CN111568936A

CN111568936A - Ginseng and antler energy peptide with function of delaying senescence, microcapsule, preparation method and application thereof

Info

Publication number: CN111568936A
Application number: CN202010377666.4A
Authority: CN
Inventors: 李娜; 胡少丹; 王继凤; 李辉
Original assignee: Changchun University of Chinese Medicine
Current assignee: Changchun University of Chinese Medicine
Priority date: 2020-05-07
Filing date: 2020-05-07
Publication date: 2020-08-25
Also published as: AU2020104149A4

Abstract

The invention provides ginseng and antler energy peptide with an anti-aging effect, a microcapsule, and a preparation method and application thereof, and belongs to the technical field of health care products. Ginseng antler energy peptide with the function of delaying senility, which comprises pilose antler polypeptide and ginseng total saponin; the mass ratio of the pilose antler polypeptide to the ginseng total saponin is (3-4): (6-7). The ginseng antler energy peptide microcapsule with the function of delaying senescence, wherein the mass ratio of a core material to a wall material is 1: (6-10), wherein the ginseng antler energy peptide is used as a core material. The invention also provides application of the ginseng antler energy peptide or the ginseng antler energy peptide microcapsule in preparing health-care products for delaying senescence. The invention establishes a mouse subacute aging model by using D-galactose, and observes the influence of the ginseng antler energy peptide, the microcapsules and the dropping pills thereof on the learning and memory abilities of aging mice and a bone biomechanics effect experiment, thereby proving that the ginseng antler energy peptide and the derivative products thereof have the curative effect of delaying aging.

Description

Ginseng and antler energy peptide with function of delaying senescence, microcapsule, preparation method and application thereof

Technical Field

The invention belongs to the technical field of health care products, and particularly relates to ginseng and antler energy peptide with an anti-aging effect, a microcapsule, and a preparation method and application thereof.

Background

Aging is an irresistible natural law, and is a critical stage of growth and development of each individual, which is often manifested as a degenerative change in structure and a decline in function. With the great increase of the life expectancy of human beings, the incidence of various aging-related diseases such as neurodegenerative diseases, cardiovascular and cerebrovascular diseases, diabetes, cancer and the like is increased year by year, so that the burden of a medical system and the social economy are increasingly heavy. Delaying aging has become a serious disease affecting the world and is highly concerned by all relevant researchers around the world.

Aging can be divided into two categories: the first is physiological aging, which refers to the physiological degradation process of the body after the maturation period and mainly reflects the degenerative change of the functions of various organs and tissues along with the age; secondly, the process of aging is accelerated by pathological aging, namely, the aging is accelerated by some diseases or the influence of external factors. The aging process is irreversible, but some drugs can delay and prevent aging. In view of the limitations of current pharmaceutical and surgical therapies, more and more researchers are looking at finding effective anti-aging drugs to reduce the risk of related diseases and prolong the health life of people, wherein some researchers focus on traditional Chinese medicine which has been used for thousands of years in clinical application history, and have made many advances. By referring to the literature, the anti-aging drugs are classified and summarized, and the anti-aging drugs can be roughly classified into traditional Chinese medicines (such as angelica sinensis, ginseng, royal jelly and the like), small molecules (such as metformin, resveratrol, procyanidine and rapamycin) and polypeptides (such as rice polypeptide, soybean polypeptide, artificially synthesized polypeptide and the like).

The compatibility and application history of ginseng and pilose antler is long, and 116 prescriptions containing ginseng and pilose antler are recorded in the prescription of Puji. Shenrong granules are collected in the national Chinese patent medicine standard compilation. Has effects of invigorating heart-qi, benefiting heart and kidney, and can be used for treating asthenia, palpitation, short breath, soreness of waist and knees, sexual impotence, and spermatorrhea. Because the ginseng and antler product preparation is safe and nontoxic, the ginseng and antler product development is concerned by people. However, because the ginseng pilose antler contains various active pharmaceutical ingredients, the compounding of the ginseng pilose antler active ingredients which play a role in resisting aging has not been reported all the time.

Disclosure of Invention

In view of the above, the present invention aims to provide a ginseng antler energy peptide (SN) with anti-aging effect, which has excellent anti-aging activity.

The invention also aims to provide the ginseng antler energy peptide microcapsule with the function of delaying senility, and the preparation method and the application thereof, which can effectively improve the stability of the medicinal ingredients.

The invention provides ginseng antler energy peptide with the function of delaying senility, which comprises antler polypeptide and ginseng total saponin; the mass ratio of the pilose antler polypeptide to the ginseng total saponin is (3-4): (6-7).

Preferably, the mass ratio of the pilose antler polypeptide to the total ginsenoside is 1: 1.8.

The invention provides a ginseng antler energy peptide microcapsule with an effect of delaying senescence, which comprises a core material and a wall material, wherein the mass ratio of the core material to the wall material is 1: (6-10); the core material is the ginseng antler energy peptide.

Preferably, the mass ratio of the core material to the wall material is 1: 8.

preferably, the raw material of the wall material comprises one or two of beta-cyclodextrin, gelatin, chitosan, acacia and carboxymethyl cellulose.

The invention provides a preparation method of the ginseng antler energy peptide microcapsule, which comprises the following steps:

1) adding the fine powder of the ginseng antler energy peptides into a saturated solution of the wall material raw material 1 under a stirring state, and dissolving to obtain an intermediate solution;

2) treating the intermediate solution with 80-240W of ultrasonic waves at 40 ℃ for 40-100 min to obtain a capsule forming solution;

3) and adding a saturated solution of the wall material raw material 2 into the capsule forming solution, stirring for 30min at 40 ℃, and freeze-drying to obtain the ginseng antler energy peptide microcapsule.

