CN100571680C

CN100571680C - The preparation method of compound kuhseng preparation for injection and medical application thereof

Info

Publication number: CN100571680C
Application number: CNB2005100748418A
Authority: CN
Inventors: 王智民; 李安平
Original assignee: Shandong Zhendong Jinjing Pharmaceutical Co Ltd; Beijing Zhendong Guangming Pharmaceutical Research Institute
Current assignee: Beijing Zhendong Guangming Pharmaceutical Research Institute Co., Ltd.; Shandong Zhendong Pharmaceutical Co., Ltd.
Priority date: 2005-06-07
Filing date: 2005-06-07
Publication date: 2009-12-23
Anticipated expiration: 2025-06-07
Also published as: CN1876016A

Abstract

The present invention relates to a kind of preparation method and the purposes in tumor and hepatitis clinical treatment thereof of compound kuhseng preparation for injection.Its prescription is to be raw material with Radix Sophorae Flavescentis and Smilax lanceaefolia Roxb. Var.opaca A.DC., can be to extract the back separately to mix or mixed extraction.Extracting method is with water or diluted acid or rare alcohol extraction, concentrated, return molten, decolouring, transfer pH to obtain the injection medicinal liquid through repeatedly precipitate with ethanol-filtration-concentrate, water again, it directly embedding be the different size injection, or add dispersant and obtain lyophilized powder through lyophilization, or the spray-dried sterile injection powder that obtains carries out packing and prepares.The characteristics of said preparation are to be main effective ingredient with oxymatrine, matrine, sophocarpine etc.Said preparation has antitumor, alleviates cancerous protuberance pain, the chemotherapeutics of tumor is had the potentiation Detoxication, also has immunological enhancement simultaneously, and antihepatitic activity can be used for the clinical treatment of tumor and hepatitis.

Description

The preparation method of compound kuhseng preparation for injection and medical application thereof

Technical field

The present invention relates to a kind of from the preparation of Radix Sophorae Flavescentis, Smilax lanceaefolia Roxb. Var.opaca A.DC. injection preparation and be used for the treatment of cancer, particularly to cancerous pain, carcino-matous hemorrhage, improve in the tumor patient life quality and the medical application in the treating hepatitis, belong to drug world.

Background technology

FUFANG KUSHEN ZHUSHEYE is made up of Radix Sophorae Flavescentis and Smilax lanceaefolia Roxb. Var.opaca A.DC., has antitumor, pain relieving, and effects such as hemostasis, because its determined curative effect on the clinical treatment of tumor, sales volume in recent years is soaring year by year.

FUFANG KUSHEN ZHUSHEYE was the key product of my company, had applied for patent on July 16th, 1997, patent No. ZL97112532.5 (notification number CN 1062181C).In view of some problems that occur in the actual production of this product, we have carried out the novelty raising of system targetedly to original production process, append the technology patent application, and have carried out enlarging protection with regard to the indication of medicine.

At present, the patent of the similar noun of compound light-yellow sophora root has 3: (1) nano medicine ' Compound Kushen ' and preparation method thereof, notification number are CN 1368220A.It discloses a kind of nano medicine ' Compound Kushen ', is to be raw material with nanometer Radix Sophorae Flavescentis, nanometer Smilax lanceaefolia Roxb. Var.opaca A.DC., and new pharmaceutical preparation is made in preparation in proportion, and its fineness of the particles reaches the 1200-1500 order, and particle diameter is 0.1-200nm, and wherein most particle diameters are less than 100nm.Adopt step preparations such as microwave extracting, concentrating under reduced pressure, supersonic jet technology spray drying.Conclusion: adopt nanometer to handle to compound light-yellow sophora root, irrelevant with the innovative content that we apply for a patent.(2) compound flavescent sophora root injection for animals, notification number CN 1048406C.It discloses a kind of compound flavescent sophora root injection for animals, and prescription is restrained into the 1000ml injection by Radix Scutellariae 300-1200 gram, Cortex Phellodendri 200-800 gram, Radix Sophorae Flavescentis 300-1200.Conclusion: this patent is different with the medicine composition of the compound light-yellow sophora root that we are applied for, does not hinder the novelty that we applied for a patent.(3) compound vagina-cleaning flavescent sophora lotion, notification number CN 1403117A.It discloses a kind of compound vagina-cleaning flavescent sophora lotion, and its prescription consists of Radix Sophorae Flavescentis, Fructus Cnidii, Radix Et Rhizoma Rhei, Rhizoma Atractylodis, crust halberd, Rhizoma Smilacis Glabrae, Cortex Dictamni, Cortex Phellodendri, Semen Coicis, the Fructus Kochiae, Spina Gleditsiae, Flos Carthami, dried Alumen; Indication is for comprising women's genital system diseases such as senile vaginitis, trichomonal vaginitis.Conclusion: this patent is formed different with the medicine of the compound light-yellow sophora root that we apply for.

Radix Sophorae Flavescentis is clinical conventional Chinese medicine, and the dry root for cassia leguminous plant Radix Sophorae Flavescentis (Sophora flavscens Alt.) has effects such as heat clearing away, dehumidifying, parasite killing, diuresis; China extensively distributes, aboundresources, Radix Sophorae Flavescentis contains alkaloid and flavones ingredient, its alkaloid composition is mainly: sophocarpine (Sophocarpine), matrine (matrine), sophoridine (sophoridine), oxymatrine (Oxymatrine), N-oxysophocarpine (Oxysophocarpine).Pharmacological evaluation and clinical practice prove that Radix Sophorae Flavescentis and preparation thereof have pharmacological action widely.Contain totally 67 of the relevant patents of Radix Sophorae Flavescentis and chemistry prescription thereof, wherein the patent of single Radix Sophorae Flavescentis is 9, and 55 of the patents of Radix Sophorae Flavescentis chemical constituent (matrine, oxymatrine etc.) contain 11 of the Chinese medicine compound patents (compound recipe that comprises chemical compound) of Radix Sophorae Flavescentis.These patents mainly concentrate in synthetic, extraction process, preparation and the pharmacological action thereof of matrine and oxymatrine.

Smilax lanceaefolia Roxb. Var.opaca A.DC. is the dry rhizome of liliaceous plant Xiao Rhizoma Smilacis Chinensis (Heterosmilax japonica Kunth.) or short column Xiao Rhizoma Smilacis Chinensis (Heterosmilaxyunnanensis Gagnep.), has dehumidifying, detoxifcation, the effect of easing joint movement is used for damp and hot stranguria with turbid discharge, leukorrhagia, carbuncle, scrofula, scabies, limbs contracture due to syphilis and the mercurialism, diseases such as bones and muscles pain; Mainly being distributed in areas such as Guangxi, Guizhou, Hunan, is medical materials traditionally used in certain regions, is usually used in replacing Rhizoma Smilacis Glabrae, but the difference that on former plant, has matter with Rhizoma Smilacis Glabrae (for the dry rhizome of liliaceous plant smilacis glabra Smilax glabra Roxb.).Its chemical constitution study seldom has the people that Smilax lanceaefolia Roxb. Var.opaca A.DC. has been carried out The Chemical Constituents recently, finds that it contains compositions such as ginsenoside and Radix Et Rhizoma Rhei anthraquinone.But, prove through our actual detected not contain these compositions in this medical material, infer that researcher is wrong in the evaluation of former plant species; We studies confirm that: the main component of Smilax lanceaefolia Roxb. Var.opaca A.DC. is saponin (total saponins about 8～9%), and the structure of chemical compound is in determining.

Summary of the invention

The object of the present invention is to provide a kind of method and medical application thereof of compound kuhseng preparation for injection, to increase clinical pharmaceutical dosage form; Simultaneously, the legal clinical using dosage that overcomes existing injection can not satisfy practical clinical needs and the unsettled shortcoming of injection formulation (oxymatrine content in put procedure reduces), reaches the purpose that improves clinical efficacy.Another object of the present invention provides the new medical application of Compound Kushen ', to satisfy patient's needs.

