The specific embodiment
The present invention is further illustrated below in conjunction with accompanying drawing, 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.
Active compound composition of the present invention is by weight the following raw material of proportioning: 1 ~ 90% Poria, 1 ~ 90% Ganoderma, 1 ~ 90% Herba Dendrobii and the macro fungi of surplus make through extraction.
Described raw material weight matches well than being: 1 ~ 60% Poria, 1 ~ 60% Ganoderma, 1 ~ 60% Herba Dendrobii and the macro fungi of surplus.
Described raw material weight matches well than being: 1 ~ 30% Poria, 1 ~ 30% Ganoderma, 1 ~ 30% Herba Dendrobii and the macro fungi of surplus.
Described active compound composition be raw material through pulverize, extract, dry, being mixed makes.
Describedly be extracted as a kind of in water extraction or the alcohol extraction.
Described macro fungi is one or more in Lentinus Edodes and Volvariella volvacea (Bull.Ex Franch.) Singer., JINZHENGU, Agaricus bisporus, Pleurotus ostreatus, Auricularia, Tremella, Caulis Bambusae In Taeniam, the Gaster caprae seu Ovis.
The preparation method of active compound composition of the present invention, it is characterized in that comprising get the raw materials ready, extraction, concentrate drying, testing sequence, specifically comprise:
A, get the raw materials ready: with the raw material after crossing 10 mesh sieves through screening, coarse pulverization by weight weighing;
B, extraction: add the purified water of 2 ~ 6 times of weight ratios in the raw material Poria, reflux, extract, 3 ~ 5 times, each 2 ~ 3h, merge extractive liquid,, filter, under agitation add ethanol, make to contain the alcohol amount and reach 70 ~ 80%, leave standstill 12 ~ 24h, separate out precipitation, filter, the collecting precipitation thing gets Poria extract; 80 ~ 99% ethanol that add 2 ~ 5 times in the raw material Ganoderma, hot bath refluxes 3 ~ 5 times, each 2 ~ 3h, merge extractive liquid,, Recycled ethanol filters to get filtrate, and concentrates to get Ganoderma extract; 90 ~ 95 ℃ of purified water lixiviates that raw material Herba Dendrobii usefulness weight ratio is 1 ~ 10 times 1 ~ 3 time, each 3 ~ 4h merges lixiviating solution, filters, the concentrated Herba Dendrobii extract that to get; 95 ~ 100 ℃ of purified water lixiviates that raw material macro fungi usefulness weight ratio is 1 ~ 20 times 2 ~ 3 times, each 2 ~ 3h merges lixiviating solution, filters, the concentrated macro fungi extract that to get;
C, drying and crushing: each extract is dried to respectively moisture content 1 ~ 9%, crosses 10 mesh sieves through coarse pulverization;
D, be mixed: each extract after coarse pulverization is carried out mixing, got objective composition through pulverizing 80 mesh sieves;
E, check: with the objective composition packing of sterilizing, pick test is provided inspection report.
Described concentration of alcohol is 90 ~ 99%.
Described Recycled ethanol is to reclaim by thermal evaporation recovery or embrane method.
The described membrane filtration that is filtered into, filter membrane aperture are 0.2 ~ 15 mm.
Described drying is constant pressure and dry or drying under reduced pressure, takes one or more the combination in spray drying, lyophilization, microwave drying, forced air drying, the hygroscopic desiccation.
Preparation of the present invention is that described active compound composition adds pharmaceutically the acceptable adjuvant and makes powder, tablet, granule, capsule, pill.
Of the present inventionly be applied as described active compound composition in the application of preparation in the antitumor drug.
Advantage of the present invention is: after composite, utilize the mutual synergism of a few class polysaccharide, can strengthen its eliminating dampness and diuresis, mind tranquilizing and the heart calming, antitumor and protect the liver dirty, body immunity, drive effects such as separating deficiency-heat.Technique of the present invention extracting method simple, that set up is strongly professional, good reproducibility, and uses purified water, alcohol extraction, not only environmental protection, and product safety is reliable simultaneously, and cost is low, is suitable for suitability for industrialized production.
