AU2020104149A4 - Ginseng-antler energy peptide having an anti-aging effect, microcapsule, and preparation method and use thereof - Google Patents

Abstract

The present disclosure provides a ginseng-antler energy peptide having anti-aging effect, a microcapsule, and a preparation method and use thereof, and belongs to the technical field of health products. The ginseng-antler energy peptide having anti-aging effect includes pilose antler 5 polypeptides and total ginsenoside; the pilose antler polypeptides and the total ginsenoside have a mass ratio of (3-4):(6-7). A ginseng-antler energy peptide microcapsule having anti-aging effect is disclosed, where the core material and the wall material have a mass ratio of 1:(6-10); the ginseng antler energy peptide serves as the core material. The present disclosure further provides use of the ginseng-antler energy peptide or the ginseng-antler energy peptide microcapsule in the preparation of 0 an anti-aging health product. In the present disclosure, D-galactose is used to establish a subacute aging mouse model to observe effects of the ginseng-antler energy peptide and the microcapsule and dropping pill thereof on learning and memory ability in aging mice and bone biomechanical efficacy. Thus, anti-aging effect of the ginseng-antler energy peptide and derivatives thereof is demonstrated. 1

Description

GINSENG-ANTLER ENERGY PEPTIDE HAVING AN ANTI-AGING EFFECT, MICROCAPSULE, AND PREPARATION METHOD AND USE THEREOF TECHNICAL FIELD The present disclosure relates to the technical field of health products, and in particular to a ginseng-antler energy peptide having an anti-aging effect, a microcapsule, and a preparation method and use thereof. BACKGROUND Senility is an irresistible natural law and is a natural stage for each individual's growth and development. Senility is often manifested as structural degenerative changes and functional decline. With the substantial increase in human life expectancy, the incidence of neurodegenerative diseases, cardiovascular and cerebrovascular diseases, diabetes, cancers and other aging-associated diseases has been increasing year by year, resulting in an increasingly heavy burden on the medical system and social economy. Anti-aging is highly concerned by all relevant researchers worldwide. Senility can be divided into two categories: one is physiological senility, which refers to the physiological degeneration process of the body after the mature period, which is mainly reflected in degenerative changes in functions of various organs and tissues with age; the other is pathological senility, which is a process accelerating the aging due to suffering from certain diseases or being affected by external factors. Aging process is irreversible, but some drugs can delay and prevent aging. In view of limitations of current drugs and surgical treatments, more and more researchers have got down to looking for effective anti-aging drugs to reduce the risk of related diseases and extend people's healthy lifespan. Some of these researchers have focused on traditional Chinese medicine with a history of thousands of years of clinical application, and many progresses have been made. By consulting the literature, anti-aging drugs are classified and summarized. Anti-aging drugs can be roughly divided into traditional Chinese medicine (such as Radix Angelicae Sinensis, Radix Ginseng and royal jelly), small molecules (such as metformin, resveratrol, procyanidine and rapamycin) and polypeptides (such as rice peptides, soybean peptides and synthetic peptides, etc.).

The compatibility of Radix Angelicae Sinensis (ginseng) and Comu Cervi Pantotrichum (pilose

antler) has a long history. There are 116 prescriptions containing ginseng and pilose antler in the

Preions for Universal Relief Shenrong Keli (Ginseng and Antler Granules) is included in the

Compilation ofNational StandardsforChinese PatentMedicine. The medicament has the effects of

replenishing heart qi and invigorating the heart and kidney. It is indicated for physical weakness,

palpitations, shortness of breath, soreness and weakness of waist and knees, impotence and

