CN1961895B

CN1961895B - A novel anticancer pharmaceutical composition and preparation method thereof

Info

Publication number: CN1961895B
Application number: CN200510045068A
Authority: CN
Inventors: 黄振华
Original assignee: Shandong Xuanzhu Pharma Co Ltd
Current assignee: Haian Su Fu Technology Transfer Center Co ltd
Priority date: 2005-11-10
Filing date: 2005-11-10
Publication date: 2010-05-12
Anticipated expiration: 2025-11-10
Also published as: CN1961895A

Abstract

The invention relates to an anti-cancer drug and relative preparation, wherein it main comprises lucid ganoderma at 50-2000 deals or relative extractives at 0.2-60 deals, and 1-50 deals of asiatic centella glycosides, while it also can contain gen-seng at 50-2000 deals or extractive gen-seng polysaccharide at 0.1-40 deals or the extractive gen-seng saponin at 0.1-40 deals. The invention can be made into oral or injection agent, with anti-cancer, antibiosis functions, etc.

Description

A kind of new anticancer pharmaceutical composition and preparation method thereof

[technical field]

The invention belongs to medical technical field, relate to a kind of pharmaceutical composition of making by Ganoderma or its extract and asiaticoside, or the pharmaceutical composition of making by Ganoderma or its extract, asiaticoside and Radix Ginseng or its extract, and its production and use.

[background technology]

Cancer is a class serious threat human life and healthy disease.Data show according to statistics, the annual newfound cancer patient of China is about about 1,000,000, and every year is seized about 6,000,000 people's life in the whole world, and 1,000 ten thousand people are placed dead edge, along with going from bad to worse of environment for human survival, the incidence rate of cancer is ascendant trend year by year.World Health Organization's prediction 21 century cancer will become human " first killer ".Modern medicine mainly is that the operative treatment cooperation is put, chemotherapy to treatment for cancer at present, though operation can be removed primary lesion, can not fundamentally stop the regeneration and the breeding of cancerous cell; Though put, chemotherapy can kill cancerous cell, simultaneously a large amount of normal tissue cells suffered damage, and brings out gastrointestinal reaction, bone marrow depression and Liver and kidney, impairment of cardiac function, makes patient's health weak more, be difficult to further treatment.And the Chinese traditional treatment cancer has long history, has formed the theoretical system and the treatment rule of own uniqueness.Work through vast medical worker has in recent years confirmed that the Chinese medicine cancer has outstanding effect, has particularly brought into play important effect to the rehabilitation behind the cancer operation and to the efficacy enhancing and toxicity reducing aspect of chemicotherapy.

Ganoderma is Polyporaceae fungus Ganoderma lucidum (Leyss. Ex Fr.) Karst. Ganoderma lucidum (Ley-ss.ex Fr.) Karst. or Ganoderma Ganoderma sinense Zhao, the dry sporophore of Xu et Zhang.The property sweet, flat, GUIXIN, lung, liver, kidney channel.Have invigorating QI and tranquilization, the effect of relieving cough and asthma is mainly used in dizzy sleeplessness, shortness of breath and palpitation, cough due to consumptive disease.Ganoderma is widely known Chinese medicine, is commonly called as " Herba mesonae chinensis ".Beginning is stated from Shennong's Herbal, and " tonifying liver QI invigorating " " the hard muscles and bones " that be considered to can " the beneficial motive " " to pacify smart soul " classified as top grade.Compendium of Material Medica thinks that Ganoderma has " strengthening by means of tonics " " life lengthening " " sharp joint " effects such as " controlling deafness ".The complex chemical composition of Ganoderma, known polysaccharide (peptide) class, triterpenes, ucleosides, alkaloid, aminoacid and the trace element etc. of containing at present.Polysaccharide compound is one of contained main chemical compositions of Ganoderma, has proved that now the ganoderan class has antitumor action, immunoregulation effect, hypoglycemic activity, effect for reducing blood fat, antioxidation and anti-aging effects.Clinical trial confirms that also ganoderan can be used as tumor chemical therapy and radiocurable effective auxiliary therapeutic agent, alleviates because of the toxic and side effects of performing the operation, putting, chemotherapy being brought, and is the very ideal medical material of Biotherapeutics.The ganoderan kind is a lot, and water soluble polysaccharide, acidic polysaccharose and alkaline polysaccharide are arranged.

Asiaticoside is a kind of white, needle-shaped crystals of extracting from Herba Centellae, and different name asiaticoside, fusing point are 235 ℃-238 ℃, and be water insoluble, is dissolved in ethanol and pyridine.Have the bibliographical information asiaticoside can suppress tumor cell proliferation, multidrug resistance tumor cells shows and the similar drug susceptibility of corresponding non-mdr cell asiaticoside, and tangible cross resistance is seen at the end, the apoptosis of energy inducing tumor cell.The asiaticoside structural formula is as follows.

Asiaticoside

Radix Ginseng is the dry root of Araliaceae Radix Ginseng Panax ginseng C.A.Mey., and sweet, the little hardship of property is flat, returns spleen, lung, heart channel, has that strongly invigorating primordial QI, multiple arteries and veins take off admittedly, an invigorating the spleen to benefit the lung, the effect of promoting the production of body fluid, calming the nerves.Radix Ginseng is one of genuine medicinal materials of abounding with of Changbaishan area, is used as medicine with its root, and be famous valuable ingredient of Chinese medicine.Mainly contain effective ingredient such as ginseng polysaccharide, Radix Ginseng total saponins, ginseng polypeptide and volatile oil, several amino acids, fatty acid and vitamin, trace element.Main component is a Radix Ginseng total saponins, measures approximately 4%, separates to such an extent that Radix Ginseng total saponins has Ra at present _1～6, Rb _1～3, Rc, Rd, Re, Rf, Rg _1～3, Rh _1～2, kind surplus the Ro etc. 20.Radix Ginseng is enhancing body's immunological function comprehensively, and its active component mainly is saponin and polysaccharide.Radix Ginseng total saponins all can strengthen engulf the clean up ability of reticuloendothelial system to carbon granules, antibacterial, chicken red blood cell etc. to multiple animal, the ginseng polysaccharide is in the external NK cells in mice activity that strengthens, ginseng polysaccharide and saponin also can make the caused by cyclophosphamide leukocyte count reduce, and it is normal that macrophage and humoral immunization and cellular immune function inhibition etc. recover; Radix Ginseng total saponins and ginseng polysaccharide all have the anti-experimental character function of tumor, the antitumor action of Radix Ginseng total saponins is relevant with the differentiation again of its inducing cancer cell, the ginseng polysaccharide has direct or indirect anti-tumor activity, vitro cytotoxicity test evidence: ginseng polysaccharide's sensitization serum all has killing and wounding and inhibitory action in various degree to the tumor cell line of people and Mus, also can make it that tangible hemorrhagic necrosis takes place to transplantation tumor in the body, think that its antitumor mechanism may be to have induced neoplasm necrosis factor TNF-N.

[summary of the invention]

In order to meet clinical needs, to the invention provides a kind of new antitumor medicine composition that is mainly used in, and its preparation method is provided.

Pharmaceutical composition of the present invention is mainly made by Ganoderma and asiaticoside, and its parts by weight are: 50～2000 parts of Ganodermas, 1～50 part of asiaticoside; Preferred umber is 200～1000 parts of Ganodermas, 2～15 parts of asiaticosides; Best umber is 400 parts of Ganodermas, 6 parts of asiaticosides.

The preparation method of aforementioned pharmaceutical compositions is: Ganoderma is obtained its extract with The suitable solvent and method through extracting processing, and extract is made arbitrary preparation with asiaticoside and mixing acceptable accessories again.Herein, The suitable solvent is meant the solvent that Chinese medicine extraction is commonly used, preferred water or alcohol, especially water or ethanol.Extracting method can adopt the conventional method of Chinese medicine extraction, as infusion process, percolation, decocting method, reflux extraction or continuous extraction.

The concrete extracting method of each crude drug is as follows:

The extraction processing technique of Ganoderma is as follows:Get the Ganoderma medical material, be ground into coarse grain, add the moistening of 3 times of amounts of water and spend the night, decoct next day three times, added 12 times of amounts of water for the first time in 3 hours, added 10 times of amounts of water for the second time in 2 hours, added 10 times of amounts of water in 1 hour for the third time, collecting decoction filters, it is 1.10～1.15 that filtrate is concentrated into relative density, puts coldly, adds ethanol and makes that to contain alcohol amount be 65%, stir evenly, placed 24 hours, filter at 4 ℃, collect filter cake, add suitable quantity of water and make dissolving, filter, add ethanol and make that to contain the alcohol amount be 85%, stir evenly, placed 24 hours at 4 ℃, filter, collect filter cake, vacuum drying, promptly.By the not low hands 50% of content of polysaccharide in the Ganoderma extract of this prepared, yield is 0.5～3%.