Embodiment 1
With through screening, the coarse pulverization raw material after crossing 10 mesh sieves by Poria 90Kg, Ganoderma 90Kg, Herba Dendrobii 90Kg and Lentinus Edodes and Volvariella volvacea (Bull.Ex Franch.) Singer., JINZHENGU, Agaricus bisporus, Pleurotus ostreatus, Auricularia, Tremella, Caulis Bambusae In Taeniam, Gaster caprae seu Ovis 100Kg; The purified water that adds 2 ~ 6 times of weight ratios in the raw material Poria, reflux, extract, 3 ~ 5 times, each 2 ~ 3h, merge extractive liquid, filters, and under agitation adds ethanol, makes to contain the alcohol amount and reach 70 ~ 80%, leaves standstill 12 ~ 24h, separates out precipitation, filters, and the collecting precipitation thing gets Poria extract; 80 ~ 99% ethanol that add 2 ~ 5 times in the raw material Ganoderma, hot bath refluxes 3 ~ 5 times, each 2 ~ 3h, merge extractive liquid,, Recycled ethanol filters to get filtrate, and concentrates to get Ganoderma extract; 90 ~ 95 ℃ of purified water lixiviates that raw material Herba Dendrobii usefulness weight ratio is 1 ~ 10 times 1 ~ 3 time, each 3 ~ 4h merges lixiviating solution, filters, the concentrated Herba Dendrobii extract that to get; 95 ~ 100 ℃ of purified water lixiviates that raw material macro fungi usefulness weight ratio is 1 ~ 20 times 2 ~ 3 times, each 2 ~ 3h merges lixiviating solution, filters, the concentrated macro fungi extract that to get; Each extract is dried to respectively moisture content 1 ~ 9%, crosses 10 mesh sieves through coarse pulverization; Each extract after coarse pulverization is carried out mixing, got objective composition through pulverizing 80 mesh sieves; With the objective composition packing of sterilizing, pick test is provided inspection report.
Embodiment 2
With through screening, the coarse pulverization raw material after crossing 10 mesh sieves by Poria 90Kg, Ganoderma 90Kg, Herba Dendrobii 90Kg and Lentinus Edodes and Volvariella volvacea (Bull.Ex Franch.) Singer., JINZHENGU, Agaricus bisporus, Pleurotus ostreatus, Auricularia, Tremella, Caulis Bambusae In Taeniam, Gaster caprae seu Ovis 100Kg; The purified water that adds 2 ~ 6 times of weight ratios in the raw material Poria, reflux, extract, 3 ~ 5 times, each 2 ~ 3h, merge extractive liquid, filters, and under agitation adds ethanol, makes to contain the alcohol amount and reach 70 ~ 80%, leaves standstill 12 ~ 24h, separates out precipitation, filters, and the collecting precipitation thing gets Poria extract; 80 ~ 99% ethanol that add 2 ~ 5 times in the raw material Ganoderma, hot bath refluxes 3 ~ 5 times, each 2 ~ 3h, merge extractive liquid,, Recycled ethanol filters to get filtrate, and concentrates to get Ganoderma extract; 90 ~ 95 ℃ of purified water lixiviates that raw material Herba Dendrobii usefulness weight ratio is 1 ~ 10 times 1 ~ 3 time, each 3 ~ 4h merges lixiviating solution, filters, the concentrated Herba Dendrobii extract that to get; 95 ~ 100 ℃ of purified water lixiviates that raw material macro fungi usefulness weight ratio is 1 ~ 20 times 2 ~ 3 times, each 2 ~ 3h merges lixiviating solution, filters, the concentrated macro fungi extract that to get; Each extract is dried to respectively moisture content 1 ~ 9%, crosses 10 mesh sieves through coarse pulverization; Each extract after coarse pulverization is carried out mixing, got objective composition through pulverizing 80 mesh sieves; With the objective composition packing of sterilizing, pick test is provided inspection report.
