CN101297853B - Bone-melon extract injection and preparation thereof - Google Patents
Bone-melon extract injection and preparation thereof Download PDFInfo
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- CN101297853B CN101297853B CN2008101151179A CN200810115117A CN101297853B CN 101297853 B CN101297853 B CN 101297853B CN 2008101151179 A CN2008101151179 A CN 2008101151179A CN 200810115117 A CN200810115117 A CN 200810115117A CN 101297853 B CN101297853 B CN 101297853B
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Abstract
The invention provides a drug of bone-melon extract for injection, mammalian bone peptides and melon seed peptides are extracted, the obtained extracts are mixed according a certain proportion, thus preparing the drug for injection. The invention adopts a special extraction technology to increase the active ingredients, the extraction efficiency is high, and the substances with high molecular weight are reduced to the minimum. The drug of the invention has the effects of regulating bone metabolism, stimulating proliferation of osteoblasts, promoting formation of new bones, regulating calcium and phosphorus metabolism, increasing bone calcium deposition and preventing osteoporosis, thus being used for prevention and treatment for rheumatism, rheumatoid arthritis, osteoarthritis, lumbar and leg pain, fracture trauma repair and other diseases.
Description
Technical field
The present invention relates to a kind of polypeptide drug composition, bone-melon extract injectable drug specifically the invention still further relates to the preparation method of this medicine.
Background technology
Bone-melon extract contains various active polypeptide and free amino acid, has the adjusting bone metabolism, and stimulating osteoblast propagation promotes new bone formation, regulates calcium, phosphorus metabolism, increases bone calcium deposition, prevents effects such as osteoporosis.
Bone is induced the effect of polypeptide class biotic factor: bone induces polypeptide class biotic factor can effectively promote to influence in the body the synthetic of bone formation and absorption and bdgf, comprise bone morphogenetic protein (BMPs), β-transforming growth factor (TGF-β), fibroblast growth factor (FGF) etc., thus multiple biological activity had.Its main pharmacological has: promote cell mitogen, differentiation, chemotaxis and molten bone active.Wherein BMPs is one group of acid low-molecular-weight glycoprotein, as a kind of efficient bone Induced substance, BMPs is that mesenchymal cell is the initially signal factor of cell differentiation to bone, the bone that the mesenchymal cell that moves about around the energy induction of vascular is converted into irreversibility is a cell, be chondrocyte and osteoblast, thereby the promotion callus formation is induced new bone formation, promotes fracture repair; In addition, BMPs is the change of scalable extracellular matrix composition also, and by inducing new bone formation better with TGF-β and FGF coordinative role each other, makes osseous tissue more ripe.TGF-β is one group of protein polypeptide with multiple function, osteoblast and chondroblast there are the dual regulation effect that promotes that differentiation or reduction are broken up, regulate somatomedin with the multiple factor such as extracellular matrix with other differentiation and work in coordination with the adjusting that the participation pair cell breaks up; TGF-β can promote extracellular matrix synthetic, can directly stimulate the synthetic of fibroblast epimatrix, and its new synthetic substrate degradation is had remarkable inhibitory action; For osteoblast, TGF-β can promote its synthetic type i collagen, bone conglutination element and osteopontin; Simultaneously, TGF-β shows that to the effect of lymphocyte and macrophage it can alleviate the destructiveness of inflammatory reaction, can assist some macrophage derived cytokine to play a role in the cell tissue reparation again.FGF is that one group of heparin binds polypeptide, but the trend of irritation cell move, rise in value and break up, increase the quantity of rubber polymer archeocyte, promote the synthetic of bone collagen and noncollagen protein, increase synthesizing of Bone Gla protein.
The effect of Fructus Melo seed extract: the Fructus Melo seed extract is to extract through special process from the ripe dry seed of cucurbitaceous plant Fructus Melo (Cucumis melo L.) to form, can reduce the local capillary permeability of fracture, reduce inflammatory exudation, promote the recovery of local blood fortune obstacle; Simultaneously can also reduce whole blood viscosity and erythrocyte aggregation degree, improve the partial blood circulation of callus, for osteocyte provides a good blood supply environment; Can also suppress the release of prostaglandin in addition, reach analgesic effect; In promoting fracture heals in early days, the Fructus Melo seed extract induces the polypeptide biotic factor to have synergism with the bone that replenishes, and promotes the effect of collagen somatomedin.
The effect of other composition: the multiple free amino acid that is rich in, supply raw materials for the synthetic BMPs of osteocyte, bone source property biotic factors such as TGF-β, FGF, promote the synthetic of bone source property biotic factor, organic calcium, phosphonium ion can participate in alcium and phosphor metabolization, keep bone volume.
