CN104127473A

CN104127473A - Pharmaceutical composition for treating bone diseases, injection thereof and preparation methods thereof

Info

Publication number: CN104127473A
Application number: CN201410370993.1A
Authority: CN
Inventors: 宋洋
Original assignee: HARBIN SANCTITY BIOLOGICAL PHARMACEUTICAL Co Ltd
Current assignee: HARBIN SANCTITY BIOLOGICAL PHARMACEUTICAL Co Ltd
Priority date: 2014-07-31
Filing date: 2014-07-31
Publication date: 2014-11-05
Anticipated expiration: 2034-07-31
Also published as: CN104127473B

Abstract

The invention relates to a pharmaceutical composition for treating bone diseases, an injection thereof and preparation methods thereof. The pharmaceutical composition is composed of an extraction liquid of pig four limbs and a melon seed extraction liquid. Further, the pharmaceutical composition is added with mixed extraction liquids respectively prepared from commen bomhax flower, flos buddlejae and herba eupatorii. The prepared pharmaceutical composition has substantially improved treatment effect on rheumatoid arthritis, degenerative osteoarthritis, ankylosing spondylitis, sciatica, lumbar disc herniation and other bone diseases, is improved in treatment effect on gout and has no toxic and side effects. Additionally, the provided injection has extremely high stability and is substantial in effect.

Description

A kind of pharmaceutical composition and injection and method for making for the treatment of osteopathia

Technical field

The invention belongs to the field of Chinese medicines, particularly a kind of pharmaceutical composition and injection and method for making for the treatment of osteopathia.

Background technology

Osteopathia is common clinical, mainly comprise rheumatoid arthritis, degenerative osteoarthritis, ankylosing spondylitis, prolapse of lumbar intervertebral disc, gout and osteoporosis etc., these common osteopathias are perplexing patient for a long time, are difficult for curing, easily recurrence, brings great misery to patient.

Bone-melon extract is the medicine that the dry mature seed of pig limbs bone and Fructus Melo is made after extracting respectively, mainly contain following composition: polypeptides bone metabolic factor, melon seed extract, multiple free amino acid, and organic calcium, inorganic calcium, phosphonium ion, inorganic salt and trace element; This product is due to the bioactive peptide containing multiple bone metabolism, therefore, there is adjusting bone metabolism, stimulated osteoblastic proliferation, promotes new bone formation, and regulates calcium, phosphorus metabolism, increase bone calcium deposition, prevent and treat osteoporosis, there is antiinflammatory and analgesic activity, be often used to treat the osteopathias such as rheumatoid arthritis; But this bone-melon extract is not remarkable to the curative effect of gout.

Secondly, the preparation method of bone-melon extract is all to adopt the techniques such as conventional extraction, water precipitating, acid-base precipitation at present, the effective ingredient in pig limbs bone and Semen Melo can not be extracted fully.

In addition, the injection of being prepared by bone-melon extract exists poor stability, effective ingredient dissolves the problems such as bad, has limited the application of bone-melon extract injection.

Summary of the invention

In order to solve the problems of the technologies described above, the invention provides a kind of pharmaceutical composition and injection and method for making for the treatment of osteopathia, this pharmaceutical composition has significant curative effect to osteopathias such as rheumatoid arthritis, degenerative osteoarthritis, ankylosing spondylitis, prolapse of lumbar intervertebral disc, sciatica or gouts; By the improvement to pharmaceutical composition preparation method, the active component of each composition fully can be extracted simultaneously, indirectly improve the curative effect of pharmaceutical composition.

The concrete technical scheme of the present invention is as follows:

The invention provides a kind of pharmaceutical composition for the treatment of osteopathia, this pharmaceutical composition is mainly made up of the component of following weight portion:

Pig limbs bone extracting solution 30-70 Semen Melo extracting solution 15-25.

Further improve, pharmaceutical composition also comprises that parts by weight are the mixed extract of 10-20 part; This mixed extract is made up of the component of following weight portion:

Flos Bombacis Malabarici 20-30 Flos Buddlejae 5-10 Herba Eupatorii 30-50.

The present invention is on the basis of original pig limbs bone extracting solution and Semen Melo extracting solution, increased Flos Bombacis Malabarici, Flos Buddlejae and three components of Herba Eupatorii, the pharmaceutical composition of preparation significantly improves osteopathia effects such as rheumatoid arthritis, degenerative osteoarthritis, ankylosing spondylitis, sciatica, prolapse of lumbar intervertebral disc; And improve the therapeutic effect to gout, had no side effect.

Preferably, this pharmaceutical composition is prepared from by the component of following weight portion:

Pig limbs bone extracting solution 50 Semen Melo extracting solution 30

Flos Bombacis Malabarici 25 Flos Buddlejae 7.5 Herba Eupatoriis 40.

The present invention provides on the other hand by the pharmaceutical composition for the treatment of osteopathia and the injection that adjuvant forms, and this injection comprises pharmaceutical composition, additives and water for injection.

Preferably, the parts by weight of pharmaceutical composition are 45-115 part, and water for injection parts by weight are 100-200 part; Additives comprise that parts by weight are the buffer agent of 2.5-7.5 part, the pH adjusting agent of 1-2.5 part, the sodium citrate of 0.2 part and the chitose of 1-3 part; Buffer agent is phosphoric acid-sodium hydrogen phosphate, and pH adjusting agent is sodium bicarbonate, sodium carbonate or sodium acetate.

The present invention selects the mixture of sodium citrate and chitose, can effectively prevent that injection is rotten, and the stability of injection is provided.

Further preferably, additives also comprise that parts by weight are the propylene glycol stearate of 0.5-1.5 part and the maltose alcohol of 0.2 part.Wherein the mixing of propylene glycol stearate and maltose alcohol can improve the dissolubility of pharmaceutical composition, makes the dissolving of pharmaceutical composition more complete, gives full play to the curative effect of pharmaceutical composition, and this mixture also plays the effect that suppresses growth of microorganism simultaneously.

The present invention also provides the preparation method of pharmaceutical composition on the other hand, and the method step is as follows:

1) preparation of pig limbs bone extracting solution:

1.1 fragmentations: raw material pig limbs bone is cleaned, extremely without naked eyes visible foreign matters, fragmentation;

1.2 extract: pig limbs bone after fragmentation is dropped into extraction pot, add purified water, hot pressing extracts twice, the amount that at every turn adds purified water is raw material weight 2 times, each 1h, controlled pressure is 0.11-0.12MPa, temperature is 121 DEG C-125 DEG C, makes extracting solution;

1.3 is concentrated: extracting solution is squeezed in settling tank by product pump, be cooled to 20 DEG C-30 DEG C, leave standstill, leave standstill to fuel-displaced, extracting solution after deoiling is concentrated in haplo-effect concentrator, and when concentrated, vacuum degree control is at-0.05Mpa to-0.06Mpa, and temperature is controlled at 70 DEG C-80 DEG C; The 60%-70% that is concentrated into raw material weight, makes concentrated solution;

1.4 acid are heavy: concentrated solution is transferred in enamel pot, is cooled to room temperature; The acid solution adjust pH of the purified water that is 1: 1 by volume ratio and the configuration of 36-38% hydrochloric acid, limit adds acid solution limit stirs, and adds acid solution at every turn and in enamel pot, stirs after 15 minutes, pH value determination; Adjust pH is 2.0-3.0, then is heated to 100 DEG C of insulations 45 minutes, cools to 0-5 DEG C of standing 12h-24h, makes the heavy liquid of acid;

1.5 alkali deposited: after the heavy liquid of acid leaves standstill and finishes, filter through rustless steel sheet frame with 300 order filter clothes, when filtration, pressure is not higher than 0.4Mpa, filtrate is squeezed in enamel pot, with 20% sodium hydroxide solution adjust pH, stir on hydro-oxidation sodium solution limit, limit, adds sodium hydroxide solution at every turn and in enamel pot, stir after 15 minutes, pH value determination; Adjust pH, to 8.9-9.1, heats 100 DEG C of insulations 45 minutes, cools, and 0-5 DEG C of standing 12h-24h, makes alkali deposited liquid;

1.6 precipitate with ethanol: after alkali deposited liquid leaves standstill and finishes, filter through rustless steel sheet frame with 300 order filter clothes, when filtration, pressure is not higher than 0.4Mpa, filtrate stirs 15min after all squeezing into enamel pot, the acid solution of the purified water that is 1: 1 by volume ratio and the configuration of 36-38% hydrochloric acid is adjusted pH to 6.5-7.0, add acid solution at every turn and need stir 15min, survey pH value; Adjusted the medicinal liquid haplo-effect concentrator of pH value to be concentrated into the 25%-30% of material quantity, when concentrated, vacuum degree control is at-0.05Mpa to-0.06Mpa, and temperature is controlled at 70 DEG C-80 DEG C; After concentrated, medicinal liquid is transferred in settling tank, is cooled to 60-70 DEG C, the amount of alcohol that by volume conversion needs, and unit is L, opens the stirring of settling tank, adds 95% ethanol, makes alcohol content reach 70%, is cooled to 0-5 DEG C, leaves standstill 12h-24h, makes alcohol deposit fluid; Add amount of alcohol computing formula:

1.7 reclaim ethanol: after alcohol deposit fluid leaves standstill and finishes, with 300 order filter clothes, through the filtration of rustless steel sheet frame, when filtration, pressure is not higher than 0.4Mpa; Medicinal liquid after filtration is transferred in haplo-effect concentrator, and normal pressure reclaims ethanol, and temperature is controlled at 60 DEG C-70 DEG C, reclaims ethanol extremely without alcohol taste, and in the time that under the liquid room temperature that haplo-effect concentrator receipts liquid bucket liquid outlet is discharged, relative density is 1.0, ethanol reclaims and finishes; Medicinal liquid Weight control is 24% ± 2% of raw material weight;

1.8 ultrafiltration: by the medicinal liquid ultrafiltration of reclaiming after ethanol, ultrafiltration condition: molecular weight 10K ultrafiltration post, do not rinse pH value 5.0-7.0 higher than 40 DEG C of injection waters by front use, the 0.4% alkali liquor envelope that reach≤1.3 μ s/cm of the water conductivity of discharge are 1kg with rear every pillar, operating pressure is not higher than 0.1Mpa, after ultrafiltration finishes, concentrated with haplo-effect concentrator, concentrated solution is controlled at the 20%-24% of raw material weight, concentrated solution mix homogeneously is made to pig limbs bone extracting solution ,-10 DEG C of following freezing depositing;

2) preparation of Semen Melo extracting solution:

2.1 decoct: the Semen Melo after pulverizing is dropped in extraction pot, add purified water, decoct three times, the amount of water of three times is respectively 8 times, 8 times, 6 times of Semen Melo weight, decocts for the first time 1h, decocts respectively for the second time, for the third time 50min; Three decoction liquor are merged, be concentrated into 1/3 of Semen Melo amount, when concentrated, vacuum degree control is-0.05 to-0.06Mpa, and temperature is controlled at 70 DEG C-80 DEG C, makes concentrated solution;

2.2 precipitate with ethanol: concentrated solution vacuum is sucked in settling tank, the amount of alcohol that by volume conversion needs, unit is L, opens stirring paddle, adds 95% ethanol, what make medicinal liquid reaches 72%V/V containing alcohol amount; Required ethanol all adds and continues to stir 20min, lowers the temperature 0-5 DEG C and leaves standstill 48 hours; One time precipitate with ethanol adds amount of alcohol computing formula:

2.3 water precipitating: by an alcohol deposit fluid through two-layer 300 order filter clothes, two-layer neutral filter paper rustless steel sheet frame filters, when filtration, pressure is not higher than 0.4Mpa, filtrate is transferred to haplo-effect concentrator, then use normal pressure, temperature is controlled at 60 DEG C-70 DEG C and reclaims ethanol extremely without alcohol taste, in the time that under the liquid room temperature that haplo-effect concentrator receipts liquid bucket liquid outlet is discharged, relative density is 1.0, ethanol reclaims and finishes, and be concentrated into 1/6 of Semen Melo weight, the volume of metering concentrated solution, add purified water by 1: 1 concentrated solution, stir 15 minutes, micro-30min that boils, feed liquid is sent into quick freezing repository freezing, freeze reality, make water precipitating material one time,

2.4 2 precipitate with ethanol: by after the negative catalysis of a water precipitating material, the negative catalysis time is controlled at 12-24 hour, with two-layer 300 order filter clothes, the box plate-and-frame filtration of two-layer neutral filter paper rustless steel, when filtration, pressure is not higher than 0.4Mpa, filtration time is controlled at 2-4h, and it is concentrated that filtrate is transferred to haplo-effect concentrator, and filtrate is concentrated into 2/5 of original volume, when concentrated, vacuum degree control is-0.05 to-0.06Mpa, and temperature is controlled at 70 DEG C-80 DEG C; Concentrated solution is transferred in settling tank, the amount of alcohol that by volume conversion needs, unit is L, opens the stirring paddle of settling tank, adds 95% ethanol, what make medicinal liquid reaches 86%V/V containing alcohol amount; Required ethanol all adds and continues to stir 20min, open cold saline turnover valve, and 0-5 DEG C leaves standstill 48 hours, makes secondary alcohol deposit fluid; Secondary precipitate with ethanol adds amount of alcohol computing formula:

2.5 2 water precipitating: by two-layer 300 order filter clothes, the box plate-and-frame filtration of two-layer neutral filter paper rustless steel for secondary alcohol deposit fluid, when filtration, pressure is not higher than 0.4Mpa; Filtrate is transferred to haplo-effect concentrator, is then controlled at 60 DEG C-70 DEG C by normal pressure, temperature, reclaim ethanol to medicinal liquid without alcohol taste, in the time that under the liquid room temperature that haplo-effect concentrator receipts liquid bucket liquid outlet is discharged, relative density is 1.0, ethanol reclaims and finishes, and is concentrated into 1/8 of Semen Melo, the volume of metering concentrated solution, add isopyknic water for injection, after mix homogeneously, by micro-feed liquid 30min that boils, feed liquid is sent into quick freezing repository freezing, freeze reality, make secondary water precipitating liquid;

2.6 subpackages: by after the negative catalysis of secondary water precipitating liquid, the negative catalysis time is controlled at 12-24h, with two-layer 300 order filter clothes, the box plate-and-frame filtration of two-layer neutral filter paper rustless steel, when filtration, pressure is not higher than 0.4Mpa, filtration time is controlled at 2-4 hour, after filtration, filtrate is stirred to 20min, make Semen Melo extracting solution;

3) by pig limbs bone extracting solution and Semen Melo extracting solution, in the ratio mix homogeneously of 5: 3, in Agitation Tank, stir 20min, heat 100 DEG C of insulation 15min, be cooled to room temperature ,-20 DEG C freeze below real 48 hours to 60 hours, make bone melon extracting solution;

4) bone melon extracting solution ultrafiltration, subpackage: take out room temperature negative catalysis, ultrafiltration by freezing real bone melon extracting solution, molecular weight 5K ultrafiltration post, do not rinse pH value 5.0-7.0, flushing water electrical conductivity≤1.3 μ s/cm higher than 40 DEG C of waters for injection by front use, operating pressure is not higher than 0.1Mpa, collect ultrafiltrate, ultrafiltrate is stirred and in Agitation Tank, stirs 20min, subpackage is in the stainless steel cask of 0.5% sodium hydroxide solution processing, handle after the assay was approved warehouse-in, make pharmaceutical composition.

Beneficial effect: the present invention is in the time preparing pig limbs bone extracting solution, regulate by the pH in and alkali deposited step heavy to acid, obtaining working as the heavy adjusting of acid pH value is 2.0-3.0, preferably regulating pH value is 2.0-2.2, it is 8.9-9.1 that alkali deposited regulates pH value, can significantly reduce the high molecular weight material in pig limbs bone extracting solution, remove acidic protein and basic protein completely, Acidity of Aikalinity gentleness, improve the content of bovine serum albumin in extracting solution, mild condition can not destroyed the content of other compositions in extracting solution simultaneously; In the time preparing Semen Melo extracting solution, by ethanol precipitation twice and twice water precipitating, and in ethanol precipitation twice process, the amount that adds ethanol is strictly controlled, effectively improve the content of active component in Semen Melo extracting solution, reduced the content of high molecular weight material, this preparation method is simple, is applicable to suitability for industrialized production.

Further improve, the preparation method of pharmaceutical composition also comprises the method for preparing mixed extract, and the method step is as follows:

3.1 decoct: the Flos Bombacis Malabarici, Flos Buddlejae and the Herba Eupatorii three taste medicines that take recipe quantity drop in extraction pot, add purified water, soak 1.5h; Then decoct with water three times, the amount of water of three times is respectively 10 times, 8 times, 5 times of three taste medicine gross weights, decocts for the first time 1h, decocts respectively for the second time, for the third time 50min; Three decoction liquor are merged, make decoction liquor;

3.2 extractions: decoction liquor is joined in extractor, decocted liquid measure by 1: 1 and add ethyl acetate, extract three times, isolate water layer, concentrated, make concentrated solution;

3.3 precipitate with ethanol: concentrated solution vacuum is sucked in settling tank, the amount of alcohol that by volume conversion needs, unit is L, opens stirring paddle, adds 95% ethanol; Required ethanol all adds and continues to stir 20min, lowers the temperature 0-5 DEG C and leaves standstill 48 hours; Precipitate with ethanol adds amount of alcohol computing formula:

3.4 water precipitating: alcohol deposit fluid is filtered through two-layer 300 order filter clothes, two-layer neutral filter paper rustless steel sheet frame, when filtration, pressure is not higher than 0.4Mpa, filtrate is transferred to haplo-effect concentrator, then be controlled at 60 DEG C-70 DEG C by normal pressure, temperature and reclaim ethanol extremely without alcohol taste, and be concentrated into 1/6 of three taste medicine gross weights, the volume of metering concentrated solution, add purified water by 1: 1 concentrated solution, stir 15 minutes, with two-layer 300 order filter clothes, the box plate-and-frame filtration of two-layer neutral filter paper rustless steel, after filtration, filtrate is stirred to 20min, make mixed extract.