Except that adopting said method, also can obtain by the following method, but be not limited only to following method:

Method one: get the Ganoderma medical material, be ground into coarse grain, decoct with water three times, added 12 times of amounts of water in 3 hours for the first time, second and third time added 10 times of amounts of water, collecting decoction in 2 hours, filter, it is 1.03～1.08 that filtrate is concentrated into relative density, puts cold, add ethanol and make that to contain alcohol amount be 65%, stir evenly, placed 24 hours at 4 ℃, filter, filtrate recycling ethanol filters to there not being the alcohol flavor, add ethanol and make that to contain alcohol amount be 80%, stir evenly, placed 24 hours, filter at 4 ℃, filtrate recycling ethanol is to there not being the alcohol flavor, placed 48 hours at 4 ℃, filter, filtrate is concentrated into the thick paste shape, spray drying, promptly.Content by polysaccharide in the Ganoderma extract of this prepared is not less than 30%, and yield is 5～8%.

Method two: get the Ganoderma medical material, be ground into coarse grain, add the moistening of 3 times of amounts of water and spend the night, decoct secondary next day, added 12 times of amounts of water for the first time in 3 hours, added 10 times of amounts of water for the second time in 2 hours, collecting decoction filters, and it is 1.02～1.06 that filtrate is concentrated into relative density, put coldly, add ethanol and make that to contain alcohol amount be 70%, stir evenly, placed 24 hours at 4 ℃, filter, filtrate recycling ethanol is to there not being the alcohol flavor, filter, add ethanol and make that to contain the alcohol amount be 85%, stir evenly, placed 24 hours at 4 ℃, filter, filtrate recycling ethanol was placed 48 hours at 4 ℃ to there not being the alcohol flavor, filter, filtrate is concentrated into the thick paste shape, spray drying, promptly.Content by ganoderan in the Ganoderma extract of this prepared is not less than 40%, and yield is 3～6%.

Asiaticoside is the clear and definite chemical constituent of structure, does not see its synthetic report as yet, at present mainly by the extracting method preparation, also can obtain by buying, or self-control.The invention provides a kind of preparation technology, as follows:

Asiaticoside can be by following prepared: get the Herba Centellae medical material, be ground into fine powder, add 10 times of amount 95% soak with ethanol and spend the night, inclining next day ethanol liquid, reuse 75% alcohol reflux secondary, each 2 hours, add 10 times of amounts of alcohol, filter, merge ethanol extract, after being evaporated to 1/3 volume, add 40 minutes after-filtration deactivation charcoals of active carbon backflow of 5%, the alcoholic solution after decolouring is evaporated to the thick paste shape, after adding 5 times of amount warm water stirring and dissolving, put coldly, add the petroleum ether of 1/2 times of amount volume, jolting is extracted 2 times, water liquid extracts 3 times with the water saturated n-butyl alcohol jolting of 1/2 times of amount volume, merge extractive liquid, washes with water 1 time, is evaporated to dried brown extractum.After it was dissolved in an amount of ethanol, the anhydrous propanone that slowly adds 8 times of amounts makes separated out precipitation, sucking filtration, washing with acetone, vacuum drying, asiaticoside.Asiaticoside yield by this prepared is not less than 0.1%, and the purity of asiaticoside is more than 80%.

In the aforementioned pharmaceutical compositions, also can replace the Ganoderma medical material to feed intake by Ganoderma extract, calculate with respect to the yield of medical material according to extract, the parts by weight of composition material medicine should be: 0.2～60 part of Ganoderma extract, 1～50 part of asiaticoside; Preferred umber is 1～30 part of Ganoderma extract, 2～15 parts of asiaticosides; Best umber is 2～15 parts of Ganoderma extracts, 6 parts of asiaticosides.Wherein, the main effective ingredient of Ganoderma extract is a polysaccharide, preferably is not less than 30%, and the purity of asiaticoside preferably is not less than 80%.Ganoderma extract and asiaticoside all can be made by oneself with reference to said method.

For reaching better antitumous effect, can also add Radix Ginseng in the crude drug of pharmaceutical composition of the present invention and make, as feeding intake with medical material, its parts by weight are: 50～2000 parts of Ganodermas, 1～50 part of asiaticoside, 50～2000 parts of Radix Ginsengs; Preferred umber is 200～1000 parts of Ganodermas, 2～15 parts of asiaticosides, 200～1000 parts of Radix Ginsengs; Best umber is 400 parts of Ganodermas, 6 parts of asiaticosides, 400 parts of Radix Ginsengs.

Ganoderma in the aforementioned pharmaceutical compositions, Radix Ginseng can be with The suitable solvent respectively or mix through extracting processing and obtain its extract, and total extract is made clinically arbitrary or pharmaceutically acceptable dosage form with mixing acceptable accessories again." extract separately " and be meant that Ganoderma, each flavor medical material of Radix Ginseng extract separately by different technology respectively and obtain extract, each extract are mixed obtaining total extract again." mixed extraction " is meant, Ganoderma, Radix Ginseng two flavor medical materials extract together and obtain total extract.Herein, The suitable solvent is meant the solvent that Chinese medicine extraction is commonly used, preferred water or alcohol, especially water or ethanol.Extracting method can adopt the conventional method of Chinese medicine extraction, as infusion process, percolation, decocting method, reflux extraction or continuous extraction.

In the above-mentioned composition, " extracting separately " method of Ganoderma can prepare with reference to preceding method.

In the above-mentioned composition,,, as follows respectively so " extracting separately " method of Radix Ginseng can adopt different extracting method because of the difference of its active component because ginseng polysaccharide and Radix Ginseng total saponins all have anti-tumor activity:

Be that (be called for short: preparation technology Radix Ginseng total saponins) is as follows for the Radix Ginseng extract of main effective ingredient with total saponins in the present composition: get the ginseng crude drug, be ground into particulate, the alcohol reflux secondary, each 3 hours, add 10 times of amounts of alcohol, merge extractive liquid,, cold preservation at every turn, filter, filtrate recycling ethanol also is concentrated into thick paste, adds water to an amount of (making every 1ml be equivalent to crude drug 1g), stirs evenly, cold preservation, filter, filtrate is added on the macroporous resin column of having handled well, and the water with 2V～3V column volume carries out eluting earlier, discard water lotion, 80% ethanol elution of 3 times of column volumes of reuse is collected eluent, reclaims ethanol and is evaporated to the thick paste of relative density 1.30～1.35, vacuum drying, promptly.The Radix Ginseng total saponins extract yield that obtains by this prepared is 1～2%, and Radix Ginseng total saponins content is not less than 50%, Radix Ginseng total saponins Re, Radix Ginseng total saponins Rg ₁Content and be not less than 30%.

Except that adopting said method, also can prepare by literature method, also can obtain by the following method, but be not limited only to following method:

Method one: get the ginseng crude drug, be ground into particulate, add the alcohol reflux secondary, each 4 hours, add 10 times of amounts of alcohol, merge extractive liquid, at every turn, cold preservation, filter, filtrate recycling ethanol also is concentrated into thick paste, and thin up to relative density is 1.15～1.20, add 1/2 times of saturated n-butanol extraction of water gaging 3 times, merge n-butyl alcohol liquid, the reclaim under reduced pressure n-butyl alcohol is to the thick paste shape, and spray drying promptly.Radix Ginseng extract yield by this prepared is 4～5%, and Radix Ginseng total saponins content is not less than 35%, Radix Ginseng total saponins Re, Radix Ginseng total saponins Rg ₁Content and be not less than 20%.

Method two: get the ginseng crude drug, be ground into particulate, add alcohol reflux three times, add for the first time 10 times of amounts of alcohol, two, three times is 8,8 times of amounts, each 2 hours, merge extractive liquid, filters, and reclaims ethanol and is concentrated into thick paste, add water in right amount, stir evenly cold preservation, filter, filtrate adds 3 concentrating under reduced pressure of 1/2 times of saturated n-butanol extraction of water gaging, merges n-butyl alcohol liquid, the reclaim under reduced pressure n-butyl alcohol is to the thick paste shape, and spray drying promptly.Radix Ginseng extract yield by this prepared is 3～5%, and Radix Ginseng total saponins content is for being not less than 40%, Radix Ginseng total saponins Re, Radix Ginseng total saponins Rg ₁Content and be no less than 20%.

In the present composition with polysaccharide be main effective ingredient Radix Ginseng extract (be called for short: the preparation technology ginseng polysaccharide) is as follows:Get Radix Ginseng, chopping is used 75% ethanol, reflux, extract, 4 hours, filter, medicinal residues dry, and decoct with water five times, merge decoction liquor, add 0.3% active carbon, stirred 30 minutes, standing over night filters, filtrate is crossed resin column, collects effluent, and concentrating under reduced pressure adds ethanol and makes and contain the alcohol amount and reach 95%, cold preservation filters, and gets precipitation and uses washing with acetone, drains acetone, taking-up is deposited in 60～80 ℃ of dryings, pulverizes, and gets powder promptly.Content by ginseng polysaccharide in ginseng polysaccharide's extract of this prepared is not less than 50%, and extract yield is 0.5～2%.