Preferably, when the wall material is beta-cyclodextrin and gelatin, the wall material raw material 1 is beta-cyclodextrin, and the mass percentage concentration of a saturated solution of the beta-cyclodextrin is 10%; the wall material raw material 2 is gelatin, and the mass percentage concentration of a saturated solution of the gelatin is 12%; the volume ratio of the saturated solution of the beta-cyclodextrin to the saturated solution of the gelatin is 1:1.

Preferably, the power of the ultrasonic treatment is 150W, and the time of the ultrasonic treatment is 70 min.

The invention provides application of the ginseng antler energy peptide or the ginseng antler energy peptide microcapsule in preparing a health-care product for delaying senescence.

The invention provides a ginseng antler energy peptide dropping pill with the function of delaying senility, which comprises a ginseng antler energy peptide microcapsule or a ginseng antler energy peptide microcapsule prepared by the preparation method.

The ginseng antler energy peptide with the function of delaying senility provided by the invention comprises antler polypeptide and ginseng total saponin; the mass ratio of the pilose antler polypeptide to the ginseng total saponin is (3-4): (6-7). The invention proves the effect of delaying senility through an efficacy experiment. During aging, the nervous system gradually declines, manifesting as a decline in learning and memory. The invention takes D-galactose to establish a mouse subacute aging model as a research object, and carries out comparative evaluation by three different behavioral experimental methods of diving tower experiment, water maze experiment and darkness avoidance (diving tower experiment and darkness avoidance experiment reflect the passive avoidance capability of animals, and water maze experiment reflects the spatial discrimination learning and memory capability of animals). The results show that: compared with the model group, the ginseng antler energy peptide group shows good learning and memory abilities in both jumping stand and dark avoidance experiments and water maze experiments. Meanwhile, with the increase of age, the aging of bone tissues can cause a series of changes including bone tissue microenvironment, bone cell morphology, signal paths in bone cells and the like, so that the mechanical response capability of bones is weakened, and then various diseases such as osteoporosis and the like are caused. The bone biomechanical efficacy experiment shows that: through the influence of aging on the mechanical response of bone cells, the biomechanical performance of aged mice can be improved by the ginseng antler energy peptide group discovered by bone biomechanical experiments.

The ginseng antler energy peptide microcapsule with the function of delaying senescence, provided by the invention, not only retains the medicinal effect of delaying senescence, but also improves the stability of polypeptide medicinal components of pilose antler through the wrapping of wall materials to a certain extent, and simultaneously covers the unpleasant odor of total ginsenoside, and the ginseng antler energy peptide microcapsule with high bioavailability, quick drug release, quick effect and convenient storage is provided and prepared into related products. The microcapsule has the advantages of simple preparation method, stable product, easily obtained raw materials and certain practical application value.

Drawings

FIG. 1 is a flow chart of a microcapsule preparation process;

FIG. 2 shows the SN shape under a transmission electron microscope;

FIG. 3 is a flow chart of a process for preparing a dripping pill;

FIG. 4 is a protein standard curve;

FIG. 5 is a standard curve of ginsenoside Rg 1;

FIG. 6 is a standard curve of ginsenoside Rb;

FIG. 7 is a protein standard curve;

FIG. 8 is an SDS-PAGE electrophoresis;

FIG. 9 shows the result of the evasion test, wherein a is the latency of the evasion test and b is the error times of the evasion test;

FIG. 10 shows the results of a positioning voyage experiment, wherein a is a graph of the distance traveled in different groups and b is a graph of the latency in different groups;

FIG. 11 is a graph showing the results of the latency and the error times in the stage jump experiment, wherein a is a graph showing the results of the latency and b is a graph showing the results of the error times in the stage jump experiment;

FIG. 12 shows the effect of energy peptide of ginseng antler on bone biomechanics.

Detailed Description

In the invention, the ginseng antler energy peptide comprises antler polypeptide. The source of the antler polypeptide of the present invention is not particularly limited, and antler polypeptide known in the art may be used, for example, by purchasing from commercial sources or by extracting according to the extraction method disclosed in the prior art. The cornu Cervi Pantotrichum polypeptide has effects of improving learning and memory function, resisting inflammation and oxidation, enhancing immunity, and preventing and treating osteoporosis.

In the invention, the ginseng antler energy peptide comprises ginseng total saponins. The source of the total ginsenoside used in the present invention is not particularly limited, and any known sources of total ginsenoside in the art may be used, for example, commercially available sources or extraction methods disclosed in the prior art. The total ginsenoside has the effects of improving memory function, resisting fatigue, enhancing immunity and delaying aging.

According to the invention, the pilose antler polypeptide and the ginseng total saponin are mixed according to a specified proportion, so that the purpose of delaying senescence can be effectively realized. The mass ratio of the pilose antler polypeptide to the ginseng total saponin is preferably 1:1.8, compared with the mixture obtained by other proportion, the compound has the advantages of obviously prolonging the latency period of a mouse, reducing the error times, obviously improving the cognitive ability of an aging mouse, obviously improving the passive avoidance response ability of the aging mouse and obviously improving the maximum load of thighbone. Meanwhile, the invention selects the pilose antler polypeptide from a plurality of active ingredients of the pilose antler, screens a plurality of active ingredients of ginseng to obtain the ginseng total saponin, and combines the pilose antler polypeptide and the ginseng total saponin for use, thereby not only defining the active ingredients in the formula and improving the utilization rate of the traditional Chinese medicine, but also simplifying the preparation process to a certain extent and reducing the production cost.

