CN102429923A - Red deer pilose antler polypeptide and application thereof to treatment of bone diseases - Google Patents
Red deer pilose antler polypeptide and application thereof to treatment of bone diseases Download PDFInfo
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- CN102429923A CN102429923A CN2011104102458A CN201110410245A CN102429923A CN 102429923 A CN102429923 A CN 102429923A CN 2011104102458 A CN2011104102458 A CN 2011104102458A CN 201110410245 A CN201110410245 A CN 201110410245A CN 102429923 A CN102429923 A CN 102429923A
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Abstract
The invention relates to the field of medicine, in particular to a polypeptide derived from red deer pilose antler and application thereof to treatment of bone diseases. The polypeptide is characterized by being a polypeptide total extract with the molecular weight of 5-10 kD from the red deer pilose antler. The polypeptide is prepared with a method consisting of the following steps of: smashing the red deer pilose antler, and leaching with a sodium acetate buffer solution; filtering a supernatant obtained by centrifuging a leaching liquor with a 0.45-mum water-series mixed filter membrane to obtain a crude total polypeptide extracting solution; precipitating the crude total polypeptide extracting solution with ethanol to remove foreign proteins; intercepting with tangential ultrafiltration membrane bags of 10,000 MWCO and 5,000 MWCO; and concentrating the intercepted part of 5,000 MWCO and performing freeze drying. The polypeptide disclosed by the invention can be used for treating the bone diseases, particularly fracture, osteoporotic fracture and osteoporosis.
Description
Technical field
The present invention relates to field of medicaments, be specifically related to a kind of polypeptide of Cervus elaphus linnaeus Cornu Cervi Pantotrichum and purposes of preparation osteopathia thereof of being derived from.
Background technology
Cornu Cervi Pantotrichum is the tender angle of stag, and the band fine hair contains blood, is a kind of valuable Chinese medicine; Cornu Cervi Pantotrichum is sweet, salty, and temperature is returned kidney, Liver Channel, has enhancing human body immunity power, the anti-ageing function of waiting for a long time.Annual trophophase at Cornu Cervi Pantotrichum; The extension of axis of bone is very fast, can reach 1～2cm/d, in 90～120 days, accomplishes from the osteocyte differentiation and development to sophisticated overall process; The respective table cortex is also wanted hypertrophy so that cover the sclerotin of new growth; Its speed of growth is that any tissue of mammal and organ are incomparable, and annual Cornu Cervi Pantotrichum all carries out periodically replacement (regeneration of Cornu Cervi Pantotrichum), generally will pass through disk stripping, give birth to cyclic process fine and soft, the young pilose antler of ossify, come off, regenerate.
The Cornu Cervi Pantotrichum peptide is not only annual 1～2 secondary growth of Cornu Cervi Pantotrichum, ripe necessary component, also is simultaneously the carrying person that pilose antler histiocyte important composition material and molecular signal transmit.As lacked the corresponding polypeptide component, and Cornu Cervi Pantotrichum will be stagnated growth, and function system will get muddled, even various chronic diseases occur.After the people took the antler polypeptide component, human body showed following characteristics to its absorption: need not digest, and direct preferential absorption, absorption is absorbed by the body fast and with complete form; Absorb peptide and need not consume body energy, peptide is to force absorption of human body with self energy.Do not have any Excreta after the absorption, for because of digestive system defective, obstacle, damage, and can not absorb nutrition person, have the advantages that to absorb preferably.This is poor for those digestion powers, malnutrition, physical weakness person, has great significance.Modern medicine also shows, mainly contains insulin like growth factor, nerve growth factor, epidermal growth factor, transforming growth factor and relevant antler polypeptide in the antler polypeptide.Pharmaceutical research shows that antler polypeptide has immune effect and antitumor action.
Cervus elaphus linnaeus (Cervus elaphus) is belong to Cervidae a kind of, a kind of for large-scale deer.The bodily form is bigger, 200 kilograms of body weight, and 2 meters of heights are surplus, and ridge is straight.Tarim Basin, Xinjiang Cervus elaphus linnaeus is the subspecies that region is extremely strong in the Chinese Cervus elaphus linnaeus strain, only is distributed in Tarimhe River middle and lower reaches and downstream, Che Erchen river area, and unique environment has been brought up the Tarim Basin Cervus elaphus linnaeus and produced fine and soft ability height.
Summary of the invention
The invention discloses a kind of Cervus elaphus linnaeus antler polypeptide, it is the polypeptide total extract of 5～10kD molecular weight of Cervus elaphus linnaeus Cornu Cervi Pantotrichum.
The Cervus elaphus linnaeus antler polypeptide total extract of 5～10kD molecular weight of the present invention is preferably with following method preparation:
The Cervus elaphus linnaeus Cornu Cervi Pantotrichum is pulverized the back use the sodium-acetate buffer lixiviate; Supernatant after lixiviating solution is centrifugal mixes filter membrane through 0.45 μ m water system and filters; Get total polypeptide crude extract; Total polypeptide crude extract removes foreign protein through ethanol precipitation, after 10000MWCO and 5000MWCO tangential ultrafiltration film bag are held back, gets that 5000MWCO holds back partial concentration, lyophilizing promptly gets earlier.
The preferred pH=3.5 of sodium-acetate buffer.
Preferred earlier through ethanol water logging bubble before wherein the Cervus elaphus linnaeus Cornu Cervi Pantotrichum is pulverized, is pulverizing the oven dry back.
The ice bath lixiviate is preferably adopted in lixiviate.
Pharmacodynamics test shows that Cervus elaphus linnaeus antler polypeptide of the present invention is better than the total polypeptide of Cervus elaphus linnaeus Cornu Cervi Pantotrichum in drug effect aspect the treatment osteopathia.Can be used for treating diseases such as fracture, osteoporotic fracture and osteoporosis.
Be part pharmacodynamics test of the present invention and result below.
One, the Cornu Cervi Pantotrichum peptide is to the research of proliferation of osteoblast:
1. experimental principle
Osteoblast needs experience, cell proliferation, cell differentiation and cell mineralization process in the bone formation process.Early stage in the cell growth, osteoblast is in vegetative state, and the secretion of 1 Collagen Type VI increases; Cell is grown mid-term, and cultivation effect slows down, and alkali phosphatase (ALP) secretion increases, and hydrolysis has inhibiting inorganic pyrophosphate to the bone mineralising, and the bone mineralising is strengthened, and regulates Ca
2+, P
3+Concentration, the enhance bone mineralising; Cell is grown late period, osteoblast secretion osteoprotegerin (OPG), and Bone Gla protein (OCN), osteonectins (ON) etc. promote the bone mineralising, and produce the mineralising knot.
2. receive reagent
Appearance A: the total peptide of Tarim Basin, Xinjiang Cervus elaphus linnaeus Cornu Cervi Pantotrichum;
Appearance B: Tarim Basin, Xinjiang Cervus elaphus linnaeus Cornu Cervi Pantotrichum molecular weight is greater than the polypeptide total extract of 10kD;
Appearance C: Tarim Basin, Xinjiang Cervus elaphus linnaeus Cornu Cervi Pantotrichum molecular weight 5～10kD polypeptide total extract;
Appearance D: the total peptide of northeast spotted deer antler;
Appearance E: northeast spotted deer antler molecular weight is greater than the polypeptide total extract of 10kD;
Appearance F: the polypeptide total extract of northeast spotted deer antler molecular weight 5～10kD;
The method for distilling of the total peptide of above-mentioned Cervus elaphus linnaeus Cornu Cervi Pantotrichum can be with reference to pertinent literature, and also available following method is extracted:
Cervus elaphus linnaeus Cornu Cervi Pantotrichum dry product is sawn into segment, immerses alcohol-water (2: 1), 4 ℃, soaked 7 days, take out section, 37 ℃ of oven dry are done fine and soft sheet and are broken into powder, weigh, and add 5mmo l/L NaA c-HAc buffer (pH 3.5) 20mL, ice bath, stirring and leaching 12h by every 1g; 4 ℃, 12000 rev/mins centrifugal 20 minutes, supernatant;-0.1mPa, 0.45 μ m water system is mixed filter membrane and is filtered, and filter liquor is the total polypeptide crude extract of Cervus elaphus linnaeus Cornu Cervi Pantotrichum dry product, and ethanol precipitation removes foreign protein, and the supernatant lyophilizing gets pale yellow powder.Obtain the total peptide of Cervus elaphus linnaeus Cornu Cervi Pantotrichum.
Above-mentioned Cervus elaphus linnaeus Cornu Cervi Pantotrichum molecular weight can be with reference to pertinent literature greater than the method for distilling of the polypeptide total extract of 10kD, and also available following method is extracted:
Cervus elaphus linnaeus Cornu Cervi Pantotrichum dry product is sawn into segment, immerses alcohol-water (2: 1), 4 ℃, soaked 7 days, take out section, 37 ℃ of oven dry are done fine and soft sheet and are broken into powder, weigh, and add 5mmo l/L NaA c-HAc buffer (pH 3.5) 20mL, ice bath, stirring and leaching 12h by every 1g; 4 ℃, 12000 rev/mins centrifugal 20 minutes, supernatant;-0.1mPa, 0.45 μ m water system is mixed filter membrane and is filtered, and filter liquor is the total polypeptide crude extract of Cervus elaphus linnaeus Cornu Cervi Pantotrichum dry product; Ethanol precipitation removes foreign protein; Every 1000mL holds back through 10000MWCO (PES) tangential ultrafiltration film bag and is concentrated into about 50mL,＞10kD component (Mr＞10000), lyophilizing gets pale yellow powder.
Tarim Basin, Xinjiang Cervus elaphus linnaeus Cornu Cervi Pantotrichum molecular weight 5～10kD polypeptide total extract of appearance C prepares with embodiment 1 method.
Appearance F is with reference to the method for embodiment 1, and just raw material changes the northeast spotted deer antler into.
3. experimental technique
1) osteoblast is cultivated
Adopt enzyme digestion culture of rat osteoblast, get newborn SD rat (male and female are not limit), take out skull, connective tissues such as cleaning periosteum, blood vessel, PBS clean skull 3 times to white, and are cut into the osteocomma of 1mm * 1mm.Add 0.25% trypsin in 37 ℃ of digestion 20min, to remove fibrous tissue; Add 0.1%II Collagen Type VI enzyme in 37 ℃ of vibration digestion 60min.Cell dissociation buffer filters through 200 eye mesh screens, and in the centrifugal 10min of 1000r/min, sedimentation cell is processed suspension with α-MEM+10%FBS, repeats above-mentioned II Collagen Type VI enzymatic digestion stage.With the cell mixing of 2 digestion acquisitions, and be inoculated in the culture bottle, place 5%CO
2Cultivate in the incubator.Changed liquid once in per 2～3 days, tight observation of cell upgrowth situation under the inverted microscope, treat cell cover with bottle at the bottom of 80%, cultivate with 0.25% pancreatin (containing 0.02%EDTA) had digestive transfer culture.Get well-grown the 3rd generation cell be used for experiment and cell function is identified.
2) rat osteoblast is identified
Alkali phosphatase (ALP) dyeing: get the 3rd generation osteoblast, with 1 * 10
6Cells/well is inoculated in 6 orifice plates.Cultivated 2 days, with the fixing 15min of 95% ethanol, PBS washing 3 times is carried out ALP with the BCIP/NBT staining and is dyeed.According to the explanation of BCIP/NBT alkali phosphatase colour reagent box, an amount of BCIP/NBT is fully covered cell sample, the room temperature lucifuge is hatched 24h, distilled water flushing.Natural drying places microscopically to observe.
The knot dyeing of cell mineralising: with the 3rd generation osteoblast, with 1 * 10
6Cells/well is inoculated in 6 orifice plates.Treat that cell 80% converges, α-MEM+10%FBS culture medium is replaced by α-MEM+10%FBS+10mM β phosphoglycerol disodium salt+50 μ g/ml vitamin C inducing cultures.Cell culture 14 days carries out alizarin red dyeing.95% ethanol is 10min fixedly, distillation washing 3 times; 37 ℃, 0.5% alizarin red dyeing 30min.Distilled water flushing, natural drying places microscopically to observe.
3) cell proliferating determining
With the 3rd generation osteoblast with 1 * 10
5Cells/well is inoculated in 96 well culture plates.Change low serum α-MEM after cultivating 24h, add 10 respectively
-7G/ml, 10
-6G/ml, 10
-5The Cornu Cervi Pantotrichum peptide of the different peptide sections of/ml, every group is repeated 5 holes.Administration 1d, 3d, 7d, every hole adds 0.5mg/ml MTT 20 μ l, in 37 ℃, 5%CO
2Continue to hatch 4h under the condition, discard culture fluid, add DMSO 150 μ l, vibration 10min, the absorbance in a mensuration hole under ELIASA 570nm wavelength.
4) alkali phosphatase (ALP) vitality test
With the 3rd generation osteoblast, with 1 * 10
6Cells/well is inoculated in 6 orifice plates.Change low serum α-MEM after cultivating 24h, add 10 respectively
-7G/ml, 10
-6G/ml, 10
-5The Cornu Cervi Pantotrichum peptide of the different peptide sections of/ml.Administration 3d, 7d, TritonX-100 extracts albumen, and detects albumen with the BCA standard measure.According to the explanation of alkaline phosphatase enzyme detection kit, in 96 orifice plates, add 20 μ l albumen supernatant and 100 μ l PNPP solution.Under 37 ℃, hatch 15min, stopped reaction, the absorbance in a mensuration hole under ELIASA 405nm wavelength.
5) statistical procedures
All experimental datas all adopt statistical method to handle.T check between relatively employing group of average differences; Data statistics is all represented with mean+SD
; With P＜0.05 (*); P＜0.01 (* *), P＜0.001 (* * *) is significant level.
4, result of the test
1) different Cornu Cervi Pantotrichum peptides influence osteoblastic proliferation
Fracture, osteoporotic fracture and osteoporosis, osteoblast are all played the part of this important role, and osteoblast needs experience, cell proliferation, cell differentiation and cell mineralization process in bone formation in fracture recovery process.Early stage in the cell growth, osteoblast is in vegetative state, and the secretion of 1 Collagen Type VI increases; Cell is grown mid-term, and cultivation effect slows down, and alkali phosphatase (ALP) secretion increases, and hydrolysis has inhibiting inorganic pyrophosphate to the bone mineralising, and the bone mineralising is strengthened, and regulates Ca
2+, P
3+Concentration, the enhance bone mineralising reaches the promotion knitting, the effect of osteanagenesis.The result sees table 1, and table 1 shows that Tarim Basin, Xinjiang Cervus elaphus linnaeus Cornu Cervi Pantotrichum peptide (appearance A, appearance B, appearance C) all significantly promotes osteoblastic proliferation effect (* P＜0.05, * * P＜0.01 or * * * P＜0.001).Drug effect obviously is better than the spotted deer antler peptide.And Tarim Basin, Xinjiang Cervus elaphus linnaeus Cornu Cervi Pantotrichum molecular weight 5～10kD polypeptide total extract drug effect obviously is better than the polypeptide total extract of the bright Cervus elaphus linnaeus Cornu Cervi Pantotrichum of total peptide of Tarim Basin, Xinjiang Cervus elaphus linnaeus Cornu Cervi Pantotrichum and Tarim Basin, Xinjiang molecular weight greater than 10kD.Prompting Tarim Basin, Xinjiang Cervus elaphus linnaeus Cornu Cervi Pantotrichum molecular weight 5～10kD polypeptide total extract can reach treatment fracture, osteoporotic fracture and osteoporotic purpose through promoting osteoblastic proliferation.
Table 1 Cornu Cervi Pantotrichum peptide is to the effect of different time points osteoblastic proliferation
Annotate: compare * P＜0.05, * * P＜0.01, * * * P＜0.001 with matched group
2) different Cornu Cervi Pantotrichum peptides are to the active influence of osteoblast ALP
Alkali phosphatase (ALP) is osteoblastic enzyme-specific, and the ALP of great expression is shown as osteocyte and rises to ripe osteoblast differentiation ability, and the bone mineralising is strengthened, and reaches the promotion knitting, the effect of osteanagenesis.The result sees Fig. 1 and table 2; Give and Tarim Basin, Xinjiang Cervus elaphus linnaeus Cornu Cervi Pantotrichum peptide 7 days; Compare Tarim Basin, the Xinjiang Cervus elaphus linnaeus Cornu Cervi Pantotrichum Toplink ALP that significantly raises in the osteoblast with matched group active, and wherein Tarim Basin, Xinjiang Cervus elaphus linnaeus Cornu Cervi Pantotrichum molecular weight 5～10kD polypeptide total extract drug effect is better than the total peptide of Tarim Basin, Xinjiang Cervus elaphus linnaeus Cornu Cervi Pantotrichum and Tarim Basin, the Xinjiang Cervus elaphus linnaeus Cornu Cervi Pantotrichum molecular weight polypeptide total extract greater than 10kD.Particularly Tarim Basin, Xinjiang Cervus elaphus linnaeus Cornu Cervi Pantotrichum molecular weight 5～10kD polypeptide total extract 10
-7G/mL, 10
-6G/mL concentration is to the active rising effect particularly evident (P＜0.01 or P＜0.001) of ALP.But the spotted deer antler peptide is not obvious to the active rising effect of ALP, and the partial peptide section also has the effect that reduces.This prompting Tarim Basin, Xinjiang Cervus elaphus linnaeus Cornu Cervi Pantotrichum 5～10kD polypeptide total extract possibly be potential treatment fracture, osteoporotic fracture and osteoporotic medicine, and comparing Cervus nippon Temminck has its original pharmacological action.
Table 2 separate sources Cornu Cervi Pantotrichum peptide is to the influence of alkaline phosphatase activities
Description of drawings
Fig. 1 is 7 days an ALP activity influence of Cornu Cervi Pantotrichum peptide effect
Fig. 2 is Tarim Basin, an Xinjiang cervus elaphus linnaeus polypeptide separation and purification product Trincine-SDS-PAGE electrophoresis pattern
Fig. 3 is that the HPLC of Tarim Basin, Xinjiang cervus elaphus linnaeus 5-10KD polypeptide total extract detects collection of illustrative plates
The specific embodiment
Embodiment 1
Cervus elaphus linnaeus Cornu Cervi Pantotrichum dry product is sawn into segment, immerses alcohol-water (2: 1), 4 ℃, soaked 7 days, take out section, 37 ℃ of oven dry are done fine and soft sheet and are broken into powder, weigh, and add 5mmol/L NaA c-HAc buffer (pH 3.5) 20mL, ice bath, stirring and leaching 12h by every 1g; 4 ℃, 12000 rev/mins centrifugal 20 minutes, supernatant;-0.1mPa, 0.45 μ m water system is mixed filter membrane and is filtered, and filter liquor is the total polypeptide crude extract of Cervus elaphus linnaeus Cornu Cervi Pantotrichum dry product; Ethanol precipitation removes foreign protein; Every 1000mL is through 10000MWCO, and 5000MWCO (PES) tangential ultrafiltration film bag is held back and is concentrated into about 50mL,＞10kD component (Mr＞10000); 5-10kD component (Mr=10000-5000) gets pale yellow powder with the lyophilizing of 5-10kD component.
Its electrophoretogram such as Fig. 2, wherein 1 is ultrafiltration component 5-10KD after the precipitate with ethanol deposition is redissolved; The 2nd, ultrafiltration component 10-30KD after the precipitate with ethanol deposition is redissolved; The 3rd, ultrafiltration component＞30KD after the precipitate with ethanol deposition is redissolved; The 4th, the standard protein band; The 5th, crude extract ultrafiltration component＜5KD; The 6th, crude extract ultrafiltration component 5-10KD; The 7th, crude extract ultrafiltration component 10-30KD; The 8th, crude extract ultrafiltration component＞30KD.
Claims (7)
1. Cervus elaphus linnaeus antler polypeptide, it is characterized in that: it is the polypeptide total extract of Cervus elaphus linnaeus Cornu Cervi Pantotrichum 5～10kD molecular weight.
2. the Cervus elaphus linnaeus antler polypeptide of claim 1; Prepare by following method: the Cervus elaphus linnaeus Cornu Cervi Pantotrichum is pulverized the back use the sodium-acetate buffer lixiviate; Supernatant after lixiviating solution is centrifugal mixes filter membrane through 0.45 μ m water system and filters, and gets total polypeptide crude extract, and total polypeptide crude extract removes foreign protein through ethanol precipitation; Earlier, get that 5000MWCO holds back partial concentration, lyophilizing promptly gets after 10000MWCO and 5000MWCO tangential ultrafiltration film bag are held back.
3. the Cervus elaphus linnaeus antler polypeptide of claim 1, wherein the Cervus elaphus linnaeus Cornu Cervi Pantotrichum is Tarim Basin, an Xinjiang Cervus elaphus linnaeus Cornu Cervi Pantotrichum.
4. the Cervus elaphus linnaeus antler polypeptide of claim 1 is used to prepare the purposes of the medicine of treating osteopathia.
5. the purposes of claim 4, wherein osteopathia is a fracture.
6. the purposes of claim 4, wherein osteopathia is an osteoporotic fracture.
7. the purposes of claim 4, wherein osteopathia is an osteoporosis.
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Cited By (2)
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| CN106860913A (en) * | 2017-02-22 | 2017-06-20 | 昆明医科大学 | The application of antler polypeptide, tooth implant and its surface treatment method |
| CN111297904A (en) * | 2020-04-10 | 2020-06-19 | 中国农业科学院特产研究所 | New application of cornu Cervi Pantotrichum aqueous extract |
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| CN106860913A (en) * | 2017-02-22 | 2017-06-20 | 昆明医科大学 | The application of antler polypeptide, tooth implant and its surface treatment method |
| CN111297904A (en) * | 2020-04-10 | 2020-06-19 | 中国农业科学院特产研究所 | New application of cornu Cervi Pantotrichum aqueous extract |
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