Except that adopting said method, also can pass through literature method (as CN200410000209) preparation, also can obtain by the following method, but be not limited only to following method:

Method one: get Radix Ginseng, chopping adds the water reflux, extract, 3 times, each 2 hours, add 10 times of amounts of water for the first time, two, three times is 8,8 times of amounts, collecting decoction, filter, it is 1.16～1.24 that filtrate decompression is concentrated into relative density, crosses macroporous resin column, collect effluent, be evaporated to the thick paste shape, vacuum drying, promptly.By the Radix Ginseng extract of this prepared, yield is 4～6%, and ginseng polysaccharide's content is not less than 40%.

Method two: get Radix Ginseng, 80% ethanol is used in chopping, reflux, extract, 3 hours filters, and medicinal residues dry, decoct with water three times, each 1.5 hours, add 10 times of amounts of water, merge decoction liquor, filter, it is 1.15～1.20 that filtrate decompression is concentrated into relative density, crosses macroporous resin, effluent is evaporated to the thick paste shape, and spray drying promptly.By the Radix Ginseng extract of this prepared, yield is 2～4%, and ginseng polysaccharide's content is not less than 45%.

In the crude drug of aforementioned pharmaceutical compositions, available Ganoderma extract, Radix Ginseng extract replace Ganoderma and Radix Ginseng to feed intake respectively making, and calculate with respect to the yield of medical material according to extract, and following two kinds of different proportionings can be arranged, and its parts by weight are respectively:

Proportioning 1: 0.2～60 part of Ganoderma extract, 1～50 part of asiaticoside, 0.1～40 part of ginseng polysaccharide; Preferred umber is 1～30 part of Ganoderma extract, 2～15 parts of asiaticosides, 0.2～20 part of ginseng polysaccharide; Best umber is 2～15 parts of Ganoderma extracts, 6 parts of asiaticosides, 1～10 part of ginseng polysaccharide.

Proportioning 2: 0.～60 part of Ganoderma extract, 1～50 part of asiaticoside, 0.1～40 part of Radix Ginseng total saponins; Preferred umber is 1～30 part of Ganoderma extract, 2～15 parts of asiaticosides, 0.2～20 part of Radix Ginseng total saponins; Best umber is 2～15 parts of Ganoderma extracts, 6 parts of asiaticosides, 1～10 part of Radix Ginseng total saponins.

In the said ratio, contain ganoderan in the Ganoderma extract and preferably be not less than 30%; Asiaticoside purity preferably is not less than 80%; The ginseng polysaccharide preferably is not less than 50%; Radix Ginseng total saponins preferably is not less than 50%, wherein ginsenoside Re and ginsenoside Rg ₁Total content preferably be not less than 30%.Ganoderma extract, asiaticoside, ginseng polysaccharide and Radix Ginseng total saponins all can be made by oneself with reference to preceding method.

Aforementioned pharmaceutical compositions, can make clinically arbitrary or pharmaceutically acceptable dosage form, as injection, oral normal release dosage form, sustained-release and controlled release dosage form, granule, pill, oral fluid agent, eye drop, nasal drop, ear drop, inhalant, suppository, ointment etc.Pharmaceutical composition preferred dosage form of the present invention is injection or oral formulations.

Pharmaceutical composition of the present invention can adopt the conventional method production in the existing pharmaceutical field, can add various pharmaceutically acceptable carriers when needing.Described carrier comprises diluent, excipient, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier, lubricant of pharmaceutical field routine etc.

The present invention in order to increase its dissolubility, can add solubilizing agents such as tween 80 when making injection.Can add the isoosmotic adjusting agent that is used to regulate osmotic pressure in the transfusion, for example, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, lactic acid are received, glucose, xylitol, sorbitol and dextran etc., preferred sodium chloride or glucose.Can add excipient in the powder pin, for example, mannitol, glucose etc.

Aforementioned pharmaceutical compositions has effects such as antitumor, infection, detoxifcation, antibiotic and enhancing human body immunity power.

The invention has the advantages that:

1. a kind of new Chinese medicine medicine for preventing compound recipe is provided, has satisfied urgent clinical needs.

2. pharmaceutical composition of the present invention has been carried out pharmaceutical research, the result shows that the present composition has obvious synergistic effect to radiotherapy; Chemotherapy is had attenuation and synergic effect, and tumour inhibiting rate is more than 50%, and the potentiation rate can reach 56%; Can obviously prolong ehrlich ascites carcinoma U ₁₄The existence natural law of mice, and its effect is compared effect very significantly (p＜0.01) with single with Ganoderma, asiaticoside and Radix Ginseng, show that Ganoderma, asiaticoside and Ganoderma, asiaticoside and two kinds of compositionss of Radix Ginseng all have the effect of Synergistic, have improved patient's life quality.

3. pass through the different proportionings of the present composition to mice S ₁₈₀The inhibiting pharmacodynamic study of tumor growth has filtered out the optimum ratio of the present composition.

4. the main content of effective of present composition extract is limited, be convenient to control the quality of product.

5. the present composition has been carried out acute toxicity test, the result shows that present composition toxicity is little, and safety range is big.

6. the stability test result that pharmaceutical composition of the present invention is carried out shows that every index is all more stable, has guaranteed safety of clinical administration.

7. pharmaceutical composition medication of the present invention is imitated definite, and dosage reduces behind the drug combination, has broad application prospects.

Below test example is further set forth the beneficial effect of medicine of the present invention, and these test examples comprise the pharmacodynamics test of pharmaceutical composition of the present invention.Below test Ganoderma basis in the example Embodiment 1Make Ganoderma extract, the Radix Ginseng basis Embodiment 2Make Radix Ginseng total saponins and and ginseng polysaccharide.Ganoderma extract and asiaticoside compositions are called for short The LJ group, Ganoderma extract, asiaticoside and ginseng polysaccharide's compositions are called for short LJR ₁ Group, Ganoderma extract, asiaticoside and Radix Ginseng total saponins compositions are called for short LJR ₂ Group

Test example 1 pharmacodynamics test one Ganoderma, asiaticoside and Radix Ginseng drug combination are to mice S ₁₈₀ The tumor growth inhibitory action

Test sample matched group: sodium chloride injection (Shangdong Changfu Jiejing Pharmaceutical Industry Co., Ltd.)

Radix Ginseng total saponins group: Radix Ginseng total saponins injection, self-control, 5ml: be equivalent to ginseng crude drug 4g.

Ginseng polysaccharide's group: ginseng polysaccharide injection, self-control, 5ml: be equivalent to ginseng crude drug 4g.

Ganoderma group: ganoderan injection 5ml: be equivalent to Ganoderma medical material 4g.

Asiaticoside group: asiaticoside injection 5ml:60mg.

The compositions group:

LJR ₁Group: self-control (preparation method is referring to the preparation of embodiment 5 aqueous injection prescription 2);

LJR ₂Group: self-control (preparation method is referring to the preparation of embodiment 5 aqueous injection prescription 3);

230 of animal subject healthy mices, body weight 16～20g, male and female half and half, 10 every group.

Tumor strain mice S ₁₈₀

Method is got and is inoculated the mice S that goes down to posterity ₁₈₀, in homogenizer, add normal saline, make mice S ₁₈₀The tumor homogenate, again with normal saline 1: 3 dilution, getting 0.2ml then, to inject oxter, a mice left side subcutaneous, weighed in 24 hours, mice gastric infusion every day once, administration volume identical (0.5ml/ is only), totally 7 days.Next day is put to death mice in drug withdrawal, and the subcutaneous tumors piece is peeled off in the also carefulness of weighing, and takes by weighing tumor in the EM50 electronic balance and weighs, and calculate tumour inhibiting rate.

Result of the test and conclusion are compared LJR with the normal saline matched group by table 1 result as can be seen ₁(Ganoderma extract+asiaticoside+ginseng polysaccharide) group, LJR ₂(Ganoderma extract+asiaticoside+Radix Ginseng total saponins) group, Radix Ginseng total saponins group, ginseng polysaccharide's group, Ganoderma group, asiaticoside group are to mice S ₁₈₀The tumor body all has inhibitory action (p＜0.05, p＜0.01), though Radix Ginseng total saponins group, ginseng polysaccharide are organized mice S ₁₈₀The tumor body also has inhibitory action, but DeGrain.Compare with Ganoderma group, asiaticoside group, when the raw material weight proportioning of compositions is: (2g～10g)+asiaticoside is (20mg～150mg)+Radix Ginseng (action effect very significantly (p＜0.01) during 2g～10g) to Ganoderma, wherein curative effect is best during Ganoderma 4g+ asiaticoside 60mg+ Radix Ginseng 4g, points out three medicines that the effect of Synergistic is arranged.