Beneficial effect: the present invention is preparing in mixed extract process, adopt extraction, precipitate with ethanol and water precipitating step, and in precipitate with ethanol process, the strict addition of controlling ethanol, the amount of its effective ingredient significantly improves, reduce the existence of high molecular weight material, made the pharmaceutical composition of preparation can bring into play better drug effect.

The present invention also provides the preparation method of injection on the other hand, and the method comprises the steps:

1) pharmaceutical compositions;

2) get water for injection, buffer agent is dissolved in water for injection, add the pharmaceutical composition of 1/2 amount, stir, then add sodium citrate and chitose, continue to stir; Add again pharmaceutical composition, propylene glycol stearate and the maltose alcohol of 1/2 amount, constantly stir, make to dissolve completely;

3) add pH adjusting agent, stir 20min;

4) through 0.22 μ m filtering with microporous membrane, 121 DEG C of sterilizing 35min, make injection.

On the other hand, pharmaceutical composition provided by the invention is used for the treatment of the osteopathias such as rheumatoid arthritis, ankylosing spondylitis, sciatica or gout.

The preparation method that the present invention further provides the pharmaceutical composition for the treatment of osteopathia and the injectable powder of adjuvant composition, described method comprises the steps:

A. bone melon extracting solution is carried out to negative catalysis at 30-40 DEG C, obtain the bone melon extracting solution after negative catalysis;

B. dense joining: the bone melon extracting solution after negative catalysis is fed in dense preparing tank 2, after pre-flock with the molecular weight that dams handled well be 5000 dalton's import diaphragms by the ultrafiltration of injection bone melon extracting solution to dilute preparing tank 2; The dextran-40 taking is put in dense preparing tank 1, be dissolved in water for injection, make 30% solution, add the active carbon of liquor capacity 0.5 ‰, be heated to 100 DEG C of insulations 15 minutes, partial circulating is more than 10 minutes; To after the decarburization of dextran-40 solution filter, squeeze in dilute preparing tank 2;

C. rare joining: after dextran-40 solution filter completes, add water for injection to dilute preparing tank 2, rare medicinal liquid of joining regulates pH value to 6.0～6.8 with 10% sodium hydroxide solution;

D. fill: rare dosing is filtered to surge tank through 0.22 μ m cartridge type sterilizing filter, gives fill;

E. lyophilizing: freezing control: setting conduction oil temperature be-45 DEG C～-50 DEG C, reaches after temperature lasting 120～240 minutes;

Rear cabinet pre-cooling: setting rear cabinet temperature is-50 DEG C;

Forvacuum: setting drying baker forvacuum degree is 0.15mbar;

F. sublimation drying:

A) setting drying baker vacuum is 0.10～0.30mbar, and conduction oil temperature is-13 DEG C, 840 minutes persistent period;

B) setting drying baker vacuum is 0.10～0.30mbar, and conduction oil temperature is-5 DEG C, 180 minutes persistent period;

C) setting drying baker vacuum is 0.10～0.30mbar, and conduction oil temperature is 0 DEG C, 180 minutes persistent period;

G. parsing-desiccation:

A) setting drying baker vacuum is 0.10～0.30mbar, and conduction oil temperature is 5 DEG C, 120 minutes persistent period;

B) setting drying baker vacuum is that 0.10～0.30mbar conduction oil temperature is 10 DEG C, 120 minutes persistent period;

C) setting drying baker vacuum is 0.10～0.30mbar, and conduction oil temperature is 20 DEG C, 120 minutes persistent period; Make injectable powder.

The pharmaceutical composition for the treatment of osteopathia provided by the present invention all has good therapeutic effect to osteopathias such as rheumatoid arthritis, ankylosing spondylitis, sciatica and gouts, and there is no the side effect such as haemolysis, anaphylaxis, blood vessel irritation; And raw materials used medicine can be realized complementing each other of each raw material of Chinese medicine medicine, the object of collaborative performance curative effect; Make after injection, can give full play to the curative effect of pharmaceutical composition, in injection, add sodium citrate, chitose propylene glycol stearate and maltose alcohol, not only improve the stability of injection, the dissolubility that has further improved pharmaceutical composition, makes it bring into play fully drug effect simultaneously; Preparation method provided by the invention can improve the content of active component significantly on the other hand, reduce the content of high molecular weight material, and preparation method is simple, is applicable to suitability for industrialized production.

Detailed description of the invention

Embodiment 1

Treat a pharmaceutical composition for osteopathia, this pharmaceutical composition is made up of the component of following weight number:

Pig limbs bone extracting solution 30 Semen Melo extracting solution 15

Preparation method:

1) preparation of pig limbs bone extracting solution:

1.2 extract: pig limbs bone after fragmentation is dropped into extraction pot, add purified water, hot pressing extracts twice, the amount that at every turn adds purified water is raw material weight 2 times, each 1h, controlled pressure is 0.12MPa, temperature is 121 DEG C, makes extracting solution;

1.3 is concentrated: extracting solution is squeezed in settling tank by product pump, be cooled to 30 DEG C, leave standstill, leave standstill to fuel-displaced, the extracting solution after deoiling is concentrated in haplo-effect concentrator, and when concentrated, vacuum degree control is at-0.05Mpa, and temperature is controlled at 80 DEG C; The 60%-70% that is concentrated into raw material weight, makes concentrated solution;

1.4 acid are heavy: concentrated solution is transferred in enamel pot, is cooled to room temperature; The acid solution adjust pH of the purified water that is 1: 1 by volume ratio and the configuration of 36-38% hydrochloric acid, limit adds acid solution limit stirs, and adds acid solution at every turn and in enamel pot, stirs after 15 minutes, pH value determination; Adjust pH is 2.2, then is heated to 100 DEG C of insulations 45 minutes, cools to 5 DEG C of standing 12h, makes the heavy liquid of acid;

1.5 alkali deposited: after the heavy liquid of acid leaves standstill and finishes, filter through rustless steel sheet frame with 300 order filter clothes, when filtration, pressure is not higher than 0.4Mpa, filtrate is squeezed in enamel pot, with 20% sodium hydroxide solution adjust pH, stir on hydro-oxidation sodium solution limit, limit, adds sodium hydroxide solution at every turn and in enamel pot, stir after 15 minutes, pH value determination; Adjust pH to 8.9, heats 100 DEG C of insulations 45 minutes, cools, and 5 DEG C of standing 12h, make alkali deposited liquid;

1.6 precipitate with ethanol: after alkali deposited liquid leaves standstill and finishes, filter through rustless steel sheet frame with 300 order filter clothes, when filtration, pressure is not higher than 0.4Mpa, filtrate stirs 15min after all squeezing into enamel pot, the acid solution of the purified water that is 1: 1 by volume ratio and the configuration of 37% hydrochloric acid is adjusted pH to 7.0, add acid solution at every turn and need stir 15min, survey pH value; Adjusted the medicinal liquid haplo-effect concentrator of pH value to be concentrated into 30% of material quantity, when concentrated, vacuum degree control is at-0.05Mpa, and temperature is controlled at 80 DEG C; After concentrated, medicinal liquid is transferred in settling tank, is cooled to 60-70 DEG C, the amount of alcohol that by volume conversion needs, and unit is L, opens the stirring of settling tank, adds 95% ethanol, makes alcohol content reach 70%, is cooled to 5 DEG C, leaves standstill 24h, makes alcohol deposit fluid; Add amount of alcohol computing formula:

1.7 reclaim ethanol: after alcohol deposit fluid leaves standstill and finishes, with 300 order filter clothes, through the filtration of rustless steel sheet frame, when filtration, pressure is not higher than 0.4Mpa; Medicinal liquid after filtration is transferred in haplo-effect concentrator, and normal pressure reclaims ethanol, and temperature is controlled at 70 DEG C, reclaims ethanol extremely without alcohol taste, and in the time that under the liquid room temperature that haplo-effect concentrator receipts liquid bucket liquid outlet is discharged, relative density is 1.0, ethanol reclaims and finishes; Medicinal liquid Weight control is 24% ± 2% of raw material weight;