CN 1742778A discloses a kind of cervus and cucumis polypeptide pharmaceutical composition, and said composition contains Os Cervi extract, Fructus Melo seed extract and one or more excipient to be formed, and wherein the weight ratio of Os Cervi polypeptide and Fructus Melo seed is 2: 1 to 6: 1.It is to prepare by the following method: the A) preparation of Os Cervi extract solution: (1) Os Cervi pretreatment: (2) pulverize pretreated clean Os Cervi; (3) add 2 times of waters for injection more than the weight in broken Os Cervi, 121 ℃ of hot pressing are extracted to leach and are obtained an Os Cervi extracting solution; (4) add weight water for injection more than 2 times in the Os Cervi slag after once extracting, continuation is extracted 121 ℃ of-123 ℃ of hot pressing, leach and obtain secondary Os Cervi extracting solution, an Os Cervi that obtains with step (3) extracts 20 liquid and merges and make the Os Cervi extracting solution: (5) are centrifugal with the Os Cervi extracting solution, collect supernatant centrifugal liquid; (6) centrifugal liquid is carried out 100KD, 10KD, 6KO ultrafiltration respectively, collect filter liquor, be evaporated to 10% of cumulative volume, collect concentrated solution, make Os Cervi extract solution: the B) preparation of Semen Melo extract solution: (1) is clean with the rinsing of drying and ripening Semen Melo: (2) add the water for injection of weight more than 2 times in clean Semen Melo, grind to form Semen Melo homogenate with colloid mill; (3) Semen Melo homogenate is extracted 121 ℃ of hot pressing, leach the Semen Melo extracting solution one time: add weight water for injection more than 2 times in (4) Semen Melo slag after once extracting, continuation is extracted 121 ℃ of-123 ℃ of hot pressing, leach secondary Semen Melo extracting solution, a Semen Melo extracting solution that obtains with step (3) merges that to make Semen Melo extracting solution 1 (5) centrifugal with the Semen Melo extracting solution, collect supernatant centrifugal liquid (6) centrifugal liquid is carried out 100KD respectively, 10KD, the 6KD ultrafiltration, collect filter liquor, be evaporated to 10% of original volume, collect concentrated solution, make Semen Melo extract solution: C) injection cervus and cucumis polypeptide preparation of drug combination: blend step A) Zhi Bei Os Cervi extract solution and step B) preparation Semen Melo extract solution and one or more excipient pharmaceutical compositions, make freeze-dried powder by Freeze Drying Technique, wherein the weight ratio of Os Cervi polypeptide and Semen Melo polypeptide is 2: 1 to 6: 1.
The extraction ratio of above-mentioned preparation method is low, and the bone-melon extract injection effective ingredient of making is lower.For this reason, people urgently seek a kind of more effective solution.
Summary of the invention
The object of the present invention is to provide a kind of bone-melon extract injectable drug, and preparation method thereof.
Bone-melon extract injectable drug of the present invention prepares by the following method:
A) preparation of bone extract
1) os purum will be got after the mammalian bone pretreatment;
2) get os purum, add the purified water of 2～5 times of weight, in 120 ℃～123 ℃, 1～3h is extracted in hot pressing under 0.11～0.13Mpa condition, filters, and gets filtrate;
3) with filtrate under 0～10 ℃ of condition static 8～16 hours, remove the upper strata oils and fats;
4) regulating PH is 8.5～9.0, is heated to 44 ℃～46 ℃, presses 0.1～0.3% of bone weight and adds pancreatin, constant temperature continuous hydrolysis 1～3 hour;
5) regulating pH is 3.5～4.0, boils, and is incubated 15～45 minutes after-filtration, and it is 6.5～7.0 that filtrate is regulated pH;
6) add proper amount of active carbon, be heated to boiling, be incubated 10～20 minutes after-filtration, filtrate in-20 ℃ dark freeze 36～50 hours after the room temperature thawing;
7) ultrafiltration under the 10KD molecular retention, filtrate after suitably concentrating, ultrafiltration under the 6KD molecular retention again, promptly;
B) preparation of Fructus Melo seed extract:
1) clean Fructus Melo seed will be got after the pretreatment of Fructus Melo seed;
2) get clean Fructus Melo seed, add the water of 1～3 times of weight, in 120 ℃～123 ℃, 1～3h is extracted in 0.11～0.13MPa hot pressing, filters to get filtrate
3) repeating step 2) 1～3 time;
4) merging filtrate is cooled to 25～35 ℃, regulates pH to 3.5～4.0, leaves standstill 8～16 hours, goes precipitation;
5) boil, be incubated 15～45 minutes, filter;
6) regulating pH is 8.5～9.0, in-20 ℃ dark freeze 36～50 hours after room temperature melt;
7) boil, be incubated 15～45 minutes, filter, regulate pH to 6.0～7.0;
8) ultrafiltration under the 10KD molecular retention, filtrate after suitably concentrating, ultrafiltration under the 6KD molecular retention again, promptly;
C) with steps A) bone extract and the step B of preparation) the Fructus Melo seed extract of preparation mixes, add an amount of excipient, be prepared into injection or lyophilized powder, wherein injection can be low capacity bone-melon extract injection, bone melon sodium chloride injection or bone melon glucose injection, wherein the weight ratio of bone polypeptide and Fructus Melo seed polypeptide is 1: 10～10: 1, be preferably 1～5: 1, more preferably 3: 1.
Preferably, injection of the present invention obtains by being prepared as follows method:
A) preparation of bone extract
1) os purum will be got after the mammalian bone pretreatment;
2) get os purum, add the purified water of 4 times of weight, in 121 ℃, 2h is extracted in hot pressing under the 0.12Mpa condition, filters, and gets filtrate;
3) with filtrate under 0～10 ℃ of condition static 12 hours, remove the upper strata oils and fats;
4) regulating pH is 8.5～9.0, is heated to 45 ℃, presses 0.2% of bone weight and adds pancreatin, constant temperature continuous hydrolysis 2 hours;
5) regulating pH is 3.5～4.0, boils, and is incubated 30 minutes after-filtration, and it is 6.5～7.0 that filtrate is regulated pH;
6) add proper amount of active carbon, be heated to boiling, be incubated 15 minutes after-filtration, filtrate in-20 ℃ dark freeze 48 hours after the room temperature thawing;
7) ultrafiltration under the 10KD molecular retention ,-0.1MPA is evaporated to 1/3 of original volume under 60 ℃ of conditions, ultrafiltration under the 6KD molecular retention again, promptly;
B) preparation of Fructus Melo seed extract:
1) clean Fructus Melo seed will be got after the pretreatment of Fructus Melo seed;
2) get clean Fructus Melo seed, add the water of 2 times of weight, in 121 ℃, 2h is extracted in 0.12MPa hot pressing, filters to get filtrate
3) repeating step 2) 2 times;
4) merging filtrate is cooled to 25～35 ℃, regulates pH to 3.5～4.0, leaves standstill 12 hours, goes precipitation;
5) boil, be incubated 30 minutes, filter;
6) regulating pH is 8.5～9.0, in-20 ℃ dark freeze 48 hours after room temperature melt;
7) boil, be incubated 15～45 minutes, filter, regulate pH to 6.0～7.0;
8) ultrafiltration under the 10KD molecular retention ,-0.1MPA is evaporated to 1/3 of original volume under 60 ℃ of conditions, ultrafiltration under the 6KD molecular retention again, promptly;
C) with steps A) bone extract and the step B of preparation) the Fructus Melo seed extract of preparation mixes, adds an amount of excipient and be prepared into injection or lyophilized powder.Wherein used excipient can be preferably mannitol for mannitol and/or dextran, and excipient is 1: 1～5 with total polypeptide by weight, is preferably 1: 2.
Mammalian bone described in the present invention is preferably the mammal bones of limbs, more preferably Os Cervi or Os Sus domestica.
Described Fructus Melo seed is the cucurbitaceous plant melon seed, is preferably the Fructus Melo seed; Extracting used water is purified water or water for injection.
Bone-melon extract injection of the present invention, its active constituent content height, diseases such as rheumatism, rheumatoid arthritis, osteoarthritis, lumbago and skelalgia, trauma fracture reparation had significant curative effect, and polymer substance content is little, comparatively stable by the injection that this method makes, the product yield height, and can make multiple dosage form for clinical use.
The specific embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that used technological means is well known to those skilled in the art among the embodiment.
The preparation of embodiment 1 Sus domestica extract
Peel off: get fresh or refrigerated pig limbs bone, remove meat and fascia, disconnect, remove bone marrow, get os purum 15kg.
Extract: get os purum, add the purified water of 4 times of weight, in 121 ℃, 0.12Mpa 2h is extracted in hot pressing under the condition, filters, and gets filtrate, filtrate was removed the upper strata oils and fats of extracting solution after under 5 ℃ of conditions static 12 hours, regulating pH with 8% sodium hydroxide solution is 8.5, be heated to about 45 ℃, press 0.2% of bone weight and add pancreatin, the constant temperature continuous hydrolysis is after 2 hours, regulating pH with 12% hydrochloric acid is 3.5, boil, be incubated 30 minutes after-filtration, it is 6.5 that filtrate is regulated pH with 8% sodium hydroxide solution.Press 0.1% of filtrate volume and add active carbon, be heated to boiling, be incubated 15 minutes after-filtration, filtrate in-20 ℃ dark freeze 48 hours after the room temperature thawing.
Ultrafiltration, concentrate: with above-mentioned solution ultrafiltration, filtrate is evaporated to 1/3 of original volume in-0.1MPA under 60 ℃ of conditions with the ultrafilter of 10,000 Dalton molecular weights.Concentrated solution through the ultrafilter ultrafiltration of 6000 Dalton molecular weights, gets filtrate 20L again, contains in sterile chamber after ultrafiltrate stirs, and preserves under-20 ℃ of conditions.
The content of the Os Sus domestica polypeptide in the extracting solution is 10.1mg/ml after testing, and free aminoacid content is 6.1mg/ml.
The preparation of embodiment 2 Os Cervi extracts
Peel off: get fresh or refrigerated Cervus nippon Temminck bones of limbs, remove meat and fascia, disconnect, remove bone marrow, get os purum 15kg.
Extract: get os purum, add the purified water of 3 times of weight, in 121 ℃, 0.12Mpa 2h is extracted in hot pressing under the condition, filters, and gets filtrate, filtrate was removed the upper strata oils and fats of extracting solution after under 4 ℃ of conditions static 12 hours, regulating pH with 8% sodium hydroxide solution is 9.0, be heated to about 45 ℃, press 0.2% of bone weight and add pancreatin, the constant temperature continuous hydrolysis is after 2 hours, regulating pH with 12% hydrochloric acid is 4.0, boil, be incubated 30 minutes after-filtration, it is 7.0 that filtrate is regulated pH with 8% sodium hydroxide solution.Press 0.1% of filtrate volume and add active carbon, be heated to boiling, be incubated 15 minutes after-filtration, filtrate in-20 ℃ dark freeze 48 hours after the room temperature thawing.