Table 1 Ganoderma, asiaticoside and Radix Ginseng drug combination are to mice S ₁₈₀The tumor growth inhibitory action (x ± s, n=10)

Annotate: compare with the normal saline matched group ^*P＜0.05, ^*P＜0.01; Compare with the Radix Ginseng total saponins group ^﹠amp;P＜0.01; Compare with ginseng polysaccharide's group ^#P＜0.01; Compare with the Ganoderma group, ^$P＜0.01; Compare with the asiaticoside group, ^aP＜0.01.

Test example 2 Ganodermas, asiaticoside drug combination are to the influence of ehrlich ascites carcinoma U14 mice increase in life span

The animal subject healthy mice, 60, body weight 20～25g, the male and female dual-purpose is divided into 6 groups at random, 10 every group.

Tumor strain mouse ascites cancer U ₁₄

Asiaticoside group: asiaticoside injection 5ml:60mg.

Compositions group: LJ group: Ganoderma+asiaticoside=4g+60mg, self-control (prescription and preparation method are referring to embodiment 5 aqueous injection prescription 1) is divided into basic, normal, high three dosage groups.

Test method mouse peritoneal inoculation ehrlich ascites carcinoma U ₁₄Tumor strain bacteria suspension (suspension concentration 2 * 10 ⁷/ ml, inoculum concentration 0.5ml/ are only).Inoculate next day, the mice random packet is weighed in, and presses table 2 intraperitoneal injection, every day 1 time, continuous 10 days.After this observe the death time of mice, the result represents [increase in life span=(test group The average survival time natural law-matched group The average survival time natural law)/matched group The average survival time natural law * 100%] with average survival natural law and increase in life span.

Result of the test and conclusion result of the test see Table 2.Compare with the sodium chloride injection matched group, but the middle and high dosage group of LJ injection utmost point significant prolongation U ₁₄The existence natural law of mice (p＜0.01, p＜0.001), LJ injection low dose group, Ganoderma injection group, asiaticoside injection group can obviously prolong U ₁₄The existence natural law of mice (p＜0.05).LJ injection low dose group is compared ehrlich ascites carcinoma U with Ganoderma injection group, asiaticoside injection group ₁₄The mice increase in life span significantly increases (p＜0.05); The middle and high dosage group of LJ injection is compared ehrlich ascites carcinoma U with Ganoderma injection group, asiaticoside injection group ₁₄The mice increase in life span extremely significantly increases (p＜0.01).Prompting, Ganoderma and asiaticoside drug combination have synergistic function, at inhibition tumor, prolongation tumor patient remarkable effect are arranged aspect the time-to-live.

Table 2 Ganoderma, asiaticoside drug combination are to ehrlich ascites carcinoma U ₁₄The influence of mice increase in life span

(meansigma methods ± standard deviation, n=10)

Compare with the normal saline matched group: ^*P＜0.05, ^*P＜0.01, ^{* *}P＜0.001;

Compare with Ganoderma injection group: ^aP＜0.05, ^bP＜0.01;

Compare with asiaticoside injection group: ^cP＜0.05, ^dP＜0.01.

Test example 3 Ganodermas, asiaticoside drug combination are to the potentiation of radiotherapy

The animal subject healthy mice, 50, body weight 20～25g, the male and female dual-purpose is divided into 5 groups at random, 10 every group.

Tumor strain mice S ₁₈₀Sarcoma.

Test sample matched group: sodium chloride injection (Shangdong Changfu Jiejing Pharmaceutical Industry Co., Ltd.);

Every mice left fore of test method oxter subcutaneous vaccination S ₁₈₀Tumor strain cell suspension (suspension concentration 2 * 10 ⁷/ ml, inoculum concentration 0.2ml/ are only), weigh in during 24h.Inoculate next day, random packet, except that the blank group, all the other each groups are the 3rd day, the 6th day usefulness after inoculation all ⁶⁰Co total irradiation, exposure dose are 0.05Gy/min.Inoculate next day, mice is pressed table 3 intraperitoneal injection, every day 1 time, continuous 10 days.Weigh in every day, observes the mice with tumor body weight change.24h after the last administration, weigh in, put to death animal, peel off the subcutaneous tumors piece, take by weighing the tumor body weight, calculate tumor control rate and potentiation rate [potentiation rate=(the average tumor of average tumor weight-radiotherapy of combination radiotherapy group and ZTJ injection therapeutic alliance group is heavy)/average tumor of combination radiotherapy group heavy * 100%].

Result of the test and conclusion result of the test see Table 3.Compare with the blank group, ⁶⁰The Co irradiation is to mice S ₁₈₀Sarcoma has significant inhibitory effect (p＜0.05); ⁶⁰Co irradiation and the therapeutic alliance of basic, normal, high dosage LJ injection are to mice S ₁₈₀Sarcoma has utmost point significant inhibitory effect (p＜0.01, p＜0.001).With ⁶⁰Co irradiation group is compared, ⁶⁰Co irradiation and the therapeutic alliance of low dosage LJ injection are to mice S ₁₈₀The inhibitory action of sarcoma significantly strengthens (p＜0.05), ⁶⁰Co irradiation and the therapeutic alliance of middle and high dosage LJ injection are to mice S ₁₈₀The inhibitory action of sarcoma extremely significantly strengthens (p＜0.01).Result of the test shows that the LJ injection can significantly strengthen ⁶⁰The radiotherapy effect of Co irradiation, prompting, Ganoderma, asiaticoside drug combination have the effect that strengthens radiotherapy effect.

Table 3 Ganoderma, asiaticoside drug combination are to the potentiation of radiotherapy

(meansigma methods ± standard deviation, n=10)

Compare with the blank group: ^*P＜0.05, ^*P＜0.01, ^{* *}P＜0.001;

With ⁶⁰Co irradiation group is compared: ^#P＜0.05, ^##P＜0.01.

Test example 4 Ganodermas, asiaticoside and Radix Ginseng drug combination are to the potentiation and the Attenuation of chemotherapy (Cy)

Tumor strain mice S ₁₈₀Sarcoma.

Cyclophosphamide for injection (positive control): commercial, 100mg, Hualian Pharmaceutical Co., Ltd., Shanghai

The compositions group:

LJR ₁Group: Ganoderma+asiaticoside+Radix Ginseng=4g+60mg+4g, self-control (prescription and preparation method are referring to embodiment 5 aqueous injection prescription 2) is divided into basic, normal, high three dosage groups;

LJR ₂Group: Ganoderma+asiaticoside+Radix Ginseng=4g+60mg+4g, self-control (prescription and preparation method are referring to embodiment 5 aqueous injection prescription 3) is divided into basic, normal, high three dosage groups.

Every mice left fore of test method oxter subcutaneous vaccination S ₁₈₀Tumor strain cell suspension (suspension concentration 2 * 10 ⁷/ ml, inoculum concentration 0.2ml/ are only).The inoculation next day, the mice random packet is weighed in, and presses table 4 intraperitoneal injection, the next day 1 time, continuous 10 days.Weigh in every day, observes the mice with tumor body weight change.24h after the last administration weighs in, and puts to death animal, peels off the subcutaneous tumors piece, takes by weighing the tumor body weight, calculates tumor control rate and potentiation rate [potentiation rate=(average tumor weight-chemotherapy of chemotherapy group and LJR ₁Or LJR ₂The average tumor of injection drug combination group is heavy)/the average tumor of chemotherapy group heavy * 100%].

Result of the test and conclusion result of the test see Table 4.

(1) to the potentiation of chemotherapy: compare with the blank group, the independent medication of low dosage Cyclophosphamide for injection is to mice S ₁₈₀Sarcoma has significant inhibitory effect (p＜0.05); Cyclophosphamide for injection and basic, normal, high dosage LJR ₁, LJR ₂The injection drug combination is to mice S ₁₈₀Sarcoma has utmost point significant inhibitory effect (p＜0.01, p＜0.001).Compare Cyclophosphamide for injection and low dosage LJR with the cyclophosphamide group ₁, LJR ₂The injection drug combination is to mice S ₁₈₀The inhibitory action of sarcoma significantly strengthens (p＜0.05), Cyclophosphamide for injection and middle and high dosage LJR ₁, LJR ₂The injection drug combination is to mice S ₁₈₀The inhibitory action of sarcoma extremely significantly strengthens (p＜0.01).Result of the test shows, LJR ₁, LJR ₂Injection can significantly strengthen the curative effect of cyclophosphamide, prompting, and Ganoderma, asiaticoside and Radix Ginseng drug combination have the effect that strengthens chemotherapeutic efficacy.