In the present invention, the mass ratio of the core material to the wall material is preferably 1: 8. the core material and the wall material are mixed according to the specified proportion, so that the stability of the active ingredients of the medicine can be effectively improved, the slow release efficiency is improved, and the bad smell is covered. The invention has no special limitation on the types of the wall materials, and the microcapsule wall materials known in the field can be adopted. The raw material of the wall material preferably comprises one or two of beta-cyclodextrin, gelatin, chitosan, Arabic gum and carboxymethyl cellulose.

In the invention, under the stirring state, the fine powder of the ginseng antler energy peptide is added into the saturated solution of the wall material raw material 1 and dissolved to obtain an intermediate solution.

In the present invention, the fine powder of the ginseng antler energy peptides is preferably sieved by a 150-mesh sieve. When the wall material is preferably beta-cyclodextrin and gelatin, the wall material raw material 1 is preferably beta-cyclodextrin, the mass percentage concentration of a saturated solution of the beta-cyclodextrin is preferably 10% (g/ml), and the wall material is preferably dissolved at 60 ℃; the wall material raw material 2 is preferably gelatin, and the mass percentage concentration of the saturated solution of the gelatin is preferably 12% (g/ml). The volume ratio of the saturated solution of beta-cyclodextrin to the saturated solution of gelatin is preferably 1:1.

After the intermediate solution is obtained, the intermediate solution is treated by 80-240W of ultrasonic waves at 40 ℃ for 40-100 min to obtain a capsule forming solution.

In the present invention, the wall material is coagulated and deposited around the core material to form a capsule by ultrasonic treatment. In embodiments of the present invention, several sets of sonication protocols are provided: the ultrasonic time is 40min, and the power is 80W; ultrasonic treatment time is 70min, and power is 150W; ultrasonic treatment time is 100min, and power is 240W; ultrasonic treatment time is 40min, and power is 150W; ultrasonic treatment time is 70min, and power is 240W; carrying out ultrasonic treatment for 100min, and decocting at power of 80W; ultrasonic treatment time is 40min, and power is 240W; ultrasonic treatment time is 70min, and power is 80W; the ultrasonic time is 100min, and the power is 150W; most preferably, the ultrasonic treatment power is 150W, and the ultrasonic treatment time is 70 min.

After a capsule forming solution is obtained, adding a saturated solution of a wall material raw material 2 into the capsule forming solution, stirring for 30min at 40 ℃, and freeze-drying to obtain the ginseng antler energy peptide microcapsule.

The method of stirring is not particularly limited in the present invention, and manual stirring or mechanical stirring well known in the art, for example, a magnetic stirrer, may be used. The stirring enables the wall material to be condensed and deposited around the capsule core material again to form capsules, and the embedding rate is increased. The invention adds wall materials for two times, aims to carry out secondary encapsulation on a primary encapsulated drug and a non-encapsulated drug, increases the encapsulation rate of the drugs, and cannot play a similar role if the two wall materials are subjected to primary ultrasonic treatment.

In the present invention, the freeze-drying is preferably performed by cold storage at 4 ℃ for 48h and pre-freezing at-80 ℃ overnight. After freeze-drying, the dried product is ground to obtain the microcapsule. The specific process flow diagram of the microcapsule preparation is shown in figure 1. The morphology of the prepared microcapsule is shown in figure 2, and the morphology of the microcapsule is regular spherical or spheroidal.

In the invention, in order to verify the encapsulation efficiency and the quality standard of the prepared microcapsule, a liquid chromatograph is adopted for determination, and the determination result shows that the ginsenoside Rg1 RSD is 1.29 percent and the ginsenoside Rb RSD is 1.07 percent, which indicates that the precision of the instrument is good; the method has good repeatability, and the sample is stable within 24 h. The sample application recovery rate test result shows that the average recovery rate of the ginsenoside is 97.1 percent, and the average recovery rate of the ginsenoside Rb is 95.8 percent, which indicates that the method has good accuracy.

In the invention, the ginseng antler energy peptide product can be added with common auxiliary materials to prepare acceptable medicines or health care products in various forms, the forms comprise tablets, granules, capsules, pills, oral liquid, liquid beverages and solid beverages, and the oral decoction, the tablets, the capsules or the granules can be prepared by adopting the conventional preparation method of corresponding preparations.

In the invention, the mass ratio of the ginseng antler energy peptide microcapsules to the dripping pill auxiliary materials is preferably 1: 1-4, more preferably 1: 4.

the preparation process flow chart of the dripping pill is shown in figure 3, and the preparation method of the ginseng antler energy peptide dripping pill is preferably as follows:

A. melting and uniformly mixing the auxiliary materials at 70 ℃, adding the ground ginseng antler energy peptide microcapsule powder while stirring, standing for thirty minutes after complete dissolution to remove bubbles, and obtaining a molten liquid;

B. dripping the molten liquid into liquid paraffin with the temperature of 4 ℃, cooling and forming, taking out, and wiping redundant liquid paraffin with filter paper;

C. drying to obtain the ginseng antler energy peptide dropping pill.

The invention has no special limitation on the types of the auxiliary materials, and the auxiliary materials well known in the field can be adopted. In the embodiment of the invention, the auxiliary material is a mixture of PEG4000 and PEG 6000. The mass ratio of PEG4000 to PEG6000 is preferably 1: 3. the mass ratio of the ginseng antler energy peptide microcapsule powder to the volume mass of the auxiliary materials is 1g: 4g of the total weight. The drop pill is more suitable for carrying. In addition, the dripping pill is taken sublingually, the active ingredients of the medicine can directly enter the blood through the mucous membrane of the human body for rapid participation in the circulation of the human body, and the quick effect can be achieved within a short time, thereby completely avoiding the defects of low bioavailability, inconvenient taking and slow effect of the traditional Chinese medicine.