Ultrafiltration, concentrate: with above-mentioned solution ultrafiltration, filtrate is evaporated to 1/3 of original volume in-0.1MPA under 60 ℃ of conditions with the ultrafilter of 10,000 Dalton molecular weights.Concentrated solution through the ultrafilter ultrafiltration of 6000 Dalton molecular weights, gets filtrate 15L again, contains in sterile chamber after ultrafiltrate stirs, and preserves under-20 ℃ of conditions.
The content of the Os Cervi polypeptide in the extracting solution is 10.2mg/ml after testing, and free aminoacid content is 7.2mg/ml.
The preparation of embodiment 3 Fructus Melo seed extracts
Washing: get the ripe dry Fructus Melo seed that gets, add the purified water rinsing 4 times, empty solid carbon dioxide branch is chosen foreign material, gets clean Fructus Melo seed 15kg.
Extract: get clean Fructus Melo seed, add the purified water of 2 times of weight, in 121 ℃, 0.12MPA 2h is extracted in hot pressing, filter, get filtrate, add the purified water of 2 times of weight again, repeat last fetched, extract so repeatedly three times, merge three times filtrate, be cooled to 30 ℃, is 3.5 with 12% hydrochloric acid with the pH regulator of above-mentioned filtrate, static 12 hours.
Remove foreign protein, said hydrolyzed liquid is boiled, be incubated 30 minutes after-filtration, filtrate is 8.5 with 8% sodium hydroxide solution with above-mentioned filtrate pH regulator, in-20 ℃ dark freeze 48 hours after room temperature melt, boil, be incubated 30 minutes after-filtration, it is 6.0 that filtrate is regulated PH with 12% hydrochloric acid.
Ultrafiltration, concentrate: with above-mentioned solution ultrafiltration, filtrate is evaporated to 1/3 of original volume in-0.1MPA under 60 ℃ of conditions with the ultrafilter of 10,000 Dalton molecular weights.Concentrated solution through the ultrafilter ultrafiltration of 6000 Dalton molecular weights, gets filtrate 30kg again, contains in sterile chamber after ultrafiltrate stirs, and preserves under-20 ℃ of conditions.
The content of the Fructus Melo seed polypeptide in the extracting solution is 4.3mg/ml after testing, and free aminoacid content is 3.8mg/ml.
The peptide algoscopy is measured according to forint phenol algoscopy.
The free amine group acidity test utilizes amino acid determining instrument or the high performance liquid chromatography that it is suitable that high performance liquid chromatography is differentiated reuse earlier to measure.
The preparation of embodiment 4 Fructus Melo seed extracts
Washing: get the ripe dry Fructus Melo seed that gets, add the purified water rinsing 3 times, empty solid carbon dioxide branch is chosen foreign material, gets clean Fructus Melo seed 15kg.
Extract: get clean Fructus Melo seed, add the purified water of 2 times of weight, in 121 ℃, 0.12MPA 2h is extracted in hot pressing, filter, get filtrate, add the purified water of 2 times of weight again, repeat last fetched, extract so repeatedly three times, merge three times filtrate, be cooled to 30 ℃, is 4.0 with 12% hydrochloric acid with the pH regulator of above-mentioned filtrate, static 12 hours.
Remove foreign protein, said hydrolyzed liquid is boiled, be incubated 30 minutes after-filtration, filtrate is 9.0 with 8% sodium hydroxide solution with above-mentioned filtrate pH regulator, in-20 ℃ dark freeze 48 hours after room temperature melt, boil, be incubated 30 minutes after-filtration, it is 6.0 that filtrate is regulated PH with 12% hydrochloric acid.
Ultrafiltration, concentrate: with above-mentioned solution ultrafiltration, filtrate is evaporated to 1/3 of original volume in-0.1MPA under 60 ℃ of conditions with the ultrafilter of 10,000 Dalton molecular weights.Concentrated solution through the ultrafilter ultrafiltration of 6000 Dalton molecular weights, gets filtrate 30kg again, contains in sterile chamber after ultrafiltrate stirs, and preserves under-20 ℃ of conditions.
The content of the Fructus Melo seed polypeptide in the extracting solution is 4.2mg/ml after testing, and free aminoacid content is 3.8mg/ml.
The polypeptide algoscopy is measured according to forint phenol algoscopy.
The free amine group acidity test utilizes amino acid determining instrument or the high performance liquid chromatography that it is suitable that high performance liquid chromatography is differentiated reuse earlier to measure.
The preparation 1 of embodiment 5 Gugua polypeptide injections
The extract 7.5g (by Os Sus domestica polypeptide) of embodiment 1
The extract 2.5g of embodiment 3 (by Fructus Melo seed polypeptide)
Water for injection adds to 2000ml
The Sus domestica extract solution of the Sus domestica extract solution of embodiment 1 preparation and embodiment 1 preparation is pressed Os Sus domestica polypeptide to be mixed with Fructus Melo seed polypeptide weight ratio at 3: 1, sodium hydroxide solution with 8% or 12% hydrochloric acid solution are regulated pH value 6.8, add to the full amount of water for injection again, in the bent neck peace of 2ml bottle, wire drawing is sealed with fill behind the filtering with microporous membrane of 0.22 μ m.Sterilized 12 minutes for 121 ℃.Through lamp inspection, labeling, packing, warehouse-in promptly.
The preparation 2 of embodiment 6 Gugua polypeptide injections
The extract 6.25 (by Os Sus domestica polypeptide) of embodiment 1
The extract 6.25g of embodiment 3 (by Fructus Melo seed polypeptide)
Water for injection adds to 2500ml
The Sus domestica extract solution of the Sus domestica extract solution of embodiment 1 preparation and embodiment 1 preparation is pressed Os Sus domestica polypeptide to be mixed with Fructus Melo seed polypeptide weight ratio at 1: 1, sodium hydroxide solution with 5% or 5% hydrochloric acid solution are regulated pH value to 6.5, add to the full amount of water for injection again, in the bent neck peace of 5ml bottle, wire drawing is sealed with fill behind the filtering with microporous membrane of 0.22 μ m.Sterilized 12 minutes for 121 ℃.Through lamp inspection, labeling, packing, warehouse-in promptly.
The preparation 3 of embodiment 7 Gugua polypeptide injections
The extract 25g (by Os Sus domestica polypeptide) of embodiment 1
The extract 5g of embodiment 3 (by Fructus Melo seed polypeptide)
Water for injection adds to 6000ml
The Sus domestica extract solution of the Sus domestica extract solution of embodiment 1 preparation and embodiment 1 preparation is pressed Os Sus domestica polypeptide to be mixed with Fructus Melo seed polypeptide weight ratio at 5: 1, regulate pH value 6.7 with rare diluted sodium hydroxide solution or dilute hydrochloric acid solution, add to the full amount of water for injection again, in the bent neck peace of 10ml bottle, wire drawing is sealed with fill behind the filtering with microporous membrane of 0.22 μ m.Sterilized 12 minutes for 121 ℃.Through lamp inspection, labeling, packing, warehouse-in promptly.
The preparation 4 of embodiment 8 Gugua polypeptide injections
The extract 37.5g (by Os Sus domestica polypeptide) of embodiment 1
The extract 12.5g of embodiment 3 (by Fructus Melo seed polypeptide)
Sodium chloride 900g
Water for injection adds to 100000ml
The Sus domestica extract solution of the Sus domestica extract solution of embodiment 1 preparation and embodiment 1 preparation is pressed Os Sus domestica polypeptide to be mixed with Fructus Melo seed polypeptide weight ratio at 3: 1, add sodium chloride 900g dissolving, regulate pH value with rare diluted sodium hydroxide solution or dilute hydrochloric acid solution, add to the full amount of water for injection again, with embedding behind the filtering with microporous membrane of 0.22 μ m in the 100ml infusion bottle.Sterilized 15 minutes for 121 ℃.Through lamp inspection, labeling, packing, warehouse-in promptly.
The preparation 5 of embodiment 9 Gugua polypeptide injections
The extract 37.5g (by Os Sus domestica polypeptide) of embodiment 1
The extract 12.5g of embodiment 3 (by Fructus Melo seed polypeptide)
Glucose 5000g
Water for injection is in adding to 100000ml
The Sus domestica extract solution of the Sus domestica extract solution of embodiment 1 preparation and embodiment 1 preparation is pressed Os Sus domestica polypeptide to be mixed with Fructus Melo seed polypeptide weight ratio at 3: 1, add glucose 5000g dissolving, regulate pH value with rare diluted sodium hydroxide solution or dilute hydrochloric acid solution, add to the full amount of water for injection again, with embedding behind the filtering with microporous membrane of 0.22 μ m in the 100ml infusion bottle.Sterilized 15 minutes for 121 ℃.Through lamp inspection, labeling, packing, warehouse-in promptly.
The preparation 6 of embodiment 10 Gugua polypeptide injections
The extract 18.75g (by Os Sus domestica polypeptide) of embodiment 1
The extract 6.25g of embodiment 3 (by Fructus Melo seed polypeptide)
Mannitol 50g
Water for injection adds to 5000ml
The Sus domestica extract solution of the Sus domestica extract solution of embodiment 1 preparation and embodiment 1 preparation is pressed Os Sus domestica polypeptide to be mixed with Fructus Melo seed polypeptide weight ratio at 3: 1, add the mannitol dissolving, regulate pH value 6.4 with rare diluted sodium hydroxide solution or dilute hydrochloric acid solution, add to the full amount of water for injection again, with fill behind the filtering with microporous membrane of 0.22 μ m, lid is rolled in lyophilizing, through labeling, packing, warehouse-in promptly.
The preparation 7 of embodiment 11 Gugua polypeptide injections
The extract 25g (by Os Sus domestica polypeptide) of embodiment 1
The extract 2.5g of embodiment 3 (by Fructus Melo seed polypeptide)
Water for injection adds to 5000ml
The Sus domestica extract solution of the Sus domestica extract solution of embodiment 1 preparation and embodiment 1 preparation is pressed Os Sus domestica polypeptide to be mixed with Fructus Melo seed polypeptide weight ratio at 10: 1, sodium hydroxide solution with 8% or 12% hydrochloric acid solution are regulated pH value 6.3, add to the full amount of water for injection again, in the bent neck peace of 5ml bottle, wire drawing is sealed with fill behind the filtering with microporous membrane of 0.22 μ m.Sterilized 12 minutes for 121 ℃.Through lamp inspection, labeling, packing, warehouse-in promptly.
