CN101068556A - Pilose antler nano-products containing enriched insulin-like growth factor and producing method thereof - Google Patents
Pilose antler nano-products containing enriched insulin-like growth factor and producing method thereof Download PDFInfo
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- CN101068556A CN101068556A CNA2006800012623A CN200680001262A CN101068556A CN 101068556 A CN101068556 A CN 101068556A CN A2006800012623 A CNA2006800012623 A CN A2006800012623A CN 200680001262 A CN200680001262 A CN 200680001262A CN 101068556 A CN101068556 A CN 101068556A
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Abstract
The invention relates to an antler extractive nanometer product which is rich with insulin-like growth factor (IGF-1) and the preparation method thereof. The content of the IGF-1 in the extractive is 100-10000 nanogram/gram, the grain diameter of the sacculus of the product is 10-1000 nanometer. In addition, the invention also relates to the preparation method of preparing antler nanometer product and the application of the product in the aspect of medicine, food, health products and cosmetics and so on.
Description
The invention belongs to the medical or edible products field of deer antler extract for pilose antler nanosizing product rich in the plain like growth factor in natural fibre line of meat island and preparation method thereof technical field.For more specifically, the present invention relates to by rich in IGF（IGF- 1) the nanosizing product that is made of deer antler extract.Another sunset is foretold, and the invention further relates to prepare the method for the pilose antler nanosizing product, and application of the product in terms of medicine, food, health products and cosmetics.Background technology pilose antler（Deer not yet ossified young horn）With deer horn as traditional Chinese medicine, the effects such as with strengthening the muscles and bones, tonifying kidney and strengthening yang, dredging governor vessel, just it is widely used in preparing since ancient times turning into various tonics, food, health products and cosmetics.Researched and analysed by Modern Pharmaceutical Chemistry, containing many kinds of physiologically active ingredients in pilose antler, including phosphatide, alkamines compound, prostaglandin, vitamin, amino acid, inorganic salts, unrighted acid, cortisol etc..With modern biotechnology high speed development, it has been found that in pilose antler in addition to containing above-mentioned various physiologically active ingredients, wherein also containing IGF (IGF- 1), insulin, human growth hormone (HGH)（HGH), GHRF（GHRF-6), nerve growth factor（NGF), EGF（EGF), fibroblast growth factor（The Large molecule active protein such as FGF).Any of the above active component, mutually especially various reactive proteins, synergy, plays effect.Wherein, IGF plays especially prominent effect.IGF-1, which has, promotes fissional effect, can be used in treating and preventing the disease in terms of the disorder of carbohydrate equal energy source Metabolism regulation, such as treats and prevents the disease in terms of glycometabolism or fat metabolism, such as diabetes.Chinese patent application CN1251531A, which is disclosed, to induce or improve tissue growth, propagation or regeneration method using IGF- 1, promote the growth of inner ear tissue especially with IGF-1；Chinese patent application CN1204263A is disclosed can treat the degenerative disease of brain or spinal nerve, such as alzheimer's disease using IGF- 1（Alzheimer's Disease；), towel platinum Sen Shi diseases (Parkinson's Disease) etc..In terms of diabetes are treated, also there is lot of documents to disclose the practice treated using IGF- 1 in terms of I types, II types and insulin-resistant diabetes（Referring to Chinese patent application CN1387440A, CN1384201A etc.）.
In traditional pilose antler process, pilose antler is taken after directly crushing, and particle is larger, causes absorption efficiency low；Sometimes, the processing of pilose antler is needed by steps such as heating decoction, drying, and high temperature can make the protein denaturations such as IGF- 1 therein, so as to reduce or even lose their effect.Another traditional processing mode is immersed in pilose antler in the wine of alcohol in high concentration content, and medicinal liquor is made and takes.But it is due to wine
In the alcohol as organic solvent containing high concentration, the protein denaturations such as iGF- Ι can be made, and be also unfavorable for from bulky grain pilose antler dissolving the protein such as IGF-1, thus the protein such as IGF-1 can be caused to be destroyed, so that the service efficiency of pilose antler is low, the wasting of resources is caused.In addition, the medicinal liquor of alcohol in high concentration content also limit each dose.
In order to improve the service efficiency of pilose antler material, people have attempted the active ingredient that various methods extract pilose antler.For example, international patent application WO2004/035069A disclose it is a kind of be used to prevent and treat deer horn product of diabetes and preparation method thereof, but only contain 18ng IGF- 1 and 0.5ng insulin in every 1 restraint product.Chinese patent CN1241943C discloses a kind of extracting methods of velvet product of ^ Γ, but the material wherein extracted does not point out that the moditied processings such as nano-package can be passed through, when oral will in face of intestines and stomach absorption barrier and digestive system proteolytic degradation problem, cause the active ingredients such as IGF-1 actual absorption efficiency it is low.
People also attempt the active ingredient that various methods extract pilose antler, are prepared into the various products for being conducive to absorbing, the product of nanosizing are especially prepared into, so as to improve the absorption efficiency of pilose antler material.For example, Chinese patent application CN1723992A discloses a kind of product containing the composition such as nanoscale pilose antler powder and walnut meat powder prepared with ad hoc fashion, antifatigue and strengthen immunity health products are used as.But, the product is during preparation, pilose antler needs to be extracted with alcohol solvent, because alcohol is a kind of organic solvent, it is low to the water soluble protein extraction efficiency such as IGF-1, and easily make these protein denaturations, destroy effect of the grade protein of IGF- 1, the active low of the protein such as IGF-1 in manufactured goods is caused, using effect is have impact on.Chinese patent CN1166369C discloses one kind and uses water, vegetable oil and/or animal oil pilose antler or deer horn are made the gel-like fluid or suspension that particle diameter is 10 ~ 1000nm, but nanosizing processing is carried out to all crushed materials of pilose antler in preparation process, the cutin composition that wherein can not be largely absorbed by human body is not removed, and can not fully extract the water soluble proteins such as IGF-1 using vegetable oil or animal oil, make the actual content of IGF-1 in the product low, and the particle of this nanosizing can not be prevented effectively from the degraded of the proteolytic enzyme of digestive system.
For defect present in prior art, by long-term and arduous research, the present inventor obtains a kind of new deer antler extract and its nanosizing product rich in IGF (IGF- 1), overcomes-drawbacks described above.The product of the present invention not only IGF-1 content is high and maintains high activity, takes full advantage of pilose antler resource, and the product of nanosizing can bring the effect of oozing of certain rush, the problem of helping to overcome the absorption barrier of intestines and stomach and enzyme barrier.In addition, the preparation technology of the product is rationally, it is easy in large-scale production control quality, and preparation process uses natural material, can be biodegradable, it is to avoid the pollution of artificial synthesized product, it is easier to receive for consumer.The content of the invention
The invention provides a kind of nanosizing product being made up of deer antler extract and preparation method thereof, the extract is rich in IGF.Present invention also offers the application of the nanosizing product in medicine, food, health products and cosmetics are prepared.
In a first aspect, the invention provides a kind of nanosizing product being made up of deer antler extract, it is 10 1000 nanometers comprising particle diameter（Nm nanocapsule ball), and IGF in the extract（IGF-1 content) is 100 ~ 10000 ng/gs.The IGF-1 contents of extract in the product of the present invention are 100 ~ 10000 ng/g extracts, significantly larger than IGF-1 contents of other known deer antler extracts, it is preferred that the IGF-1 contents are 1000 ~ 6000ng/ grams, more preferably 1500 ~ 4500ng/ grams.And the particle diameter of the nanocapsule ball in product described in the product of the present invention is preferably l (Tl000nm, more preferably 20 ~ 800nm, more preferably 40 ~ 400
The product can be obtained by following two steps：Go out the extract rich in IGF-1 with solvent extraction first from pilose antler；Then said extracted thing is prepared into nanosizing product.The present invention, as raw material, is conducive to preserving the content and activity of IGF-1 isoreactivity compositions to greatest extent preferably from fresh pilose antler or the fresh pilose antler of refrigeration.Term " fresh " used herein, which refers to gathering, to begin to be processed the nanosizing product for preparing the present invention in 6 hours after pilose antler or refrigerate, it is preferred that processing or refrigeration were earlier than 3 hours after collection, more there is choosing earlier than 1 hour, most preferably earlier than 20 minutes.Term " refrigeration " used herein refers to being stored in pilose antler in less than 0 °C of environment, is preferably stored in -20 °C of environment.
The present invention, as solvent, extracts the extract that IGF-1 contents are 100 ~ 10000ng/ grams, preferably IGF-1 contents are 100 (T6000ng/ grams, more preferably 150 (T4500 ng/ gram preferably using water.And the generation of nanosizing product can prepare nano level particle or capsule ball using supersonic jet method or polymer wrapped method.It is preferred that the product of the present invention is nano level capsule ball, it uses polymer wrapped deer antler extract to form nano level capsule ball.The polymer and aliphatic category polyester polymer of suitable polymer useful monomers synthesis.The Exemplary monomers of the polymer synthesized with monomer have acrylamide, acrylate and alkyl cyanoacrylate and their derivative.Due to mostly artificial synthesized, such many polymer can not be biodegradation, therefore the product of the present invention is preferably comprised with good biodegradable aliphatic category polyester polymer, is consequently formed nano level capsule ball.The example of this kind of polymer has PLA（PLA：), PGA（PLG), poly (glycolide-co-lactide) copolymer（PLGA), polycaprolactone（PCL), polyamino acid and their derivative etc., preferably polyamino acid.Other preferred examples are some natural polymerses and their derivative, optional natural polymerses have albumin, chitosan, glucan, alginate etc., they have good biodegradable property, and a nanometer fine gold ball parcel active component can be formed after disperseing.
The nanosizing product of the present invention is 10 (T400 particularly preferably containing particle diameternM nanocapsule ball.In a detailed embodiment, contain chitosan in nanosizing product of the invention, deer is wrapped up using chitosan
Fine and soft extract, forms the nanocapsule ball that particle diameter is 100 ~ 400nm.The deer antler extract of chitosan parcel is except having certain Penetration enhancing effect to wherein peptide matters, and because chitosan has mucoadhesive properties earlier above, also certain proteasome degradation can be suppressed, prevent the effective ingredients such as IGF- 1 to be just degraded before it is absorbed.In addition, in chitosan infix and protease inhibitors, such as EDTA metal protease inhibitors can also reduce the degradation of protease.Therefore, the nanosizing product of the invention containing chitosan is especially suitable for being further processed into oral products, such as mouth spraying agent, tablet, capsule.
The nanosizing product of the present invention is 5 (Tl 50 further preferably containing particle diameternM nanocapsule ball.In a detailed embodiment, contain polyamino acid in nanosizing product of the invention, deer antler extract is wrapped up using polyamino acid, form the nanocapsule ball that particle diameter is 50 ~ 150nm.Polyamino acid can be selected from poly-D-lysine and the polyaminoacid being made up of leucine and glutamic acid（Referring to international patent application Jane 6/029991A：).
The nanosizing product of the present invention can also further include pharmaceutically acceptable assistant agent, so as to process various formulations in addition to containing above-mentioned deer antler extract and polymer wrapped material.Pharmaceutically acceptable assistant agent includes pharmaceutically acceptable carrier, excipient, diluent etc., and they are compatible with active component.It is known for those of ordinary skills with pharmaceutically acceptable assistant agent preparation of preparation.The preparation of the present invention comprising the nanosizing product described in one or more one side of the invention as active component, by the nanosizing product and pharmaceutically acceptable assistant agent（Carrier, excipient, diluent etc. as would be known to one of ordinary skill in the art）Combine, be configured to various preparations, preferably solid pharmaceutical preparation and liquid preparation.It is preferred that the preparation of the present invention is unit dosage form, such as tablet, pill, capsule (including sustained release or sustained release form）, pulvis, supensoid agent, granule, tincture, syrup, emulsion agent, suspension, the formulation such as injection, so as to be adapted to various form of medication, such as orally, it is parenteral injection, mucous membrane, muscle, intravenous, subcutaneous, intraocular, intracutaneous or by the form of medication skin.Mouth spraying agent, capsule or injection is particularly preferably made in the nanosizing product of the present invention.Wherein carrier, excipient, diluent are pharmaceutically acceptable and compatible with active component.The suitable excipient that can be selected is preferred but is not limited only to water, physiological saline, glucose, glycerine, ethanol or its analog and combinations thereof.
In second aspect, the invention provides application of the nanosizing product of first aspect present invention in the medicine, health food and/or the cosmetics that promote cell propagation are prepared.The nanosizing product of the present invention is made up of the deer antler extract rich in IGF- 1, with the effect for promoting cell division, breeding, level available for tissue growth, propagation or the regeneration for inducing or improving patient, treat corresponding disease, the degenerative disease of such as brain or spinal nerve, such as alzheimer's disease, Parkinson's disease.Due to being natural product, and pilose antler has long usage history in itself, and Healthy People, which takes a certain amount of nanosizing product of the invention, to be helped to maintain its normal cell division, proliferation function, helps to maintain its abundant vigor.A certain amount of nanosizing product of the invention of Healthy People external application helps to maintain the division of its Skin Cell, propagation,
N2006/000841 improves external, the effect with beauty.
In the third aspect, the invention provides application of the nanosizing product of first aspect present invention in treatment and/or the disorderly medicine of prevention carbohydrate metabolism regulation and/or health food is prepared.The nanosizing product of the present invention is made up of the deer antler extract rich in IGF-1, disease or sub-health state in terms of can be used in treating and/or preventing carbohydrate equal energy source Metabolism regulation disorderly, such as treat and prevent the disease in terms of glycometabolism or fat metabolism, particularly diabetes, including I types, II types and insulin-resistant diabetes etc..
For the medicine of second and third aspect of the present invention, nanosizing product of the invention can be processed into various pharmaceutical dosage forms, to needing to promote cell propagation or the patient for needing to treat and/or preventing carbohydrate metabolism regulation disorderly to be administered.The general concrete condition by physician in view patient of the agent quantity and form of administration（Such as age, body weight, sex, the state of an illness, sick time, health）It is determined that.In general, in terms of the IGF-1 in nanosizing product, the dosage of administration is 0.001 100mg/kg weight in patients, preferably 0.01 lmg/kg, preferably 0.02 ~ 0.1mg/kg.Form of medication can be determined according to the formulation of various pharmaceutical preparations, suitable form of medication have orally, it is parenteral injection, mucous membrane, muscle, intravenous, subcutaneous, intraocular, intracutaneous or by the form of medication such as skin, preferably use that mouth disputes agent or capsule is administered orally.
In the application of second and third aspect of the present invention, nanosizing product of the invention can be used separately as medicine, health food and/or cosmetics, can also be added in other medicines, health food and/or cosmetics and be used.For example, the nanosizing product of the present invention can be added to fit applications in conventional beverage, food or cosmetics.
In fourth aspect, the invention provides the preparation method of the nanosizing product of first aspect present invention.The preparation method can include following two steps：The extract rich in IGF-1 is extracted first from pilose antler；Then said extracted thing is prepared into nanosizing product.The preparation method preferably includes the following steps：
A) pilose antler crushed with flooding, isolates molecular weight for 50 (T500000DaWater-soluble extractive, freeze-drying；
And b) with biodegradable polymer the extract obtained by step a) is rolled into nanocapsule ball.Wherein, the step of pilose antler is crushed in step a) may include the one or more cut into slices, hooked in the modes such as slurry, ultrasonic disruption." section " refers to laminating in pilose antler, preferably uses slicer and the fresh pilose antler of fresh pilose antler or refrigeration is cut into the thin slice that thickness is l ~ 5mm.Untill " homogenate " refers to pilose antler being crushed to without big tissue block, appropriate deionized water can be added in homogenization process.It by ultrasonic disruption, can fully make cell rupture, appropriate deionized water can be added in the shattering process.The step of crushing pilose antler preferably includes section, hooks slurry and ultrasonic disruption step in order, can also omit slicing step therein.The overall process for crushing pilose antler is operated preferably in low temperature environment, wherein it is low temperature precooling that the reagent used, which is preferably also, low temperature environment refers to the environment in 0 ~ 20 °C of environment, preferably 0 ~ 10 °C, most
Fortunately 4 °C.The step of the step of pilose antler crushed in step a) with flooding can be with crushing pilose antler completes simultaneously, the active ingredient of pilose antler is extracted by the water added in the steps such as section, homogenate and/or ultrasonic disruption, such as IGF- 1, extraction is naturally done with the completion of pulverising step；It can also further place and be extracted for a period of time after the step of crushing pilose antler, the time, as being no more than 36 hours, preferably places 24 hours, it is desirable to be placed in low temperature environment.The step of separating water-soluble extractive in step a) can be carried out by modes such as conventional centrifugations and/or filtering, such as ultracentrifugation and/or ultrafiltration.The preferred molecular weight that extracts is 50 (T500000Da composition, it is that 100 (Tl 00000Da compositions most preferably extract the composition that molecular weight is 1000 ~ 10000Da more to have choosing to extract molecular weight in the step.In an embodiment, the present invention first passes through the precipitation in centrifugation removal flooding product, and the composition of molecular weight is then answered from suitable milipore filter retention phase, adds the water dissolving of appropriate amount.Then it is freeze-dried, the aqueous solution of the composition dissolved with corresponding molecular weight as obtained by using freeze drier by upper step is dried with _ 30 °C ~ -50 °C of drying machine operating temperature, the water content of product is 3%-10 °/o o after drying
In step b), the final product obtained by step a) is subjected to nanosizing, it can prepare nano level particle or capsule ball using supersonic jet method or polymer wrapped method." supersonic jet method " refers to use supersonic jet equipment（Such as it is purchased from Beijing nano bio tech ltd）Deer antler extract is formed into high velocity jet, and multiply injection stream is mutually intersected at a high speed in disintegrating area, high velocity impact and grinding are produced, so as to form nano level particle.Nano level capsule ball is formed present invention preferably employs the mode of polymer wrapped deer antler extract.The polymer and aliphatic category polyester polymer of its suitable polymer useful monomers synthesis.The Exemplary monomers of the polymer synthesized with monomer have acrylamide, acrylate and alkyl cyanoacrylate and their derivative.The step of preparing nanoscale capsule ball includes together with monomer adding deer antler extract containing emulsifying agent（Such as glucan, polysorbate）Acidic aqueous media in, add polymerization initiator（Typically anion, such as 0H -) trigger the polymerisation of monomer so that and deer antler extract is embedded in polymerization process into nanoscale capsule ball.Because monomer is mostly artificial synthesized, such many polymer can not be biodegradation, therefore the product of the present invention is preferably comprised with good biodegradable aliphatic category polyester polymer, is consequently formed nano level capsule ball.The example of this kind of polymer has PLA（PLA), PGA（PLG), poly (glycolide-co-lactide) copolymer（PLGA), polycaprolactone (PCL), polyamino acid and their derivative etc., other preferred examples are some natural polymerses and their derivative, optional natural polymerses have albumin, chitosan, glucan, alginate etc., they have good biodegradable polylactic acid, and nanocapsule ball parcel active component can be formed after disperseing.This kind of polymer can wrap up deer antler extract using rear dispersion method, and specific example has to be wrapped up using modes such as solvent evaporation or spontaneous emulsification/sovent diffusions.Solvent evaporated method refers to, and polymer and deer antler extract are dissolved in organic solvent, and organic solvent is added in the aqueous systems containing emulsifying agent and emulsified, and then organic solvent evaporation is removed by the mode such as heating, depressurizing or continuously stir, and is formed
W 200
The aqueous dispersion of polymer nanocomposite capsule ball.Spontaneous emulsification/solvent diffusion method refers to, with hydrophilic organic solvent and the mixed solvent dissolving polymer and deer antler extract of hydrophobic organic solvent formation, distribute it in water, because hydrophilic organic solvent can be diffused into aqueous phase from oil phase automatically, so as to form nanocapsule ball.In a detailed embodiment, the polymer used in preparation method of the invention is chitosan, i.e., wrap up deer antler extract using chitosan, forms the nanocapsule ball that particle diameter is 100 ~ 400 Falling.In another embodiment, the polymer used in preparation method of the invention is polyamino acid, i.e., wrap up deer antler extract using polyamino acid, forms particle diameter for 5 (Tl 50nm nanocapsule balls.Polyamino acid can be selected from poly-D-lysine and the polyaminoacid being made up of leucine and glutamic acid.
The present invention refer to open source literature, and these documents are that their entire contents are included to be referred to herein in order to more clearly describe the present invention, just look like that repeated description herein has been excessively for their full text.
In order to make it easy to understand, the present invention will be described in detail by specific embodiment below.It is important to note that the description that these descriptions are merely exemplary, and be not meant to limit the scope of the invention.According to the discussion of this specification, many changes of the invention, to change for one of ordinary skill in the art be all obviously.Embodiments of the invention embodiment 1, the extraction of effective constituent of pilose antler
The fresh 5kg of pilose antler 0. is taken, pilose antler is cut into the section that thickness is wide 2mm with freezing-microtome, then added appropriate（200mL) deionized water of precooling is crushed at 4 °C with even oar machine, and homogenate operation carries out into being homogenized not having to terminate during big tissue block.Then the deionized water of 1 L precoolings is added into the product of preliminary crushing, ultrasonic disruption is carried out after mixing under condition of ice bath.Then by the suspension after broken with 5000 revs/min（Rpm) centrifuge 20 minutes, stay supernatant stand-by.Sediment after centrifugation adds the deionized water of 1L precoolings, and concussion carries out ultrasonic disruption after hooking under condition of ice bath, is then centrifuged 20 minutes with 5000 revs/min again, stays supernatant stand-by.Sediment after centrifugation repeats above-mentioned steps, adds after water, ultrasonication, centrifugation, stays supernatant stand-by.Obtained in above step 3 portions of supernatants are mixed, 2 ultrafiltration are then carried out.Ultrafiltration first is with the milipore filter that molecular cut off is l OOOOODa（Purchased from Mi l l opore, Ul trapore), retain filter liquor.Then filter liquor is subjected to ultrafiltration with the milipore filter that molecular cut off is 1000Da (being purchased from Mi l l opore, Ul trapore), discards filter liquor.The material retained with the deionized water dissolution milipore filter of 200mL precoolings, retains dissolution fluid stand-by.Milipore filter Jing Guo first time dissolution step is used to the deionized water dissolution of 200mL precoolings again, milipore filter, the dissolution fluid twice obtained by merging is discarded.Dissolution fluid is freeze-dried -30 ~ -50 using freeze drier, dried 22 grams of crude product is obtained, the wherein water content of the crude product is no more than 8%.
Use IGF-1 ELISA detection kits（Purchased from R＆D Systems companies of the U.S., catalog number：DG100 the IGF-1 contents in the crude product that above extraction process is obtained are detected).10 mg crude products accurately are weighed, are dissolved in 1 ml deionized waters, are detected according to the kit operational manual of manufacturer, the content that IGF-1 in content, above-mentioned every gram of crude product is read from the detection curves of standard IGF- 1 is 2384 nanograms（ng).
Embodiment 2, the extraction of effective constituent of pilose antler
Extracted using step similar to Example 1.The step used place different from embodiment 1 is essentially consisted in：Use the pilose antler refrigerated in -20 °C；And carried out under condition of ice bath after ultrasonic disruption, static placement suspension 24 hours, to be fully dissolved out active component.Detailed process is as follows：Refrigerated after the collection of fresh pilose antler in -20 refrigerator 10 days, take the pilose antler 0. 5kg, pilose antler is cut into the section that thickness is l ~ 2mm with freezing-microtome, then add suitable quantity of water and crushed at 4 °C with refiner.Then the deionized water of 1 L precoolings is added to the product of preliminary crushing, carries out ultrasonic disruption after mixing under condition of ice bath.Then place the suspension after broken is static 24 hours, to be fully dissolved out active material.Then, by suspension with 5000 revs/min（Rpm) centrifuge 20 minutes, stay supernatant stand-by.Sediment after centrifugation adds the deionized water of 1L precoolings, is centrifuged 20 minutes with 5000 revs/min again after concussion is uniform, stays supernatant stand-by.Sediment after centrifugation repeats above-mentioned steps, adds after water, mixing, centrifugation, stays supernatant stand-by.Obtained in above step 3 portions of supernatants are mixed, 2 ultrafiltration are then carried out.Ultrafiltration first retains filter liquor with the milipore filter that molecular cut off is l OOOOODa.Then filter liquor is subjected to ultrafiltration with the milipore filter that molecular cut off is l OOODa, discards filter liquor.The material retained with the deionized water dissolution milipore filter of 200mL precoolings, retains dissolution fluid stand-by.Milipore filter Jing Guo first time dissolution step is used to the deionized water dissolution of 200raL precoolings again, milipore filter, the dissolution fluid twice obtained by merging is discarded.Dissolution fluid is freeze-dried at -30 ~ -50 °C using freeze drier, dried 24 grams of crude product is obtained, the wherein water content of the crude product is no more than 8%.
The IGF-1 contents in the crude product that above extraction process is obtained are detected with the ELISA detection kits of IGF- 1, the content for measuring IGF-1 in above-mentioned every gram of crude product is 1832 ng。
Embodiment 3, the extraction of effective constituent of pilose antler
The fresh 5kg of pilose antler 0. is cut into slices using step same as Example 1, ultrasonic disruption, then carry out 2 ultrafiltration.Ultrafiltration first retains filter liquor with the milipore filter that molecular cut off is 10000Da (being purchased from Mi l l opore, Ul trapore).Then the milipore filter for being lOOODa with molecular cut off by filter liquor carries out ultrafiltration, discards filter liquor.The material retained with the deionized water dissolution milipore filter of 200mL precoolings, retains dissolution fluid stand-by.Milipore filter Jing Guo first time dissolution step is used to the deionized water dissolution of 200mL precoolings again, milipore filter, the dissolution fluid twice obtained by merging is discarded.Dissolution fluid is freeze-dried at -30 ~ -50 °C using freeze drier, dried 10 grams of crude product is obtained, the wherein water content of the crude product is no more than 8%.
The IGF-1 contents in the crude product that above extraction process is obtained are detected with IGF-1 ELISA detection kits, the content for measuring IGF-1 in above-mentioned every gram of crude product is 4315ng.
Embodiment 4, nanosizing velvet product is prepared with chitosan
Take the chitosan that average relative molecular weight is 45000（Purchased from Zhejiang University）Crude product 5mg made from 120mg and above-described embodiment, dissolves them in the acetums of 12mL 1%.Then the chitosan-acetic acid solution is slowly dropped to the sorbester p37 solution that 150mL contains 20% ethanol（Purchased from Solution on Chemical Reagents in Shanghai company）In, pump moisture in 40 °C of decompressions.Then product will be obtained to centrifuge 20 minutes with 5000rpm, abandoning supernatant, precipitation uses petroleum ether 2 times.Then 20 milliliters of water are added to precipitation, with ultrasonication, produces the nanocapsule ball suspension of chitosan parcel.The nanocapsule ball suspension that above-mentioned chitosan is wrapped up is diluted with water 20 times, takes in right amount on copper mesh, carries out negative staining with 2% sodium phosphotungstate, then observed using transmission electron microscope, and the particle size of the nanocapsule ball of gained is 100 ~ 400 η π ι.Repeat process made above to prepare, but be wherein added without pilose antler crude product, blank nanocapsule ball suspension is thus made and is used as blank control.
The envelop rate of above nanosizing velvet product is detected with IGF-1 ELISA detection kits.Proper amount of nano capsule ball suspension is divided into 2 parts（The dosage of IGF-1 in every part of sample is calculated as W0), a copy of it detects IGF-1 free and that absorption is in nano grain surface amount with IGF-1 ELISA detection kits（W1 ) ;Another uses 0.5U/mL chitosan enzymes（Purchased from Japan Chemical Industry company）Enzymolysis 6 hours, then detects IGF-1 amount with IGF-1 ELISA detection kits again（W2 ).According to envelop rate=(W2-W1)/W0* 100% formula, the envelop rate of the said goods measured is 68%.
Embodiment 5, nanosizing velvet product is prepared with polyamino acid
Crude product 10mg made from above-described embodiment is dissolved in 12mL pH7.4 PBS (wherein containing 0.01mol/L phosphate, 0.138mol/L NaCl and 0.0027mol/L KC1).The poly- Leu/Glu-50/50 that average relative molecular weight is 23000 is taken (Chinese Academy Of Sciences Process Engineering Research Institute to be purchased from, wherein the glutamic acid containing 50% leucine and 50%）50mg, is dispersed in above-mentioned PBS solution, automatically forms the colloidal dispersion containing nanocapsule ball（The nanosizing velvet product of polyaminoacid parcel）.The liquid can scattered light show, and be placed in 15 °C after 3 hours and do not precipitate.The nanosizing velvet product of above-mentioned polyaminoacid parcel is taken, is observed using transmission electron microscope, the particle size of the nanocapsule ball of gained is 50 ~ 150nm.
The determinations of activity of IGF- 1 in embodiment 6, nanosizing velvet product
The nanocapsule ball suspension and blank control prepared with embodiment 4 stimulates 96 orifice plate cultures respectively
The propagation of Balb-3T3 cells.96 well culture plates are coated with Collagen type-I, dilute rearmounted stand-by on ice with DMEM.After 30 minutes, 50 L nanocapsule ball suspensions and blank control are separately added into plate hole, incubator inner equilibrium is put 60 minutes.50 L MTT are subsequently added into, are incubated 4 hours at 37 °C.Then power mouthful l OO w L DMSO, warm bath 1 hour.Due to living cells can change into tetrazolium salts can be by DMSO
The color products of dissolving, its yield is directly proportional to living cells quantity, therefore surveys absorbance under 570nm with ELIASA, you can estimation viable count.Relative to blank control, the proliferation rate of Balb-3T3 cells can be significantly improved using the nanosizing velvet product of the present invention.The resistance to enzymic degradation determinations of activity of IGF- 1 in embodiment 7, nanosizing velvet product
Nanocapsule ball suspension and blank control that nanocapsule ball suspension, the pancreatin prepared with embodiment 4 is treated irritate the propagation of the Balb-3T3 cells of 96 orifice plate cultures respectively.Wherein, handled 2 hours with pancreatin 37, be prepared into the treated nanocapsule ball suspension of pancreatin.The multiplication effect of cell is detected with experimental procedure same as Example 6, absorbance is determined.Thus IGF- 1 activity remains on 81 % activity relative to what is degraded without enzyme after the said goods measured are degraded through enzyme.
Claims (1)
- ClaimsA kind of nanosizing products being made up of deer antler extract of 1, it includes the nanocapsule ball that particle diameter is lCTlOOOnm, and IGF in the extract（IGF-1 content) is 100 ~ 10000ng/ grams.2. nanosizing product according to claim 1, the wherein particle diameter of nanocapsule ball are 2 (T800nm, more preferably 4 (T400nm;IGF-1 content is 1000 ~ 6000ng/ grams, preferably 1500 ~ 4500 ng/ grams in the extract.3. nanosizing product according to claim 1, the wherein particle diameter of nanocapsule ball are that 10 (T400nm contains chitosan preferably wherein.4. nanosizing product according to claim 1, the wherein particle diameter of nanocapsule ball are that 5 (Tl 50nm, contain polyamino acid preferably wherein.5. according to claim 1-4 any nanosizing product, it is mouth spraying agent, capsule or injection.6. application of any nanosizing product of claim 1-5 in the medicine, health food and/or the cosmetics that promote cell propagation are prepared.7. claim 1-5 any nanosizing product is preparing treatment and/or prevention carbohydrate metabolism regulation disorder（Particularly diabetes）Medicine and/or health food in application.8. the preparation method of claim 1-5 any nanosizing product, it is characterised in that comprise the following steps：A) pilose antler crushed with flooding, isolates the water-soluble extractive that molecular weight is 500 ~ 500000Da, freeze-drying；And b) with biodegradable polymer the extract obtained by step a) is rolled into nanocapsule ball.9. preparation method according to claim 8, wherein biodegradable polymer are chitosan.10. preparation method according to claim 8, wherein biodegradable polymer are polyamino acid.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2006/000841 WO2007124618A1 (en) | 2006-04-28 | 2006-04-28 | Insulin-like growth factor-enriched nano-products of hairy antler and process for preparation thereof |
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| CN101068556B CN101068556B (en) | 2010-11-10 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486749A (en) * | 2017-09-11 | 2019-03-19 | 广西索芙特集团有限公司 | Velvet deerhorn cell Serium-free Culture |
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| CN1408368A (en) * | 2001-09-27 | 2003-04-09 | 成都思摩纳米技术有限公司 | Method for preparing nano pilose antler suspension |
| CN1166369C (en) * | 2001-11-07 | 2004-09-15 | 焦柏忠 | Nanometer level pilose antler and pilose antler product and their prepn process |
| CN1241943C (en) * | 2004-08-04 | 2006-02-15 | 王利忠 | Method for extracting insulin-like growth factor (IGF-1) from fresh antler |
| CN100390199C (en) * | 2005-10-25 | 2008-05-28 | 天津大学 | Method for extracting insulin type growth factor-1 from fresh pilose antler |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109486749A (en) * | 2017-09-11 | 2019-03-19 | 广西索芙特集团有限公司 | Velvet deerhorn cell Serium-free Culture |
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| CN101068556B (en) | 2010-11-10 |
| WO2007124618A1 (en) | 2007-11-08 |
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