The ginseng antler energy peptide and microcapsule with the function of delaying senility, the preparation method and the application thereof provided by the invention are explained in detail by the following examples, but the invention is not to be construed as limiting the protection scope.

Example 1

Method for extracting pilose antler polypeptide crude extract

1. Preprocessing cornu Cervi Pantotrichum

Cutting fresh cornu Cervi Pantotrichum into 1cm at low temperature³Left and right small blocks are preserved at-20 ℃ for later use.

2. Crude extraction of pilose antler polypeptide

(1) Collecting cornu Cervi Pantotrichum 100g, rapidly cleaning with 4 deg.C distilled water to remove blood color, and preserving in 4 deg.C refrigerator fresh-keeping layer.

(2) Respectively preparing acetic acid-sodium acetate buffer solution with pH value of 4.0, and refrigerating at 4 deg.C in refrigerator for use.

(3) Taking out washed cornu Cervi Pantotrichum, homogenizing with colloid mill (washing with acetate buffer solution), grinding into homogenate, storing in 4 deg.C refrigerator, intermittently stirring every 20min for 6 hr, and standing overnight.

(4) Taking out cornu Cervi Pantotrichum ground into colloid state after overnight, and centrifuging with high speed refrigerated centrifuge (8500r min)^-1，20min)。

(5) After centrifugation, the supernatant was taken and added with 90% aqueous ethanol to bring the concentration of the supernatant to 65% at the end. Left at 4 ℃ and stirred for 20 min/time, stirred for 6h, and left overnight.

(6) Centrifuging again (8500r min)^-1Centrifuging for 20min), collecting the precipitate, lyophilizing to obtain cornu Cervi Pantotrichum polypeptide crude extract, and storing at-80 deg.C.

3. Protein content determination

The concentration of polypeptide protein of pilose antler is determined by a Coomassie brilliant blue G-250 method, 1.00G of pilose antler polypeptide powder is precisely weighed, dissolved in 1mL of distilled water, and then detected by an enzyme-labeling instrument.

The method for measuring the Protein concentration by using Easy Protein Quantitative Kit comprises the following specific steps: standing Coomassie brilliant blue at room temperature for 20min, preheating enzyme labeling instrument, placing 0, 1, 5, 7, 9, 10 μ L bovine serum albumin standard solution (0.22mg/mL) in 96-well plate, adding water to make up 10 μ L, adding 100 μ L Coomassie brilliant blue dye solution into each well, shaking for 10min, measuring OD values of protein samples with different concentrations at 595nm wavelength, and calculatingThe standard curve of the protein is as follows: 0.7511x +0.1668, R²0.9992 (OD on y-axis, concentration C on x-axis). The protein standard curve is shown in FIG. 4. The protein concentration detected was 11.928 mg/ml.

Example 2

The preparation method of the total ginsenoside comprises the following steps:

cutting ginseng into thick slices, adding water, decocting for two times, the first time lasts for 2 hours, the second time lasts for 1.5 hours, filtering decoction, combining filtrates, passing through a D101 type macroporous resin column, eluting with water until the filtrate is colorless, eluting with 60% ethanol water solution, collecting 60% ethanol eluate, concentrating the filtrate to obtain clear paste with the relative density of 1.06-1.08 (80 ℃), drying, and crushing to obtain the ginseng tea. The process and process parameters are shown in Table 1.

TABLE 1 Process runs and Process parameters

Example 3

Preparation method of 1 ginseng antler energy peptide (SN) microcapsule

Grinding the pilose antler polypeptide and the ginseng total saponin into fine powder according to the weight part ratio, sieving the fine powder by a 150-mesh sieve, and uniformly mixing;

b, preparing a certain amount of beta-cyclodextrin into a solution of 10 percent (1g:10ml), and stirring and dissolving the solution in a water bath at 60 ℃ to obtain a transparent saturated solution;

and C: adding mixed powder of cornu Cervi Pantotrichum polypeptide and Ginseng radix total saponin under stirring, and dissolving completely;

step D: transferring the powder into an ultrasonic cleaner, and treating the powder by using ultrasonic waves under a certain temperature condition to enable wall materials to be condensed and deposited around the capsule core materials to form capsules;

step E: transferring the mixture to a magnetic stirrer, adding 12% (1g:12ml) gelatin saturated solution with the same volume as that of the beta-cyclodextrin, stirring at a certain temperature to enable the wall material to be condensed and deposited around the capsule core material to form capsules, and increasing the embedding rate;

step F: refrigerating at 4 deg.C for 48h, -80 prefreezing overnight, freeze-drying with freeze-dryer, and grinding the dried product to obtain Ginseng radix and cornu Cervi Pantotrichum energy peptide microcapsule.

2 orthogonal experimental design of ginseng antler energy peptide (SN) microcapsules

According to the SN property and the result of the primary screening, the factors of orthogonal experiment design investigation are determined as core wall weight ratio (A), ultrasonic time (B) and ultrasonic power (C), Rg1 and Rb encapsulation efficiency are taken as investigation indexes, an L9(3 multiplied by 3) orthogonal table is adopted for design, and the factors and the levels are shown in table 2.