The specific embodiment

For achieving the above object, the present invention is implemented by following technical proposals.

1, the preparation of injection use compound Radix Sophorae Flavescentis intermediate

The major technique of injection use compound Radix Sophorae Flavescentis intermediate preparation method is characterised in that:

(1) raw material of the compound kuhseng preparation for injection in the summary of the invention is Radix Sophorae Flavescentis and Smilax lanceaefolia Roxb. Var.opaca A.DC.; Wherein Radix Sophorae Flavescentis is the dry root of cassia leguminous plant Radix Sophorae Flavescentis (Sophora flavscens Alt.); Smilax lanceaefolia Roxb. Var.opaca A.DC. is the dry rhizome of liliaceous plant Xiao Rhizoma Smilacis Chinensis (Heterosmilaxjaponica Kunth.) or short column Xiao Rhizoma Smilacis Chinensis (Heterosmilax yunnanensis Gagnep.); The ratio of Radix Sophorae Flavescentis and Smilax lanceaefolia Roxb. Var.opaca A.DC. is 5: 2～2: 1, preferred 7: 3.

(2) extract solvent with Radix Sophorae Flavescentis and Smilax lanceaefolia Roxb. Var.opaca A.DC. decoction pieces or coarse powder utilization and distinguish and extract back merging or mixed extraction separately, obtain extracting solution.The extraction solvent of indication is water, 0.1%～60% diluted acid, 1%～80% alcohol, preferred diluted acid and water; Behind the preferred first diluted acid percolation, again with water boiling and extraction; The diluted acid percolation adopts spendable organic acid of pharmacy or mineral acid, its concentration 0.1%～60%, percolation after soaking 2～60 hours under 5 ℃～40 ℃ the condition; Sour preferred 5%～50% used acetic acid or hydrochloric acid of percolation wherein, preferred 10 ℃～30 ℃ of percolation temperature, preferred 8～48 hours of the soak time of percolation; The reflux, extract, condition of indication is to carry out reflux, extract, 1～4 time, preferred 0%～60% water-alcohol solvent, reflux, extract, 2 times with 0%～80% water-alcohol solvent; The alcohol of indication is the aliphatic alcohol of 1～5 carbon, particular methanol and ethanol.

(3) normal pressure of above-mentioned resulting extracting solution under≤100 ℃ temperature concentrates or concentrating under reduced pressure, obtains concentrate; Preferred 30～80 ℃ concentrating under reduced pressure.

(4) concentrate obtains alcohol soluble substance through the process of repeatedly precipitate with ethanol-cold preservation-filtration-recovery solvent; Precipitate with ethanol-the cold preservation of indication-filtration-recovery dissolving agent process, its precipitate with ethanol condition are to utilize the alcohol of high concentration to join in the medicine concentrated solution.For the first time during precipitate with ethanol, determining alcohol in the medicinal liquid reaches 30%～80%, preferred 50%～70%, for the second time during precipitate with ethanol, the determining alcohol in the medicinal liquid reaches 50%～90%, and preferred 70%～85%, for the third time during precipitate with ethanol, determining alcohol in the medicinal liquid reaches 80%～95%, and is preferred 85%～92%, and the precipitate with ethanol number of times is preferably three times; The refrigerated condition of alcohol deposit fluid is-20 ℃～10 ℃, 4～48 hours, and preferred-16 ℃～8 ℃, 8～36 hours; Filtercondition is plate-and-frame filtration or centrifugal filtration or membrane filtration; Filtered liquid medicine reclaims solvent, 30 ℃～90 ℃ of recovered temperatures, preferred 40 ℃～75 ℃ under reduced pressure; The relative density of each concentrated extract is 1.02～1.40 (60 ℃), preferred 1.05～1.35 (60 ℃).

(5) alcohol soluble substance returns molten, cold preservation, filtration with a certain proportion of water for injection, obtains filtrate; The water of indication return molten condition for add 1: 1～1: 10 (medicinal liquid: water liquid, W/V), preferred 1: 1.5～1: 6 (medicinal liquid: water liquid, W/V); Water returns the condition of solution cold preservation and spends the night preferred 5 ℃～-10 ℃ for being placed on 10 ℃～-20 ℃; Cold preservation liquid filtercondition is centrifugal filtration or 0.45 μ m membrane filtration; The concentrated condition of filtrate is the concentrating under reduced pressure at 45 ℃～95 ℃; The density of pharmaceutical intermediate is 1.10～1.40 (60 ℃), preferred 1.15～1.35 (60 ℃).The condition of placing the intermediate freezer is 10 ℃～-20 ℃, preferred 5 ℃～-10 ℃.

(6) filtrate is condensed into certain density, promptly prepares pharmaceutical intermediate; Measure the medicament contg and the finger printing of intermediate, be positioned in the freezer.The content of indication pharmaceutical intermediate is that the summation of oxymatrine, matrine and sophocarpine is not less than 5mg/ml, preferably is not less than 15mg/ml; Oxymatrine is a main peaks in the finger printing of indication intermediate, and is made up of matrine, sophocarpine etc.

2, the preparation of the FUFANG KUSHEN ZHUSHEYE of different size

The preparation method of the FUFANG KUSHEN ZHUSHEYE of different size is characterised in that:

(1) pharmaceutical intermediate that utilizes foregoing invention content 1 to prepare adds solvent for injection, through activated carbon decolorizing, filtration; The solvent for injection of indication comprise water for injection or normal saline or etc. ooze Glucose Liquid, add 0.1%～10% injection active carbon in 40 ℃～100 ℃ heating decolouring in 5～30 minutes, preferred 1%～8% injection active carbon was 60 ℃～100 ℃ heating 5～20 minutes; The filtercondition of destaining solution is that 0.22 μ m supermicro filtration membrane filters.

(2) through the accent pH-cold preservation-filter process more than 2 times, obtain " injection medicinal liquid ".Indication through the accent pH-cold preservation-filter process more than 2 times, be to transfer earlier pH to 7.0～9.0, preferred pH7.5～8.8, cold preservation is filtered; Transfer pH to 3.0～6.5 again, preferred pH4.0～5.5, cold preservation is filtered; Transfer pH to 4.0～7.5 again, preferred pH5.0～7.0, cold preservation is filtered; Transfer pH to 7.5～9.5 again, preferred pH7.5～9.0, cold preservation is filtered; The refrigerated condition of indication spends the night for being placed on 10 ℃～-20 ℃, preferred 5 ℃～-10 ℃; The filtercondition of indication is that 0.10～0.45 μ m supermicro filtration membrane filters preferred 0.22 μ m.

(3) " injection medicinal liquid " measures content of medicines and finger printing; The content of indication is that the summation of oxymatrine, matrine and sophocarpine in the injection is not less than 2mg/ml, preferably is not less than 10mg/ml.The finger printing of indication is based on oxymatrine, comprises matrine, sophocarpine etc.

(4) the injection medicinal liquid is through fine straining; The fine straining system of indication filters through 0.10～0.22 μ m.

(5) fill, seal, sterilize, promptly get the injection of different size; The specification of indication is 1～500ml, preferred 2ml, 5ml, 10ml, 12ml, 20ml, 25ml, 50ml, 100ml, 250ml and 500ml.

3, the preparation of compound light-yellow sophora root lyophilized powder

The principal character of invention compound light-yellow sophora root lyophilized powder is to be prepared according to certain proportioning with framework material by pharmaceutical intermediate or " injection medicinal liquid ", embedding, cold doing.

Method 1

Utilize the pharmaceutical intermediate and the framework material of 1 preparation of foregoing invention content to prepare according to certain proportioning, through activated carbon decolorizing, filtration, the accent pH-cold preservation-filter process through more than 2 times obtains the injection medicinal liquid, measures content of medicines and finger printing; Behind fine straining, embedding is put into fridge in cillin bottle again, according to the freezing curve lyophilization, takes out the compound light-yellow sophora root lyophilized powder that obtains, and aluminium lid is pressed mouth.