1.8 ultrafiltration: by the medicinal liquid ultrafiltration of reclaiming after ethanol, ultrafiltration condition: molecular weight 10K ultrafiltration post, do not rinse pH value 7.0 higher than 40 DEG C of injection waters by front use, the 0.4% alkali liquor envelope that reach≤1.3 μ s/cm of the water conductivity of discharge are 1kg with rear every pillar, operating pressure is not higher than 0.1Mpa, after ultrafiltration finishes, concentrated with haplo-effect concentrator, concentrated solution is controlled at the 20%-24% of raw material weight, concentrated solution mix homogeneously is made to pig limbs bone extracting solution ,-10 DEG C of following freezing depositing;

2) preparation of Semen Melo extracting solution:

2.1 decoct: the Semen Melo after pulverizing is dropped in extraction pot, add purified water, decoct three times, the amount of water of three times is respectively 8 times, 8 times, 6 times of Semen Melo weight, decocts for the first time 1h, decocts respectively for the second time, for the third time 50min; Three decoction liquor are merged, be concentrated into 1/3 of Semen Melo amount, when concentrated, vacuum degree control is at-0.06Mpa, and temperature is controlled at 70 DEG C-80 DEG C, makes concentrated solution;

2.3 water precipitating: by an alcohol deposit fluid through two-layer 300 order filter clothes, two-layer neutral filter paper rustless steel sheet frame filters, when filtration, pressure is not higher than 0.4Mpa, filtrate is transferred to haplo-effect concentrator, then use normal pressure, temperature is controlled at 70 DEG C and reclaims ethanol extremely without alcohol taste, in the time that under the liquid room temperature that haplo-effect concentrator receipts liquid bucket liquid outlet is discharged, relative density is 1.0, ethanol reclaims and finishes, and be concentrated into 1/6 of Semen Melo weight, the volume of metering concentrated solution, add purified water by 1: 1 concentrated solution, stir 15 minutes, micro-30min that boils, feed liquid is sent into quick freezing repository freezing, freeze reality, make water precipitating material one time,

2.4 2 precipitate with ethanol: by after the negative catalysis of a water precipitating material, the negative catalysis time is controlled at 12-24 hour, with two-layer 300 order filter clothes, the box plate-and-frame filtration of two-layer neutral filter paper rustless steel, when filtration, pressure is not higher than 0.4Mpa, filtration time is controlled at 2-4h, and it is concentrated that filtrate is transferred to haplo-effect concentrator, and filtrate is concentrated into 2/5 of original volume, when concentrated, vacuum degree control is-0.05 to-0.06Mpa, and temperature is controlled at 70 DEG C-80 DEG C; Concentrated solution is transferred in settling tank, the amount of alcohol that by volume conversion needs, unit is L, opens the stirring paddle of settling tank, adds 95% ethanol, what make medicinal liquid reaches 86%V/V containing alcohol amount; Required ethanol all adds and continues to stir 20min, open cold saline turnover valve, and 5 DEG C leave standstill 48 hours, make secondary alcohol deposit fluid; Secondary precipitate with ethanol adds amount of alcohol computing formula:

2.5 2 water precipitating: by two-layer 300 order filter clothes, the box plate-and-frame filtration of two-layer neutral filter paper rustless steel for secondary alcohol deposit fluid, when filtration, pressure is not higher than 0.4Mpa; Filtrate is transferred to haplo-effect concentrator, is then controlled at 70 DEG C by normal pressure, temperature, reclaim ethanol to medicinal liquid without alcohol taste, in the time that under the liquid room temperature that haplo-effect concentrator receipts liquid bucket liquid outlet is discharged, relative density is 1.0, ethanol reclaims and finishes, and is concentrated into 1/8 of Semen Melo, the volume of metering concentrated solution, add isopyknic water for injection, after mix homogeneously, by micro-feed liquid 30min that boils, feed liquid is sent into quick freezing repository freezing, freeze reality, make secondary water precipitating liquid;

2.6 subpackages: by after the negative catalysis of secondary water precipitating liquid, the negative catalysis time is controlled at 24h, with two-layer 300 order filter clothes, the box plate-and-frame filtration of two-layer neutral filter paper rustless steel, when filtration, pressure is not higher than 0.4Mpa, filtration time is controlled at 4 hours, after filtration, filtrate is stirred to 20min, makes Semen Melo extracting solution;

3) by pig limbs bone extracting solution and Semen Melo extracting solution, mix homogeneously stirs 20min in Agitation Tank, heats 100 DEG C of insulation 15min, is cooled to room temperature, and-20 DEG C freeze below real 48 hours to 60 hours, make bone melon extracting solution;

4) bone melon extracting solution ultrafiltration, subpackage: take out room temperature negative catalysis, ultrafiltration by freezing real bone melon extracting solution, molecular weight 5K ultrafiltration post, do not rinse pH values 7.0, flushing water electrical conductivity≤1.3 μ s/cm higher than 40 DEG C of waters for injection by front use, operating pressure is not higher than 0.1Mpa, collect ultrafiltrate, ultrafiltrate is stirred and in Agitation Tank, stirs 20min, subpackage is in the stainless steel cask of 0.5% sodium hydroxide solution processing, handle after the assay was approved warehouse-in, make pharmaceutical composition.

Embodiment 2

Pig limbs bone extracting solution 60 Semen Melo extracting solution 22.5

Preparation method: the method preparation of pressing embodiment 1.

Embodiment 3

Pig limbs bone extracting solution 50 Semen Melo extracting solution 30

Preparation method: the method preparation of pressing embodiment 1.

Embodiment 4

Treat a pharmaceutical composition for osteopathia, this pharmaceutical composition is prepared from by the component of following weight portion:

Pig limbs bone extracting solution 50 Semen Melo extracting solution 30

Flos Bombacis Malabarici 25 Flos Buddlejae 7.5 Herba Eupatoriis 40

Preparation method:

1) preparation of pig limbs bone extracting solution:

1.2 extract: pig limbs bone after fragmentation is dropped into extraction pot, add purified water, hot pressing extracts twice, the amount that at every turn adds purified water is raw material weight 2 times, each 1h, controlled pressure is 0.11Pa, temperature is 121 DEG C, makes extracting solution;

1.3 is concentrated: extracting solution is squeezed in settling tank by product pump, be cooled to 20 DEG C, leave standstill, leave standstill to fuel-displaced, the extracting solution after deoiling is concentrated in haplo-effect concentrator, and when concentrated, vacuum degree control is at-0.06Mpa, and temperature is controlled at 70 DEG C; Be concentrated into 60% of raw material weight, make concentrated solution;

1.4 acid are heavy: concentrated solution is transferred in enamel pot, is cooled to room temperature; The acid solution adjust pH of the purified water that is 1: 1 by volume ratio and the configuration of 38% hydrochloric acid, limit adds acid solution limit stirs, and adds acid solution at every turn and in enamel pot, stirs after 15 minutes, pH value determination; Adjust pH is 2.0, then is heated to 100 DEG C of insulations 45 minutes, cools to 0 DEG C of standing 24h, makes the heavy liquid of acid;

1.5 alkali deposited: after the heavy liquid of acid leaves standstill and finishes, filter through rustless steel sheet frame with 300 order filter clothes, when filtration, pressure is not higher than 0.4Mpa, filtrate is squeezed in enamel pot, with 20% sodium hydroxide solution adjust pH, stir on hydro-oxidation sodium solution limit, limit, adds sodium hydroxide solution at every turn and in enamel pot, stir after 15 minutes, pH value determination; Adjust pH to 9.1, heats 100 DEG C of insulations 45 minutes, cools, and 0 DEG C of standing 24h, makes alkali deposited liquid;

1.6 precipitate with ethanol: after alkali deposited liquid leaves standstill and finishes, filter through rustless steel sheet frame with 300 order filter clothes, when filtration, pressure is not higher than 0.4Mpa, filtrate stirs 15min after all squeezing into enamel pot, the acid solution of the purified water that is 1: 1 by volume ratio and the configuration of 38% hydrochloric acid is adjusted pH to 6.5, add acid solution at every turn and need stir 15min, survey pH value; Adjusted the medicinal liquid haplo-effect concentrator of pH value to be concentrated into 25% of material quantity, when concentrated, vacuum degree control is at-0.05Mpa, and temperature is controlled at 70 DEG C; After concentrated, medicinal liquid is transferred in settling tank, is cooled to 60 DEG C, the amount of alcohol that by volume conversion needs, and unit is L, opens the stirring of settling tank, adds 95% ethanol, makes alcohol content reach 70%, is cooled to 0 DEG C, leaves standstill 12h, makes alcohol deposit fluid; Add amount of alcohol computing formula:

1.7 reclaim ethanol: after alcohol deposit fluid leaves standstill and finishes, with 300 order filter clothes, through the filtration of rustless steel sheet frame, when filtration, pressure is not higher than 0.4Mpa; Medicinal liquid after filtration is transferred in haplo-effect concentrator, and normal pressure reclaims ethanol, and temperature is controlled at 60 DEG C, reclaims ethanol extremely without alcohol taste, and in the time that under the liquid room temperature that haplo-effect concentrator receipts liquid bucket liquid outlet is discharged, relative density is 1.0, ethanol reclaims and finishes; Medicinal liquid Weight control is 24% ± 2% of raw material weight;

1.8 ultrafiltration: by the medicinal liquid ultrafiltration of reclaiming after ethanol, ultrafiltration condition: molecular weight 10K ultrafiltration post, do not rinse pH value 5.0 higher than 40 DEG C of injection waters by front use, the 0.4% alkali liquor envelope that reach≤1.3 μ s/cm of the water conductivity of discharge are 1kg with rear every pillar, operating pressure is not higher than 0.1Mpa, after ultrafiltration finishes, concentrated with haplo-effect concentrator, concentrated solution is controlled at 20% of raw material weight, concentrated solution mix homogeneously is made to pig limbs bone extracting solution ,-10 DEG C of following freezing depositing;

2) preparation of Semen Melo extracting solution:

2.1 decoct: the Semen Melo after pulverizing is dropped in extraction pot, add purified water, decoct three times, the amount of water of three times is respectively 8 times, 8 times, 6 times of Semen Melo weight, decocts for the first time 1h, decocts respectively for the second time, for the third time 50min; Three decoction liquor are merged, be concentrated into 1/3 of Semen Melo amount, when concentrated, vacuum degree control is at-0.05Mpa, and temperature is controlled at 70 DEG C, makes concentrated solution;

2.2 precipitate with ethanol: concentrated solution vacuum is sucked in settling tank, the amount of alcohol that by volume conversion needs, unit is L, opens stirring paddle, adds 95% ethanol, what make medicinal liquid reaches 72%V/V containing alcohol amount; Required ethanol all adds and continues to stir 20min, lowers the temperature 0 DEG C and leaves standstill 48 hours; One time precipitate with ethanol adds amount of alcohol computing formula:

2.3 water precipitating: by an alcohol deposit fluid through two-layer 300 order filter clothes, two-layer neutral filter paper rustless steel sheet frame filters, when filtration, pressure is not higher than 0.4Mpa, filtrate is transferred to haplo-effect concentrator, then use normal pressure, temperature is controlled at 60 DEG C and reclaims ethanol extremely without alcohol taste, in the time that under the liquid room temperature that haplo-effect concentrator receipts liquid bucket liquid outlet is discharged, relative density is 1.0, ethanol reclaims and finishes, and be concentrated into 1/6 of Semen Melo weight, the volume of metering concentrated solution, add purified water by 1: 1 concentrated solution, stir 15 minutes, micro-30min that boils, feed liquid is sent into quick freezing repository freezing, freeze reality, make water precipitating material one time,

2.4 2 precipitate with ethanol: by after the negative catalysis of a water precipitating material, the negative catalysis time is controlled at 12 hours, with two-layer 300 order filter clothes, the box plate-and-frame filtration of two-layer neutral filter paper rustless steel, when filtration, pressure is not higher than 0.4Mpa, filtration time is controlled at 2-4h, and it is concentrated that filtrate is transferred to haplo-effect concentrator, and filtrate is concentrated into 2/5 of original volume, when concentrated, vacuum degree control is at-0.05Mpa, and temperature is controlled at 70 DEG C; Concentrated solution is transferred in settling tank, the amount of alcohol that by volume conversion needs, unit is L, opens the stirring paddle of settling tank, adds 95% ethanol, what make medicinal liquid reaches 86%V/V containing alcohol amount; Required ethanol all adds and continues to stir 20min, open cold saline turnover valve, and 0 DEG C leaves standstill 48 hours, makes secondary alcohol deposit fluid; Secondary precipitate with ethanol adds amount of alcohol computing formula:

2.5 2 water precipitating: by two-layer 300 order filter clothes, the box plate-and-frame filtration of two-layer neutral filter paper rustless steel for secondary alcohol deposit fluid, when filtration, pressure is not higher than 0.4Mpa; Filtrate is transferred to haplo-effect concentrator, is then controlled at 60 DEG C by normal pressure, temperature, reclaim ethanol to medicinal liquid without alcohol taste, in the time that under the liquid room temperature that haplo-effect concentrator receipts liquid bucket liquid outlet is discharged, relative density is 1.0, ethanol reclaims and finishes, and is concentrated into 1/8 of Semen Melo, the volume of metering concentrated solution, add isopyknic water for injection, after mix homogeneously, by micro-feed liquid 30min that boils, feed liquid is sent into quick freezing repository freezing, freeze reality, make secondary water precipitating liquid;

2.6 subpackages: by after the negative catalysis of secondary water precipitating liquid, the negative catalysis time is controlled at 12h, with two-layer 300 order filter clothes, the box plate-and-frame filtration of two-layer neutral filter paper rustless steel, when filtration, pressure is not higher than 0.4Mpa, filtration time is controlled at 2 hours, after filtration, filtrate is stirred to 20min, makes Semen Melo extracting solution;

4) bone melon extracting solution ultrafiltration, subpackage: take out freezing real bone melon extracting solution, room temperature negative catalysis, ultrafiltration, molecular weight 5K ultrafiltration post, do not rinse pH value 5.0-7.0 higher than 40 DEG C of waters for injection by front use, flushing water electrical conductivity≤1.3 μ s/cm,, operating pressure is not higher than 0.1Mpa, collect ultrafiltrate, ultrafiltrate is stirred and in Agitation Tank, stirs 20min, and subpackage, in the stainless steel cask of 0.5% sodium hydroxide solution processing, is handled warehouse-in after the assay was approved;

5) method of mixed extract is as follows:

3.3 precipitate with ethanol: concentrated solution vacuum is sucked in settling tank, the amount of alcohol that by volume conversion needs, unit is L, opens stirring paddle, adds 95% ethanol; Required ethanol all adds and continues to stir 20min, lowers the temperature 5 DEG C and leaves standstill 48 hours; Precipitate with ethanol adds amount of alcohol computing formula:

3.4 water precipitating: alcohol deposit fluid is filtered through two-layer 300 order filter clothes, two-layer neutral filter paper rustless steel sheet frame, when filtration, pressure is not higher than 0.4Mpa, filtrate is transferred to haplo-effect concentrator, then be controlled at 70 DEG C by normal pressure, temperature and reclaim ethanol extremely without alcohol taste, and be concentrated into 1/6 of three taste medicine gross weights, the volume of metering concentrated solution, add purified water by 1: 1 concentrated solution, stir 15 minutes, with two-layer 300 order filter clothes, the box plate-and-frame filtration of two-layer neutral filter paper rustless steel, after filtration, filtrate is stirred to 20min, make parts by weight and be the mixed extract of 15 parts;

6) bone melon extracting solution step 4 being made and mixed extract mix homogeneously, make pharmaceutical composition.

Embodiment 5

Pig limbs bone extracting solution 70 Semen Melo extracting solution 25

Flos Bombacis Malabarici 30 Flos Buddlejae 10 Herba Eupatoriis 50

Preparation method: the method preparation of pressing embodiment 4.

Embodiment 6

Pig limbs bone extracting solution 40 Semen Melo extracting solution 17.5

Flos Bombacis Malabarici 20 Flos Buddlejae 5 Herba Eupatoriis 30

Preparation method: the method preparation of pressing embodiment 4.

Embodiment 7 injections

The pharmaceutical composition 10.6g (in polypeptide) that embodiment 3 is prepared

Adjuvant: 5% sodium hydroxide solution is appropriate

Water for injection is appropriate

Preparation method: by the pharmaceutical composition of embodiment 3, inject dilute with water, preferably to 90% recipe quantity, with 5% sodium hydroxide solution adjusting pH value, pH is preferably 6.5-7.0, adds water for injection to 2000ml, fill is in ampoule, and 121 DEG C of sterilizings 30 minutes, obtain injection.

Embodiment 8 injections

The pharmaceutical composition 10g (in polypeptide) that embodiment 4 is prepared

Adjuvant: 5% sodium hydroxide solution is appropriate

Water for injection is appropriate

Preparation method: prepare injection by the method for embodiment 7.

Embodiment 9 injections

Preparation method:

1) press the method pharmaceutical compositions of embodiment 1;

2) get water for injection, phosphoric acid-sodium hydrogen phosphate is dissolved in water for injection, add the pharmaceutical composition of 1/2 amount, stir, then add sodium citrate and chitose, continue to stir; Add again the pharmaceutical composition of 1/2 amount constantly to stir;

3) add sodium bicarbonate, stir 20min;

Embodiment 10 injections

Preparation method: the method preparation of pressing embodiment 9.