TABLE 2 factor level table

Taking 9 parts of pilose antler polypeptide and ginseng total saponin powder according to the proportion, and carrying out tests according to orthogonal table design, wherein the specific test arrangement is as follows:

sample No. 1: beta-cyclodextrin 3 times, ultrasonic time 40min, power 80W, gelatin 3 times, magnetic stirring 30min at 40 ℃.

Sample No. 2: beta-cyclodextrin 3 times, ultrasonic time 70min, power 150W, gelatin 3 times, magnetic stirring 30min at 40 ℃.

Sample No. 3: beta-cyclodextrin 3 times, ultrasonic time 100min, power 240W, gelatin 3 times, magnetic stirring 30min at 40 ℃.

Sample No. 4: 4 times of beta-cyclodextrin, 40min of ultrasonic time, 150W of power and 4 times of gelatin, and magnetically stirring for 30min at 40 ℃.

Sample No. 5: 4 times of beta-cyclodextrin, ultrasonic time of 70min, power of 240W and gelatin of 4 times, and magnetic stirring at 40 ℃ for 30 min.

Sample No. 6: beta-cyclodextrin 4 times, ultrasonic treatment time 100min, power decoction 80W, gelatin 4 times, and magnetic stirring at 40 deg.C for 30 min.

Sample No. 7: 5 times of beta-cyclodextrin, 40min of ultrasonic time, 240W of power and 5 times of gelatin, and 30min of magnetic stirring at 40 ℃.

Sample No. 8: 5 times of beta-cyclodextrin, ultrasonic time of 70min, power of 80W, 5 times of gelatin, and magnetic stirring at 40 ℃ for 30 min.

Sample No. 9: 5 times of beta-cyclodextrin, 100min of ultrasonic time, 150W of power and 5 times of gelatin, and magnetically stirring for 30min at 40 ℃.

The results of the orthogonality are shown in Table 3.

TABLE 3 Quadrature results

Conclusion

According to orthogonal experimental results, A is more than C and more than B, A2 is more than A3 is more than A1, B2 is more than B4 and more than B1, and C2 is more than C3 and more than C1, and the optimal process is that the core-wall weight ratio is 1:8, the ultrasonic treatment is carried out for 70min, the ultrasonic power is 150W, and the magnetic stirring is carried out for 30min at 40 ℃.

3. Process verification test

Preparing three SN microcapsules according to the optimal scheme, and detecting the encapsulation efficiency of the ginsenoside by a high performance liquid chromatograph. Substituting into formula I to obtain drug loading rate, and substituting into formula II to obtain encapsulation efficiency (calculated by ginsenoside Rg1 and ginsenoside Rb).

Drug loading (amount of drug contained in the microcapsule/total amount of the microcapsule) × 100% public indicator I

Encapsulation efficiency i amount of drug encapsulated in system/total amount of drug encapsulated and not encapsulated in system 100% is disclosed in II

The results showed that the average drug loading of 3 samples was (86.44. + -. 0.001)%, and the average encapsulation efficiency was (91.09. + -. 0.49)%. The preparation method is stable and feasible.

4. Measurement of SN potential and morphology

Weighing 200mg SN microcapsules, adding 1mL distilled water solution, dissolving by vortex oscillation, centrifuging, taking supernate, and researching the surface morphology of the micelle by using a field emission scanning electron microscope. The sample was mounted on the surface of a sample stub and sputter coated with gold-palladium under an argon atmosphere. The acceleration voltage used was 3kV and the morphology was regular spherical or spheroidal as shown in figure 2.

Example 4

Preparation method of ginseng and pilose antler energy peptide containing dripping pills

Respectively weighing 3.6g of pilose antler polypeptide and 6.4g of ginseng total saponin, grinding into fine powder, sieving with a 150-mesh sieve, uniformly mixing, preparing 40g of beta-cyclodextrin into a 10% (1g:10ml) solution, stirring and dissolving in a water bath at 60 ℃ to obtain a transparent saturated solution, adding mixed powder of pilose antler polypeptide and ginseng total saponin while stirring, and completely dissolving. Transferring to ultrasonic cleaner, and treating with 150W ultrasonic wave at 40 deg.C for 70 min. Transferring to a magnetic stirrer, adding 40g of 12% (1g:12ml) gelatin saturated solution, stirring at 40 deg.C for 30min, and taking out. Refrigerating at 4 deg.C for 48h, pre-freezing at-80 deg.C overnight, freeze drying with freeze drier, and grinding to obtain Ginseng and cornu Cervi Pantotrichum energy peptide microcapsule.

Weighing 10g of PEG4000 and 30g of PEG6000, melting and uniformly mixing in a 70 ℃ water bath kettle, adding the prepared ginseng antler energy peptide microcapsules while stirring, standing for 30min after complete dissolution, removing bubbles, sucking molten liquid by a dropper, dripping into liquid paraffin, keeping the distance between the dropper and the liquid paraffin at 10cm, the condensation temperature at 5 ℃ and the dripping speed at 50 drops/min, cooling and forming, taking out, wiping redundant liquid paraffin by filter paper, and drying in a 50 ℃ oven to obtain the ginseng antler energy peptide dripping pills.

Example 5

Method for measuring SN microencapsulation rate prepared in example 4 and quality standard

1. Chromatographic conditions and System suitability test

The column was Zorbax SB-C18(4.6 x 250mm, 5 μm); flow rate 0.4 mL/min^-1(ii) a The column temperature is 30 ℃; the detection wavelength is 203 nm; the sample injection amount is 5 mu L; the mobile phase is acetonitrile: the elution was carried out with a water gradient and the elution procedure is shown in Table 4.