(1) ratio range of pharmaceutical intermediate and framework material is that (intermediate: framework material, proportioning W/W) was prepared, preferred 1: 1 in 1: 0.5～1: 3.

(2) framework material is mannitol or sodium chloride or glucose.

(3) specification requirement of each preparation process of above-mentioned indication is with (1) in the summary of the invention 2～(4).

Method 2

Utilize " the injection medicinal liquid " of 2 preparations of foregoing invention content to prepare according to certain proportioning, behind fine straining, measure content of medicines and finger printing with framework material; Embedding is put into fridge in cillin bottle, according to the freezing curve lyophilization, take out the compound light-yellow sophora root lyophilized powder that obtains, and aluminium lid is pressed mouth.

(1) ratio range of " injection medicinal liquid " and framework material is that (solid content of injection medicinal liquid: framework material, W/W) proportioning was prepared, preferred 1: 1 and 1: 1.5 in 1: 0.5～1: 3.

(2) framework material is mannitol or sodium chloride or glucose.

(3) fine straining of indication ties up in 100 grades the GMP workshop and filters preferred 0.22 μ m with 0.10～0.45 μ m supermicro filtration membrane.

Method 3

Utilize " the injection medicinal liquid " of 2 preparations of foregoing invention content, embedding is put into fridge in cillin bottle, according to the freezing curve lyophilization, obtain the compound light-yellow sophora root lyophilized powder.

Indication common technology feature in the method 1～3:

(1) the cillin bottle fill is every cillin bottle fill 2ml or 5ml or 10ml or 15ml or 20ml, preferred 5ml and 10ml;

(2) lyophilization of indication comprises pre-freeze and sublimation stage; The pre-freeze stage: pre-freeze temperature-10 ℃～-60 ℃, preferred-20 ℃～-50 ℃; Sublimation stage: vacuum is reached below the 30Pa, heat up to product according to 0.5～2 ℃ programming rate per hour, when product temperature reaches-5 ℃～-10 ℃, heat up with 1～5 ℃ speed per hour, temperature until product is raised to 21 ℃～31 ℃, continue to keep 2～8 hours, make temperature of articles overlap (differing in 1 ℃) with shelf temperature, in 100 grades GMP workshop, put into aseptic filtration gas, chamber door can be opened, unpack, seal rapidly, promptly get the compound light-yellow sophora root freeze-dried powder.

(3) total amount of oxymatrine, matrine and the sophocarpine in the indication freeze-dried powder is that 0.01～0.3g/ props up, and preferred 0.03～0.2g/ props up.

4, the preparation of injection use compound Radix Sophorae Flavescentis powder pin

The principal character of invention injection use compound Radix Sophorae Flavescentis powder pin be by " injection medicinal liquid " directly spray or with framework material according to certain proportioning prepare the back spray drying, packing prepares.

Method 1:

After being adjusted to certain relative density, " the injection medicinal liquid " that utilizes summary of the invention 2 to prepare carry out spray drying, being sent to the whirlwind platform through the dry product of aerosol apparatus ejection makes dampness separate with dry powder, dry powder closes in the catcher, under aseptic condition, be sub-packed in the sterilization Carbenicllin bottle, seal, promptly obtain injection use compound Radix Sophorae Flavescentis powder pin.Its important technological parameters:

(1) relative density of indication is 1.01～1.30, preferred 1.05～1.15.

(2) the spray drying condition of indication is that whiff pressure is adjusted to 2～5kg/cm ², draft 4～8kg/cm ², 150～300 ℃ of inlet temperatures, 50～100 ℃ of outlet temperatures.

(3) total amount of oxymatrine, matrine and the sophocarpine in the indication injection use compound Radix Sophorae Flavescentis powder pin is that 0.01～0.3g/ props up, and preferred 0.03～0.2g/ props up.

Method 2:

Utilize " the injection medicinal liquid " of summary of the invention 2 preparations to prepare according to certain proportioning, behind fine straining, measure content of medicines and finger printing with framework material; Spray drying is sent to the whirlwind platform through the dry product of aerosol apparatus ejection dampness is separated with dry powder, and dry powder closes in the catcher, is sub-packed under aseptic condition in the Carbenicllin bottle of sterilizing, and seals, and promptly obtains injection use compound Radix Sophorae Flavescentis powder pin.

(1) indication obtain injection medicinal liquid and framework material by 1: 0.5～1: 3 (solid content of injection medicinal liquid: framework material, W/W) proportioning is prepared, preferred 1: 1; Framework material is mannitol or sodium chloride or glucose.

(2) fine straining of indication is to filter preferred 0.22 μ m in 100 grades GMP workshop with 0.10～0.45 μ m supermicro filtration membrane.

5, the new medical application of compound kuhseng preparation for injection

Each medicine in the summary of the invention 1～4 has antitumor action, chemotherapeutic is had efficacy enhancing and toxicity reducing effect, analgesic activity, leukogenic effect, raising immunization, antihepatitic activity etc.

The advantage of foregoing invention and effect are to have strengthened stability of drug, can not produce the reduction of tiring, and more can bring into play the due clinical effectiveness of medicine, are convenient to transportation simultaneously, stock, and reduce breakage rate, reduce cost, and have enriched the dosage form of Compound Kushen '.

The following examples are used to further specify the present invention, but they are not to attempt in office where face limits the scope of the invention.

Experimental example 1

Prescription: Radix Sophorae Flavescentis 7000g Smilax lanceaefolia Roxb. Var.opaca A.DC. 3000g makes injection use compound Radix Sophorae Flavescentis intermediate

Preparation method:

Radix Sophorae Flavescentis and Smilax lanceaefolia Roxb. Var.opaca A.DC. are ground into coarse powder, mixing, put in the percolation cylinder, add the 1% acetic acid dipping 24 hours that is equivalent to 6～8 times of doses (according to the difference of seasonal temperature and difference, 24 hours winters, 8 hours summers), carry out percolation, collect percolate, percolate concentrating under reduced pressure (below 75 ℃) is to 900～1200ml[or relative density 1.20 (60 ℃)], device is deposited in addition.

Medicinal residues add 6～8 times of amount waters for injection, decoct 2 times, each 1 hour, filter, merge decoction liquor, are evaporated to 900～1200ml[or relative density 1.20 (60 ℃)], to put coldly, filtration obtains supernatant.

The concentrated solution of supernatant and above-mentioned percolate merges, and uses ethanol precipitate with ethanol 3 times.Add for the first time ethanol and make the alcohol amount that contains of solution reach 60%, standing over night or cold preservation (0～5 ℃, more than the 24h), 0.45 μ m microporous filter membrane filters, and the supernatant decompression recycling ethanol is concentrated into relative density 1.20 (60 ℃); Add ethanol again and make that to contain alcohol amount be 80%, the same processing; Add ethanol for the third time and make that to contain alcohol amount be 90%, the same processing obtains the alcohol soluble substance extractum of relative density 1.30 (60 ℃).

Alcohol soluble substance extractum with the water for injection of 3000～3500ml return molten, spend the night 0 ℃ of cold preservation, 0.45 μ m membrane filtration, obtain filtrate.Filtrate promptly obtains pharmaceutical intermediate 1000ml at relative 1.40 (60 ℃) that are evaporated to below 80 ℃; The freezer that is positioned over-5 ℃～5 ℃ is preserved.

The determining and to determine according to the total amount of wherein oxymatrine, matrine and sophocarpine of pharmaceutical intermediate relative density, require its content greater than 100mg/ml, oxymatrine is a main peaks in the finger printing of intermediate, and is made up of matrine, sophocarpine etc.