(2) to the Attenuation of chemotherapy: compare with the blank group, during the independent medication of Cyclophosphamide for injection, the peripheral leukocytes number of mice, thymus index, spleen index all extremely significantly reduce (p＜0.01).Compare Cyclophosphamide for injection and LJR with the Cyclophosphamide for injection group ₁, LJR ₂During the injection drug combination, LJR ₁, LJR ₂The injection low dosage can significantly suppress the reduction (p＜0.05) of mice peripheral leukocytes number, thymus index, spleen index, LJR ₁, LJR ₂The middle and high dosage of injection can extremely significantly suppress the reduction (p＜0.01) of mice peripheral leukocytes number, thymus index, spleen index.Result of the test shows, LJR ₁, LJR ₂Injection can significantly reduce the toxicity of cyclophosphamide, prompting, and Ganoderma, asiaticoside and Radix Ginseng drug combination have the effect that reduces chemotherapeutic toxicity.

Table 4 Ganoderma, asiaticoside and Radix Ginseng drug combination to the potentiation of chemotherapy and Attenuation (meansigma methods ± standard deviation, n=10)

Annotate: compare with the blank group: ^*P＜0.05, ^*P＜0.01, ^{* *}P＜0.001; Compare with the Cyclophosphamide for injection group: ^#P＜0.05, ^##P＜0.01.

The 5 injected in mice administration acute toxicity tests of test example

(1) test method

Test sample: the LJ injection, specification 2ml derives from embodiment 5 aqueous injection prescription; LJR ₁, LJR ₂Injection, specification 5ml derives from embodiment 5 aqueous injection prescription 2, prescription 3.

Animal subject: mice, each 60 of every group of male and female, male body weight 25～28g, female body weight 21～24g.

Route of administration: intravenous injection, lumbar injection.

Observe special project: death toll, general state, body weight, cut open inspection, half lethal dose.

(2) result of the test

Require to carry out prerun according to acute toxicity test, lumbar injection and intravenous injection two route of administration all can't be measured the median lethal dose(LD 50) of medicine, also do not see tangible toxic reaction, so carry out a day maximum dosage-feeding test.Dosage: tail vein injection 0.05ml/10g, lumbar injection 0.05ml/10g, 2 times on the one.

Death toll: do not occur dead.

General state: no abnormality seen changes.

Body weight: in administration preceding 1 day, administration day, measured in 2,4,6,8,10,12,14 days after the administration; No abnormality seen changes.

Cut open inspection: the heart, liver, lung, kidney etc. organize no abnormality seen to change.

(3) conclusion

Death, LJ, LJR appear in this experiment ₁, LJR ₂Injection is 0.1ml/10g to the maximum tolerated dose of male and female mouse vein and intraperitoneal injection, is equivalent to 120 times of maximum consumption 5ml of the 60kg body weight day for human beings.Show this product low toxicity, safe.

Test example 6 composite injection stability tests

Test sample: the LJ injection, specification 2ml derives from the preparation of embodiment 5 aqueous injection prescription 1; LJR ₁, LJR ₂Injection, specification 5ml derives from embodiment 5 aqueous injection prescription 2,3.

Investigation project: character, pH value, clarity, related substance, sign content; And at accelerated test 6 months and the aseptic and pyrogen test of long term test end of term increase.

1, influence factor's test

The strong illumination test: get test sample, putting illumination is interior the placement 10 days of lighting box of 4500Lx.

Hot test: get test sample, place respectively under 40 ℃, the 60 ℃ conditions and placed 10 days.

Low-temperature test: get test sample, in 4 ℃ of refrigerators, placed 10 days.

Above-mentioned test was respectively at the 5th, 10 day sampling and measuring.Relatively test every index after the character, and with result and comparison in 0 day.

The result: placed 10 days under the illumination 4500Lx condition, except that related substance slightly raise, all other indexs had no significant change.Placed 10 days under 60 ℃ of conditions of high temperature, every index does not have significant change.Placed 10 days under 40 ℃ of high temperature, 4 ℃ of conditions of low temperature, every index does not have significant change.

2, accelerated test

Method: put under the condition of 40 ℃ ± 2 ℃ of temperature, relative humidity 75% ± 5% and placed 6 months.Respectively at taking a sample 1st month, 2 months, 3 months, 6 the end of month, relatively after the outward appearance, test every index at duration of test, with result and comparison in 0 month; And at 6 aseptic and pyrogen tests of increase at the end of month.

Result: placed 6 months under the condition of 40 ℃ ± 2 ℃ of temperature, relative humidity 75% ± 5%, removing related substance slightly increases, and outside sign content slightly descended, all other indexs had no significant change, at 6 the end of month of accelerated test, pyrogen, sterility test are all up to specification.

3, long term test

Method: put under the condition of 25 ℃ ± 2 ℃ of temperature, relative humidity 60% ± 10% and placed 18 months.Respectively at 3rd month, 6 months, 9 months, 12 months, 18 months, relatively after the outward appearance, test every index, with result and comparison in 0 month; And at 18 aseptic and pyrogen tests of increase at the end of month.

The result: placed under the condition of 25 ℃ ± 2 ℃ of temperature, relative humidity 60% ± 10% 18 months, every index has no significant change, and at 18 the end of month of long term test, pyrogen, sterility test are all up to specification.

Conclusion: reach a conclusion by above-mentioned investigation result, in every test, LJ, LJR ₁, LJR ₂Injection is all more stable.

In sum, Ganoderma provided by the invention and asiaticoside or Ganoderma, asiaticoside and ginseng composition have synergistic function, obviously are better than the individually dosed drug effect of Ganoderma, asiaticoside or Radix Ginseng.To LJ, LJR ₁, LJR ₂The stability test result that injection carries out shows that every index of the injection that compositions provided by the invention is made is all more stable, can be used for amplifying producing.

[specific embodiment]

Come by the following examples further to set forth preparation of drug combination method of the present invention, but this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following embodiment.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.The adjuvant of each dosage form can be replaced with acceptable accessories in following examples, perhaps reduces, increases.

Among Ganoderma extract in following examples 4～12, asiaticoside and the Radix Ginseng total saponins embodiment 1,2,3 in Ganoderma extract, the Radix Ginseng total saponins second batch.Ginseng polysaccharide in following examples 4～12 derives from first among the ginseng polysaccharide among the embodiment 1,2,3.

The preparation of embodiment 1 Ganoderma extract

Get Ganoderma medical material 40kg, be ground into coarse grain, add the moistening of 3 times of amounts of water and spend the night, decoct next day three times, added 12 times of amounts of water for the first time in 3 hours, added 10 times of amounts of water for the second time in 2 hours, added 10 times of amounts of water in 1 hour for the third time, collecting decoction filters, it is 1.10～1.15 that filtrate is concentrated into relative density, puts coldly, adds ethanol and makes that to contain alcohol amount be 65%, stir evenly, placed 24 hours, filter at 4 ℃, collect filter cake, add suitable quantity of water and make dissolving, filter, add ethanol and make that to contain the alcohol amount be 85%, stir evenly, placed 24 hours at 4 ℃, filter, collect filter cake, vacuum drying, promptly.

Prepare three batches of extracts respectively, extract yield and content see the following form 5.

Three batches of Ganoderma extract yield of table 5 and assay result

The assay of Ganoderma extract

It is an amount of that the preparation precision of reference substance solution takes by weighing 105 ℃ of glucose reference substances that are dried to constant weight, adds water and make the solution that every 1ml contains 0.1mg, promptly.

The preparation of standard curve is quiet close absorption reference substance solution 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, 1.2ml respectively, put in the 10ml tool plug test tube, add water to 2.0ml, quiet close adding sulphuric acid anthrone solution (the quiet close anthrone 0.1g that takes by weighing, add 80% sulfuric acid solution 100ml and make dissolving, shake up) 6ml, shake up, put in the water-bath and heated 15 minutes, take out, put into water-bath cooling 15 minutes, with the corresponding solvent is blank, measures absorbance under 625nm, is vertical coordinate with the absorbance, concentration is abscissa, the drawing standard curve.

This product 20mg is got in the preparation of need testing solution, puts in the 50ml measuring bottle, is dissolved in water, and is diluted to scale, shakes up promptly.

The accurate need testing solution 2ml that draws of algoscopy puts in the 10ml tool test tube, and assay method under the sighting target directrix curve is measured absorbance in accordance with the law, from the weight that standard curve is read the glucose of need testing solution, calculates, promptly.

The discriminating of Ganoderma extract

Get this product 50mg, add ethanol 25ml, reflux 30 minutes filters, and filtrate evaporate to dryness, residue add methanol 2ml makes dissolving, as need testing solution.Other gets Ganoderma control medicinal material 2g, shines medical material solution in pairs with legal system.According to the thin layer chromatography test, draw above-mentioned two kinds of each 5ul of solution, put respectively on same silica gel G plate, upper solution with petroleum ether (60～90 ℃)-Ethyl formate-formic acid (15: 5: 1) is developing solvent, launches, and takes out, dry, put under the ultra-violet lamp (365nm) and inspect.In the test sample chromatograph, with the corresponding position of control medicinal material chromatograph on, show the fluorescence speckle of same color.