Embodiment 3
With through screening, the coarse pulverization raw material after crossing 10 mesh sieves by Poria 90Kg, Ganoderma 90Kg, Herba Dendrobii 90Kg and Lentinus Edodes and Volvariella volvacea (Bull.Ex Franch.) Singer., JINZHENGU, Agaricus bisporus, Pleurotus ostreatus, Auricularia, Tremella, Caulis Bambusae In Taeniam, Gaster caprae seu Ovis 100Kg; The purified water that adds 2 ~ 6 times of weight ratios in the raw material Poria, reflux, extract, 3 ~ 5 times, each 2 ~ 3h, merge extractive liquid, filters, and under agitation adds ethanol, makes to contain the alcohol amount and reach 70 ~ 80%, leaves standstill 12 ~ 24h, separates out precipitation, filters, and the collecting precipitation thing gets Poria extract; 80 ~ 99% ethanol that add 2 ~ 5 times in the raw material Ganoderma, hot bath refluxes 3 ~ 5 times, each 2 ~ 3h, merge extractive liquid,, Recycled ethanol filters to get filtrate, and concentrates to get Ganoderma extract; 90 ~ 95 ℃ of purified water lixiviates that raw material Herba Dendrobii usefulness weight ratio is 1 ~ 10 times 1 ~ 3 time, each 3 ~ 4h merges lixiviating solution, filters, the concentrated Herba Dendrobii extract that to get; 95 ~ 100 ℃ of purified water lixiviates that raw material macro fungi usefulness weight ratio is 1 ~ 20 times 2 ~ 3 times, each 2 ~ 3h merges lixiviating solution, filters, the concentrated macro fungi extract that to get; Each extract is dried to respectively moisture content 1 ~ 9%, crosses 10 mesh sieves through coarse pulverization; Each extract after coarse pulverization is carried out mixing, got objective composition through pulverizing 80 mesh sieves; With the objective composition packing of sterilizing, pick test is provided inspection report.
Embodiment 4
With through screening, the coarse pulverization raw material after crossing 10 mesh sieves by Poria 90Kg, Ganoderma 90Kg, Herba Dendrobii 90Kg and Lentinus Edodes and Volvariella volvacea (Bull.Ex Franch.) Singer., JINZHENGU, Agaricus bisporus, Pleurotus ostreatus, Auricularia, Tremella, Caulis Bambusae In Taeniam, Gaster caprae seu Ovis 100Kg; The purified water that adds 2 ~ 6 times of weight ratios in the raw material Poria, reflux, extract, 3 ~ 5 times, each 2 ~ 3h, merge extractive liquid, filters, and under agitation adds ethanol, makes to contain the alcohol amount and reach 70 ~ 80%, leaves standstill 12 ~ 24h, separates out precipitation, filters, and the collecting precipitation thing gets Poria extract; 80 ~ 99% ethanol that add 2 ~ 5 times in the raw material Ganoderma, hot bath refluxes 3 ~ 5 times, each 2 ~ 3h, merge extractive liquid,, Recycled ethanol filters to get filtrate, and concentrates to get Ganoderma extract; 90 ~ 95 ℃ of purified water lixiviates that raw material Herba Dendrobii usefulness weight ratio is 1 ~ 10 times 1 ~ 3 time, each 3 ~ 4h merges lixiviating solution, filters, the concentrated Herba Dendrobii extract that to get; 95 ~ 100 ℃ of purified water lixiviates that raw material macro fungi usefulness weight ratio is 1 ~ 20 times 2 ~ 3 times, each 2 ~ 3h merges lixiviating solution, filters, the concentrated macro fungi extract that to get; Each extract is dried to respectively moisture content 1 ~ 9%, crosses 10 mesh sieves through coarse pulverization; Each extract after coarse pulverization is carried out mixing, got objective composition through pulverizing 80 mesh sieves; With the objective composition packing of sterilizing, pick test is provided inspection report.