Experimental example 2

Preparation method:

Radix Sophorae Flavescentis powder is broken into coarse powder, mixing, put in the percolation cylinder, add the 1% acetic acid dipping 24 hours that is equivalent to 8～10 times of doses (according to the difference of seasonal temperature and difference, 24 hours winters, 8 hours summers), carry out percolation, collect percolate, percolate concentrating under reduced pressure (below 75 ℃) is to 600～700ml[or relative density 1.20 (60 ℃)], device is deposited in addition.

Medicinal residues and Smilax lanceaefolia Roxb. Var.opaca A.DC. coarse powder add 6～8 times of amount waters for injection, decoct 2 times, each 1 hour, filter, merge decoction liquor, are evaporated to 800～1000ml[or relative density 1.20 (60 ℃)], to put coldly, filtration obtains supernatant.

The concentrated solution of supernatant and Radix Sophorae Flavescentis percolate merges, with ethanol precipitate with ethanol 3 times.Add for the first time ethanol and make the alcohol amount that contains of solution reach 60%, standing over night or cold preservation (0～5 ℃, more than the 24h), 0.45 μ m microporous filter membrane filters, and the supernatant decompression recycling ethanol is concentrated into relative density 1.20 (60 ℃); Add ethanol again and make that to contain alcohol amount be 80%, the same processing; Add ethanol for the third time and make that to contain alcohol amount be 90%, the same processing obtains the alcohol soluble substance extractum of relative density 1.30 (60 ℃).

Alcohol soluble substance extractum with the water for injection of 2000～3000ml return molten, spend the night 0 ℃ of cold preservation, 0.45 μ m membrane filtration, obtain filtrate.Filtrate promptly obtains pharmaceutical intermediate 1000ml at relative 1.40 (60 ℃) that are evaporated to below 80 ℃; The freezer that is positioned over-5 ℃～5 ℃ is preserved.

Experimental example 3

Preparation method:

Radix Sophorae Flavescentis powder is broken into coarse powder, and mixing is put in the percolation cylinder, add the 0.5% salt acid dip 24 hours that is equivalent to 8～10 times of doses, carry out percolation, collect percolate, percolate concentrating under reduced pressure (below 75 ℃) is to 600～700ml[or relative density 1.20 (60 ℃)], device is deposited in addition.

The concentrated solution of supernatant and Radix Sophorae Flavescentis percolate merges, with ethanol precipitate with ethanol 3 times.Add for the first time ethanol and make the alcohol amount that contains of solution reach 60%, standing over night or cold preservation (0～5 ℃, more than the 24h), 0.45 μ m microporous filter membrane filters, and the supernatant decompression recycling ethanol is concentrated into relative density 1.25 (60 ℃); Add ethanol again and make that to contain alcohol amount be 80%, the same processing; Add ethanol for the third time and make that to contain alcohol amount be 90%, the same processing obtains the alcohol soluble substance extractum of relative density 1.30 (60 ℃).

Alcohol soluble substance extractum with the water for injection of 2000～3000ml return molten, spend the night 0 ℃ of cold preservation, 0.45 μ m membrane filtration, obtain filtrate.Filtrate promptly obtains pharmaceutical intermediate at relative 1.40 (70 ℃) that are evaporated to below 80 ℃; The freezer that is positioned over-5 ℃～5 ℃ is preserved.

The determining and to determine according to the total amount of wherein oxymatrine, matrine and sophocarpine of pharmaceutical intermediate relative density, require its content greater than 300mg/ml, oxymatrine is a main peaks in the finger printing of intermediate, and is made up of matrine, sophocarpine etc.

Experimental example 4

Prescription: Radix Sophorae Flavescentis 1400g Smilax lanceaefolia Roxb. Var.opaca A.DC. 600g

Preparation method:

Any method preparation according to example 1～3 becomes pharmaceutical intermediate.The total amount of the oxymatrine in the pharmaceutical intermediate, matrine and sophocarpine should be main peaks with the oxymatrine in the finger printing of intermediate, and be made up of matrine, sophocarpine etc. greater than 100mg/ml.

Qualified pharmaceutical intermediate adds water for injection to 900ml, filters through 3% injection active carbon reflux decolouring 10 minutes, 0.22 μ m supermicro filtration membrane.

Filtrate is transferred pH-cold preservation-filter process through 4 times.For the first time transfer to pH7.8, spend the night 0 ℃ of left and right sides cold preservation, 0.22 μ m supermicro filtration membrane filters; For the second time transfer to pH5.0, handle with method; Transfer to pH6.8 for the third time, handle with method.Transfer pH to carry out with 20%NaOH and 40% acetic acid.

After transferring pH to 8.8 the 4th time, add the injection water and transfer to 1000ml, spend the night-5 ℃ of left and right sides cold preservations, 0.22 μ m supermicro filtration membrane filters.Embedding becomes the 2ml/ bottle respectively, 5ml/ bottle, 10ml/ bottle, 12ml/ bottle, 20ml/ bottle, 25ml/ bottle.Seal and, pack, passed examination, warehouse-in 105 ℃ of sterilizations 30 minutes.

Experimental example 5

Prescription: Radix Sophorae Flavescentis 1400g Smilax lanceaefolia Roxb. Var.opaca A.DC. 600g sodium chloride 18g

Preparation method:

Sodium chloride adds qualified pharmaceutical intermediate after dissolving with proper amount of water for injection, and adds water for injection to 900ml, filters through 3% injection active carbon reflux decolouring 10 minutes, 0.22 μ m supermicro filtration membrane.

After transferring pH to 8.8 the 4th time, add the injection water and transfer to 2000ml, spend the night-5 ℃ of left and right sides cold preservations, 0.22 μ m supermicro filtration membrane filters.Embedding becomes the 50ml/ bottle respectively, 100ml/ bottle, 250ml/ bottle, 500ml/ bottle.Seal and, pack, passed examination, warehouse-in 105 ℃ of sterilizations 30 minutes.

Experimental example 6

Prescription: Radix Sophorae Flavescentis 1400g Smilax lanceaefolia Roxb. Var.opaca A.DC. 600g glucose 100g

Preparation method:

Glucose adds qualified pharmaceutical intermediate after dissolving fully with proper amount of water for injection, and with water for injection to 900ml, filter through 3% injection active carbon reflux decolouring 10 minutes, 0.22 μ m supermicro filtration membrane.

After transferring pH to 7.8 the 4th time, add injection water water and transfer to 2000ml, spend the night-5 ℃ of left and right sides cold preservations, 0.22 μ m supermicro filtration membrane filters.Embedding becomes the 50ml/ bottle respectively, 100ml/ bottle, 250ml/ bottle, 500ml/ bottle.Seal and, pack, passed examination, warehouse-in 105 ℃ of sterilizations 30 minutes.

Experimental example 7

Preparation method:

In 100 grades GMP workshop, after framework material (mannitol 200g) adding proper amount of water for injection is dissolved fully, add qualified pharmaceutical intermediate 200g, add water for injection to 450ml, be heated to 80 ℃ and be incubated 30 minutes through 3g injection active carbon, take off charcoal 0.22 μ m supermicro filtration membrane when being chilled to 45 ℃ and filter.

After transferring pH to 7.8 the 4th time, add the injection water and transfer to 500ml, spend the night, filter, semi-finished product inspection (measuring content of medicines and finger printing) with 0.22 μ m supermicro filtration membrane-5 ℃ of left and right sides cold preservations.Embedding in the 7ml cillin bottle, every bottled 2ml; Or embedding is in the 10ml cillin bottle, every bottled 5ml; Or embedding is in the 20ml cillin bottle, and every bottled 10ml covers butyl rubber bung, keeps venthole, puts into freeze dryer and carries out lyophilization according to the freeze-drying curve of design; When the vacuum in the drying baker and pumping hole vacuum near and products temperature when overlapping with plate temperature, close refrigeration system and vacuum system, compress bottle stopper, sample is taken out in venting, aluminium lid is pressed mouth, packs, passed examination is put in storage; Promptly get compound light-yellow sophora root freeze-drying injection.