Embodiment 11 injections

Preparation method:

1) press the method pharmaceutical compositions of embodiment 3;

2) get water for injection, phosphoric acid-sodium hydrogen phosphate is dissolved in water for injection, add the pharmaceutical composition of 1/2 amount, stir, then add sodium citrate and chitose, continue to stir; Add again pharmaceutical composition, propylene glycol stearate and the maltose alcohol of 1/2 amount, constantly stir, make to dissolve completely;

3) add sodium acetate and sodium carbonate, stir 20min;

Embodiment 12 injections

Preparation method: prepare injection by the method for embodiment 11.

Embodiment 13 injections

Preparation method: prepare injection by the method for embodiment 11.

Embodiment 14

Rear cabinet pre-cooling: setting rear cabinet temperature is-50 DEG C;

Forvacuum: setting drying baker forvacuum degree is 0.15mbar;

F. sublimation drying:

G. parsing-desiccation:

Control Example 1 injection

Preparation method: according to the method preparation of embodiment 9.

Control Example 2 injections

Preparation method: according to the method preparation of embodiment 9.

Control Example 3 injections

Preparation method: according to the method preparation of embodiment 9.

Test example 1 preparation method is investigated

In 1.1 pig limbs bone preparation methoies, in heavy, the alkali deposited step of acid, pH changes the impact on polypeptides matter in extracting solution (in bovine serum albumin) content

Assay method: precision measures pig limbs bone extracting solution, puts in 25ml measuring bottle, is diluted with water to scale, shakes up, and as need testing solution, looks for forint phenol algoscopy to measure;

The heavy pH value that regulates of acid is respectively 1.9,2.0,2.1,2.2 and 2.5; Alkali deposited regulates pH value to be respectively 8.8,8.9,9.0,9.1 and 9.3; All identical with embodiment 1 of all the other preparation processes; Measure the amount (mg) containing bovine serum albumin in every ml extracting solution, testing result is in table 1.

Table 1 acid is heavy, pH changes the impact on bovine serum albumin content in alkali deposited step

As can be seen from the table, in the time that the heavy adjusting of acid pH is 2.1, it is 9.0 o'clock that alkali deposited regulates pH, the amount that contains bovine serum albumin in every ml pig limbs bone extracting solution is the highest, when the heavy adjusting of acid, pH is between 2.0-2.1, it is between 8.9-9.1 time that alkali deposited regulates pH, the amount that contains bovine serum albumin in every ml pig limbs bone extracting solution all exceed 6.5mg, and when sour heavy, the pH value regulating in alkali deposited process exceeds above-mentioned scope, the amount that contains bovine serum albumin in every ml pig limbs bone extracting solution significantly declines, show in the time preparing pig limbs bone extracting solution, in the heavy step of acid and alkali deposited step, the variation of pH value has significant impact to the measurer of bovine serum albumin in extracting solution.

Test example 2 stability tests

1. accelerated test

In order to investigate mixing and the impact of weight proportion on injection stability thereof of additives sodium citrate of the present invention and chitose, the present invention is by the injection of embodiment 9, control Example 1, control Example 2 and control Example 3, all under the condition of 40 DEG C ± 2 DEG C of temperature, place 6 months, sample respectively once 0 month, 1 month, 2 months, 3 months, 6 the end of month at duration of test, by the regulation in Chinese Pharmacopoeia, the character, pH value, visible foreign matters and the labelled amount % containing bovine serum albumin that detect injection, testing result is in table 2;

The accelerated test result of table 2 injection

The injection that the embodiment of the present invention 9 provides as can be seen from the table, known through accelerated test result, to place after 6 months, the character of described injection, visible foreign matters, pH value and the amount that contains bovine serum albumin all do not change; And the injection of control Example 1, control Example 2 and control Example 3 was placed after 6 months, character changes, and has visible foreign matters, and variation has all occurred for pH value and the amount that contains bovine serum albumin; Wherein, the pH value of control Example 3 has reduced by 1.97, and the pH of control Example 2 has reduced by 1.38, and the pH value of control Example 1 has reduced by 0.69; Control Example 3 has declined 3.31% containing the labelled amount of bovine serum albumin, and control Example 2 has declined 1.8% containing the labelled amount of bovine serum albumin, and the labelled amount containing bovine serum albumin of control Example 1 has declined 0.83%; The injection of control Example 2 and control Example 3 was placed after 2 months simultaneously, and character changes, and has visible foreign matters; Draw the injection of the embodiment of the present invention 9 from above result, at high temperature place after 6 months, stability is significantly better than control Example 1, control Example 2 and control Example 3; And the stability of the injection of control Example 1 is higher than control Example 2 and control Example 3; Show that thus sodium citrate and chitose exist simultaneously, and proportion is limited in sodium citrate: chitose=0.2: when 1-3 (weight portion), can significantly improve the stability of injection.

2. long term test

Get the present invention by the injection of embodiment 9, control Example 1, control Example 2 and control Example 3 25 DEG C ± 2 DEG C of temperature, humidity is to place 24 months under 60% ± 10% condition, sample respectively once at duration of test 0 month, 3 months, 6 months, 9 months, 12 months, 18 months and 24 the end of month, by the regulation in Chinese Pharmacopoeia, the character, pH value, visible foreign matters and the labelled amount % containing bovine serum albumin that detect injection, testing result is in table 3;

The long-term test results of table 3 injection

The injection that the embodiment of the present invention 9 provides as can be seen from the table, known through long-term test results, to place after 24 months, the character of described injection, visible foreign matters, pH value and the amount that contains bovine serum albumin all do not change; And the injection of control Example 1, control Example 2 and control Example 3 was placed after 24 months, character changes, and has visible foreign matters, and variation has all occurred for pH value and the amount that contains bovine serum albumin.Draw the injection of the embodiment of the present invention 9 from above result, can place for a long time, show that thus sodium citrate and chitose exist simultaneously, and proportion is limited in sodium citrate: chitose=0.2: when 1-3 (weight portion), can significantly improve the stability of injection, and extend the shelf-life of injection.

Test example 3 safety testings

3.1 hemolytic test

1. tested medicine

1 group of administration: the injection of intravenous drip embodiment 11,25mg/ props up, and adds in 250ml normal saline;

2 groups of administrations: the injection of intravenous drip embodiment 12,25mg/ props up, and adds in 250ml normal saline; Embodiment 12

Blank group: 0.9% chloride injection agent.

2. animal subject

Japan large ear rabbit, body weight 3.0kg, male;

3. test method

The preparation of 2% rabbit erythrocyte suspension: gets ear vein of large ear rabbit about 10ml that takes a blood sample, put in the conical flask of tool bead in clean dried, and jolting is except defibrinating, and adds normal saline and shakes up, 3000 revs/min centrifugal 10 minutes, abandoning supernatant; Normal saline washing three times for the erythrocyte of precipitation, till not taking on a red color to supernatant; Gained erythrocyte is mixed with to 2% red blood cell suspension with normal saline, is for experiment;

The preparation of 1 group of diluent of administration: get 4 of embodiment 11 injections, add normal saline to 5ml, get 4ml and add normal saline to 5ml, obtain 4mg/ml solution;

The preparation of 2 groups of diluents of administration: get 4 of embodiment 12 injections, add normal saline to 5ml, get 4ml and add normal saline to 5ml, obtain 4mg/ml solution;

Get 7 of clean dry test-tubes; Add successively 2% red blood cell suspension and normal saline by table 4 proportional quantity, after shaking up, in 37 DEG C of calorstats, place 30 minutes, then add respectively 2 groups of diluents of 1 group of administration and administration, volume is respectively 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml; The 6th pipe is physiology saline control, and the 7th pipe is distilled water contrast, shakes up in rearmounted 37 DEG C of calorstats, observes each test tube solution in 4 hours and has or not haemolysis and erythroagglutination, and criterion is in table 5.

Table 4 injection bone-melon extract (4mg/ml) hemolytic test

Table 5 haemolysis criterion

4. result of the test

In 4 hours, observe, 1-6 pipe solution erythrocyte sinks, supernatant liquid achromatism and clarity, after jolting, erythrocyte can disperse completely, show that 1 group of diluent of administration, 2 groups of diluents of administration and normal saline are without haemolysis and erythroagglutination, No. 7 pipe is red clear and bright, and the pipe end shows whole haemolysis without erythrocyte is residual, the results are shown in Table 6 and table 7.

Table 6 embodiment 11 injections (4mg/ml) hemolytic test result

Table 7 embodiment 12 injections (4mg/ml) hemolytic test result

Note: "-" be haemolysis not, does not also condense; "+" cohesion; " ++ " part haemolysis; " +++ " complete hemolysis.

5. conclusion (of pressure testing)

The injection of embodiment 11 and embodiment 12 to family's rabbit erythrocyte without haemolysis and coacervation.