TABLE 4 elution procedure

2. Preparation of control solutions

Accurately weighing appropriate amount of ginsenoside Rg1 reference substance and ginsenoside Rb reference substance, and adding methanol to obtain 1ml mixed solution containing ginsenoside Rg 10.026mg and ginsenoside Rb 0.026 mg.

3. Preparation of test solution

Taking 150mg of the product, precisely weighing, placing in a 10ml measuring flask, adding methanol, ultrasonically dissolving, diluting to scale, filtering, and taking the subsequent filtrate to obtain the negative sample solution by the same method.

4. Assay method

Precisely sucking 5 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

5. Methodology investigation

5.1 creation of Standard Curve

Precisely sucking 0, 1, 3, 4, 5 and 70 μ L of SN reference substance solution, measuring according to the chromatographic conditions, and obtaining a regression equation by taking the peak area (y) as a vertical coordinate and the SN concentration (x) as a horizontal coordinate: the results of the ginsenoside rg1 and ginsenoside rb of 105535x +0.02 and 92020x-0.0938 show that SN ginsenoside rg1 is 0-0.00183 mg.mL^-1Has good linear relationship in the range shown in figure 5, and ginsenoside rg1 is 0-0.0039 mg/mL^-1The range is well linear, see fig. 6.

5.2 precision test

Taking 1 part of SN sample, preparing into test solution, measuring according to the above chromatographic conditions, injecting 5 μ L each time, and repeating the injection for 6 times. And (3) measuring knots: ginsenoside rg1 RSD is 1.29%, ginsenoside Rb RSD is 1.07%, indicating that the precision of the instrument is good.

5.3 repeatability test

6 parts of SB sample is prepared, a sample solution is prepared, the sample amount is 5 microliter per time according to the chromatographic conditions, and the determination result shows that the ginsenoside Rg1 RSD is 1.51 percent and the ginsenoside Rb RSD is 1.03 percent, which indicates that the method has good repeatability.

5.4 stability test

Taking 1 part of SN sample, preparing a sample solution, measuring according to the chromatographic conditions, wherein the sample amount is 5 mu L each time, and carrying out analytical measurement for 0h, 2h, 4h, 8h, 12h and 24h respectively, and the results show that the ginsenoside Rg1 RSD is 3.02% and the ginsenoside Rb RSD is 3.25%, which indicates that the sample is stable within 24 h.

5.5 sample application recovery test

A sample of SN with a known content is precisely added with an appropriate amount of a reference substance to prepare a test solution, and the sample volume is 5 muL per time according to the chromatographic conditions of the section 1 in the example 3. The result shows that the average recovery rate of the ginsenoside is 97.1 percent, and the average recovery rate of the ginsenoside Rb is 95.8 percent, which indicates that the method has good accuracy.

5.6SN ginsenoside Rg1 and ginsenoside Rb content determination results

Preparing SN3 parts, precisely weighing 20mg, adding 10mL of methanol for ultrasonic dissolution, and filtering by using a 0.22 mu m microporous filter membrane. And (3) after the sample is prepared, performing high performance liquid detection, and measuring that the average value of the total content of the ginsenoside Rg1 and the ginsenoside Rb in the SN is 0.44 mg/g.

Example 6

Determination of protein concentration in SN prepared in example 4

1. Drawing of standard curve

The EasyProtein Quantitative Kit method is used for measuring the protein concentration, and the specific detection method comprises the following steps: preheating a microplate reader for 30min, standing the Coomassie brilliant blue at room temperature for 20min, adding 0, 1, 5, 7, 9 and 10 mu L of bovine serum albumin standard solution (0.22mg/mL) into a 96-well plate respectively, adding distilled water into each well to make up the volume to 10 mu L, adding 100 mu L of Coomassie brilliant blue dye solution into each well, shaking the mixture in a shaking table for 10min, measuring the OD (optical density) values of different groups of protein samples at the wavelength of 595nm, and calculating a protein standard curve as follows: y is 0.919x +0.2346, R²0.9907 (concentration C on the y-axis and OD on the x-axis). The protein standard curve is shown in FIG. 7.

2. Preparation of test solution

Taking 100mg of SN sample, adding 1ml of lysis solution, carrying out lysis on ice for 1h, then centrifuging for 15min at 4 ℃, 14000rpm, taking supernatant, diluting ten times, adding 10ul of test sample and 100ul of Coomassie brilliant blue, shaking and mixing for 5min, and measuring absorbance at 595nm of a microplate reader. The standard curve is substituted and the content is calculated. The protein content in SN was determined to be 16. mu.g/mg.

3. SDS-PAGE electrophoretic analysis

And (3) carrying out electrophoresis by adopting discontinuous vertical plate polyacrylamide gel, and observing the protein distribution in the SN microcapsule.

3.1 protein sample treatment

The loading amount of protein in each hole is 20 mug, Buffer solution and protein are mixed according to the volume ratio of 4:1, processed samples are placed in a water bath kettle at 100 ℃ to be boiled for 10min, and the samples are centrifuged to be loaded.

3.2SDS-PAGE electrophoresis

Cleaning the glass plate, blowing with blower, assembling the glass plate into a gel plate, preparing 12% separation gel, preparing 5% concentrated gel after 30min of gel, and performing gel for 30 min. And (3) assembling the rubber plate in an electrophoresis tank, filling electrophoresis liquid, and sequentially adding protein samples. The blank wells were filled with buffer. The upper layer gel is electrophoresed for 40min at constant voltage of 90V, and the lower layer gel is electrophoresed for 120min at constant voltage of 110V.