Can not form solid skeleton after the simple compound light-yellow sophora root solution lyophilizing, vibrations back sample is promptly powdered a little, may influence its redissolution, so need it is carried out the skeleton screening with mannitol or sodium chloride or glucose etc. with different ratios, the ratio of compound light-yellow sophora root intermediate and framework materials such as mannitol is within 1: 0.5～3 (W/W) scope, choose 1: 1 and 1: 1.5, sample appearance is all good after the lyophilizing, and easily dissolving.

Freeze-drying curve comprises pre-freeze and two stages of distillation:

The pre-freeze stage: the eutectic point of this product is-15～-20 ℃, and freeze-drying process generally is reduced to sample temperature below the eutectic point-10 ℃, begins distillation; The pre-freeze temperature is low more, and sample is difficult for flying upward in intensification, sublimation process, the particle uniform and smooth of lyophilized products, and dissolution velocity is fast.Therefore, select that the pre-freeze temperature begins to heat up, distillation is preferable when-30 ℃ or lower temperature.

Sublimation stage: vacuum is reached below the 30Pa driving condenser, preferred 9～20Pa heats up to product according to 0.5～2 ℃ programming rate per hour, when product temperature reaches-5 ℃～-10 ℃, heat up with 1～5 ℃ speed per hour, be raised to 21 ℃～31 ℃, continue to keep 2～8 hours until the temperature of product, make temperature of articles overlap (differing in 1 ℃) with shelf temperature, put into aseptic filtration gas, chamber door can be opened, unpack, seal rapidly, promptly get the cold-dry powder pin.For making the aqueous vapor distillation fully, the residual moisture of dried frozen aquatic products is low, but intensification and temperature retention time proper extension.

Adopt the test of compound light-yellow sophora root freeze-drying injection to rabbit, the result shows that this product does not produce obvious irritation to the rabbit auricular vein, and by giving guinea pig intraperitoneal injection, the result shows that Cavia porcellus does not cause the systemic anaphylaxis reaction; The hemolytic test method is observed the 2mg/ml injection routinely, and the result was presented in 4 hours does not have obvious haemolysis to tame rabbit erythrocyte, also show cell agglutination phenomenon not.

The compound light-yellow sophora root goods skeleton that the present invention makes is loose, and the particle uniform and smooth jolts non-friablely, and adding water can dissolve rapidly.The total amount of the oxymatrine in the freeze-dried powder, matrine and sophocarpine is 0.01～0.3g/ bottle, preferred 0.03～0.2g/ bottle.

Experimental example 8

Framework material is selected sodium chloride for use, presses the ratio 1: 1.5 of compound light-yellow sophora root intermediate and framework material (sodium chloride), and is surplus all with embodiment 7.

Experimental example 9

Framework material is selected glucose for use, presses the ratio 1: 1 of compound light-yellow sophora root intermediate and framework material, and is surplus all with embodiment 7.

Experimental example 10

According to the qualified injection medicinal liquid of preparation among the embodiment 4, directly (whiff pressure is adjusted to 4kg/cm in spraying ², draft 6kg/cm ²180 ℃ of inlet temperatures, 80 ℃ of outlet temperatures), be sent to the whirlwind platform through the dry product of aerosol apparatus ejection dampness is separated with dry powder, dry powder closes in the catcher, under aseptic condition, be sub-packed in the sterilization Carbenicllin bottle, seal packing, passed examination, warehouse-in promptly obtains injection use compound Radix Sophorae Flavescentis powder pin.The total amount of oxymatrine, matrine and sophocarpine in the injection use compound Radix Sophorae Flavescentis powder pin is that 0.01～0.3g/ props up, and preferred 0.03～0.2g/ props up.

Experimental example 11

Qualified injection medicinal liquid and framework material according to preparation among the embodiment 4 are prepared according to certain proportioning, behind the 0.22 μ m fine straining, and directly spraying, all the other preparation process such as spray condition are with embodiment 10.

Injection medicinal liquid and framework material by 1: 0.5～1: 3 (solid content of injection medicinal liquid: framework material, W/W) proportioning is prepared, preferred 1: 1 and 1: 1.5; Framework material is mannitol or sodium chloride or glucose.The total amount of oxymatrine, matrine and sophocarpine in the injection use compound Radix Sophorae Flavescentis powder pin is that 0.01～0.3g/ props up, and preferred 0.03～0.2g/ props up.

Experimental example 12

The analgesic test of compound kuhseng preparation for injection

One. measure the analgesic activity of FUFANG KUSHEN ZHUSHEYE with the mice hot plate method

1. intravenous injection FUFANG KUSHEN ZHUSHEYE is to the influence of mice analgesic activity

Mice is all with female, laboratory temperature remains on 20 ℃, mice placed on 55 ℃ the constant temperature hot plate, to lick the index of metapedes as the pain reaction, put hot plate to the pain threshold of sufficient response time occurring licking with stopwatch record white mice as this Mus, the mice of picking out pain threshold 5-30 second is as experimental subject, retrial in second day, with allergy, late pure or like dancing animal all to reject need not, mice is divided into 5 groups at random by body weight and basic pain threshold, normal control group (normal saline 0.2ml/ only), three various dose groups of FUFANG KUSHEN ZHUSHEYE (0.8g/kg, 1.6g/kg and 3.2g/kg) and positive controls (pethidine 25mg/kg).Except that the pethidine intramuscular injection, all the other respectively organize equal tail vein injection.Measured the threshold of pain respectively once in 0.5,1,2,3,4 hour after the administration, totally 5 times.Calculate the pain threshold of matched group and administration group and organize a t check, pain threshold surpasses 60 seconds after the medication, promptly gives taking-up for preventing to scald, and still calculates with 60 seconds.

As seen from Table 1, FUFANG KUSHEN ZHUSHEYE three dosage 0.8g/kg, 1.6g/kg and 3.2g/kg all can obviously improve pain threshold, half an hour after the administration, all show analgesic activity, along with the increase of FUFANG KUSHEN ZHUSHEYE dosage, its acting duration also increases, and analgesia intensity also strengthens along with the increase of dosage, high dose group pain threshold raising rate can reach 83.74%, sustainable three hours of analgesia time.Experimental result proves that FUFANG KUSHEN ZHUSHEYE has significant analgesia role to the pain that thermostimulation causes.

Table 1 FUFANG KUSHEN ZHUSHEYE iv is to the influence of mice hot plate pain threshold (X ± SD)

Compare with matched group, ^*P＜0.05, ^*P＜0.01, ^{* *}P＜0.001

2. the lumbar injection FUFANG KUSHEN ZHUSHEYE is to the influence of mice analgesic activity

Experimental technique, date processing are with test 1, only changing route of administration is lumbar injection, mice is divided into 5 groups, normal control group (normal saline 0.2ml/ is only given in the abdominal cavity), three various dose groups of FUFANG KUSHEN ZHUSHEYE (1.6,3.2,6.4g/kg) with positive control pethidine group (25mg/kg), except that the pethidine subcutaneous administration, all the other respectively organize equal intraperitoneal injection.

Table 2 FUFANG KUSHEN ZHUSHEYE ip is to the influence of mice hot plate pain threshold (X ± SD)

Annotate: compare * P＜0.01, * * P＜0.001 with matched group

The results are shown in Table 2, three dosage groups of FUFANG KUSHEN ZHUSHEYE and matched group more all have marked difference, all can obviously improve the threshold of pain, after the administration between the 0.5-2h analgesic activity the strongest, threshold of pain raising rate reaches as high as 60.29%.Along with the prolongation analgesic activity of time weakens the sustainable 3h of analgesia time gradually.Medicine analgesia intensity strengthens along with the increase of concentration, and all tangible dose-effect relationship of each time point finds out that from experimental result FUFANG KUSHEN ZHUSHEYE ip has significant analgesia role to the pain that thermostimulation causes, its analgesia time is similar to pethidine with intensity.

Two. stimulate writhing method to measure the analgesic activity of FUFANG KUSHEN ZHUSHEYE with acid to mice

Get healthy mice, male female half and half, be divided into 5 groups at random by body weight: normal control group (the tail vein is only given normal saline 0.2ml/), three various dose groups of FUFANG KUSHEN ZHUSHEYE (0.8g/kg, 1.6g/kg and 3.2g/kg) and positive control pethidine group (25mg/kg).Except that the positive controls intramuscular injection, all the other respectively organize equal tail vein injection administration, inject 1.2% acetic acid 0.2ml for respectively after half an hour every mouse peritoneal, calculate behind the 5min body number of times (elongation hind leg, abdominal part shrink and the distortion body) takes place typically to turn round, write down in each mice 15min and turn round the body number of times, relatively each administration group and matched group turn round the body number of times, organize a t check, and calculate the slip that the administration group is turned round the body number of times.

The influence that table 3 FUFANG KUSHEN ZHUSHEYE iv reacts mouse writhing (X ± SD)

Compare with matched group: * P＜0.05, * * P＜0.01, * * * P＜0.001

As shown in Table 3, after the injected in mice FUFANG KUSHEN ZHUSHEYE, can obviously reduce with acetic acid and cause the incidence rate of turning round body, the body slip of turning round of three various dose groups reaches 38.29%, 54.63%, 60.52% respectively, and its slip increases along with the increase of drug dose.

2. intramuscular injection FUFANG KUSHEN ZHUSHEYE is to the influence of mice analgesic activity

Experimental technique, date processing is with " 2.1 ", only changing route of administration is intramuscular injection, and mice is divided into 5 groups: the normal control group gives normal saline 0.2ml/ only, three various dose groups of FUFANG KUSHEN ZHUSHEYE (2.4,4.8,9.6g/kg) and positive control pethidine group (25mg/kg).Except that the positive controls subcutaneous injection, all the other respectively organize equal administered intramuscular.

The influence that table 4 FUFANG KUSHEN ZHUSHEYE im reacts mouse writhing (X ± SD)

The results are shown in Table 4, the FUFANG KUSHEN ZHUSHEYE group is compared with matched group can obviously reduce because of what acetic acid caused and is turned round the body incidence rate, the slip of three various dose groups is respectively 50.00%, 57.91% and 89.55% its inhibitory action increases and strengthens along with drug level, relatively has the difference of highly significant with matched group, illustrate that the FUFANG KUSHEN ZHUSHEYE Dichlorodiphenyl Acetate stimulates the pain that causes to have significant analgesia role, the heavy dose of group of compound light-yellow sophora root injection is turned round body number of times slip and is compared with pethidine, statistically there was no significant difference.

Experimental example 13

Antitumor Effects

1. FUFANG KUSHEN ZHUSHEYE is to mice S ₁₈₀The influence of solid tumor growth

40 of mices, every at right armpit subcutaneous vaccination S ₁₈₀Ascites tumor 0.1 (contains 10 ⁴Individual cell) transfers solid tumor to.Animal was divided into 5 groups, 0.2ml/ positive drug group of matched group iv normal saline ip cyclophosphamide 35mg/kg at random in second day; Give FUFANG KUSHEN ZHUSHEYE for three groups in addition, difference ip 3.2,6.4,12.8g/kg, be administered once the next day of positive drug, all the other are respectively organized and are administered once every day, amount to administration 11 days, and animal is dissected in drug withdrawal next day, the taking-up tumor is also weighed, relatively tumor is heavy with matched group for each group, organizes a t check, calculates the heavy suppression ratio of tumor.

Table 5 FUFANG KUSHEN ZHUSHEYE is to mouse tumor S ₁₈₀The influence of growth (X ± SD)

Compare * P＜0.05, * * P＜0.01, * * * P＜0.001 with matched group

The results are shown in Table 5, FUFANG KUSHEN ZHUSHEYE is little, in, big three dosage groups, the heavy suppression ratio of tumor is respectively 38.9%, 40.9%, 58.5%, with matched group significant difference is arranged relatively, illustrates that FUFANG KUSHEN ZHUSHEYE has obvious inhibition S ₁₈₀The effect of tumor growth.Experiment repeats three batches, and the result is similar.

2. compound light-yellow sophora root is to rat liver cancer H ₂₂The influence of solid tumor growth

Tumor source H ₂₂Replace S ₁₈₀, other method comprises that animal, grouping, drug dose, observation index etc. and this tests 1 identical.

Table 6 FUFANG KUSHEN ZHUSHEYE is to rat liver cancer H ₂₂The influence of growth (X ± SD)

Annotate: with matched group ratio, * P＜0.05, * * P＜0.01, * * * P＜0.001

The results are shown in Table 6, FUFANG KUSHEN ZHUSHEYE is little, in, big three dosage groups, the heavy suppression ratio of tumor is respectively 50.0%, 51.4%, 51.2%, with matched group significant difference is arranged relatively, illustrates that FUFANG KUSHEN ZHUSHEYE has the effect of obvious inhibition H22 tumor growth.Repeated experiments, the result is similar.

Experimental example 14

Effect research to immunologic function

1, the influence that the tumor-bearing mice splenocyte is transformed

Method: 54 of BALB/C male mices, body weight 18-20g is divided into 6 groups at random, and 9 every group, II (contains 10 at the right axil of mice/subcutaneous vaccination S180 ascites tumor 0.1ml in advance ⁶Individual oncocyte), beginning administration in second day, normal control group, lotus tumor group ip normal saline 0.2ml/20g.The positive drug group is po QINGCHUN BAO 0.4ml/20g, three groups of difference of FUFANG KUSHEN ZHUSHEYE ip3.2,6.4,12.8g/kg (volume is 0.2ml/20g).Successive administration 12 days, 24 hours broken end sacrificed by exsanguination mices are taken out spleen after the drug withdrawal under aseptic condition, make splenocyte suspension with RPMI-1640 (containing 10% calf serum), and mouse boosting cell is adjusted to 5 * 10 ⁶Cell/ml is added in 96 well culture plates, and every hole 100 μ l, each spleen add double (promptly adds ConA, and does not add ConA).ConA 20 μ l (concentration is 100 μ g/ml), RPMI-1640 80 μ l make every hole to 200 μ l volume, in 5%CO ²Cultivated 72 hours in 37 ℃ of calorstats, stop cultivating preceding 24 hours, every hole adds ³H-TdR 3.7 * 109 (iucr).With cell harvestor (TITERTEK CELL HARVESTER 550) collecting cell on 49 type glass fiber filter paper, use distilled water, 5% 3 ammonia acetic acid, absolute ethanol washing successively, filter paper is put 60 ℃ of incubator oven dry, be added in the cup that contains the 5ml scintillation solution, participate in radioactivity (cpm) through liquid scintillation instrument (BECKMEN LS 9800) survey, calculate stimulation index (SI) then, that is:

The influence that table 7 FUFANG KUSHEN ZHUSHEYE transforms the tumor-bearing mice splenocyte (X ± SD)

With lotus tumor matched group ratio, * P＜0.05, * * P＜0.01, * * * P＜0.001

The results are shown in Table 7, behind the mouse inoculation oncocyte, stimulation index is starkly lower than normal group (P≤0.05).Give QINGCHUN BAO and FUFANG KUSHEN ZHUSHEYE is little, in, behind big three dosage, stimulation index obviously rises, and significant difference is relatively arranged, P＜0.05 with lotus tumor matched group.The results suggest FUFANG KUSHEN ZHUSHEYE stimulates the tumor-bearing mice lymphocyte transformation that facilitation is arranged to COnA, promptly the T lymphocyte immunologic function is had potentiation.

2. the influence that hemolysis plaque (PFC) is formed

Grouping, administration drench to be changeed experiment with " three .1 ", in administration in the 8th day after 10 minutes, with 2 * 10 ⁸/ ml SRBC the mice of saluting, every Mus iv0.1ml.Dissect after 5 days and get spleen, preparation splenocyte suspension and counting change experiment with drenching.Get that test tube is some to be gone, every pipe adds splenocyte 0.2%SRBC, 1: 10 each 0.5ml of guinea pig serum successively, and mixing was put in 30 ℃ of water-baths incubation 1 hour, and 3000rpm gets supernatant after centrifugal 5 minutes, writes down trap under UV-754 spectrophotometer 413nm wavelength.

Table 8 FUFANG KUSHEN ZHUSHEYE is to the influence of tumor-bearing mice PFC (X ± SD)

Compare with lotus tumor matched group: * P＜0.05, * * P＜0.01, * * * P＜0.001

The results are shown in Table 8, behind the mouse inoculation oncocyte, optical density is starkly lower than normal group (P＜0.05).Given QINGCHUN BAO and FUFANG KUSHEN ZHUSHEYE little, in, behind big three dosage, optical density obviously rises, and significant difference is more all arranged, P＜0.05 with lotus tumor matched group.The results suggest FUFANG KUSHEN ZHUSHEYE produces anti-bright and beautiful sheep red blood cell antibody haemolysis to the tumor-bearing mice splenocyte and have facilitation, promptly the bone-marrow-derived lymphocyte humoral immune function is had potentiation.

Experimental example 15

The research of anastalsis

1, to the influence of clotting time of mice

The healthy mice weight average is divided into 5 groups, be respectively three various dose groups of normal control group, positive controls and FUFANG KUSHEN ZHUSHEYE, the normal saline that normal control group ip in mice is identical with the test group volume, positive controls ip in mice 0.75g/kg etamsylate.Test group is ip1.5,3.0,6.0g/kg FUFANG KUSHEN ZHUSHEYE respectively, successive administration three days, behind the last administration 30min, with internal diameter is that the capillary glass tube of 1mm is taken a blood sample from mouse orbit, be full of capillary tube to blood, and begin to clock, a bit of every the 30s capillary tube that fractures, check to have or not the blood clotting silk to occur.Calculating is full of blood to the time that the blood clotting silk occurs from capillary tube, is clotting time, compares with the normal saline matched group, to declare drug effect.

Table 9 FUFANG KUSHEN ZHUSHEYE is to the influence of clotting time of mice (X ± SD)

The results are shown in Table 9, after the FUFANG KUSHEN ZHUSHEYE of ip in mice various dose, its clotting time is compared with the normal control group, and significant difference is all arranged, and this has shown that FUFANG KUSHEN ZHUSHEYE can shorten the clotting time of mice.

2. to the influence in mice bleeding time

The mensuration in bleeding time, naturally stop as the hemorrhage time index of test through hemorrhage, get the 18-20g healthy male mice, be divided into normal control group, three different dosage groups of positive controls and FUFANG KUSHEN ZHUSHEYE, the normal saline that normal control group ip in mice is identical with the test group volume by weight average.Positive controls ip in mice 0.75g/kg etamsylate, test group is ip 1.5 respectively, 3.0 the 6.0g/kg FUFANG KUSHEN ZHUSHEYE is behind the administration 30min, cross-section in order to cutting respectively with mouse tail point 3mm place, treat that blood begins to clock when overflowing voluntarily, inhale to dehematize with filter paper every 30s and drip once, when filter paper is inhaled till the depletion of blood, be the bleeding time, the gained data are carried out time t check.

Table 10 FUFANG KUSHEN ZHUSHEYE is to the influence in mice bleeding time (X ± SD)

The results are shown in Table 10, after the FUFANG KUSHEN ZHUSHEYE of mouse peritoneal injection various dose, heavy dose of and the middle dosage group bleeding time is compared with the normal control group, has significant difference, the mice bleeding time of small dose group and normal control group no significant difference, experimental result shows that FUFANG KUSHEN ZHUSHEYE can shorten the bleeding time of mice.

Experimental example 16

FUFANG KUSHEN ZHUSHEYE is to the potentiation and the Attenuation of cyclophosphamide chemotherapy

1. FUFANG KUSHEN ZHUSHEYE is to the potentiation of cyclophosphamide

40 of mices, body weight 22.3 ± 1.3g inoculates oncocyte, gets the S in the week of growing ₁₈₀Ascites tumor tumor source, (counting is 3 * 10 with normal saline (N.S) dilution in 1: 4 ⁴/ mm ³) inoculation 0.1ml is subcutaneous in the right axil of every mice, grouping is divided into into 4 groups after 24 hours.The 1st group is matched group, to N.S combination Cy 20mg/kg; 3 groups is Cy 20mg/kg+ FUFANG KUSHEN ZHUSHEYE 6.4g/kg; 4 groups is Cy 20mg/kg+ FUFANG KUSHEN ZHUSHEYE 12.8g/kg; More than each group be intraperitoneal injection, continuous 9 days of every day 1 time of FUFANG KUSHEN ZHUSHEYE wherein, cyclophosphamide 1 time totally 4 times every other day, the 10th puts to death in all disconnected cervical vertebras and to get tumor, uses scales/electronic balance weighing, the results are shown in Table 11.

By table 11 as seen, it is heavy that Cy 20mg/kg and 35mg/kg all can suppress tumor, gives N.S respectively with the matched group ratio; 2 groups is after Cy 100mg/kg adds FUFANG KUSHEN ZHUSHEYE 6.4,12.8g/kg two dosage respectively, has to suppress the heavy potentiation of tumor, difference P＜0.05, P＜0.001.

2. FUFANG KUSHEN ZHUSHEYE is to the effect of chemotherapy attenuation

51 of mices, body weight 19.4 ± 0.78g is divided into 5 groups at random.The 1st group is that matched group is given N.S; 2 groups is Cy100mg/kg; 3 groups is Cy 100mg/kg+ FUFANG KUSHEN ZHUSHEYE 12.8g/kg; 4 groups is Cy 100mg/kg+ FUFANG KUSHEN ZHUSHEYE 6.4g/kg; 5 groups is Cy 100mg/kg+ FUFANG KUSHEN ZHUSHEYE 3.2g/kg.More than each group be intraperitoneal injection, continuous 7 days of every day 1 time of FUFANG KUSHEN ZHUSHEYE wherein, Cy is all in each administration in per 6,7 days 1 time, gets blood and femur from eye socket and gets bone marrow and do not have WBC with the hemocyte automatic counter for counting, bone marrow nucleated cell in the 8th day.The results are shown in Table 12.

By table 12 as seen, Cy 100mg/kg all reduces WBC, bone marrow nucleated cell, after adding with FUFANG KUSHEN ZHUSHEYE, bone marrow nucleated cell has tangible increase, after adding with FUFANG KUSHEN ZHUSHEYE 3.2g/kg, WBC obviously rises, equal P＜0.05, and wherein two groups of FUFANG KUSHEN ZHUSHEYE 6.4g/kg, 3.2g/kg and simple Cy group compare P＜0.05.

Table 11 FUFANG KUSHEN ZHUSHEYE is to the potentiation of cyclophosphamide (X ± SD)

(1) compares with matched group ^*P＜0.05

(2) compare * P＜0.05, * * P＜0.001 with Cy

Table 12 FUFANG KUSHEN ZHUSHEYE is to chemotherapy Attenuation (X ± SD)

(1) compares with matched group ^*P＜0.001

(2) compare * P＜0.05 with Cy

Experimental example 17

The clinical research of FUFANG KUSHEN ZHUSHEYE treatment chronic hepatitis B

1. case selects 431 routine chronic hepatitis B diagnosis to meet national standard.Patient HBV infects and surpasses half a year, liver function ALT and AST are unusual above 3 months, weak, poor appetite in various degree etc. is arranged, Serum ALT (142 ± 106) U/L, AST (110 ± 97) U/L, serum total bilirubin (51 ± 39) μ mol/L, the HBeAg positive, serum albumin is got rid of liver cirrhosis and other viral hepatitis clinically in normal range.431 routine patients are divided into FUFANG KUSHEN ZHUSHEYE+interferon group and interferon group at random.FUFANG KUSHEN ZHUSHEYE+interferon group 222 examples, male 122 examples, women 100 examples; Interferon group 209 examples, male 119 examples, women 90 examples.Two groups of patient's ordinary circumstance difference do not have significance.