3.2 vascular stimulation tests

1. tested medicine

2 groups of administrations: the injection of intravenous drip embodiment 12,25mg/ props up, and adds in 250ml normal saline;

Blank group: 0.9% chloride injection agent.

2. animal subject

Japan large ear rabbit, body weight 3.0kg, male.

3. test method

The preparation of 1 group of diluent of administration: get 4 of embodiment 11 injections, add normal saline to 25ml, obtain 4mg/ml solution;

The preparation of 2 groups of diluents of administration: get 4 of embodiment 12 injections, add normal saline to 25ml, obtain 4mg/ml solution;

Get 6 of Japan large ear rabbits, body weight 2.5～2.8kg, male and female dual-purpose, from auricular vein drug administration by injection group 1 diluent 3ml/kg and 2 groups of diluents of administration, blank group is given isopyknic normal saline once-a-day, and continuous 3 days, in last administration sacrificed by exsanguination after 2 hours; Take off the rabbit ear in last injection site distal end 0.5cm and injection site proximal part 1.5cm mono-segment mark basis, fix by 10% formalin, carry out tissue slice inspection.

4. result of the test

Visual inspection: after diluent and normal saline that 1 group of rabbit auricular vein drug administration by injection and administration are 2 groups, it is red and swollen that local organization perusal has no, and oozes out, downright bad grade stimulates performance.

5. conclusion

The injection (4mg/ml) of embodiment 11 and embodiment 12 has no stimulation to rabbit blood vessel.

3.3 sensitivity test

1. tested medicine

Positive control solution: bovine serum albumin; Be mixed with 10mg/ml with normal saline before use;

Blank medicine: 0.9% chloride injection agent.

2. animal subject

Cavia porcellus, male and female dual-purpose.

3. experimental technique

The preparation of 1 group of diluent of administration: get 1 of embodiment 11 injection, add normal saline to 5ml, get 4ml and add normal saline to 5ml, obtain 4mg/ml solution;

The preparation of 2 groups of diluents of administration: get 1 of embodiment 12 injection, add normal saline to 5ml, get 4ml and add normal saline to 5ml, obtain 4mg/ml solution;

Get 24 of body weight 250-350g Cavia porcelluss, male and female dual-purpose, is divided into 4 groups at random, 6 every group; 1 group of diluent 0.5ml/ of first group of intraperitoneal injection only; 2 groups of diluent 0.5ml/ of second group of intraperitoneal injection only; The 3rd group of intraperitoneal injection of saline 0.5ml/ is only as negative control; The 4th group of lumbar injection 10mg/ml bovine serum albumin 0.5ml/ is only as positive control; The next day that above lumbar injection being, injection, carries out altogether 3 times; After sensitization first the 14th day and the 21st day, get respectively 3 Cavia porcellus ear veins for every group and inject each group of medicinal liquid 1ml/ and only attack, observe Cavia porcellus in latter 15 minutes of injection and whether grab the allergic symptoms such as nose, sneeze, perpendicular hair, tic; Mark by table 8 standards of grading.

Table 8 Cavia porcellus anaphylaxis standards of grading

Note: 2 points of reaction score values above (comprising 2 points), think that tested drug anaphylaxis is defective.

4. result of the test

1 group of administration, 2 groups of administrations and negative control group are not grabbed the anaphylaxiss such as nose, sneeze, tic after continuous 3 lumbar injections and twice attack; Positive controls Cavia porcellus is after continuous 3 lumbar injections, and twice attack starts to occur dyspnea, tic, death in 5 minutes, the results are shown in Table 9 and table 10;

2 groups of injections of 1 group of table 9 administration and administration (4mg/ml) anaphylaxis result of the test that vein is attacked for the first time

2 groups of injection 4mg/ml of 1 group of table 10 administration and administration) for the second time vein attack anaphylaxis result of the test

5. conclusion

The injection of embodiment 11 and embodiment 12 does not produce without anaphylaxis Cavia porcellus.

3.4 local excitation tests

1. tested medicine

Blank medicine: 0.9% chloride injection agent.

2. animal subject

Japan's white big ear rabbit, male and female dual-purpose, body weight 2.6-2.9kg.

3. test method

The preparation of 1 group of diluent of administration: get 1 of embodiment 11 injection, add normal saline to 4ml, obtain 12.5mg/ml solution;

The preparation of 2 groups of diluents of administration: get 1 of embodiment 12 injection, add normal saline to 4ml, obtain 12.5mg/ml solution;

Get 6 of Japanese white big ear rabbits, be divided at random three groups, 2 every group; 2 groups of 1 group of administration and administrations are respectively injected test liquid 1ml at its left and right lower limb quadriceps femoris respectively, blank group is to equivalent 0.9% chloride injection agent, inject and within latter 48 hours, put to death animal, dissect and take out quadriceps femoris, longitudinally cut, observe the irritant reaction of injection site muscle, mark by table 11 standards of grading; Result of the test is in table 12.

Table 11 rabbit quadriceps femoris irritant reaction standards of grading

Note: the highest and minimum difference of each group reaction level is greater than at 2 o'clock, answers retry; 4 quadriceps femoris order of reaction sums are less than at 10 o'clock, the local excitation agreement with experimental regulation of administration group.

Table 12 injection bone-melon extract local excitation result of the test

4. conclusion

Under this experimental condition, the irritant test of the injection (12.5mg/ml) of embodiment 11 and embodiment 12 to rabbit quadriceps femoris, 4 quadriceps femoris order of reaction sums of 2 rabbit are less than 10, so think the local excitation test determination of injection (12.5mg/ml) of embodiment 11 and embodiment 12.

Test example 4 clinical trials

The curative effect of 4.1 treatment rheumatoid arthritiss and ankylosing spondylitis

1. test case

Accept 133 routine patients for medical treatment and all meet rheumatoid arthritis and ankylosing spondylitis diagnostic criteria, wherein patient with rheumatoid arthritis 60 examples, patients with ankylosing spondylitis 70 examples, age 35-60 year, the course of disease 1 month-8 years.

2. grouping and administration

60 routine patient with rheumatoid arthritis are divided into 1 group of 23 example for the treatment of at random, treat 2 group of 23 example, matched group 17 examples; 73 routine patients with ankylosing spondylitis are divided into 1 group of 28 example for the treatment of at random, treat 2 group of 27 example, matched group 18 examples; Treat 1 group of injection described in the equal intravenous drip embodiment of the present invention 7, a 15ml (adding 250ml 0.9% chloride injection agent), every day 1 time, 20th is a course for the treatment of; Treat 2 groups of injections described in the equal intravenous drip embodiment of the present invention 8, a 15ml (adding 250ml 0.9% chloride injection agent), every day 1 time, 20th is a course for the treatment of; The equal intravenous drip ibuprofen injection of matched group, a 400mg (adding 100ml0.9% chloride injection dilution agent), every day 1 time, 20th is a course for the treatment of.

3. diagnostic criteria

The diagnostic criteria of rheumatoid arthritis:

(1) symptom: arthroncus or little joint symmetry swell and ache, morning deadlock;

(2) sign: the arthroncus tenderness of getting involved, movable function is limited or lopsided, or tetanic;

(3) lab testing: the RF positive, ESR speeds more;

(4) x-ray inspection: the emphasis joint of getting involved has the performance of typical rheumatoid arthritis x-ray.

The diagnostic criteria of ankylosing spondylitis:

(1) spinal column deadlock in morning, arthralgia, swelling;

(2) lab testing: serum albumin reduces, serum immune globulin lgG, lgA and lgM increase, and serum complement C3 and C4 increase;

(3) sacro-iliac joints x-ray performance.

4. efficacy assessment standard

Recovery from illness: symptom all disappears, main experimental index is normal;

Effective: cardinal symptom is clearly better, main experimental index is clearly better;

Take a turn for the better: cardinal symptom takes a turn for the better to some extent, main experimental index takes a turn for the better to some extent;

Invalid: before and after treatment, not change.

5. treatment is analyzed

Patient clinical data, through finishing analysis, is compared 1 group for the treatment of, 2 groups of treatments and matched group in recovery from illness, effective, effective and invalid four standards, check to verify the difference between treatment group and matched group by t.

6. result

Treat 1 group, 2 courses for the treatment of of the equal administration of patient for the treatment of 2 groups and matched group, three groups to the therapeutic effect of rheumatoid arthritis and ankylosing spondylitis more respectively in table 13 and table 14;

Three groups, table 13 is to the comparison of rheumatoid arthritis treatment effect

With matched group comparison, P ^a< 0.01, compares P with 1 group for the treatment of ^b< 0.05.

Three groups, table 14 is to the comparison for the treatment of ankylosing spondylitis effect

With matched group comparison, P ^a< 0.01, with 1 group of comparison for the treatment of, P ^b< 0.05.

