CN103655671A - Cervus and cucumis polypeptide pharmaceutical composition and preparation method thereof - Google Patents
Cervus and cucumis polypeptide pharmaceutical composition and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a cervus and cucumis polypeptide pharmaceutical composition for injection. The pharmaceutical composition contains a deer bone extract, a muskmelon seed extract and one or more of excipients, wherein the weight ratio of deer bone polypeptide to muskmelon seed polypeptide is (2:1)-(6:1). The invention further discloses an application of the cervus and cucumis polypeptide pharmaceutical composition for injection in preparation of drugs for treating rheumatism, rheumatoid arthritis, ankylosing spondylitis, various types of fractures, wound repair and low back and leg pain. As shown in experimental results, the cervus and cucumis polypeptide pharmaceutical composition for injection has excellent functions of alleviating pain, eliminating swelling and promoting fracture healing.
Description
Technical field
The present invention relates to Cervus and Cucumis Polypeptide for injection pharmaceutical composition and preparation method thereof.More specifically, the present invention relates to the Cervus and Cucumis Polypeptide for injection pharmaceutical composition that contains Os Cervi polypeptide and Semen Melo polypeptide.The invention still further relates to the preparation method of Cervus and Cucumis Polypeptide for injection pharmaceutical composition.
Background technology
Cervus and cucumis polypeptide is formulated with the extract of Os Cervi and Semen Melo, motherland's medical science is thought, Os Cervi sweet in the mouth, slight fever, there is tonify deficiency win, bone and muscle strengthening, the effect of dispelling the wind and dampness pathogens limbs pain and the cold numbness of muscles and bones, and Semen Melo has eliminating stagnation, the effects such as pain disappear, both are used for cold expelling, wind-damp dispelling, analgesia at preparation, thereby rheumatic arthritis or rheumatoid arthritis are had to therapeutical effect.
Some are disclosed in prior art about cervus and cucumis polypeptide extract or its preparation technology.For example CN1470256A discloses and has promoted pharmaceutical composition that union of fracture and osteoarticular injury are repaired and its preparation method and application.The pharmaceutical composition that the compositions of this Patent Application Publication is comprised of Os Cervi extract and Semen Melo extract and one or more carriers of pharmaceutically accepting or excipient; Os Cervi extract and Semen Melo extract are respectively that Os Cervi and Semen Melo crude drug raw material obtain through extracting, and the weight ratio of Os Cervi and Semen Melo crude drug raw material is 1: 4 to 4: 1.This patent application also discloses the preparation method of pharmaceutical composition, and it comprises (1) by fresh Os Cervi after pretreatment, the water of weight such as in Os Cervi (400-100Kg), adds, and at the temperature of 121 ℃, heating extraction is 3 times; The water extraction solution that merges gained, regulates pH value to be transferred to 3.2 extracting solution that obtain after Acid precipitation with 2M hydrochloric acid; Extracting solution after heated and boiled Acid precipitation, removes by filter impurity the centrifugal Acid precipitation of removing; Collect after filtrate and centrifugal supernatant, add sodium hydroxide solution that PH is transferred to 8.5, thereby obtain required Os Cervi extracting solution; (2), in rustless steel container, to the water of weight such as adding in Semen Melo (100-400Kg) after cleaning, at the temperature of 121 ℃, heating extraction is 3 times; Merge water extraction solution, with 2M hydrochloric acid, regulate PH value to be transferred to 3.2; Extracting solution after heated and boiled Acid precipitation, the centrifugal acid deposit of removing; Collect after centrifugal supernatant, add caustic lye of soda PH to be transferred to 8.3, thereby obtain required Semen Melo extracting solution; (3) blend step (1) and (2) obtain Os Cervi extracting solution and Semen Melo extracting solution, heating is concentrated into 1/3 of original volume.After the PH of this concentrated solution is transferred to 7.2, using successively the molecular weight that dams is that 10,000 and 8,000 hollow fiber column carries out hyperfiltration treatment, and the mixed liquor obtaining and one or more carriers of pharmaceutically accepting or excipient are made pharmaceutical composition.
In addition, CN1579542A discloses a kind of cervus and cucumis polypeptide for injection and its prepration technology.This cervus and cucumis polypeptide comprises polypeptides matter and excipient, wherein polypeptides matter is to extract and form from animal in deer family skeleton and cucurbitaceous plant muskmelon seeds, its parts by weight are polypeptides matter 5-100, excipient 20-90, polypeptides matter molecular weight is 3000-10000 dalton, and pH value is 3.5-9.0.
This patent application also discloses the preparation technology of cervus and cucumis polypeptide, comprise (1) by weight mark get Os Cervi, add the water for injection that 3-8 doubly measures, hot pressing (80-120 ℃) is extracted 2-4 time.Merge extractive liquid,, adjusts pH value to 3.5-4.5, removes sediment, and supernatant adjusts alkaline ph value to 8.5-9.0, removes sediment, and supernatant is adjusted after neutral PH, uses ultrafilter membrane ultrafiltration, and molecular cut off is 3000-10000 dalton relatively, from making Os Cervi extracting solution; (2) mark is got Semen Melo 3000-10000 by weight, adds the water for injection that 3-8 doubly measures, and hot pressing (80-120 ℃) is extracted 2-4 time, merge extractive liquid,, removes sediment, ultrafiltration, molecular cut off is 3000-10000 dalton relatively, makes Semen Melo extracting solution; (3) by the Os Cervi of step (1) and (2) and Semen Melo extracting solution in 1-10: 1 ratio is mixed, and obtains cervus and cucumis polypeptide solution; (4) get cervus and cucumis polypeptide solution, add excipient (mannitol, Polyethylene Glycol, glucosan, lactose, glycine and colloidal substance) aqueous solution, lyophilization obtains off-white color block or amorphous powder.