2. Therapeutic Method FUFANG KUSHEN ZHUSHEYE+interferon group gives 60ml+10% Glucose Liquid 250mL intravenous drip, 3 totally months every day 1 time, α-2b interferon 50 μ g intramuscular injection, every day 1 time is 10d altogether, later 50 μ g intramuscular injection, the next day 1 time totally 90 times; The interferon group is single with α-2b interferon therapy, and its dosage, usage, the course of treatment are consistent with the treatment group.Two groups of patients use liver-protecting medicine treatments such as potenlin or diammonium glycyrrhizinate, Gu Lading simultaneously.

3. result

3.1 ALT and AST change before two groups of patient treatments and 3 weeks of treatment before and after the treatment, in 6 weeks, when 12 weeks and 24 weeks, the variation of its ALT and AST comprises fall, as if the time of recovery has any different, and handles the equal nonsignificance of difference but learn by statistics.See Table 13.

When 3.2 the patient treatment of variation FUFANG KUSHEN ZHUSHEYE+interferon group of HBeAg, anti-HBe and HBV-DNA finishes in the serum before and after two groups of patient treatments, serum HBV-DNA, the negative conversion rate of HBeAg is respectively 73% and 68%, anti-HBe sun rate of rotation is 61%, when list finished with the treatment of interferon group, These parameters was respectively 46%, 43% and 36%, the curative effect of treatment group obviously is better than matched group, sees Table 14.

ALT and AST change X ± SD, U/L before and after table 13 treatment

The variation of anti-HBe of HBeAg and HBV-DNA before and after table 14 treatment

Group	HBeAg is cloudy to be changeed	Anti-HBe sun changes	HBV-DNA is cloudy to be changeed
Group	HBeAg is cloudy to be changeed	Anti-HBe sun changes	HBV-DNA is cloudy to be changeed	FUFANG KUSHEN ZHUSHEYE+interferon	68％	61％	73％
Interferon	43％	36％	46％	FUFANG KUSHEN ZHUSHEYE+interferon	68％	61％	73％

Experimental example 18

Toxic and side effects

In the clinical observation of this product through 2588 examples, do not find any toxic and side effects, and portion's case is after receiving treatment greatly, general situation such as spirit, appetite, sleep is also improved thereupon, and the scoring of whole body performance status is risen, raising patient's life quality.Thereby, regardless of the general situation of patient, all can safety use, can be applicable to the treatment of middle and advanced stage cancer widely.

Claims

1, a kind of preparation method of compound kuhseng preparation for injection is characterized in that:

(1) crude drug is: Radix Sophorae Flavescentis and Smilax lanceaefolia Roxb. Var.opaca A.DC. weight ratio are 7: 3;

(2) Radix Sophorae Flavescentis powder is broken into coarse powder, mixing is put in the percolation cylinder, add the salt acid dip 24 hours of 8～10 times 1% acetic acid being equivalent to dose or 0.5%, carry out percolation, collect percolate, percolate is evaporated to 1.20/60 ℃ of relative density at 75 ℃, and device is deposited in addition; Radix Sophorae Flavescentis medicinal residues and Smilax lanceaefolia Roxb. Var.opaca A.DC. coarse powder add 6～8 times of amount waters for injection, decoct 2 times, and each 1 hour, filter, merge decoction liquor, be evaporated to 1.20/60 ℃ of relative density, to put coldly, filtration obtains supernatant; The concentrated solution of supernatant and Radix Sophorae Flavescentis percolate merges, and with ethanol precipitate with ethanol 3 times, adds ethanol for the first time and makes the alcohol amount that contains of solution reach 60%, left standstill liquid or 0～5 ℃ of cold preservation more than 24 hours, 0.45 μ m microporous filter membrane filters, the supernatant decompression recycling ethanol is concentrated into 1.20/60 ℃ of relative density; Add ethanol again and make that to contain alcohol amount be 80%, left standstill liquid or 0～5 ℃ of cold preservation more than 24 hours, 0.45 μ m microporous filter membrane filters, and the supernatant decompression recycling ethanol is concentrated into 1.20/60 ℃ of relative density; Add ethanol for the third time and make that to contain alcohol amount be 90%, left standstill liquid or 0～5 ℃ of cold preservation more than 24 hours, 0.45 μ m microporous filter membrane filters, and the supernatant decompression recycling ethanol is concentrated into the alcohol soluble substance extractum of 1.30/60 ℃ of relative density; Alcohol soluble substance adds the injection water and returns moltenly, crosses liquid, 0.45 μ m microporous filter membrane 0 ℃ of cold preservation and filters, and obtains filtrate, and filtrate is being evaporated to 1.40/60 ℃ of relative density below 80 ℃, obtains pharmaceutical intermediate, and the freezer that is positioned over-5 ℃～5 ℃ is preserved;

(3) injection preparation: the thing intermediate of getting it filled adds water for injection, filter through 3% injection active carbon reflux decolouring 10 minutes, 0.22 μ m supermicro filtration membrane, filtrate is transferred pH-cold preservation-filter process through 4 times, transfers to pH7.8 for the first time, spend the night 0 ℃ of cold preservation, 0.22 μ m supermicro filtration membrane filters; For the second time transfer to pH5.0, handle with method; Transfer to pH6.8 for the third time, handle, transfer pH to carry out with 20%NaOH and 40% acetic acid with method; After transferring pH to 8.8 the 4th time, add the injection water, spend the night-5 ℃ of left and right sides cold preservations, 0.22 μ m supermicro filtration membrane filters; Embedding is respectively sealed and 105 ℃ of sterilizations 30 minutes, is packed, passed examination, and warehouse-in obtains injection;

Or injection preparation: sodium chloride or glucose add pharmaceutical intermediate after dissolving with water for injection, and add water for injection, filter through 10 minutes 0.22 μ m supermicro filtration membranes of 3% injection active carbon reflux decolouring; Filtrate is transferred pH-cold preservation-filter process through 4 times, transfers to pH7.8 for the first time, spends the night 0 ℃ of left and right sides cold preservation, and 0.22 μ m supermicro filtration membrane filters; For the second time transfer to pH5.0, handle with method; Transfer to pH6.8 for the third time, handle with method; Transfer pH to carry out with 20%NaOH and 40% acetic acid; After transferring pH to 8.8 the 4th time, add the injection water, spend the night-5 ℃ of left and right sides cold preservations, 0.22 μ m supermicro filtration membrane filters; Embedding respectively, seal, and 105 ℃ of sterilizations 30 minutes, packing, passed examination, warehouse-in obtains injection;

Or lyophilized injectable powder preparation: in 100 grades GMP workshop, after framework material mannitol adding water for injection dissolves fully, add pharmaceutical intermediate, add water for injection, be heated to 80 ℃ and be incubated 30 minutes through the injection active carbon, take off charcoal 0.22 μ m supermicro filtration membrane when being chilled to 45 ℃ and filter, filtrate is transferred pH-cold preservation-filter process through 4 times; For the first time transfer to pH7.8, spend the night 0 ℃ of left and right sides cold preservation, 0.22 μ m supermicro filtration membrane filters; For the second time transfer to pH5.0, handle with method; Transfer to pH6.8 for the third time, handle, transfer pH to carry out with 20%NaOH and 40% acetic acid with method; After transferring pH to 7.8 the 4th time, add the injection water, spend the night-5 ℃ of left and right sides cold preservations, filter with 0.22 μ m supermicro filtration membrane, in the embedding cillin bottle, cover butyl rubber bung, keep venthole, lyophilization promptly gets lyophilized injectable powder.