The preparation 8 of embodiment 12 Gugua polypeptide injections
The extract 2.5g (by Os Sus domestica polypeptide) of embodiment 1
The extract 25g of embodiment 3 (by Fructus Melo seed polypeptide)
Water for injection adds to 2000ml
The Sus domestica extract solution of the Sus domestica extract solution of embodiment 1 preparation and embodiment 1 preparation is pressed Os Sus domestica polypeptide to be mixed with Fructus Melo seed polypeptide weight ratio at 1: 10, sodium hydroxide solution with 8% or 12% hydrochloric acid solution are regulated pH value 6.8, add to the full amount of water for injection again, in the bent neck peace of 5ml bottle, wire drawing is sealed with fill behind the filtering with microporous membrane of 0.22 μ m.Sterilized 12 minutes for 121 ℃.Through lamp inspection, labeling, packing, warehouse-in promptly.
Experimental example 1 high molecular weight material assay
High effective liquid chromatography for measuring according to regulation in " two appendix VD of Chinese Pharmacopoeia version in 2005 ".
Reference substance is insulin (molecular weight 5800), adds mobile phase (trifluoracetic acid-acetonitrile-water 0.05: 10: 90) and makes the solution that every 1ml contains 1mg.
Test article is the preparation among the embodiment 5, adds mobile phase and makes the solution that contains polypeptide 1mg among the 1ml.
Assay method: get reference substance solution and test solution 20 a μ l, inject chromatograph of liquid respectively, the record chromatogram, after testing, the polymer substance content of test solution is lower than 1.5%.
Illustrate that this product is stable, can not cause allergy.
The effect of experimental example 2 preparation for treating osteoarthritis of the present invention
Method: 60 routine OA patients (meeting the OA diagnostic criteria of U.S. ACR revision) are divided into 2 groups at random, and the each patient of observation group uses the continuous 10d treatment of bone-melon extract injection, 12mg intravenous drip every day.Matched group is only used calcium preparation and active alpha-D3 and pain management, the course of treatment 10d,
The therapeutic evaluation standard:
1. the pain observation index is represented than scale (VAS) with vision.
2. tenderness, swelling, dysfunction are observed, and estimate every day 1 time.Concrete point system is pressed the score of table 1 content.
3. efficacy determination produce effects: swelling and tenderness obviously alleviate, and joint motion improves obviously, and the total score in treatment back descends 〉=2/3; Effectively: swelling and tenderness alleviate, and joint motion has obviously, and the total score in treatment back descends 〉=1/3; Invalid: symptom does not have improvement, and the total score in treatment back descends＜1/3.
The statistical procedures measurement data adopts the t check, and enumeration data adopts variance analysis, and ranked data are checked with Ridit, and all data are handled with the SPSS8.0 software kit.
Table 1 tenderness, swelling, dysfunction score standard
| Degree | Tenderness | Swelling | Dysfunction | The result |
| Do not have | Do not have | Do not have | Do not have | 0 |
| Slightly | Push back pain | Slightly, dermatoglyph shoals | Slightly, normal activity | 1 |
| Degree | Tenderness | Swelling | Dysfunction | The result |
| Moderate | It is painful to push the back expression | Moderate, dermatoglyph tail off | Pain when moderate, activity | 2 |
| Severe | Tenderness | Seriously, skin is thin, bright | Severe, function of joint are limited | 3 |
Result 1. observation group's pain score before and after treatment is respectively (5.7 ± 1.5) branch and (4.2 ± 1.4) branch, and the two comparing difference has significance (P＜0.05).Matched group pain score before and after treatment is respectively (4.9 ± 1.1) divides and (4.3 ± 1.0) branch, and the two comparing difference has significance (P＜0.05).2. 2 groups of curative effects relatively: observation group's produce effects total effective rate is 80.00%, and the matched group total effective rate is 53.34%, and observation group's curative effect is good than matched group, and difference has significance (P＜0.05).Conclusion bone-melon extract injection has preferably detumescence, analgesic effect, and the effect of certain adjusting bone metabolism is arranged, safety is good, can be used as a kind of auxiliary treatment measure of OA.
Claims (8)
1. bone-melon extract injectable drug for the treatment of osteoarthritis, it prepares by the following method:
A) preparation of bone extract
1) os purum will be got after the mammalian bone pretreatment;
2) get os purum, add the purified water of 2～5 times of weight, in 120 ℃～123 ℃, 1～3h is extracted in hot pressing under 0.11～0.13Mpa condition, filters, and gets filtrate;
3) with filtrate under 0～10 ℃ of condition static 8～16 hours, remove the upper strata oils and fats;
4) regulating pH is 8.5～9.0, is heated to 44 ℃～46 ℃, presses 0.1～0.3% of bone weight and adds pancreatin, constant temperature continuous hydrolysis 1～3 hour;
5) regulating pH is 3.5～4.0, boils, and is incubated 15～45 minutes after-filtration, and it is 6.5～7.0 that filtrate is regulated pH;
6) add proper amount of active carbon, be heated to boiling, be incubated 10～20 minutes after-filtration, filtrate in-20 ℃ dark freeze 36～50 hours after the room temperature thawing;
7) ultrafiltration under the 10KD molecular retention, filtrate after suitably concentrating, ultrafiltration under the 6KD molecular retention again, promptly;
B) preparation of Fructus Melo seed extract:
1) clean Fructus Melo seed will be got after the pretreatment of Fructus Melo seed;
2) get clean Fructus Melo seed, add the water of 1～3 times of weight, in 120 ℃～123 ℃, 1～3h is extracted in 0.11～0.13MPa hot pressing, filters to get filtrate
3) repeating step 2) 1～3 time;
4) merging filtrate is cooled to 25～35 ℃, regulates pH to 3.5～4.0, leaves standstill 8～16 hours, goes precipitation;
5) boil, be incubated 15～45 minutes, filter;
6) regulating pH is 8.5～9.0, in-20 ℃ dark freeze 36～50 hours after room temperature melt;
7) boil, be incubated 15～45 minutes, filter, regulate pH to 6.0～7.0;
8) ultrafiltration under the 10KD molecular retention, filtrate after suitably concentrating, ultrafiltration under the 6KD molecular retention again, promptly;
C) with steps A) bone extract and the step B of preparation) the Fructus Melo seed extract of preparation mixes, and adds an amount of excipient and is prepared into injectable drug, and wherein the weight ratio of bone polypeptide and Fructus Melo seed polypeptide is 1: 10 to 10: 1,
Wherein said mammiferous bone is Os Cervi or Os Sus domestica.