Embodiment 5
With through screening, the coarse pulverization raw material after crossing 10 mesh sieves by Poria 90Kg, Ganoderma 90Kg, Herba Dendrobii 90Kg and Lentinus Edodes and Volvariella volvacea (Bull.Ex Franch.) Singer., JINZHENGU, Agaricus bisporus, Pleurotus ostreatus, Auricularia, Tremella, Caulis Bambusae In Taeniam, Gaster caprae seu Ovis 100Kg; The purified water that adds 2 ~ 6 times of weight ratios in the raw material Poria, reflux, extract, 3 ~ 5 times, each 2 ~ 3h, merge extractive liquid, filters, and under agitation adds ethanol, makes to contain the alcohol amount and reach 70 ~ 80%, leaves standstill 12 ~ 24h, separates out precipitation, filters, and the collecting precipitation thing gets Poria extract; 80 ~ 99% ethanol that add 2 ~ 5 times in the raw material Ganoderma, hot bath refluxes 3 ~ 5 times, each 2 ~ 3h, merge extractive liquid,, Recycled ethanol filters to get filtrate, and concentrates to get Ganoderma extract; 90 ~ 95 ℃ of purified water lixiviates that raw material Herba Dendrobii usefulness weight ratio is 1 ~ 10 times 1 ~ 3 time, each 3 ~ 4h merges lixiviating solution, filters, the concentrated Herba Dendrobii extract that to get; 95 ~ 100 ℃ of purified water lixiviates that raw material macro fungi usefulness weight ratio is 1 ~ 20 times 2 ~ 3 times, each 2 ~ 3h merges lixiviating solution, filters, the concentrated macro fungi extract that to get; Each extract is dried to respectively moisture content 1 ~ 9%, crosses 10 mesh sieves through coarse pulverization; Each extract after coarse pulverization is carried out mixing, got objective composition through pulverizing 80 mesh sieves; With the objective composition packing of sterilizing, pick test is provided inspection report.
Embodiment 6
With through screening, the coarse pulverization raw material after crossing 10 mesh sieves by Poria 90Kg, Ganoderma 90Kg, Herba Dendrobii 90Kg and Lentinus Edodes and Volvariella volvacea (Bull.Ex Franch.) Singer., JINZHENGU, Agaricus bisporus, Pleurotus ostreatus, Auricularia, Tremella, Caulis Bambusae In Taeniam, Gaster caprae seu Ovis 100Kg; The purified water that adds 2 ~ 6 times of weight ratios in the raw material Poria, reflux, extract, 3 ~ 5 times, each 2 ~ 3h, merge extractive liquid, filters, and under agitation adds ethanol, makes to contain the alcohol amount and reach 70 ~ 80%, leaves standstill 12 ~ 24h, separates out precipitation, filters, and the collecting precipitation thing gets Poria extract; 80 ~ 99% ethanol that add 2 ~ 5 times in the raw material Ganoderma, hot bath refluxes 3 ~ 5 times, each 2 ~ 3h, merge extractive liquid,, Recycled ethanol filters to get filtrate, and concentrates to get Ganoderma extract; 90 ~ 95 ℃ of purified water lixiviates that raw material Herba Dendrobii usefulness weight ratio is 1 ~ 10 times 1 ~ 3 time, each 3 ~ 4h merges lixiviating solution, filters, the concentrated Herba Dendrobii extract that to get; 95 ~ 100 ℃ of purified water lixiviates that raw material macro fungi usefulness weight ratio is 1 ~ 20 times 2 ~ 3 times, each 2 ~ 3h merges lixiviating solution, filters, the concentrated macro fungi extract that to get; Each extract is dried to respectively moisture content 1 ~ 9%, crosses 10 mesh sieves through coarse pulverization; Each extract after coarse pulverization is carried out mixing, got objective composition through pulverizing 80 mesh sieves; With the objective composition packing of sterilizing, pick test is provided inspection report.
Embodiment 7
With through screening, the coarse pulverization raw material after crossing 10 mesh sieves by Poria 90Kg, Ganoderma 90Kg, Herba Dendrobii 90Kg and Lentinus Edodes and Volvariella volvacea (Bull.Ex Franch.) Singer., JINZHENGU, Agaricus bisporus, Pleurotus ostreatus, Auricularia, Tremella, Caulis Bambusae In Taeniam, Gaster caprae seu Ovis 100Kg; The purified water that adds 2 ~ 6 times of weight ratios in the raw material Poria, reflux, extract, 3 ~ 5 times, each 2 ~ 3h, merge extractive liquid, filters, and under agitation adds ethanol, makes to contain the alcohol amount and reach 70 ~ 80%, leaves standstill 12 ~ 24h, separates out precipitation, filters, and the collecting precipitation thing gets Poria extract; 80 ~ 99% ethanol that add 2 ~ 5 times in the raw material Ganoderma, hot bath refluxes 3 ~ 5 times, each 2 ~ 3h, merge extractive liquid,, Recycled ethanol filters to get filtrate, and concentrates to get Ganoderma extract; 90 ~ 95 ℃ of purified water lixiviates that raw material Herba Dendrobii usefulness weight ratio is 1 ~ 10 times 1 ~ 3 time, each 3 ~ 4h merges lixiviating solution, filters, the concentrated Herba Dendrobii extract that to get; 95 ~ 100 ℃ of purified water lixiviates that raw material macro fungi usefulness weight ratio is 1 ~ 20 times 2 ~ 3 times, each 2 ~ 3h merges lixiviating solution, filters, the concentrated macro fungi extract that to get; Each extract is dried to respectively moisture content 1 ~ 9%, crosses 10 mesh sieves through coarse pulverization; Each extract after coarse pulverization is carried out mixing, got objective composition through pulverizing 80 mesh sieves; With the objective composition packing of sterilizing, pick test is provided inspection report.