3.3 dyeing and decolorizing

After the electrophoresis is finished, the gel is put into a prepared Coomassie brilliant blue R250 solution, the gel is soaked in a staining solution with the volume not less than 5 times of the volume of the gel, and the gel is placed on a shaking table which is shaken slowly and is stained for 50min at room temperature. After dyeing, the gel is soaked in a destaining solution (methanol: glacial acetic acid: water: 4: 1: 5 by volume), gently shaken, and the destaining solution is replaced at intervals until clear bands can be seen by naked eyes. After the completion of the decolorization, the gel was immersed in distilled water to terminate the decolorization.

3.4 gel imaging

The gel was placed in a gel imager to observe the detection results and photographed, and the results are shown in fig. 8. The result shows that the protein molecular weight of the SN microcapsule is between 25 and 70 KDa.

Example 7

1. Laboratory animals and groups

60 ICR mice are selected, half of each sex (provided by the hundred million animals center) and the weight (20 +/-2) g, and all the animals are raised under the standard conditions of the temperature of 25 +/-1 ℃ and the relative humidity of 45-50%. Randomly dividing the mice into a blank group (K), a model group (M), a positive group (Y) and a ginseng antler energy peptide group: the mass ratio of the pilose antler polypeptide to the ginseng total saponin is 1:1(SN1), the mass ratio of the pilose antler polypeptide to the ginseng total saponin is 1:1.5(SN2), the mass ratio of the pilose antler polypeptide to the ginseng total saponin is 1:2.3(SN3), and the mass ratio of the pilose antler polypeptide to the ginseng total saponin is 1:2.8(SN 4).

After the ICR mice are adapted for 3 days, the positive group and the ginseng antler energy peptide group are as follows: SN1, SN2, SN3, SN4, vitamin E for preventive administration (150mg/kg), ginseng antler energy peptide group: SN1, SN2, SN3 and SN4, the dosage is 150mg/kg based on polypeptide of pilose antler, the administration lasts for 8 days, the blank group is filled with physiological saline with the same volume as the stomach, after 8 days, the mice in other groups except the blank group are injected with D-galactose (1000mg/kg) subcutaneously in the neck and back every day, and the blank group is injected with physiological saline subcutaneously for 60 days continuously. The weight of the rat is reweighed every 3-5 days, and the administration dosage is adjusted according to the weight.

2. Behavioral experiments

2.1 mouse dark avoidance experiment

An adaptation stage: on the first day, the mice of each group are respectively placed in a bright room according to the serial numbers, a switch connected with a dark room is turned on, the mice freely move in the bright room and the dark room for about 2-3 min, the mice are placed back to the rearing cage after fully adapting to the environment of the darkness-avoiding instrument, and the training on the next day is prepared. A learning stage: the next day, the switch to the dark and light chambers was closed and the mouse placed in the light chamber. After the mouse is placed, the switch of the electric shock device is turned on at the same time as the bright and dark room connection switch is turned on. Because of the darkness of the mouse, the mouse quickly enters the dark room and is shocked by the shock machine after entering the dark room. The time from the beginning of the experiment to the first electric shock of the mouse is the dark-avoiding latent period, and the time is not more than 5min at most. After the experiment is finished, the total number of times of electric shock of each mouse in 5min is recorded, namely the number of times of dark avoidance errors.

2.2 Water maze experiment

Positioning navigation experiment: and (3) randomly placing the platform in the middle of one of the four quadrants of the water maze, wherein the platform is not higher than the water surface and is positioned 2cm under water. An appropriate amount of titanium dioxide is added into the water maze and stirred evenly and fully to ensure that the mouse can be marked smoothly. The mice were placed in the water maze from each of the four quadrants, respectively, with their heads facing the pool wall of the pool. After the experiment starts, if the mouse finds the platform, the instrument can automatically record the time when the mouse finds the platform, namely the time when the mouse finds the platform, the timing is stopped, and the experiment is ended. In the learning and training process of the previous five days, if the platform can not be found in the mice for 2min, the mice are placed on the platform for 15-30 s, and the latency period of the mice which can not find the platform is 120 s. After completion of the experiment, the mice were returned to the animal room after being air-dried.

2.3 diving platform experiment

And (3) carrying out a diving platform experiment on the 40 th day of administration, opening a mouse diving platform test box, putting the mouse into a diving platform instrument, and then carrying out 32V alternating current to ensure that the animal is subjected to electric shock, wherein the escape reaction is the jumping-up platform. Most animals jump to the copper grid again or repeatedly, jump back to the platform quickly after being shocked, train for 5min, and record the shocked times of each mouse as the learning achievement (EN 1). On day 41, mice were placed directly under power. Recording the time from the mouse being put on the platform to the 1 st jump as the Latency (LT) on the platform; the mice jump to the platform evasively after being shocked, and then the number of times that the mice jump down again within 5min (EN2) is recorded as the error number, and LT and EN2 are jointly used as the memory achievement.

2.4 determination of the biomechanical Properties of bone

Utilize bone biomechanics tester to carry out three point bending test survey, in changchun traditional chinese medicine university research and development center, place the thighbone that is surveyed on biomechanics measuring apparatu's support, and soak gauze with normal saline, wrap around the thighbone, make the convex surface downward, guarantee that the mid point of thighbone and the center and the pressure point of both sides fulcrum are all on a straight line, and span L between two branches equals 27mm, and the falling speed is 5.0mm/min, at the uniform velocity descends, and the afterburning is on the thighbone mid point until the thighbone fracture. From the pressure sensor data, the maximum load that the femur can withstand is determined.