Above-mentioned technology is to take crude drug raw material weight than extract forming as formula and preparation method thereof, owing to there is the place of production and the difference on the collection phase in crude drug raw material Os Cervi and Semen Melo, contained active ingredient differs greatly, with crude drug raw material weight, liken formula to, the effective component content of every batch of cervus and cucumis polypeptide extracting solution that very difficult assurance is produced is consistent, thereby cannot guarantee produced drug quality.Simultaneously, owing to being difficult to accomplish the in full accord of every batch of extraction conditions, even if therefore the same place of production, the active ingredient that crude drug is carried of collection phase also there are differences, take crude drug raw material weight ratio as formula forms, be difficult to guarantee that the content of peptides of every batch of preparation of cervus and cucumis polypeptide is consistent.
Above-mentioned technology adopts respectively 8,000 hollow fiber column carries out hyperfiltration technique or adopts 10,000 ultrafilter membrane hyperfiltration technique, can not effectively remove high molecular weight material in extracting solution (in pharmaceutical production, molecular weight is greater than 5800 material and is called high molecular weight material), very easily cause allergy.Os Cervi and Semen Melo extract contain multiple free amino acid, and it is supplied raw materials for bone source property biotic factors such as the synthetic BMPs of osteocyte, TGF-β, FGF, promotes the synthetic of bone source property biotic factor.But most of aminoacid is easily separated out in alkaline solution, and above-mentioned technology all adopts alkaline pH value in leaching process, be not all inconsistent with the national drug standards.Therefore, in prior art in the urgent need to providing a kind of active ingredient to keep stable Cervus and Cucumis Polypeptide for injection and the production technology of corresponding simplification, Cervus and Cucumis Polypeptide for injection can be raw material by the different places of production and the Os Cervi of different acquisition phase and Semen Melo, and product quality is not affected by extraction process condition, safe and effective, meet the relevant pharmaceutical production standard of country.
Summary of the invention
The present invention, through making great efforts for many years research and exploring, has solved the demand, has completed the present invention.
The invention provides a kind of Cervus and Cucumis Polypeptide for injection pharmaceutical composition, it contains Os Cervi extract, Semen Melo extract and one or more excipient, and wherein the weight ratio of Os Cervi polypeptide and Semen Melo polypeptide is 2: 1 to 6: 1.
The present invention also provides a kind of Cervus and Cucumis Polypeptide for injection pharmaceutical composition, and the method comprises the steps:
A) preparation of Os Cervi extract solution:
(1) Os Cervi pretreatment;
(2) pretreated clean Os Cervi is pulverized;
(3) in broken Os Cervi, add the water for injection of 2 times of weight, 121 ℃ of hot pressing are extracted to leach and are obtained an Os Cervi extracting solution;
(4) in the Os Cervi slag after once extracting, add 2 times of weight waters for injection, continue to extract 121 ℃ of-123 ℃ of hot pressing, leach and obtain secondary Os Cervi extracting solution, the Os Cervi extracting solution obtaining with step (3) merges makes Os Cervi extracting solution;
(5) Os Cervi extracting solution is centrifugal, collect supernatant centrifugal liquid;
(6) centrifugal liquid is carried out respectively to 100KD, 10KD, 6KD ultrafiltration, collect filter liquor, be evaporated to 10% of cumulative volume, collect concentrated solution, make Os Cervi extract solution;
B) preparation of Semen Melo extract solution:
(1) rinsing of drying and ripening Semen Melo is clean;
(2) in clean Semen Melo, add the water for injection of more than 2 times weight, with colloid mill, grind to form Semen Melo homogenate;
(3) Semen Melo homogenate is extracted 121 ℃ of hot pressing, leach Semen Melo extracting solution one time;
(4) in the Semen Melo slag after once extracting, add more than 2 times weight water for injection, continuation is extracted 121 ℃ of-123 ℃ of hot pressing, leach secondary Semen Melo extracting solution, make Semen Melo extracting solution with the Semen Melo extracting solution merging that step (3) obtains;
(5) Semen Melo extracting solution is centrifugal, collect supernatant centrifugal liquid;
(6) centrifugal liquid is carried out respectively to 100KD, 10KD, 6KD ultrafiltration, collect filter liquor, be evaporated to 10% of original volume, collect concentrated solution, make Semen Melo extract solution;
C) preparation of Cervus and Cucumis Polypeptide for injection pharmaceutical composition:
Blend step A) the Os Cervi extract solution of preparing and step B) the Semen Melo extract solution prepared and one or more excipient pharmaceutical compositions, by Freeze Drying Technique, make freeze-dried powder, wherein the weight ratio of Os Cervi polypeptide and Semen Melo polypeptide is 2: 1 to 6: 1.
The preparation method of the preferred Cervus and Cucumis Polypeptide for injection pharmaceutical composition of the present invention:
A) preparation of Os Cervi extract solution:
(1) Os Cervi boiled after 30 minutes in boiling water, removed fascia and residual meat, completed pretreatment;
(2) pretreated clean deer bone powder is broken to the broken bone below 1cm;
(3) in broken Os Cervi, add the water for injection of 2 times of weight, 121 ℃ of hot pressing are extracted 2 hours, leach and obtain an Os Cervi extracting solution;
(4) in the Os Cervi slag after once extracting, add 2 times of weight waters for injection, continuation is extracted 2 hours 121 ℃ of-123 ℃ of hot pressing, leach and obtain secondary Os Cervi extracting solution, make Os Cervi extracting solution with the Os Cervi extracting solution merging that step (3) obtains, placement is spent the night;
(5) by Os Cervi extracting solution with 4000 revs/min centrifugal 15 minutes, collect supernatant centrifugal liquid;
(6) centrifugal liquid is carried out respectively to 100KD, 10KD, 6KD ultrafiltration, collect filter liquor, be evaporated to 10% of cumulative volume, collect concentrated solution, with 0.22 μ m membrane filtration, make Os Cervi extract solution;
B) preparation of Semen Melo extract solution:
(1) by water for injection rinsing 3-4 time for drying and ripening Semen Melo;
(2) in clean Semen Melo, add the water for injection of 2 times of weight, with colloid mill, grind to form Semen Melo homogenate;
(3) Semen Melo homogenate is extracted 2 hours 121 ℃ of hot pressing, leach Semen Melo extracting solution one time;
(4) in the Semen Melo slag after once extracting, add 2 times of weight waters for injection, continuation is extracted 2 hours 121 ℃ of-123 ℃ of hot pressing, leach secondary Semen Melo extracting solution, make Semen Melo extracting solution with the Semen Melo extracting solution merging that step (3) obtains;
(5) by Semen Melo extracting solution with 4000 revs/min centrifugal 15 minutes, collect supernatant centrifugal liquid;
(6) centrifugal liquid is carried out respectively to 100KD, 10KD, 6KD ultrafiltration, collect filter liquor, be evaporated to 10% of original volume, collect concentrated solution, with 0.22 μ m membrane filtration, make Semen Melo extract solution;
C) preparation of injection Os Cervi polypeptide pharmaceutical composition:
Blend step A) the Os Cervi extract solution of preparing and step B) the Semen Melo extract solution prepared and one or more excipient pharmaceutical compositions, by Freeze Drying Technique, make freeze-dried powder, wherein the weight ratio of Os Cervi polypeptide and Semen Melo polypeptide is 2: 1 to 4: 1, excipient is selected from Dextran 40, glucose or lactose, and the weight ratio of polypeptide total amount and excipient is 1: 5-30.
As the raw material for pharmaceutical composition Os Cervi extract of the present invention and Semen Melo extract, use animal in deer family, especially the dry mature seed of the skeleton of Cervus nippon Temminck (Cervus nippon Temmick) and cucurbitaceous plant Fructus Melo (Cucumis melo L.), after extracting respectively, mix, and make pharmaceutical composition of the present invention with one or more mixed with excipients.According to the preferred embodiments of the invention, in Cervus and Cucumis Polypeptide for injection pharmaceutical composition, the weight ratio of Os Cervi polypeptide and Semen Melo polypeptide is 2: 1 to 4: 1.Excipient as pharmaceutical composition of the present invention can be Dextran 40, glucose, lactose, Polyethylene Glycol, glucosan, glycine etc., consider the impact that content of peptides in final products is detected, wherein preferred excipient is Dextran 40, glucose or lactose.Polypeptide total amount and excipient weight ratio should be 1: 5～and 30.
Cervus and Cucumis Polypeptide for injection pharmaceutical composition of the present invention can be used for treating disease or the symptoms such as rheumatism, rheumatoid arthritis, ankylosing spondylitis, all kinds fracture, repair in trauma and lumbago and skelalgia.Although do not intend to be so limited, but we think, Os Cervi polypeptide in pharmaceutical composition of the present invention is extracted by Os Cervi, bone wherein induction polypeptide class biotic factor can effectively promote affect in body the synthesizing of bdgf of bone formation and absorption, comprises bone morphogenetic protein (BMP
s), β-transforming growth factor (TGF-β), fibroblast growth factor (FGF) etc., thus there is multiple biological activity.Its main pharmacological has: promote cell mitogen, differentiation, chemotaxis and molten bone active.BMP wherein
sone group of acid low-molecular-weight glycoprotein, as a kind of efficient bone Induced substance, BMP
sthat mesenchymal cell is the initially signal factor of cell differentiation to bone, the bone that the mesenchymal cell can induction of vascular around moving about is converted into irreversibility is cell, i.e. chondrocyte and osteoblast, thus promote callus formation, induce new bone formation, promote fracture repair; In addition BMP,
sthe also change of scalable extracellular matrix composition, and by inducing better new bone formation with TGF-β and FGF coordinative role each other, make osseous tissue more ripe.TGF-βZe Shi gang has the protein polypeptide of several functions, osteoblast and chondroblast are had the differentiation of promotion or fall PD dual regulation effect, with the multiple factor as the adjusting of collaborative participation to cell differentiation together with extracellular matrix and other differentiation growth regulation factors; TGF-β can promote extracellular matrix synthetic, can directly stimulate the synthetic of fibroblast epimatrix, and its new synthetic substrate degradation is had to remarkable inhibitory action; For osteoblast, TGF-β can promote its synthetic type i collagen, osteonectin and osteopontin; Meanwhile, TGF-β shows the effect of lymphocyte and macrophage, and it can alleviate the destructiveness of inflammatory reaction, can assist again some macrophage derived cytokine to play a role in cell tissue reparation.FGF is that one group of heparin binds polypeptide, and trend that can irritation cell moves, and increment and differentiation increase the quantity of rubber polymer archeocyte, promote the synthetic of bone collagen and noncollagen protein, increase the synthetic of Bone Gla protein.
Semen Melo polypeptide extract in pharmaceutical composition of the present invention is to extract from the dry seed of the maturation of cucurbitaceous plant Fructus Melo, can reduce the local capillary permeability of fracture, reduces inflammatory exudation, promotes the recovery of local blood fortune obstacle; Can also reduce whole blood viscosity and erythrocyte aggregation degree, improve the blood circulation of callus part, for osteocyte provides a good blood supply environment simultaneously; Can also suppress the release of prostaglandin in addition, reach analgesic effect; In promoting fracture heals in early days, Semen Melo extract has synergism with supplementary bone induction polypeptide biotic factor, promotes synthesizing of bdgf.In addition injection Os Cervi polypeptide pharmaceutical composition of the present invention is rich in multiple free amino acid, is the synthetic BMP of osteocyte
s, the bone source property biotic factor such as TGF-β, FGF supplies raw materials, and promotes the synthetic of bone source property biotic factor, organic calcium, phosphonium ion can participate in alcium and phosphor metabolization, maintain bone volume.Thereby can effectively treat disease or the symptoms such as rheumatism, rheumatoid arthritis, ankylosing spondylitis, all kinds fracture, repair in trauma and lumbago and skelalgia.
The present invention further discloses a kind of pharmaceutical composition, mainly contain cervus and cucumis polypeptide and one or more pharmaceutic adjuvants.Described compositions is solid preparation or liquid preparation.Described wherein solid preparation is capsule, soft capsule, tablet, buccal tablet, granule or drop pill; Liquid preparation is oral liquid or injection.The present invention's adjuvant used refers to the compound that pharmaceutically acceptable carrier, excipient and other additive form, can adopt lactose or starch to do carrier, gelatin, sodium carboxymethyl cellulose, methylcellulose, polyvinylpyrrolidone etc. is suitable bonding agent or becomes granular agent.As disintegrating agent, can select starch or microcrystalline Cellulose, often with Pulvis Talci, santocedl, tristerin, calcium stearate or magnesium etc. are as suitable antiadhesives and lubricant.For example, can prepare tablet by compacting wet granular.Active component and carrier and optionally with a disintegrate additive composition mixture; the aqueous solution of this mixture and binding agent; alcohol or aqueous alcohol solution carry out granulating in suitable equipment; dried particles adds other disintegrating agent subsequently, and lubricant and antiplastering aid are by this mixture tabletting.
The dosage of Cervus and Cucumis Polypeptide for injection pharmaceutical composition of the present invention is: when for intramuscular injection, and a 4-8mg, daily dose is 8-16mg, with intramuscular injection after appropriate water for injection dissolved dilution; When for intravenous injection, every day, 16-24mg, instiled with 5% glucose injection or 0.9% sodium chloride injection 250-500ml dissolved dilution posterior vein.Accurately dosage will be determined according to factors such as patient's age, body weight, the symptom for the treatment of, patient body situations by clinician, and general 10-15 day is a course for the treatment of.The packing specification of cervus and cucumis polypeptide pharmaceutical composition of the present invention has 4mg, 8mg, 16mg, 24mg.
In more particular embodiment of the present invention, it provides a kind of preparation method of Cervus and Cucumis Polypeptide for injection pharmaceutical composition, and it comprises the steps:
A) preparation of Os Cervi extract solution:
(1) Os Cervi boiled after 30 minutes in boiling water, removed fascia and residual meat, completed pretreatment;
(2) pretreated clean deer bone powder is broken to the broken bone below 1cm;
(3) in broken Os Cervi, add the water for injection of 2 times of weight, 121 ℃ of hot pressing are extracted 2 hours, leach and obtain an Os Cervi extracting solution;
(4) in the Os Cervi slag after once extracting, add 2 times of weight waters for injection, continuation is extracted 2 hours 121 ℃ of-123 ℃ of hot pressing, leach and obtain secondary Os Cervi extracting solution, make Os Cervi extracting solution with the Os Cervi extracting solution merging that step (3) obtains, placement is spent the night;
(5) by Os Cervi extracting solution with 4000 revs/min centrifugal 15 minutes, collect supernatant centrifugal liquid;
(6) centrifugal liquid is carried out respectively to 100KD, 10KD, 6KD ultrafiltration, collect filter liquor, be evaporated to 10% of cumulative volume, collect concentrated solution, with 0.22 μ m membrane filtration, make Os Cervi extract solution;
B) preparation of Semen Melo extract solution:
(1) by water for injection rinsing 3-4 time for drying and ripening Semen Melo;
(2) in clean Semen Melo, add the water for injection of 2 times of weight, with colloid mill, grind to form Semen Melo homogenate;
(3) Semen Melo homogenate is extracted 2 hours 121 ℃ of hot pressing, leach Semen Melo extracting solution one time;
(4) in the Semen Melo slag after once extracting, add 2 times of weight waters for injection, continuation is extracted 2 hours 121 ℃ of-123 ℃ of hot pressing, leach secondary Semen Melo extracting solution, make Semen Melo extracting solution with the Semen Melo extracting solution merging that step (3) obtains;
(5) by Semen Melo extracting solution with 4000 revs/min centrifugal 15 minutes, collect supernatant centrifugal liquid;
(6) centrifugal liquid is carried out respectively to 100KD, 10KD, 6KD ultrafiltration, collect filter liquor, be evaporated to 10% of original volume, collect concentrated solution, with 0.22 μ m membrane filtration, make Semen Melo extract solution;
C) preparation of Cervus and Cucumis Polypeptide for injection pharmaceutical composition:
Blend step A) the Os Cervi extract solution of preparing and step B) the Semen Melo extract solution prepared and one or more excipient pharmaceutical compositions, by Freeze Drying Technique, make freeze-dried powder, wherein the weight ratio of Os Cervi polypeptide and Semen Melo polypeptide is 2: 1 to 4: 1, excipient is selected from Dextran 40, glucose or lactose, and polypeptide total amount and excipient weight ratio are 1: 5-30.
The method of pharmaceutical compositions step C) is method known in pharmaceutical technology field, and those skilled in the art will choose concrete technology method and condition accurately according to the excipient of prepared dosage form and selection.
As mentioned above, Cervus and Cucumis Polypeptide for injection pharmaceutical composition provided by the invention and preparation method thereof tool has the following advantages:
(1) take Os Cervi polypeptide and Semen Melo polypeptide as unit of account, can guarantee that every batch of preparation content of peptides is consistent, batch difference can be down to minimum, guarantees product quality;
(2) take Os Cervi polypeptide and Semen Melo polypeptide as unit of account, the final products content difference of having avoided the place of production, collection phase, extraction conditions difference to occur;
(3) pulverize in advance Os Cervi and prepare Semen Melo homogenate, improving the extraction ratio that Os Cervi polypeptide and Semen Melo polypeptide are extracted in hot pressing;
(4) adopt pure hot pressing extracting method, avoid acid or alkali treatment, improve the extraction ratio of active ingredient;
(5) adopt the method for classification ultrafiltration, can effectively remove the molecular weight existing in extract and surpass 5800 high molecular weight material, allergic probability is down to minimum, improved constant product quality;
(6) adopting Dextran 40, glucose or lactose is excipient, can avoid content of peptides to detect the impact bringing.
The specific embodiment
Below in conjunction with embodiment, the present invention is described, the scheme of embodiment described here, do not limit the present invention, one of skill in the art can make improvements and change according to spirit of the present invention, these described improvement and variation all should be considered as within the scope of the invention, and scope of the present invention and essence are limited by claim.The present invention's various reagent used all has commercially available.
Embodiment 1: the preparation of Os Cervi extract solution
Os Cervi is placed in to boiling water and boils 30min, take out, reject fascia and residual meat, obtain clean Os Cervi.Get clean Os Cervi, be crushed to that to be less than the broken bone of 1cm standby.Get broken bone 30kg, add 60kg water for injection, 121 ℃ of hot pressing, extract 2h, leach extracting solution one time; Add 60kg water for injection, 2h is extracted in 121 ℃ of hot pressing, leaches secondary raffinate again, merging filtrate, and placement is spent the night.Get Os Cervi extracting solution, centrifugal (4000 revs/min, 15 minutes), get supernatant, abandon precipitation.Get supernatant, carry out classification ultrafiltration, 100KD, 10KD, 6KD get filtered solution at every turn, abandon concentrated solution.Get filtered solution, be evaporated to 10% of cumulative volume, collect concentrated solution, regulate pH value 6.0-6.5,0.22 μ m filter membrane aseptic filtration, obtains Os Cervi extract solution 10L.Through high performance liquid chromatography, Forint phenol method, measure, have Os Cervi polypeptide, its content is 6.1mg/ml.
Embodiment 2: the preparation of Semen Melo extract solution
Get Semen Melo, put in water for injection, rinsing is 3-4 time repeatedly, and empty solid carbon dioxide part, chooses foreign material, obtains clean Semen Melo standby.Get clean Semen Melo 15kg, add 30kg water for injection, with colloid mill, grind to form Semen Melo homogenate.Get Semen Melo homogenate, 2h is extracted in 121 ℃ of hot pressing, leaches extracting solution one time; Add 30kg water for injection, 121 ℃ of ℃ of hot pressing continue to extract 2h, leach secondary raffinate again, merging filtrate, and placement is spent the night.Get Semen Melo extracting solution, centrifugal (4000 revs/min, 15 minutes), get supernatant, abandon precipitation.Get supernatant, carry out classification ultrafiltration: 100KD, 10KD, 6KD, get filtered solution at every turn, abandon concentrated solution.Get final filtered solution, be evaporated to 10% of cumulative volume, collect concentrated solution, regulate pH value 6.0-6.5,0.22 μ m filter membrane aseptic filtration, obtains the about 5L of Semen Melo extract solution.Through spectrophotography, Forint phenol method, measure, have Semen Melo polypeptide, its content is 4.2mg/ml.
Embodiment 3: pharmaceutical composition 1
Preparation prescription:
The Os Cervi extract solution 26.7g (by Os Cervi polypeptide) of embodiment 1
The Semen Melo extract solution 13.3g (by Semen Melo polypeptide) of embodiment 2
20% excipient solution 5000ml
5%NaOH (or 5%HCl) is appropriate
Water for injection adds to 20000ml
The Semen Melo extract solution of the Os Cervi extract solution of embodiment 1 preparation and embodiment 2 preparations is mixed in 2: 1 ratios of Os Cervi polypeptide and Semen Melo polypeptide weight ratio, add 5000ml excipient (Dextran 40, glucose or lactose) solution, with appropriate 5%NaOH or 5%HCl commissioning solution pH value to 5.5-7.0, benefit adds to the full amount of water for injection, by canned, lyophilizing, roll the steps such as lid, make the preparation of every 4mg.
Embodiment 4: pharmaceutical composition 2
Preparation prescription:
The Os Cervi extract solution 30g (by Os Cervi polypeptide) of embodiment 1
The Semen Melo extract solution 10g (by Semen Melo polypeptide) of embodiment 2
20% excipient solution 5000ml
5%NaOH (or 5%HCl) is appropriate
Water for injection adds to 20000ml
Except the consumption of Os Cervi extract and Semen Melo extract is different, pharmaceutical composition is prepared as described in example 3 above, by canned, lyophilizing, roll the steps such as lid, makes the preparation of every 4mg.
Embodiment 5: pharmaceutical composition 3
Preparation prescription:
The Os Cervi extract solution 53.4g (by Os Cervi polypeptide) of embodiment 1
The Semen Melo extract solution 26.6g (by Semen Melo polypeptide) of embodiment 2
20% Dextran 40 solution 10000ml
5%NaOH (or 5%HCl) is appropriate
Water for injection adds to 40000ml, and except the consumption of Os Cervi extract and Semen Melo extract is different, pharmaceutical composition is prepared as described in example 3 above, by canned, lyophilizing, roll the steps such as lid, makes the preparation of every 8mg.
Embodiment 6: pharmaceutical composition 4
Preparation prescription:
The Os Cervi extract solution 64g (by Os Cervi polypeptide) of embodiment 1
The Semen Melo extract solution 16g (by Semen Melo polypeptide) of embodiment 2
20% Dextran 40 solution 10000ml
5%NaOH (or 5%HCl) is appropriate
Water for injection adds to 40000ml
Except the consumption of Os Cervi extract and Semen Melo extract is different, pharmaceutical composition is prepared as described in example 3 above, by canned, lyophilizing, roll the steps such as lid, makes every 8mg preparation.
Embodiment 7: preparation active ingredient is measured
1, polypeptide active ingredient is determined
(1) preparation of sampling embodiment 3, adds water and makes the solution that contains polypeptides matter 2mg in every 1ml, gets 2ml, adds indenes three bronze medal test solution 1ml, and heating, should show bluish violet.
(2) according to the method described in two appendix IV A > > of < < Chinese Pharmacopoeia version in 2000, with the preparation of embodiment 3, add water and make in every 1ml the solution containing polypeptides matter 2mg, get 2ml, put in 50ml measuring bottle, add water to scale, shake up, according to spectrophotometry, at the wavelength place of 258nm, have absorption maximum.As above presentation of results, the absorption maximum of embodiment 3 preparations is differentiated and is set up.
2, content of high molecule weight substance is measured
According to the high effective liquid chromatography for measuring of stipulating in two appendix V D > > of < < Chinese Pharmacopoeia version in 2000.
Chromatographic condition and system suitability: with gel chromatographic columns (TSKgelG2000SW 7.8mm * 300mm, 5 μ m); Mobile phase is trifluoracetic acid-acetonitrile-water (0.05: 10: 90); Detection wavelength is 214nm.Number of theoretical plate must not calculate lower than 3000 by insulin spikes.
The preparation of reference substance solution: get insulin reference substance (molecular weight 5800) appropriate, add mobile phase and make the solution that contains 1mg in every 1ml, in contrast product solution.
The preparation of test solution: add mobile phase and make the solution that contains polypeptide 1mg in every 1ml in the preparation of embodiment 3, as test solution.
Algoscopy: get each 20 μ l of reference substance solution and test solution, injection liquid chromatography, records chromatogram respectively, peak prior to insulin spikes retention time is considered as to high molecular weight material, by area normalization method, calculate, result demonstration, the polymer substance content of test solution is lower than 2.0%.
3, Contents of Amino Acids
By high performance liquid chromatography, differentiate respectively and quantitative contained free amino acid in embodiment 5 preparations, the results are shown in Table A.
Table A: Cervus and Cucumis Polypeptide for injection free amino acid check result:
As above result shows, Cervus and Cucumis Polypeptide for injection preparation contains 18 kinds of free amino acids.
Embodiment 8:
The difference of polypeptide total amount between each batch in production Cervus and Cucumis Polypeptide for injection
The present embodiment explanation drops into identical crude drug raw material at every batch, while preparing polypeptide extract with identical process, and the difference of polypeptide total amount in every batch of extract.
By the method for embodiment 1, drop into the Os Cervi of identical weight, prepare Os Cervi extract, its Os Cervi polypeptide total amount is shown in following form B.
Table B: Os Cervi extract solution compolision is analyzed
| Lot number | Drop into clean Os Cervi | Cumulative volume | The many content of Os Cervi | Polypeptide amount |
| 20040301 | 150Kg | 56L | 7.32mg/ml | 409.9g |
| 20040302 | 150Kg | 53L | 5.25mg/ml | 278.3g |
| 20040303 | 150Kg | 62L | 5.33mg/ml | 330.5g |
By the method for embodiment 2, drop into the Semen Melo of identical weight, prepare Semen Melo extract, its Semen Melo polypeptide total amount is shown in following form C.
Table C: Semen Melo extract solution compolision is analyzed
| Lot number | Drop into Semen Melo | Cumulative volume | Os Cervi polypeptide content | Polypeptide total amount |
| 20040301 | 50Kg | 19L | 3.50mg/ml | 66.5g |
| 20040302 | 50Kg | 22L | 4.35mg/ml | 95.7g |
| 20040303 | 50Kg | 23L | 3.54mg/ml | 81.4g |
From table B result, every batch is dropped into 150Kg Os Cervi, and Os Cervi polypeptide maximum extracted amount and minimum difference reach 131g; From table C result, every batch is dropped into 50Kg Semen Melo, and Semen Melo polypeptide maximum extracted amount and minimum difference reach 29.2g.
This embodiment explanation, if produce with crude drug weight ratio according to background technology, not only criticizes a content very large, and in final Cervus and Cucumis Polypeptide for injection product Os Cervi polypeptide and Semen Melo polypeptide also without definite ratio.This is explanation just, has only with the Os Cervi polypeptide in extract separately and the weight ratio production control Cervus and Cucumis Polypeptide for injection pharmaceutical composition of Semen Melo polypeptide active ingredient, can guarantee that the content difference of final preparation is minimum.The specification that explanation is prepared by the method for embodiment 5 as following table D is that the difference that 8mg/ props up is criticized effective component content explanation between preparation.
Table D: Cervus and Cucumis Polypeptide for injection
As above showing data can find out, method of the present invention can be prepared with the stable preparation of effective constituent, has guaranteed product quality, meets the national drug standards.
Embodiment 9:
The effect of preparation for treating rheumatism of the present invention, rheumatoid arthritis
Random rheumatic arthritis 21 examples, rheumatoid arthritis 8 examples selected from inpatient; Leukocyte count, the rheumatoid factor in clinical examination erythrocyte sedimentation rate, blood.
Therapeutic Method: with the preparation of embodiment 6, dosage is to use Cervus and Cucumis Polypeptide for injection 16-24mg every day, with 0.9% sodium chloride injection 250-500ml dissolved dilution posterior vein, instils, and within 15th, be a course for the treatment of, treat altogether 2 courses for the treatment of, during treatment, stop using other therapies and medicine.
Curative effect judging standard:
1. effective: arthralgia and swelling disappear, motion is freely without sense of discomfort, and every inspection all recovers normal person.
2. effective: arthralgia and swelling disappear substantially, but still sense of motion is uncomfortable, or have 1～2 inspection not recover normal person.
3. invalid: the person of being not improved before clinical symptoms and treatment.
Therapeutic effect is shown in as in following table E.
Table E: this product treatment rheumatism, rheumatoid arthritis clinical efficacy
The result demonstration of table E, preparation of the present invention has significant therapeutic effect to treatment rheumatism, rheumatoid arthritis tool.
Embodiment 10:
Preparation of the present invention is to the result that promotes that bone heals
Random membra closure type bone fracture 57 examples of selecting, are divided into treatment group 42 examples, matched group 15 examples at random.
Conventional methods such as all adopting traction after patient is admitted to hospital, reset, be fixing, and give antibiotic 3 days with prevention infection.Matched group gives routine administration simultaneously, and treatment group gives the Cervus and Cucumis Polypeptide for injection 16-24mg of embodiment 5, with 0.9% sodium chloride injection 250-500ml dissolved dilution posterior vein, instil, and within 15th, be a course for the treatment of.
After observing medication, two groups of patient's swelling regression times, pain weaken time, callus formation time, healing time.The results are shown in table F.Curative effect statistics:
Table F: the clinical effectiveness of preparation of the present invention to the pain of subsiding a swelling, subtract, promotion union of fracture
Show F and show, Cervus and Cucumis Polypeptide for injection preparation has the effect that slows down significantly pain, elimination swelling, promotes union of fracture.
Claims (8)
1. a Cervus and Cucumis Polypeptide for injection pharmaceutical composition, is characterized in that it contains Os Cervi extract, Semen Melo extract and one or more excipient, and wherein the weight ratio of Os Cervi polypeptide and Semen Melo polypeptide is 2: 1 to 6: 1.
2. the preparation method of Cervus and Cucumis Polypeptide for injection pharmaceutical composition described in claim 1, it comprises the steps:
A) preparation of Os Cervi extract solution:
(1) Os Cervi pretreatment;
(2) pretreated clean Os Cervi is pulverized;
(3) in broken Os Cervi, add the water for injection of more than 2 times weight, 121 ℃ of hot pressing are extracted to leach and are obtained an Os Cervi extracting solution;
(4) in the Os Cervi slag after once extracting, add more than 2 times weight water for injection, continue to extract 121 ℃ of-123 ℃ of hot pressing, leach and obtain secondary Os Cervi extracting solution, make Os Cervi extracting solution with the Os Cervi extracting solution merging that step (3) obtains;
(5) Os Cervi extracting solution is centrifugal, collect supernatant centrifugal liquid;
(6) centrifugal liquid is carried out respectively to 100KD, 10KD, 6KD ultrafiltration, collect filter liquor, be evaporated to 10% of cumulative volume, collect concentrated solution, make Os Cervi extract solution;
B) preparation of Semen Melo extract solution:
(1) rinsing of drying and ripening Semen Melo is clean;
(2) in clean Semen Melo, add the water for injection of more than 2 times weight, with colloid mill, grind to form Semen Melo homogenate;
(3) Semen Melo homogenate is extracted 121 ℃ of hot pressing, leach Semen Melo extracting solution one time;
(4) in the Semen Melo slag after once extracting, add more than 2 times weight water for injection, continuation is extracted 121 ℃ of-123 ℃ of hot pressing, leach secondary Semen Melo extracting solution, make Semen Melo extracting solution with the Semen Melo extracting solution merging that step (3) obtains;
(5) Semen Melo extracting solution is centrifugal, collect supernatant centrifugal liquid; (6) centrifugal liquid is carried out respectively to 100KD, 10KD, 6KD ultrafiltration, collect filter liquor, be evaporated to 10% of original volume, collect concentrated solution, make Semen Melo extract solution;
C) the Semen Melo extract solution of the preparation of injection Os Cervi polypeptide pharmaceutical composition: the Os Cervi extract solution of blend step A) preparing and step B) preparing and one or more excipient pharmaceutical compositions, by Freeze Drying Technique, make freeze-dried powder, wherein the weight ratio of Os Cervi polypeptide and Semen Melo polypeptide is 2: 1 to 6: 1.
3. preparation method claimed in claim 1, wherein the weight ratio of Os Cervi polypeptide and Semen Melo polypeptide is 2: 1 to 4: 1.
4. preparation method claimed in claim 2, wherein excipient is selected from Dextran 40, glucose or lactose.
5. a preparation method for Cervus and Cucumis Polypeptide for injection pharmaceutical composition, it comprises the steps:
A) preparation of Os Cervi extract solution:
(1) Os Cervi boiled after 30 minutes in boiling water, removed fascia and residual meat, completed pretreatment;
(2) pretreated clean deer bone powder is broken to the broken bone below 1cm;
(3) in broken Os Cervi, add the water for injection of 2 times of weight, 121 ℃ of hot pressing are extracted 2 hours, leach and obtain an Os Cervi extracting solution;
(4) in the Os Cervi slag after once extracting, add 2 times of weight waters for injection, continuation is extracted 2 hours 121 ℃ of-123 ℃ of hot pressing, leach and obtain secondary Os Cervi extracting solution, make Os Cervi extracting solution with the Os Cervi extracting solution merging that step (3) obtains, placement is spent the night;
(5) by Os Cervi extracting solution with 4000 revs/min centrifugal 15 minutes, collect supernatant centrifugal liquid;
(6) centrifugal liquid is carried out respectively to 100KD, 10KD, 6KD ultrafiltration, collect filter liquor, be evaporated to 10% of cumulative volume, collect concentrated solution, with 0.22 μ m membrane filtration, make Os Cervi extract solution;
B) preparation of Semen Melo extract solution:
(1) by water for injection rinsing 3-4 time for drying and ripening Semen Melo;
(2) in clean Semen Melo, add the water for injection of 2 times of weight, with colloid mill, grind to form Semen Melo homogenate;
(3) Semen Melo homogenate is extracted 2 hours 121 ℃ of hot pressing, leach Semen Melo extracting solution one time;
(4) in the Semen Melo slag after once extracting, add 2 times of weight waters for injection, continuation is extracted 2 hours 121 ℃ of-123 ℃ of hot pressing, leach secondary Semen Melo extracting solution, make Semen Melo extracting solution with the Semen Melo extracting solution merging that step (3) obtains;
(5) by Semen Melo extracting solution with 4000 revs/min centrifugal 15 minutes, collect supernatant centrifugal liquid;
(6) centrifugal liquid is carried out respectively to 100KD, 10KD, 6KD ultrafiltration, collect filter liquor, be evaporated to 10% of original volume, collect concentrated solution, with 0.22 μ m membrane filtration, make Semen Melo extract solution;
C) preparation of injection Os Cervi polypeptide pharmaceutical composition:
Blend step A) the Os Cervi extract solution of preparing and step B) the Semen Melo extract solution prepared and one or more excipient pharmaceutical compositions, by Freeze Drying Technique, make freeze-dried powder, wherein the weight ratio of Os Cervi polypeptide and Semen Melo polypeptide is 2: 1 to 4: 1, excipient is selected from Dextran 40, glucose or lactose, and the weight ratio of polypeptide total amount and excipient is 1: 5-30.
6. compositions claimed in claim 1, wherein said compositions is solid preparation or liquid preparation.
7. compositions claimed in claim 3, wherein said solid preparation is capsule, soft capsule, tablet, buccal tablet, granule or drop pill; Liquid preparation is oral liquid or injection.
8. the application of Cervus and Cucumis Polypeptide for injection pharmaceutical composition aspect preparation treatment rheumatism, rheumatoid arthritis, ankylosing spondylitis, all kinds fracture, repair in trauma and lumbago and skelalgia medicine described in claim 1.
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| CN110339335A (en) * | 2019-07-01 | 2019-10-18 | 哈尔滨誉衡制药有限公司 | A kind of cervus and cucumis polypeptide ejection preparation |
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| CN110339335A (en) * | 2019-07-01 | 2019-10-18 | 哈尔滨誉衡制药有限公司 | A kind of cervus and cucumis polypeptide ejection preparation |
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