2. bone-melon extract injectable drug as claimed in claim 1, it prepares by the following method:
A) preparation of bone extract
1) os purum will be got after the mammalian bone pretreatment;
2) get os purum, add the purified water of 4 times of weight, in 121 ℃, 2h is extracted in hot pressing under the 0.12Mpa condition, filters, and gets filtrate;
3) with filtrate under 0～10 ℃ of condition static 12 hours, remove the upper strata oils and fats;
4) regulating pH is 8.5～9.0, is heated to 45 ℃, presses 0.2% of bone weight and adds pancreatin, constant temperature continuous hydrolysis 2 hours;
5) regulating pH is 3.5～4.0, boils, and is incubated 30 minutes after-filtration, and it is 6.5～7.0 that filtrate is regulated pH;
6) add proper amount of active carbon, be heated to boiling, be incubated 15 minutes after-filtration, filtrate in-20 ℃ dark freeze 48 hours after the room temperature thawing;
7) ultrafiltration under the 10KD molecular retention ,-0.1MPA is evaporated to 1/3 of original volume under 60 ℃ of conditions, ultrafiltration under the 6KD molecular retention again, promptly;
B) preparation of Fructus Melo seed extract:
1) clean Fructus Melo seed will be got after the pretreatment of Fructus Melo seed;
2) get clean Fructus Melo seed, add the water of 2 times of weight, in 121 ℃, 2h is extracted in 0.12MPa hot pressing, filters to get filtrate
3) repeating step 2) 2 times;
4) merging filtrate is cooled to 25～35 ℃, regulates pH to 3.5～4.0, leaves standstill 12 hours, goes precipitation;
5) boil, be incubated 30 minutes, filter;
6) regulating pH is 8.5～9.0, in-20 ℃ dark freeze 48 hours after room temperature melt;
7) boil, be incubated 15～45 minutes, filter, regulate pH to 6.0～7.0;
8) ultrafiltration under the 10KD molecular retention ,-0.1MPA is evaporated to 1/3 of original volume under 60 ℃ of conditions, ultrafiltration under the 6KD molecular retention again, promptly;
C) with steps A) bone extract and the step B of preparation) the Fructus Melo seed extract of preparation mixes, and adds an amount of excipient and is prepared into injectable drug, and wherein the weight ratio of bone polypeptide and Fructus Melo seed polypeptide is 1～5: 1.
3. bone-melon extract injectable drug as claimed in claim 1 or 2 is characterized in that described Fructus Melo seed is the cucurbitaceous plant melon seed.
4. bone-melon extract injectable drug as claimed in claim 1 or 2, it is injection or lyophilized powder.
5. bone-melon extract injectable drug as claimed in claim 4 is characterized in that, described injection is bone-melon extract injection, sodium chloride injection or glucose injection.
6. bone-melon extract injectable drug as claimed in claim 4, used excipient is mannitol and/or dextran when it is characterized in that being prepared into lyophilized powder, excipient is 1: 1～5 with total polypeptide by weight.
7. method for preparing each described bone-melon extract injectable drug of claim 1～6, it comprises step:
A) preparation of bone extract
1) os purum will be got after the mammalian bone pretreatment;
2) get os purum, add the purified water of 2～5 times of weight, in 120 ℃～123 ℃, 1～3h is extracted in hot pressing under 0.11～0.13Mpa condition, filters, and gets filtrate;
3) with filtrate under 0～10 ℃ of condition static 8～16 hours, remove the upper strata oils and fats;
4) regulating pH is 8.5～9.0, is heated to 44 ℃～46 ℃, presses 0.1～0.3% of bone weight and adds pancreatin, constant temperature continuous hydrolysis 1～3 hour;
5) regulating pH is 3.5～4.0, boils, and is incubated 15～45 minutes after-filtration, and it is 6.5～7.0 that filtrate is regulated pH;
6) add proper amount of active carbon, be heated to boiling, be incubated 10～20 minutes after-filtration, filtrate in-20 ℃ dark freeze 36～50 hours after the room temperature thawing;
7) ultrafiltration under the 10KD molecular retention, filtrate after suitably concentrating, ultrafiltration under the 6KD molecular retention again, promptly;
B) preparation of Fructus Melo seed extract:
1) clean Fructus Melo seed will be got after the pretreatment of Fructus Melo seed;
2) get clean Fructus Melo seed, add the water of 1～3 times of weight, in 120 ℃～123 ℃, 1～3h is extracted in 0.11～0.13MPa hot pressing, filters to get filtrate
3) repeating step 2) 1～3 time;
4) merging filtrate is cooled to 25～35 ℃, regulates pH to 3.5～4.0, leaves standstill 8～16 hours, goes precipitation;
5) boil, be incubated 15～45 minutes, filter;
6) regulating pH is 8.5～9.0, in-20 ℃ dark freeze 36～50 hours after room temperature melt;
7) boil, be incubated 15～45 minutes, filter, regulate pH to 6.0～7.0;
8) ultrafiltration under the 10KD molecular retention, filtrate after suitably concentrating, ultrafiltration under the 6KD molecular retention again, promptly;
C) with steps A) bone extract and the step B of preparation) the Fructus Melo seed extract of preparation mixes, adds an amount of excipient and be prepared into injectable drug.
8. method as claimed in claim 7, it prepares by the following method:
A) preparation of bone extract
1) os purum will be got after the mammalian bone pretreatment;
2) get os purum, add the purified water of 4 times of weight, in 121 ℃, 2h is extracted in hot pressing under the 0.12Mpa condition, filters, and gets filtrate;
3) with filtrate under 0～10 ℃ of condition static 12 hours, remove the upper strata oils and fats;
4) regulating pH is 8.5～9.0, is heated to 45 ℃, presses 0.2% of bone weight and adds pancreatin, constant temperature continuous hydrolysis 2 hours;
5) regulating pH is 3.5～4.0, boils, and is incubated 30 minutes after-filtration, and it is 6.5～7.0 that filtrate is regulated pH;
6) add proper amount of active carbon, be heated to boiling, be incubated 15 minutes after-filtration, filtrate in-20 ℃ dark freeze 48 hours after the room temperature thawing;
7) ultrafiltration under the 10KD molecular retention ,-0.1MPA is evaporated to 1/3 of original volume under 60 ℃ of conditions, ultrafiltration under the 6KD molecular retention again, promptly;
B) preparation of Fructus Melo seed extract:
1) clean Fructus Melo seed will be got after the pretreatment of Fructus Melo seed;
2) get clean Fructus Melo seed, add the water of 2 times of weight, in 121 ℃, 2h is extracted in 0.12MPa hot pressing, filters to get filtrate
3) repeating step 2) 2 times;
4) merging filtrate is cooled to 25～35 ℃, regulates pH to 3.5～4.0, leaves standstill 12 hours, goes precipitation;
5) boil, be incubated 30 minutes, filter;
6) regulating pH is 8.5～9.0, in-20 ℃ dark freeze 48 hours after room temperature melt;
7) boil, be incubated 15～45 minutes, filter, regulate pH to 6.0～7.0;
8) ultrafiltration under the 10KD molecular retention ,-0.1MPA is evaporated to 1/3 of original volume under 60 ℃ of conditions, ultrafiltration under the 6KD molecular retention again, promptly;
C) with steps A) bone extract and the step B of preparation) the Fructus Melo seed extract of preparation mixes, adds an amount of excipient and be prepared into injectable drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101151179A CN101297853B (en) | 2008-06-17 | 2008-06-17 | Bone-melon extract injection and preparation thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101151179A CN101297853B (en) | 2008-06-17 | 2008-06-17 | Bone-melon extract injection and preparation thereof |
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| CN101297853A CN101297853A (en) | 2008-11-05 |
| CN101297853B true CN101297853B (en) | 2010-09-15 |
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| CN2008101151179A Expired - Fee Related CN101297853B (en) | 2008-06-17 | 2008-06-17 | Bone-melon extract injection and preparation thereof |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103040911A (en) * | 2011-12-08 | 2013-04-17 | 赵联华 | Composition for injection for treating orthopedic diseases |
| CN103070906A (en) * | 2011-12-08 | 2013-05-01 | 赵联华 | Quality control method of injection composition for treating orthopedic diseases |
| CN105688182A (en) * | 2014-11-28 | 2016-06-22 | 西藏誉衡阳光医药有限责任公司 | A Lugua polypeptide injection |
| CN105688181A (en) * | 2014-11-28 | 2016-06-22 | 西藏誉衡阳光医药有限责任公司 | Injection preparation prepared from beer bones and muskmelon seeds |
| CN104561210A (en) * | 2015-01-27 | 2015-04-29 | 张恒 | Extraction method of deer bone peptide |
| CN107674113A (en) * | 2017-10-20 | 2018-02-09 | 广东医科大学 | The preparation method and its purposes of the transdermal patch of a kind of small active peptides and preparation method thereof and use active peptide |
| CN110339335B (en) * | 2019-07-01 | 2023-03-24 | 哈尔滨誉衡制药有限公司 | Lugua polypeptide injection |
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Assignee: Kaifeng Kangnuo Pharmaceutical Co., Ltd. Assignor: Yang Shuanding Contract record no.: 2011120000051 Denomination of invention: Bone-melon extract injection and preparation thereof Granted publication date: 20100915 License type: Exclusive License Open date: 20081105 Record date: 20110413 |
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