Embodiment 8
With through screening, the coarse pulverization raw material after crossing 10 mesh sieves by Poria 90Kg, Ganoderma 90Kg, Herba Dendrobii 90Kg and Lentinus Edodes and Volvariella volvacea (Bull.Ex Franch.) Singer., JINZHENGU, Agaricus bisporus, Pleurotus ostreatus, Auricularia, Tremella, Caulis Bambusae In Taeniam, Gaster caprae seu Ovis 100Kg; The purified water that adds 2 ~ 6 times of weight ratios in the raw material Poria, reflux, extract, 3 ~ 5 times, each 2 ~ 3h, merge extractive liquid, filters, and under agitation adds ethanol, makes to contain the alcohol amount and reach 70 ~ 80%, leaves standstill 12 ~ 24h, separates out precipitation, filters, and the collecting precipitation thing gets Poria extract; 80 ~ 99% ethanol that add 2 ~ 5 times in the raw material Ganoderma, hot bath refluxes 3 ~ 5 times, each 2 ~ 3h, merge extractive liquid,, Recycled ethanol filters to get filtrate, and concentrates to get Ganoderma extract; 90 ~ 95 ℃ of purified water lixiviates that raw material Herba Dendrobii usefulness weight ratio is 1 ~ 10 times 1 ~ 3 time, each 3 ~ 4h merges lixiviating solution, filters, the concentrated Herba Dendrobii extract that to get; 95 ~ 100 ℃ of purified water lixiviates that raw material macro fungi usefulness weight ratio is 1 ~ 20 times 2 ~ 3 times, each 2 ~ 3h merges lixiviating solution, filters, the concentrated macro fungi extract that to get; Each extract is dried to respectively moisture content 1 ~ 9%, crosses 10 mesh sieves through coarse pulverization; Each extract after coarse pulverization is carried out mixing, got objective composition through pulverizing 80 mesh sieves; With the objective composition packing of sterilizing, pick test is provided inspection report.
Embodiment 9
Get any one active compound composition among the embodiment 1 ~ 8, add pharmaceutic adjuvant, packing is powder.
Embodiment 10
Get any one active compound composition among the embodiment 1 ~ 8, add pharmaceutic adjuvant, through granulating, drying, be filled in the capsule, namely get capsule.
Embodiment 11
Get any one active compound composition among the embodiment 1 ~ 8, add 1% magnesium stearate, mixing is filled in the capsule, namely gets capsule.
Embodiment 12
Get any one active compound composition among the embodiment 1 ~ 8, add pharmaceutic adjuvant, through granulating, drying, namely get granule.
Embodiment 13
Get any one active compound composition among the embodiment 1 ~ 8, add pharmaceutic adjuvant, through granulation, tabletting, namely get tablet.
Embodiment 14
Get any one active compound composition among the embodiment 1 ~ 8, add pharmaceutic adjuvant, pill, oven dry namely get pill.
Experimental example 1
The contrast test of active compound composition and Ganoderma
1 experiment material
1.1 animal Kunming mouse: weight 18 ~ 22 g, male and female have (every batch of a kind of sex of test) concurrently, and quality certification SCXK (army) 2007-004 is provided by Test Animal Centre, Academy of Military Medical Sciences, P.L.A.
1.2 tumor strain and cell strain: hepatocarcinoma H
22The tumor strain, the institute of oncology, Shenyang City provides, Kunming kind white mice conservation.
1.3 medicine and reagent: active compound composition (the luxuriant Pharmacy stock Co., Ltd in the river in Jiangsu Province which flows into the Huangpu River of Shanghai, Pu'er provides).The drying at room temperature place preserves, before use with dissolved in distilled water and be diluted to desired concn.Ganoderma (the luxuriant Pharmacy stock Co., Ltd in the river in Jiangsu Province which flows into the Huangpu River of Shanghai, Pu'er provides); Cyclophosphamide (CTX), Hualian Pharmaceutical Co., Ltd., Shanghai.
1.4 key instrument: ware electronic balance on the FA/JA1004 series: balance equipment factory of upper Nereid section; PH050A type incubator/drying baker: Shanghai one permanent experimental apparatus company limited; XDS-1B type inverted biological microscope: Chongqing Photoelectric Instruments Corp..
2 methods and result
2.1 active compound composition is to mouse bearing liver cancer H
22Affects on the growth
The hepatocarcinoma H of the 7d that goes down to posterity gets in the sterile working
22Mouse ascites, cell counting is 2 * 107/ml with 0.9% normal saline dilution to concentration, every the right axil subcutaneous vaccination of mice 0.2ml (4 * 106 /).At random be divided into 5 group and administration by weight next day.The administration volume is 20ml/kg.24h after the last administration, animal is put to death in the cervical vertebra dislocation, and minute another name weight, tumor weigh, and calculate tumour inhibiting rate, carry out therapeutic evaluation.The result carries out statistical procedures with Excel software, and this experiment repeats 3 batches.The result shows, active compound composition dose dependent ground inhibition transplanted hepatoma
H22Growth, and administration group Mice Body steady quality increases.See Table 1.
Table 1 active compound composition is to mouse bearing liver cancer H
22Affects on the growth (x ± s, n=3)
Annotate: compare * P<0.05, * * P<0.01 with model group
2.2 active compound composition is on the impact of immunologic hypofunction mice serum hemolysin level
Choose 60 of healthy Kunming mouses, be divided at random 6 groups, be respectively active compound composition 1.04,0.52,
0.26g/kg dosage group and Ganoderma 5.2mg/kg group, model group (giving cyclophosphamide) and blank group.Each organizes successive administration, in administration the 5th day, gives every Mus lumbar injection 5% chicken erythrocyte suspension 0.2ml.Except the blank group, all the other each groups were pressed 75mg/kg dosage intraperitoneal injection of cyclophosphamide respectively at the 4th day, the 9th day, caused the immunologic hypofunction model.Successive administration 10d tested on the 11st day.Each is organized mice eyeground vein clump and gets blood, centrifugalize serum, with normal saline serum is diluted 50 times, get dilute serum 1ml and add in the reaction tube, add again 0.5% chicken erythrocyte suspension 0.5ml, then in frozen water, add 0.5ml complement (10%3 Cavia porcellus pooled serums), solution in the reaction tube is shaken up, place 37 ℃ of waters bath with thermostatic control to react 30min, jolting is 2 times in the course of reaction, and reaction places 0 ℃ of refrigerator cessation reaction with reaction tube after finishing.Centrifugal, get supernatant in 722 type spectrophotometer 540nm place colorimetrics, measure absorbance (transferring 0 with the blank pipe that does not add mice serum), represent the height of mice serum hemolysin level with the absorbance of supernatant.Each organizes as a result, and Excel software carries out statistical procedures.Result's demonstration, model group mice serum hemolysin level significantly reduces than the blank group; Compare with model group, active compound composition group 1.04,0.52g/kg can significantly improve the serum hemolysin of mice, improve the inhibitory action that cyclophosphamide produces the mice serum hemolysin level.See Table 2.
Table 2 active compound composition is on the impact of immunologic hypofunction mice serum hemolysin level (x ± S)
Annotate: compare * P<0.05, * * P<0.01 with model group; Compare ##P<0.01 with the blank group
Originally studies show that, active compound composition can significantly reduce hepatocarcinoma H than Ganoderma
22The average tumor of mice is heavy.By observing the variation of a plurality of indexs of immune system, investigated the impact of active compound composition on body's immunity, the result shows, active compound composition is the enhancing human body immunity function by the immune different links of impact, thereby produces anti-tumor activity.
Experimental example 2
1 experiment material
1.1 animal Kunming mouse: weight 36-44g, male and female have (every batch of a kind of sex of test) concurrently, and quality certification SCXK (army) 2007-004 is provided by Test Animal Centre, Academy of Military Medical Sciences, P.L.A.
1.2 tumor strain and cell strain: hepatocarcinoma H
22The tumor strain, the institute of oncology, Shenyang City provides, Kunming kind white mice conservation.
1.3 medicine and reagent: the active compound composition powder that makes with embodiment 9, the active compound composition capsule that makes with embodiment 11, the active compound composition granule that makes with embodiment 12, the active compound composition tablet that makes with embodiment 13, the active compound composition pill (the luxuriant Pharmacy stock Co., Ltd in the river in Jiangsu Province which flows into the Huangpu River of Shanghai, Pu'er provides) that makes with embodiment 14.The drying at room temperature place preserves, before use with dissolved in distilled water and be diluted to desired concn.Cyclophosphamide (CTX), Hualian Pharmaceutical Co., Ltd., Shanghai.
1.4 key instrument: ware electronic balance on the FA/JA1004 series: balance equipment factory of upper Nereid section; PH050A type incubator/drying baker: Shanghai one permanent experimental apparatus company limited; XDS-1B type inverted biological microscope: Chongqing Photoelectric Instruments Corp..
2 methods and result
2.1 active compound composition is to mouse bearing liver cancer H
22Affects on the growth
The hepatocarcinoma H of the 7d that goes down to posterity gets in the sterile working
22Mouse ascites, cell counting is 2 * 107/ml with 0.9% normal saline dilution to concentration, every right axil subcutaneous vaccination 0.2 ml of mice (4 * 106 /).At random be divided into 5 group and administration by weight next day.The administration volume is 20ml/kg.24h after the last administration, animal is put to death in the cervical vertebra dislocation, and minute another name weight, tumor weigh, and calculate tumour inhibiting rate, carry out therapeutic evaluation.The result carries out statistical procedures with Excel software, and this experiment repeats 3 batches.The result shows, active compound composition powder, capsule, granule, tablet, pill dependency ground inhibition transplanted hepatoma H
22Growth, and administration group Mice Body steady quality increases.And the result of the test of each dosage form is very approaching.
2.2 active compound composition is on the impact of immunologic hypofunction mice serum hemolysin level
Choose 60 of healthy Kunming mouses, be divided at random 6 groups, be respectively active compound composition powder, capsule, granule, tablet, pill group, model group (giving cyclophosphamide) and blank group.Each organizes successive administration, in administration the 5th day, gives every Mus lumbar injection 5% chicken erythrocyte suspension 0.2ml.Except the blank group, all the other each groups were pressed 75mg/kg dosage intraperitoneal injection of cyclophosphamide respectively at the 4th day, the 9th day, caused the immunologic hypofunction model.Successive administration 10d tested on the 11st day.Each is organized mice eyeground vein clump and gets blood, centrifugalize serum, with normal saline serum is diluted 50 times, get dilute serum 1ml and add in the reaction tube, add again 0.5% chicken erythrocyte suspension 0.5ml, then in frozen water, add 0.5 ml complement (10%3 Cavia porcellus pooled serums), solution in the reaction tube is shaken up, place 37 ℃ of waters bath with thermostatic control to react 30min, jolting is 2 times in the course of reaction, and reaction places 0 ℃ of refrigerator cessation reaction with reaction tube after finishing.Centrifugal, get supernatant in 722 type spectrophotometer 540nm place colorimetrics, measure absorbance (transferring 0 with the blank pipe that does not add mice serum), represent the height of mice serum hemolysin level with the absorbance of supernatant.Each organizes as a result, and Excel software carries out statistical procedures.Result's demonstration, model group mice serum hemolysin level significantly reduces than the blank group; Compare with model group, each dosage form group 1.04 of active compound composition, 0.52g/kg can significantly improve the serum hemolysin of mice, improve the inhibitory action that cyclophosphamide produces the mice serum hemolysin level.And the result of the test of each dosage form is very approaching.