The preparation of embodiment 2 asiaticosides

Get the Herba Centellae medical material, be ground into fine powder, add 10 times of amount 95% soak with ethanol and spend the night, inclining next day ethanol liquid, reuse 75% alcohol reflux secondary, each 2 hours, add 10 times of amounts of alcohol, filter, merge ethanol extract, after being evaporated to 1/3 volume, add 40 minutes after-filtration deactivation charcoals of active carbon backflow of 5%, the alcoholic solution after decolouring is evaporated to the thick paste shape, after adding 5 times of amount warm water stirring and dissolving, put coldly, add the petroleum ether of 1/2 times of amount volume, jolting is extracted 2 times, water liquid extracts 3 times with the water saturated n-butyl alcohol jolting of 1/2 times of amount volume, merge extractive liquid, washes with water 1 time, is evaporated to dried brown extractum.After it was dissolved in an amount of ethanol, the anhydrous propanone that slowly adds 8 times of amounts makes separated out precipitation, sucking filtration, washing with acetone, vacuum drying, asiaticoside.

Prepare three batches of asiaticosides respectively, its yield and purity see the following form 6.

The discriminating of asiaticoside

Get this product 30mg, add methanol 20ml, supersound process 30 minutes, filter, filtrate evaporate to dryness, residue add methanol 1ml makes dissolving, as need testing solution. other gets the asiaticoside reference substance, add methanol and make the solution that every 1ml contains 1mg, in contrast product solution. draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of adhesive with the sodium carboxymethyl cellulose, with chloroform-methanol-water (7: 3: 0.5) is developing solvent, launch, take out, dry, spray is with 1% ethanol solution of sulfuric acid, it is clear to be heated to speckle colour developing at 105 ℃. in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show the speckle of same color.

The assay of asiaticoside

Chromatographic condition and system suitability test are filler with the octadecylsilane chemically bonded silica; With acetonitrile-water (60: 40) is mobile phase; The monitoring wavelength is 205nm; 30 ℃ of column temperatures; 0.6ml/min flow velocity is.Number of theoretical plate calculates by the asiaticoside peak should be not less than 2000.

It is an amount of that the asiaticoside reference substance is got in the preparation of reference substance solution, adds 50% methanol and make the solution that every 1ml contains 1mg, promptly.

This product 50mg is got in the preparation of need testing solution, and accurate the title decides, and puts in the tool plug conical flask quiet close adding 50% methanol 30ml, claim to decide weight, supersound process (power 100kw, frequency 40kHz) 30 minutes is put cold, weight decided in title again, supplies with 50% methanol to subtract weight loss, filters, and gets subsequent filtrate promptly.

Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid, measure, promptly.

The purity and the yield of three batches of asiaticosides of table 6

The preparation of embodiment 3 Radix Ginseng extracts

The preparation of Radix Ginseng total saponins

Get ginseng crude drug 40kg, be ground into particulate, add 8 times of amount alcohol reflux 2 times, each 3 hours, merge extractive liquid,, cold preservation filters, and filtrate recycling ethanol also is concentrated into thick paste, add water to an amount of (making every 1ml be equivalent to crude drug 1g), stir evenly, cold preservation filters, filtrate is added on the macroporous resin column of having handled well, water with 2V～3V column volume carries out eluting earlier, discards water lotion, 80% ethanol elution of 3 times of column volumes of reuse, collect eluent, reclaim ethanol and be evaporated to the thick paste of relative density 1.30～1.35, vacuum drying, promptly.

Make three batches of Radix Ginseng total saponins extracts respectively, extract yield and content see the following form 7.

Three batches of Radix Ginseng total saponins extract yields of table 7 and assay result

Determination of Total Saponin Content in Panax Ginseng

Reference substance solution preparation: get ginsenoside R _G1The about 10mg of reference substance through 60 ℃ of vacuum dryings 2 hours, accurately claims surely, puts in the 100ml measuring bottle, with anhydrous alcohol solution and be diluted to scale, shakes up, and makes every 1ml and contains R _G1The solution of reference substance 0.1mg, promptly.

The need testing solution preparation: precision takes by weighing this product 50mg, puts in the 50ml measuring bottle, adds dehydrated alcohol and is diluted to scale, shakes up, and precision is measured 2ml, puts in the 10ml measuring bottle, adds dehydrated alcohol and is diluted to scale, shakes up, promptly.

The algoscopy precision is measured need testing solution and each 1ml of reference substance solution, puts respectively in the 10ml tool plug test tube, and evaporate to dryness in water-bath is put cold, add 5% vanillin glacial acetic acid solution 0.2ml, add perchloric acid 0.8ml again, in 60 ℃ of insulations 15 minutes, be cooled to room temperature, add glacial acetic acid 5ml, shake up; Make blank simultaneously.According to spectrophotography (an appendix V of Chinese Pharmacopoeia version in 2005 B), measure trap at 560nm wavelength place, calculate, promptly.

Ginsenoside Re, ginsenoside Rg ₁Assay

Chromatographic condition and system suitability test are filler with the octadecylsilane chemically bonded silica; With acetonitrile-water (22: 78) is mobile phase; The detection wavelength is 203nm.Number of theoretical plate calculates by Radix Ginseng total saponins Re peak should be not less than 2000.

The preparation precision of reference substance solution takes by weighing the ginsenoside Rg ₁Reference substance, Re reference substance are an amount of, add methanol and make the mixed solution that every 1ml contains 0.2mg.

This product 0.2g is got in the preparation of need testing solution, and accurate the title decides, the accurate water-saturated n-butanol 30ml that adds, close plug, placement is spent the night, supersound process (power 250W, frequency 50kHz) 30 minutes, filter, discard filtrate just, precision is measured subsequent filtrate 15ml, puts evaporate to dryness in the evaporating dish, and residue adds dissolve with methanol and is transferred in the 5ml volumetric flask, add methanol and be diluted to scale, shake up, filter, get subsequent filtrate promptly.

Accurate respectively reference substance and each 20ul of need testing solution of drawing of algoscopy injects chromatograph of liquid, measures, promptly.

The discriminating of Radix Ginseng total saponins

Get this product 0.5g, add water 0.5ml and stir moistening, add water-saturated n-butanol 10ml, supersound process 30 minutes is drawn supernatant and is added 3 times of amount ammonia solutions, shakes up, and places layering, gets upper strata liquid evaporate to dryness, and residue adds methanol 1ml makes dissolving, as need testing solution.Other gets ginsenoside Re, Rg ₁Reference substance adds methanol and makes the mixed solution that every 1ml contains 2mg, in contrast product solution.Draw above-mentioned two kinds of each 2ul of solution, put respectively on same silica gel g thin-layer plate, (15: 40: 22: 10) lower floor's solution of placing below 10 ℃ was developing solvent with chloroform-ethyl acetate-methanol-water, launch, take out, dry, spray is with 10% ethanol solution of sulfuric acid, it is clear to be heated to speckle colour developing at 105 ℃, puts under the ultra-violet lamp (365nm) and inspects.In the test sample chromatograph, with reference substance chromatograph relevant position on show the fluorescence speckle of same color.

Ginseng polysaccharide's preparation

Get Radix Ginseng 10kg, chopping is used 75% ethanol, reflux, extract, 4 hours, filter, medicinal residues dry, and decoct with water five times, merge decoction liquor, add 0.3% active carbon, stirred 30 minutes, standing over night filters, filtrate is crossed resin column, collects effluent, and concentrating under reduced pressure adds ethanol and makes and contain the alcohol amount and reach 95%, cold preservation filters, and gets precipitation and uses washing with acetone, drains acetone, taking-up is deposited in 60～80 ℃ of dryings, pulverizes, and gets powder promptly.

Make three batches of ginseng polysaccharide's extracts respectively, extract must be filtered with content and see the following form 8.

Ginseng polysaccharide's assay

The preparation precision of reference substance solution takes by weighing 60 ℃ of vacuum dryings to the about 100mg of the galacturonic acid of constant weight, puts in the 100ml measuring bottle, adds water to scale, shakes up.Precision is measured 10ml, puts in the 100ml measuring bottle, adds water to scale, shakes up, promptly.

The preparation precision of need testing solution takes by weighing this product 50mg, puts in the 100ml measuring bottle, is dissolved in water and is diluted to scale, shakes up, promptly.

The algoscopy precision is measured each 1ml of reference substance solution, need testing solution and water, the accurate respectively 0.25mol/L Borax sulfuric acid solution 6ml that adds, put in the water-bath heating 30 minutes, put cold, the accurate respectively again 0.125% carbazole ethanol solution 0.4ml that adds, put in the water-bath and heated 10 minutes, put and be chilled to room temperature,, measure trap at 530nm wavelength place according to the spectrophotography test, calculate, promptly.