7. conclusion

With matched group comparison, injection provided by the invention has significant curative effect to rheumatoid arthritis and ankylosing spondylitis; Treat 1 group and 2 groups of comparisons for the treatment of, the therapeutic effect of rheumatoid arthritis and ankylosing spondylitis be there are differences; The curative effect of the injection that shows can to improve adding of Flos Bombacis Malabarici, Flos Buddlejae and Herba Eupatorii embodiment 7 to rheumatoid arthritis and ankylosing spondylitis.

The curative effect of 4.2 treatment sciatica

1. test case

Accept the equal sciatica diagnostic criteria of 68 routine patient for medical treatment, age 30-60 year, the course of disease 2 months-4 years.

2. grouping and administration

68 routine sciatica patients are divided into 1 group of 24 example for the treatment of at random, treat 2 group of 28 example, matched group 16 examples; Treat 1 group of injection described in the intravenous drip embodiment of the present invention 7, a 15ml (adding 250ml 0.9% chloride injection agent), every day 1 time, 20th is a course for the treatment of; Treat 2 groups of injections described in the intravenous drip embodiment of the present invention 8, a 15ml (adding the agent of 250ml0.9% chloride injection), every day 1 time, 20th is a course for the treatment of; Matched group intravenous drip Moschus injection (composition: formed by artificial Moschus, Radix Curcumae, Herba Pogostemonis, Rhizoma Acori Graminei, Borneolum Syntheticum, Mentholum), a 15ml (adding 250ml 0.9% chloride injection dilution agent), 20th be a course for the treatment of every day 1 time.

3. efficacy assessment standard

Recovery from illness: pain all disappears;

Effective: pain is clearly better;

Take a turn for the better: pain takes a turn for the better to some extent;

Invalid: before and after treatment, not change.

4. treatment is analyzed

5. result

Treat 1 group, 2 courses for the treatment of of the equal administration of patient for the treatment of 2 groups and matched group, three groups to the therapeutic effect of sciatica more respectively in table 15;

Three groups, table 15 is to the comparison of sciatica therapeutic effect

With matched group comparison, P ^a< 0.05, P ^b< 0.01; Compare P with 1 group for the treatment of ^c< 0.01.

6. conclusion

With matched group comparison, injection provided by the invention has significant curative effect to sciatica; Treat 1 group and 2 groups of comparisons for the treatment of, the therapeutic effect of sciatica is existed to significant difference; The injection of the embodiment of the present invention 8 is the injection significantly better than embodiment 7 to the curative effect of sciatica; Show that the injection that can significantly improve embodiment 7 adding of Flos Bombacis Malabarici, Flos Buddlejae and Herba Eupatorii is to the curative effect of sciatica.

The curative effect of 4.3 treatment gouts

Accept patient with gout totally 82 examples for medical treatment, the age is all between 20-60 year, get rid of gestation and age of sucking patient.

2. grouping and administration

75 routine patient with gout are divided into 1 group of 28 example for the treatment of at random, treat 2 group of 30 example, matched group 17 examples; Treat 1 group of injection described in the intravenous drip embodiment of the present invention 7, a 15ml (adding 250ml 0.9% chloride injection agent), every day 1 time, 20th is a course for the treatment of; Treat 2 groups of injections described in the intravenous drip embodiment of the present invention 8, a 15ml (adding the agent of 250ml0.9% chloride injection), every day 1 time, 20th is a course for the treatment of; Matched group intravenous drip antondin injection, 7th is a course for the treatment of at every day 1 time.

3. check

Clinical manifestation: lower limb red and swollen heat pain, can not land, inconvenient activity;

Lab testing: blood uric acid 650-800 μ mol/L.

4. efficacy assessment standard

Recovery from illness: after treatment, disease all disappears, and reaches clinical cure standard, it is normal that blood uric acid recovers;

Effective: after treatment, state of an illness sign is obviously improved, and Uric Acid Content reduces more than 2/3;

Effective: after treatment, state of an illness sign makes moderate progress, and Uric Acid Content reduces less than 2/3;

Invalid: unchanged before and after treatment.

5. treatment is analyzed

Patient clinical data, through finishing analysis, is compared treatment group and matched group in recovery from illness, effective, effective and invalid four standards, check to verify the difference between treatment group and matched group by t.

6. result

Treat 1 group, 2 courses for the treatment of of the equal administration of patient for the treatment of 2 groups and matched group, three groups to the therapeutic effect of gout relatively in table 16;

Three groups, table 16 is to the comparison of gout treatment effect

7. conclusion

With matched group comparison, injection provided by the invention has significant curative effect to gout; Treat 1 group and 2 groups of comparisons for the treatment of, the therapeutic effect of gout be there are differences; The injection of the embodiment of the present invention 8 is the injection significantly better than embodiment 7 to the curative effect of gout; Show that the injection that can significantly improve embodiment 7 adding of Flos Bombacis Malabarici, Flos Buddlejae and Herba Eupatorii is to the curative effect of gout.

Claims

1. a pharmaceutical composition for the treatment of osteopathia, is characterized in that, described pharmaceutical composition is mainly made up of the component of following weight portion:

Pig limbs bone extracting solution 30-70 Semen Melo extracting solution 15-25.

2. the pharmaceutical composition for the treatment of osteopathia as claimed in claim 1, is characterized in that, described pharmaceutical composition also comprises that parts by weight are the mixed extract of 10-20 part; Described mixed extract is made up of the component of following weight portion:

Flos Bombacis Malabarici 20-30 Flos Buddlejae 5-10 Herba Eupatorii 30-50.

3. the pharmaceutical composition for the treatment of osteopathia as claimed in claim 2, is characterized in that, described pharmaceutical composition is prepared from by the component of following weight portion:

Pig limbs bone extracting solution 50 Semen Melo extracting solution 30

Flos Bombacis Malabarici 25 Flos Buddlejae 7.5 Herba Eupatoriis 40.

4. by the pharmaceutical composition of the arbitrary described treatment osteopathia of claim 1-3 and the injection that adjuvant forms, it is characterized in that, described injection comprises pharmaceutical composition, additives and water for injection.

5. injection as claimed in claim 4, is characterized in that, the parts by weight of described pharmaceutical composition are 45-115 part, and described water for injection parts by weight are 100-200 part; Described additives comprise that parts by weight are the buffer agent of 2.5-7.5 part, the pH adjusting agent of 1-2.5 part, the sodium citrate of 0.2 part and the chitose of 1-3 part; Described buffer agent is phosphoric acid-sodium hydrogen phosphate, and described pH adjusting agent is sodium bicarbonate, sodium carbonate or sodium acetate.

6. injection as claimed in claim 5, is characterized in that, described additives also comprise that parts by weight are the propylene glycol stearate of 0.5-1.5 part and the maltose alcohol of 0.2 part.

7. a preparation method for the pharmaceutical composition for the treatment of osteopathia claimed in claim 1, is characterized in that, described method step is as follows:

1) preparation of pig limbs bone extracting solution:

1.2 extract: pig limbs bone after fragmentation is dropped into extraction pot, add purified water, hot pressing extracts twice, the amount that at every turn adds purified water is raw material weight 2 times, each 1h, controlled pressure is 0.11-0.12MPa, temperature is 121-125 DEG C, makes extracting solution;

2) preparation of Semen Melo extracting solution:

4) bone melon extracting solution ultrafiltration, subpackage: take out room temperature negative catalysis, ultrafiltration by freezing real bone melon extracting solution, molecular weight 5K ultrafiltration post, do not rinse pH value 5.0-7.0, flushing water electrical conductivity≤1.3 μ s/cm higher than 40 DEG C of waters for injection by front use, operating pressure is not higher than 0.1Mpa, collect ultrafiltrate, ultrafiltrate is stirred and in Agitation Tank, stirs 20min, subpackage is in the stainless steel cask of 0.5% sodium hydroxide solution processing, handle after the assay was approved warehouse-in, make pharmaceutical composition;

Preferably, described preparation method also comprises the method for preparing mixed extract, and described method step is as follows:

8. a preparation method for injection claimed in claim 6, is characterized in that, described method comprises the steps:

1) pharmaceutical compositions;

3) add pH adjusting agent, stir 20min;

9. the pharmaceutical composition for the treatment of osteopathia as claimed in claim 3, is characterized in that, the application of described pharmaceutical composition in the medicine of preparation rheumatoid arthritis, ankylosing spondylitis, sciatica or gout.

10. the preparation method of the injectable powder being made up of pharmaceutical composition and the adjuvant for the treatment of osteopathia claimed in claim 1, is characterized in that, described method comprises the steps:

Rear cabinet pre-cooling: setting rear cabinet temperature is-50 DEG C;

Forvacuum: setting drying baker forvacuum degree is 0.15mbar;

F. sublimation drying:

G. parsing-desiccation: