WO2007124618A1

WO2007124618A1 - Insulin-like growth factor-enriched nano-products of hairy antler and process for preparation thereof

Info

Publication number: WO2007124618A1
Application number: PCT/CN2006/000841
Authority: WO
Inventors: Lizhong Wang
Original assignee: Lizhong Wang
Priority date: 2006-04-28
Filing date: 2006-04-28
Publication date: 2007-11-08
Also published as: CN101068556B; CN101068556A

Abstract

Nano-products prepared from insulin-like growth factor (IGF-1)-enriched extracts of hairy antler. IGF-1 is present in said extracts in amount of 100 to 10,000 nanogram per gram. The nanocapsule or nanosphere in said products is in size of 10 to 1,000 nanometer. Additionally, the process for preparation of the nano-products of hairy antler, use of the products in medicaments, food, health food and cosmetics etc. are provided.

Description

TECHNICAL FIELD The present invention relates to the field of medical or edible products of velvet antler extract. More specifically, the present invention relates to nanosized articles made from velvet extract rich in insulin-like growth factor (IGF-1). In addition, the present invention also relates to a method of preparing the antler nano-product, and the use of the article in medicines, foods, health care products, cosmetics, and the like. BACKGROUND OF THE INVENTION Velvet antler (the horns of the deer have not been ossified) and antlers are traditional Chinese medicinal materials, and have the functions of strengthening the muscles and strengthening the bones, invigorating the kidney and strengthening the yang, and dredging the veins. Since ancient times, they have been widely used in the preparation of various nourishing medicines and foods. , health products and cosmetics. Through modern medicinal chemistry research, antler has many physiologically active ingredients, including phospholipids, biogenic amines, prostaglandins, vitamins, amino acids, inorganic salts, unsaturated fatty acids, cortisol and the like. With the rapid development of modern biotechnology, it has been found that in addition to the above various physiologically active ingredients, antler contains insulin-like growth factor (IGF-1), insulin, human growth hormone (HGH), and growth-promoting factor ( Macromolecular active proteins such as GHRF-6), nerve growth factor (NGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). The above various active ingredients, especially various active proteins, act synergistically to exert their effects. Among them, insulin-like growth factor plays a prominent role. IGF-1 has a function of promoting cell division and can be used for the treatment and prevention of diseases such as disorders such as carbohydrate metabolism regulation, such as treatment and prevention of diseases such as glucose metabolism or fat metabolism, such as diabetes. Chinese patent application CN1251531A discloses a method for inducing or enhancing tissue growth, proliferation or regeneration by using IGF-1, in particular, promoting growth of inner ear tissue by using IGF-1; Chinese patent application CN1204263A discloses that brain or spinal cord can be treated with IGF-1 Neurodegenerative diseases, such as Alzheimer's Disease; Parkinson's Disease. In the treatment of diabetes, a large number of literatures have also disclosed the practice of using IGF-1 to treat type I, type II and insulin resistance diabetes (see Chinese patent application CN1387440A, CN1384201A, etc.).

In the traditional processing of antler, the antler is directly pulverized and taken, and the granules are large, resulting in low absorption efficiency. Sometimes, the processing of antler needs to be subjected to heating, boiling and drying, and the high temperature will denature proteins such as IGF-1. , thereby reducing or even losing their efficacy. Another traditional method of processing is to immerse the velvet in a high concentration of alcohol and make it into a medicinal wine. But because of the wine The high concentration of alcohol as an organic solvent denatures proteins such as iGF-Ι, and it is not conducive to the dissolution of proteins such as IGF-1 from large-particle velvet antlers, which may result in the destruction of proteins such as IGF-1. The use is inefficient and wastes resources. In addition, high-alcohol content of medicinal liquor also limits the amount of each dose.

In order to improve the efficiency of the use of velvet antler materials, various methods have been tried to extract the effective components of velvet antler. For example, International Patent Application No. WO 2004/035069 A discloses an antler article for preventing and treating diabetes mellitus and a method for preparing the same, but contains only 18 ng of IGF-1 and 0.5 ng of insulin per gram of the product. Chinese patent CN1241943C discloses a method for extracting a antler product, but the extracted substance does not suggest that it can be modified by nano-encapsulation, and the oral absorption barrier and the proteolytic enzyme degradation of the digestive system are faced orally. The problem is that the actual absorption efficiency of active ingredients such as IGF-1 is low.

Various methods have also been tried to extract the active ingredients of velvet antler, and to prepare various products which are advantageous for absorption, especially to prepare nano-sized products, thereby improving the absorption efficiency of antler materials. For example, Chinese Patent Application No. CN1723992A discloses a product prepared by a specific method containing components such as nano-sized antler powder and walnut kernel powder as a health care product for anti-fatigue and immunity enhancement. However, in the preparation process, the velvet antler needs to be extracted with an ethanol solvent. Since alcohol is an organic solvent, the extraction efficiency of water-soluble proteins such as IGF-1 is low, and the proteins are easily denatured and the IGF-1 is destroyed. The efficacy of such proteins, resulting in low activity of proteins such as IGF-1 in the finished product, affects the use effect. Chinese patent CN1166369C discloses a colloidal liquid or suspension of velvet antler or antler with a particle size of 10 to 1000 nm using water, vegetable oil and/or animal oil, but nanometering all pulverized materials of velvet antler during preparation. The treatment does not remove a large amount of keratinous components which cannot be absorbed by the human body, and the use of vegetable oil or animal oil will not sufficiently extract water-soluble proteins such as IGF-1, so that the actual content of IGF-1 in the preparation is low, and the nanometerized The particles are not effective in preventing degradation of the proteolytic enzymes of the digestive system.

In view of the shortcomings in the prior art, after long and arduous research, the inventors have obtained a new antler extract rich in insulin-like growth factor (IGF-1) and its nano-products, which overcomes the above-mentioned defects. . The product of the invention not only has the content of IGF-1 but also maintains high activity, fully utilizes the resources of antler, and the nano-formed product can bring about a certain penetration-promoting effect, and helps to overcome the absorption barrier and enzyme barrier of the gastrointestinal tract. The problem. In addition, the preparation process of the product is reasonable, and the quality is controlled in large-scale production, and the preparation process uses natural raw materials, can be biodegraded, avoids contamination of synthetic products, and is more easily accepted by consumers. Summary of the invention The present invention provides a nanosized article made from antler extract and a preparation method thereof, the extract being rich in insulin-like growth factor. The invention also provides the use of the nanosized article in the preparation of pharmaceuticals, foods, health care products and cosmetics.

In a first aspect, the present invention provides a nanosized article made from velvet antler extract comprising nanocapsules having a particle size of 10 1000 nanometers (nm), and insulin-like growth factor (IGF) in said extract The content of -1) is from 100 to 10,000 ng/g. The extract of the product of the invention has an IGF-1 content of 100~10000 ng/g extract, which is much higher than the IGF-1 content of other known velvet antler extracts, preferably the IGF-1 content is 1000~ 6000 ng / gram, more preferably 1500 ~ 4500 ng / gram. Further, the particle diameter of the nanocapsules in the article of the article of the present invention is preferably 1 (Tl000 nm, more preferably 20 to 800 nm, still more preferably 40 to 400).

The article can be obtained by the following two steps: First, an extract rich in IGF-1 is extracted from a velvet antler with a solvent; and the above extract is then prepared into a nanosized article. The present invention preferably uses fresh antler or chilled fresh antler as a raw material, which is beneficial for maximally preserving the content and activity of active ingredients such as IGF-1. The term "fresh" as used herein refers to the preparation of the nanostructured article of the present invention within 6 hours after the collection of velvet antler or for refrigerating, preferably processed or refrigerated no later than 3 hours after collection, more preferably It is later than 1 hour, most preferably no later than 20 minutes. As used herein, the term "refrigerated" refers to the preservation of velvet antlers in an environment below 0 ° C, preferably in an environment of -20 ° C.

In the present invention, water is preferably used as a solvent to extract an extract having an IGF-1 content of 100 to 10000 ng/g, preferably an IGF-1 content of 100 (T6000 ng/g, more preferably 150 (T4500 ng/g.) The production of nano-sized particles or capsules can be carried out by supersonic jet method or polymer encapsulation method. Preferably, the article of the invention is a nano-sized capsule ball, and the polymer-packed antler extract is used to form nano-sized capsules. Suitable polymers are polymers synthesized from monomers and aliphatic polyester polymers. Examples of monomers of polymers synthesized from monomers are acrylamide, acrylate and alkyl cyanoacrylate and their derivatives. Since many of the polymers are not biodegradable, the articles of the present invention preferably comprise an aliphatic biopolymer having good biodegradability, thereby forming nanoscale capsules. Examples of such polymers are polylactic acid (PLA:), polyglycolide (PLG), polyglycolide lactide copolymer (PLGA), polycaprolactone (PCL), polyamino acids and their derivatives. Other preferred examples are natural high molecular polymers and derivatives thereof, and optional natural high molecular polymers include albumin, chitosan, dextran, alginate, and the like. They have good biodegradable properties and, upon dispersion, form nano-pillar-coated active ingredients.

The nanosized article of the present invention particularly preferably contains nanocapsules having a particle size of 10 (T400 _n m. In a specific embodiment, the nanosized article of the present invention contains chitosan, which is coated with chitosan The velvet extract forms a nanocapsule sphere having a particle diameter of 100 to 400 nm. The chitosan-coated antler extract has a certain osmotic effect on the peptides, and because chitosan has the same mucoadhesive properties, it can also inhibit certain protease degradation and prevent active ingredients such as IGF-1. It is degraded before absorption. In addition, chitosan conjugates and protease inhibitors, such as metalloproteinase inhibitors such as EDTA, can also reduce the degradation of proteases. Accordingly, the chitosan-containing nanosized articles of the present invention are particularly suitable for further processing into oral preparations such as mouth sprays, tablets, capsules and the like.

The nanosized article of the present invention preferably further comprises a nanocapsule sphere having a particle size of 5 (Tl 50 _n m. In a specific embodiment, the nanosized article of the present invention contains a polyamino acid, and the polyanthotic acid is used to wrap the antler extract The nanocapsules having a particle diameter of 50 to 150 nm are formed. The polyamino acid may be selected from the group consisting of polylysine and a polyamino acid composed of leucine and glutamic acid (see International Patent Application No. 6/029991 A:).

The nanosized article of the present invention may further comprise a pharmaceutically acceptable adjuvant in addition to the above-mentioned antler extract and polymer encapsulating material, thereby processing various dosage forms. Pharmaceutically acceptable adjuvants include pharmaceutically acceptable carriers, excipients, diluents and the like which are compatible with the active ingredient. The preparation of formulations using pharmaceutically acceptable adjuvants is well known to those of ordinary skill in the art. The formulations of the present invention comprise one or more of the nanosized articles of the first aspect of the invention as an active ingredient, the nanosized article and a pharmaceutically acceptable adjuvant (such as a carrier well known to those of ordinary skill in the art) The excipients, diluents, and the like are combined and formulated into various preparations, preferably solid preparations and liquid preparations. Preferably, the preparation of the present invention is in unit dosage form, such as tablets, pills, capsules (including sustained release or delayed release forms), powders, suspensions, granules, elixirs, syrups, emulsions, suspensions, injections, and the like. Thus, it is suitable for administration forms such as oral administration, parenteral injection, mucosa, muscle, intravenous, subcutaneous, intraocular, intradermal or transdermal. The nanosized article of the present invention is particularly preferably formulated as a mouth spray, a capsule or an injection. Wherein the carrier, excipient, diluent are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients which may be selected are preferably, but not limited to, water, physiological saline, dextrose, glycerol, ethanol or the like and combinations thereof.

In a second aspect, the present invention provides the use of the nanosized article of the first aspect of the invention for the manufacture of a medicament, health food and/or cosmetic for promoting cell proliferation. The nano-sized product of the invention is made of antler extract rich in IGF-1, has the function of promoting cell division and proliferation, and can be used for inducing or improving the level of tissue growth, proliferation or regeneration of a patient, and treating a corresponding disease, such as a brain. Or degenerative diseases of the spinal nerves, such as Alzheimer's disease, Parkinson's disease, etc. Because it is a natural product, and the antler itself has a long history of use, a healthy person taking a certain amount of the nano-sized product of the present invention helps maintain its normal cell division and proliferation, and helps to maintain its vigorous vitality. A healthy person uses a certain amount of the nano-sized product of the invention to help maintain the division and proliferation of skin cells. N2006/000841 Improves the external image and has a cosmetic effect.

In a third aspect, the present invention provides the use of the nanosized article of the first aspect of the invention for the manufacture of a medicament and/or health food for treating and/or preventing a disorder of carbohydrate metabolism regulation. The nanosized preparation of the invention is made of IGF-1-rich antler extract and can be used for treating and/or preventing diseases or sub-health states such as disorders of energy metabolism regulation such as carbohydrates, such as treating and preventing glucose metabolism or Diseases such as fat metabolism, especially diabetes, include type I, type II, and insulin-resistant diabetes.

For the pharmaceuticals of the second and third aspects of the invention, the nanosized preparations of the invention can be processed into a variety of pharmaceutical dosage forms for administration to a patient in need of promoting cell proliferation or in need of treatment and/or prevention of disorders of carbohydrate metabolism regulation. The dosage and form of administration will generally be determined by the physician based on the particular circumstances of the patient (e.g., age, weight, sex, condition, time of illness, physical condition, etc.). In general, the dose is 0.001 to 100 mg/kg of the patient's body weight, preferably 0.01 to 1 mg/kg, preferably 0.02 to 0.1 mg/kg, based on IGF-1 in the nanosized preparation. The administration form can be determined according to the dosage form of each pharmaceutical preparation, and the suitable administration forms are oral, parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular, intradermal or transdermal. Oral administration is carried out using an oral or capsule.

In the application of the second and third aspects of the invention, the nanosized article of the invention may be used alone as a medicine, a health food and/or a cosmetic, or may be added to other medicines, health foods and/or cosmetics. For example, the nanosized articles of the present invention can be added to conventional beverages, foods or cosmetics for use in conjunction.

In a fourth aspect, the invention provides a method of making a nanosized article of the first aspect of the invention. The preparation method may comprise the following two steps: first extracting an IGF-1 rich extract from velvet antler; and then preparing the above extract into a nanosized product. The preparation method preferably comprises the following steps:

a) pulverizing the velvet antler with water, separating _a water-soluble extract having _a molecular weight of 50 (T500000D _a , freeze-dried;

And b) wrapping the extract obtained in step a) into a nanocapsule ball with a biodegradable polymer. The step of crushing the antler in the step a) may include one or more of slicing, pulping, ultrasonication and the like. "Slicing" refers to the velvet velvet being sliced, preferably using a microtome to cut fresh velvet antler or refrigerated fresh antler into a sheet having a thickness of 1 to 5 mm. "Homogeneous" refers to the smashing of velvet antlers until there is no large tissue block. An appropriate amount of deionized water can be added during the homogenization process. After ultrasonication, the cells can be fully broken, and an appropriate amount of deionized water can be added during the crushing process. The step of pulverizing the velvet antler preferably includes the steps of slicing, grouting and sonication in order, and the slicing step therein may be omitted. The whole process of crushing velvet antler is best to operate in a low temperature environment. The reagents used are preferably low temperature precooled. The low temperature environment refers to an environment of 0 to 20 ° C, preferably 0 to 10 ° C. Fortunately at 4° C. The step of extracting the pulverized velvet antler with water in step a) may be carried out simultaneously with the step of pulverizing the velvet antler, that is, extracting the active ingredient of the velvet antler by water added in the steps of slicing, homogenizing and/or ultrasonication, such as IGF- 1, the leaching is completed naturally with the completion of the pulverization step; it can also be further immersed for a period of time after the step of pulverizing the velvet antler, if the time is not more than 36 hours, preferably placed for 24 hours, preferably placed at In a low temperature environment. The step of separating the water-soluble extract in step a) can be carried out by conventional centrifugation and/or filtration, such as ultracentrifugation and/or ultrafiltration. In this step, it is preferred to extract a component having a molecular weight of 50 (T500000Da, more preferably an extracting molecular weight of 100 (Tl 00000Da, most preferably a component having an extracted molecular weight of 1000 to 10000 Da. In one embodiment, the present invention first removes water by centrifugation) The precipitate in the product is extracted, and then a suitable ultrafiltration membrane is used to intercept the component of the corresponding molecular weight, and then dissolved in an appropriate amount of water, and then freeze-dried, such as using a freeze dryer to dissolve the component of the corresponding molecular weight obtained in the previous step. The aqueous solution is dried at a dryer operating temperature of _30 ° C ~ -50 ° C. The moisture content of the product after drying is 3% -10 ° / oo

In step b), the final product obtained in step a) is nano-sized, which can be prepared by supersonic jet method or polymer encapsulation method to prepare nano-sized particles or capsules. "Supersonic jet method" refers to the use of supersonic jet equipment (such as commercially available from Beijing Nano Biotechnology Co., Ltd.) to form a high-speed jet of velvet antler extract, and to allow multiple jets to meet each other at high speed in the crushing zone. Rapid collision and grinding to form nanoscale particles. Preferably, the present invention forms a nano-sized capsule by means of a polymer-packed antler extract. Suitable polymers thereof are polymers synthesized from monomers and aliphatic polyester polymers. Examples of the monomer of the polymer synthesized by the monomer are acrylamide, acrylate and alkyl cyanoacrylate and derivatives thereof. The step of preparing the nano-sized capsules comprises adding the velvet antler extract together with the monomer to an acidic aqueous medium containing an emulsifier (such as dextran, polysorbate, etc.), and adding a polymerization initiator (usually an anion such as 0H). The polymerization of the monomer is initiated to cause the antler extract to be embedded in the nano-sized capsule during the polymerization. Since many of the monomers are synthetic, such polymers are not biodegradable, and thus the articles of the present invention preferably comprise an aliphatic biopolymer having good biodegradability, thereby forming nanoscale capsules. Examples of such polymers are polylactic acid (PLA), polyglycolide (PLG), polyglycolide lactide copolymer (PLGA), polycaprolactone (PCL), polyamino acids and derivatives thereof. Etc. Other preferred examples are some natural high molecular polymers and their derivatives. The optional natural high molecular polymers are albumin, chitosan, dextran, alginate, etc., which have good biota. The polylactic acid is degraded and dispersed to form a nanocapsule ball encapsulating active ingredient. Such polymers may be coated with antler extract by post-dispersion method, and specific examples are encapsulation by solvent evaporation or spontaneous emulsification/solvent diffusion. The solvent evaporation method refers to dissolving the polymer and the velvet antler extract in an organic solvent, adding the organic solvent to an aqueous system containing an emulsifier for emulsification, and then evaporating the organic solvent by heating, decompression or continuous stirring. Forming W 200

A water dispersion of polymer nanocapsules. The spontaneous emulsification/solvent diffusion method refers to dissolving a polymer and a velvet antler extract in a mixed solvent formed of a hydrophilic organic solvent and a hydrophobic organic solvent, and dispersing it into water, since the hydrophilic organic solvent is automatically diffused from the oil phase to The aqueous phase forms a nanocapsule. In a specific embodiment, the polymer used in the preparation method of the present invention is chitosan, that is, a chitin-coated antler extract is used to form a nanocapsule having a particle diameter of 100 to 400 Å. In another embodiment, the polymer used in the preparation method of the present invention is a polyamino acid, that is, a poly-amino acid-packed antler extract is used to form a nanocapsule having a particle size of 5 (Tl 50 nm. Polyamino acid can be It is selected from the group consisting of polylysine and a polyamino acid consisting of leucine and glutamic acid.

The present invention is hereby incorporated by reference in its entirety in its entirety in its entirety in its entirety in its entirety in the extent of the disclosure of the disclosure of the disclosure of the entire disclosure.

For ease of understanding, the present invention will be described in detail below by way of specific examples. It is to be understood that the description is not to be construed as limiting Many variations and modifications of the invention will be apparent to those skilled in the <RTIgt; EXAMPLES OF THE INVENTION Example 1, Extraction of Active Components of Antler

Take fresh velvet antler 0. 5kg, cut the velvet antler into slices with a thickness of 2mm by using a cryostat, then add appropriate amount (200mL) of pre-cooled deionized water to pulverize with a paddle machine at 4 °C, and homogenize the operation until It ends when there is no large tissue block in the homogenate. Then, 1 L of pre-cooled deionized water was added to the initially pulverized product, and after mixing, ultrasonication was carried out under ice bath conditions. The disrupted suspension was then centrifuged at 5000 rpm for 20 minutes, leaving the supernatant ready for use. The precipitate after centrifugation was added with 1 L of pre-cooled deionized water, shaken and then ultrasonically disrupted under ice bath conditions, and then centrifuged at 5000 rpm for 20 minutes, leaving the supernatant for use. The precipitate after centrifugation is repeated for the above steps, and after adding water, ultrasonic treatment, and centrifugation, the supernatant is left for use. The three supernatants obtained in the above procedure were mixed together, followed by two ultrafiltrations. For the first ultrafiltration, an ultrafiltration membrane (purchased from Mi ll opore, Ul trapore) with a molecular weight cut off of 10,000 OODa was used, and the filtrate was retained. The filtrate was then ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 1000 Da (purchased from Mi ll opore, Ul trapore) and the filtrate was discarded. The material trapped by the ultrafiltration membrane was dissolved in 200 mL of pre-cooled deionized water, and the eluate was retained for use. The ultrafiltration membrane subjected to the first dissolution step was further dissolved in 200 mL of pre-cooled deionized water, the ultrafiltration membrane was discarded, and the resulting two dissolution solutions were combined. The eluate was freeze-dried at -30 to -50 Torr using a freeze dryer to obtain 22 g of the dried crude product, wherein the crude product had a water content of not more than 8%. The IGF-1 ELISA test kit (purchased from R&D Systems, USA, catalog number: DG100) was used to detect the IGF-1 content in the crude product obtained by the above extraction process. Accurately weigh 10 mg of crude product, dissolved in 1 ml of deionized water, according to the manufacturer's kit operating instructions, read the content from the standard IGF-1 detection curve, the above IGF-1 content per gram of crude product is 2384 Nuck (ng).

Example 2, extraction of active ingredients of pilose antler

Extraction was carried out using a procedure similar to that of Example 1. The steps used differed from Example 1 mainly in that: velvet antlers refrigerated at - 20 ° C were used; and after ultrasonication in an ice bath condition, the suspension was allowed to stand still for 24 hours to sufficiently dissolve the active ingredient. The specific process is as follows: Fresh velvet antler is collected and refrigerated in -20 Ό refrigerator for 10 days, take the velvet velvet 0. 5kg, use a cryostat to cut the velvet antler into slices of thickness 1-2mm, then add appropriate amount of water at 4 °C The pulper is pulverized. Then, 1 L of pre-cooled deionized water was added to the initially pulverized product, and the mixture was homogenized and ultrasonically broken under ice bath conditions. The disrupted suspension was then left to stand for 24 hours to fully dissolve the active material. Next, the suspension was centrifuged at 5000 rpm for 20 minutes, and the supernatant was left for use. The precipitate after centrifugation was added with 1 L of pre-cooled deionized water, shaken evenly, and then centrifuged at 5000 rpm for 20 minutes, leaving the supernatant for use. Repeat the above steps after centrifuging the precipitate, add water, mix, centrifuge, and leave the supernatant for use. The three supernatants obtained in the above procedure were mixed together, followed by two ultrafiltrations. For the first time, ultrafiltration was carried out with an ultrafiltration membrane with a molecular weight cut off of l OOOOODa, and the filtrate was retained. The filtrate was then ultrafiltered through an ultrafiltration membrane with a molecular weight cut off of 0.01 Da, and the filtrate was discarded. The material trapped by the ultrafiltration membrane was dissolved in 200 mL of pre-cooled deionized water, and the eluate was retained for use. The ultrafiltration membrane subjected to the first dissolution step was further dissolved in 200 laL of pre-cooled deionized water, the ultrafiltration membrane was discarded, and the resulting two dissolution solutions were combined. The eluate was freeze-dried at - 30 to -50 °C using a freeze dryer to obtain 24 g of the dried crude product, wherein the crude product had a water content of not more than 8%.

With IGF- 1 ELISA assay kit to detect the IGF-1 content in the crude product obtained above in the extraction process, measured above the crude product per gram of IGF-1 content was 1832 n _g.

Example 3, extraction of active ingredients of pilose antler

5公斤 slices, ultrasonically disrupted, and then subjected to 2 ultrafiltrations using the same procedure as in Example 1. For the first ultrafiltration, an ultrafiltration membrane with a molecular weight cut-off of 10,000 Da (purchased from Mi ll opore, Ul trapore) was used to retain the filtrate. The filtrate was then ultrafiltered through an ultrafiltration membrane having a molecular weight cut off of 100 ODA, and the filtrate was discarded. The material trapped by the ultrafiltration membrane was dissolved in 200 mL of pre-cooled deionized water, and the eluate was retained for use. The ultrafiltration membrane subjected to the first dissolution step was further dissolved in 200 mL of pre-cooled deionized water, the ultrafiltration membrane was discarded, and the resulting two dissolution solutions were combined. The eluate was freeze-dried at - 30 to -50 ° C using a freeze dryer to obtain 10 g of the dried crude product, wherein the crude product had a water content of not more than 8%. The IGF-1 ELISA kit was used to detect the IGF-1 content in the crude product obtained by the above extraction process, and the content of IGF-1 per gram of the crude product was determined to be 4,315 ng.

Example 4, Preparation of Nanosized Deer Velvet Products Using Chitosan

120 mg of chitosan (purchased from Zhejiang University) having an average relative molecular weight of 45,000 and 5 mg of the crude product obtained in the above-mentioned examples were taken, and they were dissolved in 12 mL of a 1% acetic acid solution. Then, the chitosan acetic acid solution was slowly added dropwise to 150 mL of a 20% ethanol-containing Span 85 solution (purchased from Shanghai Chemical Reagent Co., Ltd.), and the water was removed under reduced pressure at 40 °C. The obtained product was then centrifuged at 5000 rpm for 20 minutes, the supernatant was discarded, and the precipitate was washed twice with petroleum ether. Then, 20 ml of water was added to the precipitate, and ultrasonic treatment was performed to obtain a chitosan-coated nanocapsule ball suspension. The above-mentioned chitosan-coated nanocapsule suspension was diluted 20 times with water, and the amount was applied to a copper mesh, negatively stained with 2% sodium phosphotungstate, and then observed by a transmission electron microscope to obtain a particle size of the nanocapsule sphere. The size is 100~400ηπι. The above preparation was repeated, but no velvet crude product was added, and a blank nanocapsule suspension was prepared as a blank control.

The encapsulation efficiency of the above nanosized velvet antler products was detected by an IGF-1 ELISA test kit. The appropriate amount of nanocapsule suspension was divided into 2 parts (the dose of IGF-1 in each sample was W0), and one of them was used to detect free and adsorbed IGF on the surface of nanoparticles by IGF-1 ELISA test kit. The amount of -1 (W1); the other was digested with 0.5 U/mL chitosanase (purchased from Nippon Chemical Industry Co., Ltd.) for 6 hours, and then the amount of IGF-1 was detected by the IGF-1 ELISA test kit ( W2). According to the formula of the encapsulation ratio = (W2-W1) / W0* 100%, the encapsulation efficiency of the above products was 68%.

Example 5: Preparation of nanosized antler products using polyamino acids

10 mg of the crude product obtained in the above examples was dissolved in 12 mL of PBS pH 7.4 containing 0.01 mol/L of phosphate, 0.138 mol/L of NaCl and 0.0027 mol/L of KC1. Take PolyLeu/Glu-50/50 (purchased from Institute of Process Engineering, Chinese Academy of Sciences, containing 50% leucine and 50% glutamic acid) with an average molecular weight of 23,000, and disperse it in the above PBS solution. In the middle, a colloidal dispersion containing nanocapsules (a nano-sized deer antler product coated with polyamino acid) is automatically formed. The liquid was able to scatter light and did not precipitate after 3 hours at 15 °C. The nano-sized antler products coated with the above polyamino acid were observed by a transmission electron microscope, and the obtained nanocapsules had a particle size of 50 to 150 nm.

Example 6. Determination of IGF-1 activity in nanosized velvet antler products

The nanocapsule ball suspension prepared in Example 4 and the blank control were respectively stimulated in 96-well plate culture.

Proliferation of Balb-3T3 cells. The 96-well culture plate was coated with rat tail collagen, diluted with DMEM, and placed on ice for use. After 30 minutes, 50 L of nanocapsule suspension and blank control were added to the wells and equilibrated for 60 minutes in the incubator. Then 50 L MTT was added and incubated for 4 hours at 37 °C. Then, the pressure was 10000 w L DMSO and the bath was warmed for 1 hour. Because living cells can convert tetrazolium salts into DMSO The dissolved colored product, whose yield is proportional to the number of living cells, can be estimated by measuring the absorbance at 570 nm with a microplate reader. The use of the nanosized antler products of the present invention can significantly increase the proliferation rate of Balb-3T3 cells relative to the blank control. Example 7, Determination of Anti-enzymatic Degradation Activity of IGF-1 in Nanosized Antler Products

The proliferation of Balb-3T3 cells cultured in 96-well plates was stimulated by the nanocapsule ball suspension prepared in Example 4, the trypsin-treated nanocapsule ball suspension, and the blank control, respectively. Among them, pancreatin-treated nanocapsule suspension was prepared by treating with trypsin for 37 hours. The proliferation effect of the cells was measured by the same experimental procedure as in Example 6, and the absorbance was measured. The activity of IGF-1 detected by the above-mentioned products after enzyme degradation was still 81% active relative to that which was not degraded by the enzyme.

Claims

Claim

What is claimed is: 1. A nanosized article made of antler extract comprising a nanocapsule having a particle size of lCT100Onm, and wherein the extract has an insulin-like growth factor (IGF-1) content of 100 to 10000 ng/g.

2. The nanosized article according to claim 1, wherein the nanocapsules have a particle size of 2 (T800 nm, more preferably 4 (T400 nm _; the content of IGF-1 in the extract is 1000 to 6000 ng/g, preferably 1500). ~4500 ng / gram.

The nanosized article according to claim 1, wherein the nanocapsules have a particle diameter of 10 (T400 nm, preferably containing chitosan therein).

The nanosized article according to claim 1, wherein the nanocapsules have a particle diameter of 5 (Tl 50 nm, preferably containing a polyamino acid therein.

5. A nanosized article according to any of claims 1-4 which is a mouth spray, capsule or injection.

6. Use of a nanosized article according to any of claims 1 - 5 for the preparation of a medicament, a health food and/or a cosmetic which promotes cell proliferation.

7. Use of a nanosized article according to any of claims 1-5 for the manufacture of a medicament and/or health food for the treatment and/or prevention of disorders of carbohydrate metabolism regulation, in particular diabetes.

8. A method of making a nanosized article according to any of claims 1-5, characterized by comprising the steps of:

a) pulverizing the velvet antler with water, separating the water-soluble extract with a molecular weight of 500-500,000 Da, and lyophilizing;

And b) wrapping the extract obtained in step a) into a nanocapsule ball with a biodegradable polymer.

9. The production method according to claim 8, wherein the biodegradable polymer is chitosan.

10. The production method according to claim 8, wherein the biodegradable polymer is a polyamino acid.