Ginseng polysaccharide's discriminating

(1) gets the about 0.2g of this product, after adding water 5ml dissolving, add 5 of alkaline cupric tartrate test solutions, heating promptly produces red precipitate, cooling, filter, get filtrate add 1 of hydrochloric acid make acid, heating in water bath 10 minutes, put cold, regulate pH value to neutral, add alkaline cupric tartrate test solution 0.5ml, heating in water bath promptly produces red copper oxidule precipitation.

(2) get the about 0.2g of this product, add water 2ml dissolving after, add 5% alpha-Naphthol alcoholic solution 0.5ml and shake up, slowly add sulphuric acid 3ml, two liquid level intersection displaing amaranth rings.

Table 8 ginseng polysaccharide extract yield and assay result

The preparation of embodiment 4 present composition injectable powder

Prescription:

Prescription 1

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Polyoxyethylene sorbitan monoleate 50g

Mannitol 300g

Sterile water for injection adds to 3000ml

Prepare 1000 altogether

Prescription 2

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Ginseng polysaccharide 48.4g (being equivalent to ginseng crude drug 4kg)

Polyoxyethylene sorbitan monoleate 50g

Mannitol 300g

Sterile water for injection adds to 3000ml

Prepare 1000 altogether

Prescription 3

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Radix Ginseng total saponins 49.2g (being equivalent to ginseng crude drug 4kg)

Polyoxyethylene sorbitan monoleate 50g

Mannitol 300g

Sterile water for injection adds to 3000ml

Prepare 1000 altogether

Preparation technology:

1) vessel of at first dosing being used and antibiotic glass bottle, plug etc. carry out aseptic process.

2) take by weighing raw material and adjuvant according to recipe quantity.

3) get the water for injection of dosing amount 40%, add the polyoxyethylene sorbitan monoleate dissolving earlier fully, add the asiaticoside of recipe quantity again, the heated and stirred dissolving fully.Chinese medicine extract adds dosing amount 30% water for injection stirring and dissolving.Merge above-mentioned solution, add the dissolving of mannitol heated and stirred more fully, add sterile water for injection to full dose.

4) needle-use activated carbon of adding dosing amount 0.1%, heated and stirred 15 minutes.

5) through sand filtration rod filtering decarbonization.Measure the also pH value of regulator solution.

6) through the microporous filter membrane fine straining of 0.22um.

7) clarity of inspection solution, the semi-finished product chemical examination.

8) be sub-packed in the antibiotic glass bottle half tamponade.Sample is put into the freeze dryer lyophilization.Pre-freeze-45 ℃ 5 hours, low-temperature vacuum drying-45 ℃～0 ℃ 20 hours was warming up to 25 ℃ of vacuum dryings 4 hours then.

9) lyophilizing finishes, and lid is rolled in tamponade.

10) finished product is examined entirely, the packing warehouse-in

The preparation of embodiment 5 present composition aqueous injection

Prescription:

Prescription 1

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Polyoxyethylene sorbitan monoleate 100g

Water for injection adds to 2000ml

Prepare 1000 altogether

Prescription 2

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Ginseng polysaccharide 48.4g (being equivalent to ginseng crude drug 4kg)

Polyoxyethylene sorbitan monoleate 100g

Water for injection adds to 5000ml

Prepare 1000 altogether

Prescription 3

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Radix Ginseng total saponins 49.2g (being equivalent to ginseng crude drug 4kg)

Polyoxyethylene sorbitan monoleate 100g

Water for injection adds to 5000ml

Prepare 1000 altogether

Preparation technology:

1) carries and handle the previous day such as pipeline that dosing uses and container etc., face with the fresh water for injection flushing of preceding reuse.

2) get the water for injection of dosing amount 40%, add the polyoxyethylene sorbitan monoleate dissolving earlier fully, add the asiaticoside of recipe quantity again, the heated and stirred dissolving fully.Chinese medicine extract adds dosing amount 30% water for injection stirring and dissolving.

3) merge above-mentioned solution, benefit adds to the full amount of water for injection.

6) through the microporous filter membrane fine straining of 0.45um.

8) with the solution sealing by fusing in glass ampule.

9) 100 ℃ of flowing steam sterilizations are 30 minutes.

10) heat is put into 0.01% methylene blue solution with sample and is hunted leak.

11) lamp inspection, finished product is examined entirely, the packing warehouse-in.

The preparation of embodiment 6 present compositions transfusion

The sodium chloride transfusion:

Prescription:

Prescription 1

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Polyoxyethylene sorbitan monoleate 50g

Sodium chloride 900g

Water for injection adds to 100000ml

Prepare 1000 bottles altogether

Prescription 2

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Ginseng polysaccharide 48.4g (being equivalent to ginseng crude drug 4kg)

Polyoxyethylene sorbitan monoleate 50g

Sodium chloride 900g

Water for injection adds to 100000ml

Prepare 1000 bottles altogether

Prescription 3

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Radix Ginseng total saponins 49.2g (being equivalent to ginseng crude drug 4kg)

Polyoxyethylene sorbitan monoleate 50g

Sodium chloride 900g

Water for injection adds to 100000ml

Prepare 1000 bottles altogether

Preparation technology:

1) handles the previous day such as pipeline that dosing uses and container etc., face with the fresh water for injection flushing of preceding reuse.

2) get the water for injection of dosing amount 40%, add the polyoxyethylene sorbitan monoleate dissolving earlier fully, the asiaticoside that adds recipe quantity again, the heated and stirred dissolving is fully. and Chinese medicine extract adds dosing amount 30% water for injection stirring and dissolving. and sodium chloride is complete with the water for injection dissolving of dosing amount 20%.

6) through the microporous filter membrane fine straining of 0.45um.

8) fill is in the infusion bottle of 100ml.

9) 115 ℃ of pressure sterilizings are 30 minutes.

10) lamp inspection, finished product is examined entirely, the packing warehouse-in.

Glucose infusion liquid:

Prescription:

Prescription 1

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Polyoxyethylene sorbitan monoleate 50g

Glucose 5000g

Water for injection adds to 100000ml

Prepare 1000 bottles altogether

Prescription 2

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Ginseng polysaccharide 48.4g (being equivalent to ginseng crude drug 4kg)

Polyoxyethylene sorbitan monoleate 50g

Glucose 5000g

Water for injection adds to 100000ml

Prepare 1000 bottles altogether

Prescription 3

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Radix Ginseng total saponins 49.2g (being equivalent to ginseng crude drug 4kg)

Polyoxyethylene sorbitan monoleate 50g

Glucose 5000g

Water for injection adds to 100000ml

Prepare 1000 bottles altogether

Preparation technology:

2) get the water for injection of dosing amount 40%, add the polyoxyethylene sorbitan monoleate dissolving earlier fully, add the asiaticoside of recipe quantity again, the heated and stirred dissolving fully.Chinese medicine extract adds dosing amount 30% water for injection stirring and dissolving.Glucose is complete with the water for injection dissolving of dosing amount 20%.

6) through the microporous filter membrane fine straining of 0.45um.

8) fill is in the infusion bottle of 100ml.

9) 115 ℃ of pressure sterilizings are 30 minutes.

The preparation of embodiment 7 present composition tablets

Prescription:

Prescription 1

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Starch 150.0g

Microcrystalline Cellulose 50.0g

The 2%HPMC aqueous solution is an amount of

Magnesium stearate 4.0g

Carboxymethylstach sodium 8.0g

Prepare 1000 altogether

Prescription 2

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Ginseng polysaccharide 48.4g (being equivalent to ginseng crude drug 4kg)

Starch 150.0g

Microcrystalline Cellulose 50.0g

The 2%HPMC aqueous solution is an amount of

Magnesium stearate 4.0g

Carboxymethylstach sodium 8.0g

Prepare 1000 altogether

Prescription 3

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Radix Ginseng total saponins 49.2g (being equivalent to ginseng crude drug 4kg)

Starch 150.0g

Microcrystalline Cellulose 50.0g

The 2%HPMC aqueous solution is an amount of

Magnesium stearate 4.0g

Carboxymethylstach sodium 8.0g

Prepare 1000 altogether

Preparation technology:

1) it is standby Chinese medicine extract and asiaticoside to be pulverized 100 mesh sieves.

2) take by weighing raw material and adjuvant according to recipe quantity.

3) hypromellose 2% the aqueous solution made soluble in water is standby.

4) with Chinese medicine extract, asiaticoside, starch, microcrystalline Cellulose mix homogeneously, adding 2%HPMC aqueous solution is an amount of, stirs, and makes suitable soft material.

5) cross 20 mesh sieve system granules.

6) granule is dried under 60 ℃ condition.

7) dry good granule adds magnesium stearate and carboxymethylstach sodium, crosses 18 mesh sieve granulate, mix homogeneously.

8) sampling, the semi-finished product chemical examination.

9) determine sheet weight sheet by result of laboratory test.

10) finished product is examined entirely, the packing warehouse-in.

The preparation of embodiment 8 present composition capsules

Prescription:

Prescription 1

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Starch 80.0g

Microcrystalline Cellulose 40.0g

The 2%HPMC aqueous solution is an amount of

Magnesium stearate 3.0g

Prepare 1000 altogether

Prescription 2

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Ginseng polysaccharide 48.4g (being equivalent to ginseng crude drug 4kg)

Starch 80.0g

Microcrystalline Cellulose 40.0g

The 2%HPMC aqueous solution is an amount of

Magnesium stearate 3.0g

Prepare 1000 altogether

Prescription 3

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Radix Ginseng total saponins 49.2g (being equivalent to ginseng crude drug 4kg)

Starch 80.0g

Microcrystalline Cellulose 40.0g

The 2%HPMC aqueous solution is an amount of

Magnesium stearate 3.0g

Prepare 1000 altogether

Preparation technology:

2) take by weighing raw material and adjuvant according to recipe quantity.

3) hypromellose 2% the aqueous solution made soluble in water is standby.

5) cross 20 mesh sieve system granules.

6) granule is dried under 60 ℃ condition.

7) dry good granule adds magnesium stearate, crosses 18 mesh sieve granulate, mix homogeneously.

8) sampling, the semi-finished product chemical examination.

9) loading amount of determining according to chemical examination incapsulates.

10) finished product is examined entirely, the packing warehouse-in.

The preparation of embodiment 9 present composition granules

Prescription:

Prescription 1

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Icing Sugar 2000.0g

The 2%HPMC50% alcoholic solution is an amount of

Prepare 1000 bags altogether

Prescription 2

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Ginseng polysaccharide 48.4g (being equivalent to ginseng crude drug 4kg)

Icing Sugar 2000.0g

The 2%HPMC50% alcoholic solution is an amount of

Prepare 1000 bags altogether

Prescription 3

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Radix Ginseng total saponins 49.2g (being equivalent to ginseng crude drug 4kg)

Icing Sugar 2000.0g

The 2%HPMC50% alcoholic solution is an amount of

Prepare 1000 bags altogether

Preparation technology:

1) it is standby sucrose to be pulverized 100 mesh sieves.It is standby that Chinese medicine extract, asiaticoside were pulverized 100 mesh sieves.

2) take by weighing raw material and adjuvant according to recipe quantity.

3) the method mix homogeneously that Chinese medicine extract, asiaticoside and Icing Sugar are progressively increased with equivalent, adding 2%HPMC50% alcoholic solution is an amount of, stirs, and makes suitable soft material.

4) cross 20 mesh sieve system granules.

5) granule is dried under 60 ℃ condition.

6) dried granule is crossed 18 mesh sieve granulate.

7) sampling, the content of principal agent is determined loading amount in the semi-finished product chemical examination granule.

8) packing, finished product is examined entirely, the packing warehouse-in.

The preparation of embodiment 10 present composition soft capsules

Prescription:

Prescription 1

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Soybean oil 500.0g

Soybean phospholipid 50g

Cera Flava 50g

Prepare 1000 altogether

Prescription 2

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Ginseng polysaccharide 48.4g (being equivalent to ginseng crude drug 4kg)

Soybean oil 500.0g

Soybean phospholipid 50g

Cera Flava 50g

Prepare 1000 altogether

Prescription 3

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Radix Ginseng total saponins 49.2g (being equivalent to ginseng crude drug 4kg)

Soybean oil 500.0g

Soybean phospholipid 50g

Cera Flava 50g

Prepare 1000 altogether

Preparation technology:

Chinese medicine extract, asiaticoside pulverize separately are crossed 100 mesh sieves, and with the soybean oil of recipe quantity and soybean phospholipid, Cera Flava heating and melting, mixing is put coldly, adds Chinese medicine extract, asiaticoside grinds well, and is pressed into soft capsule and gets final product.

The preparation of embodiment 11 present composition oral liquids

Prescription:

Prescription 1

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Polyoxyethylene sorbitan monoleate 50g

Sodium benzoate 15g

Stevioside 10g

Water adds to 10000ml

Prepare 1000 altogether

Prescription 2

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Ginseng polysaccharide 48.4g (being equivalent to ginseng crude drug 4kg)

Polyoxyethylene sorbitan monoleate 50g

Sodium benzoate 15g

Stevioside 10g

Water adds to 10000ml

Prepare 1000 altogether

Prescription 3

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Radix Ginseng total saponins 49.2g (being equivalent to ginseng crude drug 4kg)

Polyoxyethylene sorbitan monoleate 50g

Sodium benzoate 15g

Stevioside 10g

Water adds to 10000ml

Prepare 1000 altogether

Preparation technology:

1) earlier that polyoxyethylene sorbitan monoleate is complete with the water dissolution of dosing amount 30%, again asiaticoside is added the heated and stirred dissolving fully.Chinese medicine extract adds dosing amount 50% water heating back dissolving fully.

2) sodium benzoate and stevioside is complete with the water dissolution of dosing amount 10%.

3) merge above-mentioned solution, mend and add water to full dose.

4) filtering with microporous membrane of mistake 0.8um.

5) semi-finished product chemical examination.

6) fill.Finished product is examined entirely, the packing warehouse-in.

The preparation of embodiment 12 present composition drop pills

Prescription:

Prescription 1

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Polyethylene glycol 6000 600g

Prescription 2

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Ginseng polysaccharide 48.4g (being equivalent to ginseng crude drug 4kg)

Polyethylene glycol 6000 600g

Prescription 3

Ganoderma extract 61.6g (being equivalent to Ganoderma medical material 4kg)

Asiaticoside 60g

Radix Ginseng total saponins 49.2g (being equivalent to ginseng crude drug 4kg)

Polyethylene glycol 6000 600g

Preparation technology:

With polyethylene glycol 6000 heating and melting in water-bath, treat to add Radix Ginseng extract, Ganoderma extract, asiaticoside after whole fusions, stirring and dissolving, 60 mesh sieves filter, and keep 60 ℃ to splash in the liquid paraffin that is chilled to below 10 ℃ and make ball.

Claims

1. a pharmaceutical composition is characterized in that, said composition is mainly made by following bulk drugs: 50～2000 parts of Ganodermas or 0.2～60 part of its extract, 1～50 part of asiaticoside, wherein the polysaccharide of Ganoderma extract is not less than 30%.

2. pharmaceutical composition as claimed in claim 1 is characterized in that said composition mainly made by following bulk drugs: 200～1000 parts of Ganodermas or 1～30 part of its extract, 2～15 parts of asiaticosides.

3. pharmaceutical composition as claimed in claim 2 is characterized in that said composition mainly made by following bulk drugs: 400 parts of Ganodermas or 2～15 parts of its extracts, 6 parts of asiaticosides.

4. pharmaceutical composition as claimed in claim 1 is characterized in that the said composition crude drug also has: 0.1～40 part of 0.1～40 part of 50～2000 parts of Radix Ginsengs or ginseng polysaccharide or Radix Ginseng total saponins.

5. pharmaceutical composition as claimed in claim 4 is characterized in that said composition is to be made by following bulk drugs:

0.2～20 part of 200～1000 parts of Ganodermas or 1～30 part of its extract, 2～15 parts of asiaticosides, 200～1000 parts of Radix Ginsengs or ginseng polysaccharide; Perhaps be: 0.2～20 part of 200～1000 parts of Ganodermas or 1～30 part of its extract, 2～15 parts of asiaticosides, 200～1000 parts of Radix Ginsengs or Radix Ginseng total saponins.

6. as claim 4,5 described arbitrary pharmaceutical compositions, it is characterized in that described ginseng polysaccharide's content is not less than 50%; Described Radix Ginseng total saponins content is not less than 50%, wherein ginsenoside Re and ginsenoside Rg ₁Total content be not less than 30%.

7. as the described arbitrary pharmaceutical composition of claim 1～5, it is characterized in that described Ganoderma extract contains ganoderan and is not less than 30%; Asiaticoside purity is not less than 80%.

8. as the described arbitrary preparation of drug combination method of claim 1～5, it is characterized in that, medical material is wherein singly carried with The suitable solvent and method or is mixed to obtain through refining and obtains extract fully, and total extract is made arbitrary preparation with asiaticoside and mixing acceptable accessories again.

9. preparation of drug combination method as claimed in claim 8, contained main effective ingredient is a ganoderan in the described total extract, perhaps is: ganoderan and ginseng polysaccharide; Perhaps be: ganoderan and Radix Ginseng total saponins.

10. as the described arbitrary pharmaceutical composition of claim 1～5, it is characterized in that said composition makes clinically any or pharmaceutically acceptable dosage form.