3. Results of the experiment

3.1 results of darkness avoidance experiments

The dark avoidance experiment result shows that in the memory test of the next day, as shown in a in figure 9, the latency of the model group mice is obviously shortened (P <0.01) compared with the blank control group, the latency of the positive group mice and the ginseng antler energetic peptide group mice is obviously prolonged (P <0.01) compared with the model control group, and the latency of the SN2 group mice and the SN3 group mice are obviously superior to that of the SN1 group and the SN4 group (P <0.05 and P < 0.01). As shown in b of FIG. 9, the number of errors was significantly increased within 5min in the mice of the model group (P <0.01) compared to the blank control group; compared with the model control group, the number of errors of the positive group and the ginseng antler energy peptide group is reduced within 5min (P is less than 0.05).

3.2 Water maze

The results of the positioning navigation experiment are shown in fig. 10, and the moving distance of each group of mice for finding the platform is gradually shortened along with the increase of the training time. The distance of movement of model mice to find the platform on day 5 of training (P <0.05) was increased compared to the blank control group. Compared with the model group, on the 5 th training day, the platform latency of the ginseng antler energy peptide group mice is obviously shortened (P < 0.01). The ginseng antler energy peptide is suggested to improve the cognitive ability of aged mice induced by D-galactose, and the SN2 group and the SN3 group are both significantly better than the SN1 group and the SN4 group (P <0.05, P < 0.01).

3.3 diving platform experiment

As shown in fig. 11, compared with the blank control group, the number of errors in the model group is significantly increased, the latency is significantly reduced (P <0.05), after learning for 5min, the number of errors in the positive group and the ginseng antler energy peptide group is significantly reduced (P <0.05, P <0.01), the latency is prolonged, and the results show that the ginseng antler energy peptide group can improve the passive avoidance response capability of the aged mice, and the SN2 group and the SN3 group are significantly better than the SN1 group and the SN4 group (P <0.05, P < 0.01).

3.4 Effect of ginseng antler energy peptides on bone biomechanics the results are shown in FIG. 12. The maximal load of femur was significantly decreased in the model group compared to the blank group (P <0.01), increased in the ginseng antler energy peptide group compared to the model group (P <0.05), and the SN2 group and SN3 group were significantly better than the SN1 and SN4 groups (P <0.05, P < 0.01). The ginseng and antler energy peptide can improve the biomechanical performance of the aging mice and delay bone aging.

Compared with the blank control group, the composition of the composition,^#P<0.05,^##P<0.01; comparison with model group<0.05,**P<0.01; comparing with SN2 set ^ P<0.01,^^P<0.01; in comparison with the set of SN3,^&P<0.01，^&&P<0.01。

according to the embodiments, the invention proves that the ginseng antler energy peptide and the microcapsule and the dropping pill thereof have the curative effect of delaying senility by establishing a mouse subacute senescence model by using D-galactose and observing the influence of the ginseng antler energy peptide on the learning and memory ability of senescent mice and a bone biomechanics effect experiment.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. An energy peptide of ginseng antler for delaying senility is characterized by comprising antler polypeptide and ginseng total saponin; the mass ratio of the pilose antler polypeptide to the ginseng total saponin is (3-4): (6-7).

2. The ginseng antler energy peptide of claim 1, wherein the mass ratio of the antler polypeptide to the total saponins of panax ginseng is 1: 1.8.

3. the ginseng antler energy peptide microcapsule with the function of delaying senescence comprises a core material and a wall material, and is characterized in that the mass ratio of the core material to the wall material is 1: (6-10); the core material is the ginseng antler energy peptide of claim 1 or 2.

4. The ginseng antler energy peptide microcapsule according to claim 3, wherein the mass ratio of the core material to the wall material is 1: 8.

5. the ginseng antler energy peptide microcapsule according to claim 3 or 4, wherein the raw material of the wall material comprises one or two of beta-cyclodextrin, gelatin, chitosan, gum arabic, and carboxymethyl cellulose.

6. The method for preparing the ginseng antler energy peptide microcapsules of any one of claims 3 to 5, which is characterized by comprising the following steps:

1) adding fine powder of the ginseng antler energy peptide of claim 1 or 2 into a saturated solution of the wall material raw material 1 under stirring, and dissolving to obtain an intermediate solution;

7. The preparation method of claim 6, wherein when the wall material is beta-cyclodextrin and gelatin, the wall material 1 is beta-cyclodextrin, and the mass percentage concentration of the saturated solution of beta-cyclodextrin is 10%; the wall material raw material 2 is gelatin, and the mass percentage concentration of a saturated solution of the gelatin is 12%; the volume ratio of the saturated solution of the beta-cyclodextrin to the saturated solution of the gelatin is 1:1.

8. The method according to claim 6 or 7, wherein the power of the ultrasonic treatment is 150W, and the time of the ultrasonic treatment is 70 min.

9. Use of the ginseng antler energy peptide according to claim 1 or 2 or the ginseng antler energy peptide microcapsule according to any one of claims 3 to 5 in the preparation of a health care product for delaying senescence.

10. A ginseng antler energy peptide dropping pill with the function of delaying senility is characterized by comprising ginseng antler energy peptide microcapsules as claimed in any one of claims 3 to 5 or ginseng antler energy peptide microcapsules prepared by the preparation method as claimed in claims 6 to 8.