above-mentioned 20 (R)-ginsenoside Rg3 water solublity intermediate has the inhibition growth and metastasis of tumours, the raising immune function of human body reaches and the effect of radiation and chemotherapy drug combination efficacy enhancing and toxicity reducing.Therefore, this 20 (R)-ginseng sapoglycoside Rg 3 medicinal soluble intermediate lyophilized injectable powder can be used as preparation antitumor, the curative effect that increases chemotherapeutics and the application that alleviates its toxicity and improve the body's immunity medicine.
The present invention has following advantage compared to existing technology:
1. 20 (R)-ginsenoside Rg3 water solublity intermediate of the present invention can obtain the powder that is dissolved in water fully, do not contain residual organic solvent after drying, can be made into thus the freezing-dried powder injection that meets the injection prescription, can 100% enter in the blood of animal and human body through injection, its absolute bioavailability is 100%, obviously improves than the bioavailability of the existing oral capsule (Shenyi capsule) of being made by 20 (R)-ginsenoside Rg3s.
2. the injection of 20 (R)-ginsenoside Rg3 water solublity intermediate preparations of the present invention, input 20 (R)-ginsenoside Rg3 crude drug can be made the water solublity intermediate in 100% ground, make its cost for preparing injection be starkly lower than the clathrate of this composition.
3. after the injection intravenous injection of being made by water solublity intermediate of the present invention, the absolute bioavailability of medicine is 100%, and is higher at liver, the gastrointestinal wall distributed density of rat and dog, makes it have good therapeutical effect to digestive tract tumor and transfer.
Description of drawings
Fig. 1 is 20 (R)-ginsenoside Rg3 standard substance high performance liquid chromatography spectrograms.
Fig. 2 is 20 (R)-ginsenoside Rg3 water solublity intermediate high performance liquid chromatography assay spectrogram 1mg/ml.
Fig. 3 is 20 (R)-ginsenoside Rg3 water solublity intermediate direct dried frozen aquatic products high performance liquid chromatography assay spectrograms (5mg/ props up).
Fig. 4 is 20 (R)-ginsenoside Rg3 water solublity intermediate direct dried frozen aquatic products high performance liquid chromatography assay spectrograms (5mg/ props up).
Fig. 5 is dried frozen aquatic products high performance liquid chromatography assay spectrogram (5mg/ props up) again after 20 (R)-ginsenoside Rg3 water solublity intermediate vacuum dryings.
Fig. 6 is dried frozen aquatic products high performance liquid chromatography assay spectrogram (5mg/ props up) again after 20 (R)-ginsenoside Rg3 water solublity intermediate spray dryinges.
Fig. 7 is dried frozen aquatic products high performance liquid chromatography assay spectrogram (5mg/ props up) again after 20 (R)-ginsenoside Rg3 water solublity intermediate vacuum dryings.
The specific embodiment
Embodiment 1
100 gram 20 (R)-ginsenoside Rg3s are used chloroform: ethyl acetate: methanol: water=20: 40: 25: 10, lower floor; 100 liters of dissolvings of mixed organic solvents.Get 22000 gram 2-HP-BETA-CDs, add 40 liters of distilled water dissolvings, afterwards this solution is heated to 40 ℃, under stirring condition, at the uniform velocity drip 20 (R)-ginsenoside Rg3 solution with 80 ml/min, drip this solution and namely stop stirring, reclaim under reduced pressure is closely dried on rotary evaporator.Add 50 liters of distilled water it is dissolved, then carry out the decompression and solvent recovery operation, repeat twice.With 100 liters of water for injection dissolving concentrate, the aqueous solution that obtains is 20 (R)-ginsenoside Rg3 water solublity intermediate.Products measure: accurate 20 (R)-ginsenoside Rg3 solution and direct accurate each 20 microlitres of 20 (R)-ginsenoside Rg3 water solublity midbody solutions of drawing drawn, inject high performance liquid chromatograph, measure peak area, calculate this water solublity intermediate take external standard method and contain the ginsenoside Rg3 as 1 mg/ml, containing altogether the ginsenoside Rg3 in 100 premium on currency dissolubility intermediate is 100 grams, and yield is 100%.Its measurement result is seen Fig. 2.
get 50 liters of 20 (R)-ginsenoside Rg3 water solublity intermediate that embodiment 1 makes, add 50 gram injection-use activated carbon mix homogeneously, filter except thermal source with 0.45 μ m filter membrane after 30 minutes in 80 ℃ of insulations, under aseptic condition with 0.22 μ m microporous filter membrane Entkeimung, be filled in the aseptic cillin bottle of 10ml control in lower minute in aseptic condition, every bottle of loading amount is 5ml approximately, and the cillin bottle after installing adds half plug, put on the freeze dryer dish, start freeze dryer, prior to-45 ℃ of pre-freezes after 5 hours, start vacuum drying, and follow procedure heats up :-45～-15 ℃ of sublimation dryings 20 hours,-15～30 ℃ of vacuum dryings 10 hours.Aluminium lid is rolled in tamponade after lyophilization, and sampling detects, and qualified rear packing namely makes 10000 freeze-dried powder injections.Products measure: precision weighing dried frozen aquatic products 1.10 grams, with distilled water dissolving and standardize solution in 100 milliliters of volumetric flasks, accurate 20 (R)-ginsenoside Rg3 solution and each 20 microlitres of 20 (R)-ginsenoside Rg3 water solublity midbody solutions drawn, inject high performance liquid chromatograph, measure peak area, calculate the water solublity intermediate take external standard method and contain 20 (R)-ginsenoside Rg3s as 1 mg/ml, its measurement result is seen Fig. 3.
Embodiment 3
Get 5 liters of 20 (R)-ginsenoside Rg3 water solublity intermediate that embodiment 1 makes, in dry 48 hours of 30 ℃, 0.01Mpa vacuum under pressure, get water-soluble powder.add again 2.5 liters of waters for injection to redissolve, and then repeat embodiment 2 except thermal source, degerming and lyophilization operating procedure, make 11000 gram lyophilized powders, aseptic subpackaged, can make 500 freeze-dried powder injections, products measure: precision weighing dried frozen aquatic products 2.20 grams, with distilled water dissolving and standardize solution in 100 milliliters of volumetric flasks (A), 5 milliliters of dilutions of accurate absorption and standardize solution are in 10 milliliters of volumetric flasks (B) from (A), accurate draw 20 (R)-ginsenoside Rg3 solution and (B) in each 20 microlitres of 20 (R)-ginsenoside Rg3 water solublity midbody solutions, inject high performance liquid chromatograph, measure peak area, calculate the water solublity intermediate take external standard method and contain 20 (R)-ginsenoside Rg3s as 2 mg/ml, its measurement result is seen Fig. 4.
Embodiment 4
Get 10 liters of 20 (R)-ginsenoside Rg3 water solublity intermediate that embodiment 1 makes, in dry 48 hours of 60 ℃, 0.08Mpa vacuum under pressure, get water-soluble powder.add again 10 liters of waters for injection to redissolve, and then repeat embodiment 2 except thermal source, degerming and lyophilization operating procedure, make 22000 gram lyophilized powders, aseptic subpackaged, can make 2000 freeze-dried powder injections, products measure: precision weighing dried frozen aquatic products 1.10 grams, with distilled water dissolving and standardize solution in 100 milliliters of volumetric flasks, accurate 20 (R)-ginsenoside Rg3 solution and each 20 microlitres of 20 (R)-ginsenoside Rg3 water solublity midbody solutions drawn, inject high performance liquid chromatograph, measure peak area, calculate the water solublity intermediate take external standard method and contain the ginsenoside Rg3 as 1 mg/ml, its measurement result is seen Fig. 5.
Embodiment 5
Get 10 liters of 20 (R)-ginsenoside Rg3 water solublity intermediate that embodiment 1 makes, carry out spray drying with extractum spray dryer, employing supersonic jet technology, effluxvelocity 300 meter per seconds, with the instantaneous drying of hypersonic velocity, get water-soluble powder under 30 ℃ of temperature, 0.01Mpa pressure.add again 20 liters of waters for injection to redissolve, and then repeat embodiment 2 except thermal source, degerming and lyophilization operating procedure, make 22000 gram lyophilized powders, aseptic subpackaged, can make 4000 freeze-dried powder injections, products measure: precision weighing dried frozen aquatic products 1.10 grams, with distilled water dissolving and standardize solution in 100 milliliters of volumetric flasks, 5 milliliters of dilutions of accurate absorption and standardize solution are in 10 milliliters of volumetric flasks (B) from (A), accurate 20 (R)-ginsenoside Rg3 solution and (B) each 10 microlitres of 20 (R)-ginsenoside Rg3 water solublity midbody solutions drawn, inject high performance liquid chromatograph, measure peak area, calculate the water solublity intermediate take external standard method and contain the ginsenoside Rg3 as 0.5 mg/ml, its measurement result is seen Fig. 6.
Get 20 liters of 20 (R)-ginsenoside Rg3 water solublity intermediate that embodiment 1 makes, carry out spray drying with extractum spray dryer, employing supersonic jet technology, effluxvelocity 990 meter per seconds, with the instantaneous drying of hypersonic velocity, get water-soluble powder under 60 ℃ of temperature, 0.05Mpa pressure.add again 20 liters of waters for injection to redissolve, and then repeat embodiment 2 except thermal source, degerming and lyophilization operating procedure, make 44000 gram lyophilized powders, aseptic subpackaged, can make 4000 freeze-dried powder injections, products measure: precision weighing dried frozen aquatic products 1.10 grams, with distilled water dissolving and standardize solution in 100 milliliters of volumetric flasks, accurate 20 (R)-ginsenoside Rg3 solution and each 20 microlitres of 20 (R)-ginsenoside Rg3 water solublity midbody solutions drawn, inject high performance liquid chromatograph, measure peak area, calculate the water solublity intermediate take external standard method and contain the ginsenoside Rg3 as 1 mg/ml, its measurement result is seen Fig. 7.
The test of embodiment 21-20 (R) ginsenoside Rg3 lyophilized injectable powder antitumor drug effect
1 tested medicine
1.1 title ginsenoside Rg3's lyophilized injectable powder and corresponding adjuvant.
1.2 the living natural drug of Dalian richness development corporation, Ltd. of the unit of providing.
1.3 lot number ginsenoside Rg3 lyophilized injectable powder 20030519, corresponding adjuvant.
1.4 content ginsenoside Rg3 lyophilized injectable powder: 5mg/ props up.
1.5 the compound method precision measures a certain amount of ginsenoside Rg3's lyophilized injectable powder or corresponding adjuvant adds normal saline to desired concn to get final product, administration volume 0.5ml/ Mus.
2 experiment materials
2.1 solvent normal saline.
2.2 positive reference substance Cyclophosphamide for injection (CTX), Hua Lian, Shanghai pharmacy group produces; Once a day, continuous seven days; The 5Fu injection, Shanghai Xudong Hipu Medicine Co., Ltd produces; Mitomycin for inj (MMC), Japanese Kyowa Hakkokogyo Co., Ltd produces.
2.3 tumor source Human Breast Cancer Bcap-
37Model, Human Lung Cancer A
549Tumor more than model, human body intestinal cancer LOVO model, Human Gastric Cancer MGC model and the equal second filial generation of human hepatoma QGY is as the tumor source; Mice B
16Melanoma cell, Mice Bearing Lewis Lung Cancer model, murine sarcoma S
180Models etc. go down to posterity by pharmacological room of Shanghai Institute of Pharmaceutical Industry and keep.
3 laboratory animals
3.1 the source nude mouse is provided by Shanghai City Chinese Academy of Sciences Experimental Animal Center, the quality certification number: SCXK2003-0003.C
57BL/6 and Kunming mouse are provided by the court's laboratory animal group, laboratory animal occupancy permit number: SYXK (Shanghai) 2004-0015.
3.2 the body weight nude mouse was 6 ages in week, Kunming mouse and C
57The BL/6 mice is 18～22 grams.
3.3 the sex male and female all can, the same sex is used in every batch of experiment.
3.4 every group of 6 mices of number of animals test group and positive controls nude mouse, every group of 8-10 of other mices, negative control is respectively two groups.
4 EXPERIMENTAL DESIGN
4.1 dosage arranges ginsenoside Rg3's lyophilized injectable powder dosage and is made as: 1.5mg/kg/d, 0.75mg/kg/d, 0.375mg/kg/d.
4.2 the dosage regimen intravenously administrable, every day 2 times, the mouse model iv * 14bid of human tumor model and cell inoculation, the Regimen Chemotherapy of the model iv * 10bid of mouse tumor inoculation.
4.3 experimental control negative control: give adjuvant corresponding to test group, the same test group of dosage regimen; Positive control: cyclophosphamide CTX 30mg/kg, MMC
2Mg/kg and 5Fu30mg/kg abdominal cavity or intravenously administrable once a day, continuous seven days.
5 test methods and result
5.1 test method
5.1.1 antitumor test
5.1.1.1 stomach orthotopic model
Get the MGC gastric cancer that passed for 2 generations in eugonic body under aseptic condition, standby into about 2 * 10 with the homogenate legal system
7/ ml cell suspension, underwent operative are injected cell suspension 0.05ml in the greater gastric curvature flesh layer of nude mouse, press the administration of experimental design scheme next day, calculate lotus tumor host increase in life span by following formula:
Sky, increase in life span %=administration group the average survival time sky/matched group the average survival time * 100%
5.1.1.2 liver orthotopic model
Get biography second filial generation QGY tumor source in eugonic body under aseptic condition, prepare into about 1-2 * 10 at 1: 6 with the homogenate method
7/ ml cell suspension, homogenate is through 100 order stainless steel sift net filtrations, and is standby.The nude mouse routine disinfection is anaesthetized and cut skin and abdominal cavity under the xiphoid-process of abdominal cavity center, exposes liver, and the 28ga 1/2ml injector to inject 0.05ml cell suspension through liver parenchyma section with import is closed the abdominal cavity, layer-by-layer suture abdominal cavity and skin.Nude mouse is placed in laminar-flow rack and raises, and the apparatus of feedstuff used, bedding and padding, cage tool and contact etc. all should use after autoclave sterilization.Press the administration of experimental design scheme next day, observe the life span in each treated animal 45 days, compare with negative control group, the statistics increase in life span.
5.1.2 synergism test
The dosage of ginsenoside Rg3's lyophilized injectable powder and chemotherapeutic sees the result of the test table for details.The tumor inoculation method is as follows.
5.1.2.1 axil subcutaneous vaccination model
Get eugonic tumor source under aseptic condition, standby into about 1～2 * 10 with the homogenate legal system
7/ ml cell suspension; In the every Mus of corresponding host's axil subcutaneous vaccination 0.2ml/, press the administration of experimental design scheme next day, each treated animals are put to death in three about weeks, cut open to get tumor and weigh, and calculate tumor control rate by following formula:
The average tumor of the average tumor weight-administration of tumor control rate %=[(matched group group is heavy)/the average tumor of matched group is heavy] * 100%
5.1.2.2 tail intravenous inoculation model
The B of trophophase takes the logarithm under aseptic condition
16The murine melanoma cultured cell, preparation is into about 2.5 * 10
5/ ml cell suspension is in C
57BL/6 mouse tail vein inoculation 0.2ml/ Mus is pressed the administration of experimental design scheme next day, puts to death each treated animal after three weeks, cut open and get the lungs of respectively organizing mice, the colony number that the every Mus lungs of instrumentation shift, with the average colony number of each group tumor, calculate tumor control rate by following formula:
The average colony number of tumor control rate %=[(matched group-average colony number of administration group)/average colony number of matched group] * 100%
5.1.2 attenuation test
The impact of observation on ginsenoside Rg3's lyophilized injectable powder chemotherapeutic induced mice White blood cell.Get C
5780 of BL/6 mices are got blood from every rathole venous plexus, leukocyte slide counting method routinely, and each Mus leukocyte count of instrumentation is selected 7500 ± 300 leukocyte/mm
3Mice, random packet, every group of 10 mices.0th, all the other each groups were all given chemotherapeutic CTX100mg/kg ip * 2 except the blank group in 2 days.Press the such scheme administration, began in the 0th day to survey and respectively organize leukocyte count, then experimental session every three days, detects and respectively organizes the murine interleukin number, respectively organizes mutually leukocyte count average and the standard deviation of mice when calculating each, until till the positive controls leukocyte recovers normal number.
5.1.4 immunity test
5.1.4.1 to lotus Lewis lung cancer C
57The NK cytoactive test of BL/6 mice
Get C
57The BL/6 mice, the Lewis lung cancer suspension 0.05ml/ Mus of the aseptic preparation of toe subcutaneous vaccination (approximately 1 * 10
6Individual tumor cell).Next day, random packet, pressed the experimental design administration, and after the last administration, get mouse spleen next day under aseptic condition, makes single splenocyte suspension with 100 eye mesh screens, and the hypotonic erythrocyte of removing changes cell suspension in culture bottle over to, 37 ℃ of 5%CO
2Condition is cultivated after 1 hour and is removed attached cell, living cell counting and to adjust cell concentration be 3 * 10
6Individual cell/ml action effect cell.Target cell is got L
929Cultured cell in vitro, cellar culture 24 hours is adjusted cell concentration as 1.5 * 10 take culture fluid
5Individual cell/ml, making the effect target cell ratio is 20: 1.Get 96 well culture plates and add respectively effector lymphocyte and target cell, another laying effect cell and target cell contrast are in 37 ℃ of 5%CO
2Condition was cultivated after 4 hours, added the MTT dyeing liquor, then cultivated and add Digestive system to survey each hole OD value next day after 2 hours, by formula calculated the NK cytotoxic activity.
NK cytotoxic activity %={[target cell matched group OD average-(experimental group OD average-effector lymphocyte OD average)]/target cell matched group OD average } * 100%
5.1.4.2 the Peritoneal Macrophage Phagocytosis test to normal Kunming mouse
Get the male mice in kunming random packet, press the administration of experimental design scheme, only respectively organize mouse peritoneal injection 0.5% hydrolyzed protein 1.5ml/ after the last administration, every milliliter 1 * 10 of pneumoretroperitoneum injection in 24 hours
6Chicken erythrocyte suspension 0.2ml/ only; Use the normal saline eluting after 40 minutes, collect mouse peritoneal liquid; Centrifugal, get cell precipitation liquid and make smear, to fix with methanol, the Giemsa staining mounting with engulfing the erythrocytic macrophage number of chicken and engulf the erythrocytic sum of chicken in 100 macrophages of oily mirror counting, calculates percentage phagocytosis and phagocytic index by following formula.
Percentage phagocytosis %=(engulfing the erythrocytic macrophage number of chicken/100 macrophages in 100 macrophages) * 100%
Engulf the erythrocytic sum of chicken/100 macrophages in the macrophage of phagocytic index=100
Result of the test
1. ginsenoside Rg3's lyophilized injectable powder anti-alimentary tract tumor clinical trial the results are shown in Table 1-2;
2. ginsenoside Rg3's lyophilized injectable powder increase chemotherapeutical medicine curative effect result of the test sees Table 3-4;
3. ginsenoside Rg3's lyophilized injectable powder alleviates chemotherapeutical medicine curative effect toxic and side effects clinical trial and the results are shown in Table 5;
4. ginsenoside Rg3's lyophilized injectable powder raising body's immunity clinical trial the results are shown in Table 6-7.
Table 1 ginsenoside Rg3 lyophilized injectable powder is to Human Gastric Cancer MGC (in-situ inoculating) clinical trial (n=3)
Table 2 ginsenoside Rg3 lyophilized injectable powder is to human hepatoma QGY (in-situ inoculating) clinical trial (n=3)
Table 3 ginsenoside Rg3 lyophilized injectable powder merges 5Fu to murine sarcoma S180 clinical trial (n=3)
Table 4 ginsenoside Rg3 lyophilized injectable powder merges CTX to mouse melanin B16 clinical trial (n=3)
The impact test (n=3) of table 6 ginsenoside Rg3 lyophilized injectable powder on lotus Lewis lung cancer mice NK activity
Compare * * * P＜0.01 with negative control group; 2# refers to (effector lymphocyte+target cell)-effector lymphocyte's OD value; The 3 positive contrasts of lentinan.
Table 7 ginsenoside Rg3 lyophilized injectable powder is tested (n=3) to normal Kunming mouse Peritoneal Macrophage Phagocytosis
Compare * * * P＜0.01 with negative control group
Claims (1)
1. ginsenoside Rg3's lyophilized injectable powder improves the application of the medicine of lotus Lewis lung cancer mice NK activity in preparation, and wherein ginsenoside Rg3's lyophilized injectable powder prepares by the following method:
100 gram 20 (R)-ginsenoside Rg3s are used chloroform: ethyl acetate: methanol: water=20: 40: 25: 100 liters of dissolvings of 10 mixed organic solvents; Get 22000 gram 2-HP-BETA-CDs, add 40 liters of distilled water dissolvings, afterwards this solution is heated to 40 ℃, under stirring condition, at the uniform velocity drip 20 (R)-ginsenoside Rg3 solution with 80 ml/min, drip this solution and namely stop stirring, reclaim under reduced pressure is closely dried on rotary evaporator; Add 50 liters of distilled water it is dissolved, then carry out the decompression and solvent recovery operation, repeat twice; With 100 liters of water for injection dissolving concentrate, the aqueous solution that obtains is 20 (R)-ginsenoside Rg3 water solublity intermediate; With
get 50 liters of above-mentioned 20 (the R)-ginsenoside Rg3 water solublity intermediate that makes, add 50 gram injection-use activated carbon mix homogeneously, filter except thermal source with 0.45 μ m filter membrane after 30 minutes in 80 ℃ of insulations, under aseptic condition with 0.22 μ m microporous filter membrane Entkeimung, be filled in the aseptic cillin bottle of 10ml control in lower minute in aseptic condition, every bottle of 5ml of loading amount, and the cillin bottle after installing adds half plug, put on the freeze dryer dish, start freeze dryer, prior to-45 ℃ of pre-freezes after 5 hours, start vacuum drying, and follow procedure heats up :-45～-15 ℃ of sublimation dryings 20 hours,-15～30 ℃ of vacuum dryings 10 hours, aluminium lid is rolled in tamponade after lyophilization, and sampling detects, and qualified rear packing namely makes 10000 freeze-dried powder injections.
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