spermatorrhea. Since products and preparations containing ginseng and pilose antler are safe and non toxic, much attention has been paid to the development of ginseng-antler products. However, since the ginseng and the pilose antler contain a plurality of active pharmaceutical ingredients (APIs), there has been no report on the compounding of APIs of the ginseng and the pilose antler that exert anti aging effect. SUMMARY In view of this, an objective of the present disclosure is to provide a ginseng-antler energy peptide (SN) having anti-aging effect, which has excellent anti-aging activity. Another objective of the present disclosure is to provide a ginseng-antler energy peptide microcapsule having anti-aging effect, and a preparation method and use thereof, effectively improving the stability of APIs. The present disclosure provides a ginseng-antler energy peptide having anti-aging effect, including pilose antler polypeptides and total ginsenoside; the pilose antler polypeptides and the total ginsenoside have a mass ratio of (3-4): (6-7). Preferably, the pilose antler polypeptides and the total ginsenoside may have a mass ratio of 1:1.8. The present disclosure provides a ginseng-antler energy peptide microcapsule having anti-aging effect, including a core material and a wall material, where the core material and the wall material have a mass ratio of 1:(6-10); the core material is the ginseng-antler energy peptide. Preferably, the core material and the wall material may have a mass ratio of 1:8. Preferably, raw materials of the wall material may include one or two of p-cyclodextrin, gelatin, chitosan, arabic gum and carboxymethyl cellulose. The present disclosure provides a method for preparing the ginseng-antler energy peptide microcapsule, including the following steps: 1) under stirring, adding and dissolving fine powders of the ginseng-antler energy peptide in a saturated solution of a wall material 1 to obtain an intermediate solution; 2) sonicating the intermediate solution at 80-240 W for 40-100 min to obtain an encapsulation solution; and 3) adding a saturated solution of a wall material 2 to the encapsulation solution, stirring for 30 min, and freeze-drying to obtain ginseng-antler energy peptide microcapsules. Preferably, when the wall material includes p-cyclodextrin and gelatin, the wall material 1 may be p-cyclodextrin, and the saturated solution of p-cyclodextrin may have a concentration of 10 wto; the wall material 2 may be gelatin, and the saturated solution of gelatin may have a concentration of 12 wt%; the saturated solution of p-cyclodextrin and the saturated solution of gelatin may have a volume ratio of 1:1. Preferably, the sonication may be conducted at 150 W for 70 min. The present disclosure provides use of the ginseng-antler energy peptide or the ginseng-antler energy peptide microcapsule in the preparation of an anti-aging health product. The present disclosure provides a ginseng-antler energy peptide dripping pill having anti-aging effect, including the ginseng-antler energy peptide microcapsule or the ginseng-antler energy peptide microcapsule prepared by the preparation method. The ginseng-antler energy peptide having anti-aging effect provided by the present disclosure includes pilose antler polypeptides and total ginsenoside; the pilose antler polypeptides and the total ginsenoside have a mass ratio of (3-4): (6-7). The present disclosure proves the anti-aging effect thereof through efficacy experiments. In the aging process, the nervous system gradually declines, which is prominently manifested by the decline in learning and memory. The present disclosure uses D-galactose to establish a subacute aging mouse model as a research object, and conducts comparative evaluation through three different behavioral experimental methods: step-down test, Morris water maze test, and step-through test (the step-down test and the step-through test reflect animal's passive avoidance response, while the Morris water maze test reflects animal's spatial discriminability and learning and memory). The results showed that compared with the model group, the ginseng-antler energy peptide group showed excellent learning and memory no matter in the step down and step-through tests or the Morris water maze test. Meanwhile, with age, aging of bone tissue will cause a range of changes in bone microenvironment, osteocyte morphology, signaling pathways in osteocytes, etc., which will weaken the skeletal biomechanics and response and thus cause osteoporosis and other diseases. In the aging process, the structural aging and hypofunction of the skeletal system are important causes of aging. Through bone biomechanical efficacy tests, the present disclosure shows that: with the effect of aging on the mechanical response of osteocytes, bone biomechanical tests have found that the ginseng-antler energy peptide group can improve the biomechanical properties of aging mice. The ginseng-antler energy peptide microcapsule having anti-aging effect provided by the present disclosure not only retains the anti-aging effect, but also partly improves the stability of pilose antler polypeptides through the encapsulation of the wall material; meanwhile, the microcapsule masks the unpleasant odor of the total ginsenoside, provides a ginseng-antler energy peptide microcapsule with high bioavailability, rapid drug release, rapid acting, and convenient storage and is made into related products. The method for preparing the microcapsule is simple, the product is stable, and raw materials are easily available, having certain practical application value. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates a process flow of microcapsule preparation; FIG. 2 shows the morphology of SN under transmission electron microscope (TEM); FIG. 3 illustrates a process flow of dropping pill preparation; FIG. 4 shows a protein standard curve; FIG. 5 is a standard curve of ginsenoside Rgl;

FIG. 6 is a standard curve of ginsenoside Rd; FIG. 7 is a protein standard curve; FIG. 8 illustrates sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); FIG. 9 shows the results of step-through tests, where a illustrates the latency of the step-through test, and b illustrates the error frequency in the step-through test; FIG. 10 shows the results of place navigation tasks, where a illustrates swim distances of different groups, and b illustrates latencies of different groups; FIG. 11 shows the latency and the error frequency in the step-down test, where a is the results of the latency in the step-down test, and b is the results of the error frequency in the step-down test; FIG. 12 shows the effect of ginseng-antler energy peptide on bone biomechanics. DETAILED DESCRIPTION The present disclosure provides a ginseng-antler energy peptide having an anti-aging effect, including pilose antler polypeptides and total ginsenoside; the pilose antler polypeptides and the total ginsenoside have a mass ratio of (3-4): (6-7). In the present disclosure, the ginseng-antler energy peptide includes pilose antler polypeptides. Sources of the pilose antler polypeptides are not particularly limited in the present disclosure, and the pilose antler polypeptides well known in the art may be used, for example, purchased through commercial purchase channels or extracted by extraction methods disclosed in the prior art. The efficacy of the pilose antler polypeptides is to improve learning and memory functions, exert anti inflammatory and anti-oxidant effects, improve immunity, and prevent and treat osteoporosis. In the present disclosure, the ginseng-antler energy peptide includes total ginsenosides. Sources of the total ginsenoside are not particularly limited in the present disclosure, as long as the sources of total ginsenosides are well-known in the art, for example, purchased through commercial purchase channels or extracted by extraction methods disclosed in the prior art. The efficacy of the total ginsenosides is to improve memory function, resist fatigue, boost immunity, and delay aging. In the present disclosure, the objective of delaying aging can be effectively achieved by mixing the pilose antler polypeptides and the total ginsenoside in a prescribed ratio. Preferably, the pilose antler polypeptides and the total ginsenoside may have a mass ratio of 1:1.8. Compared with mixtures obtained in other ratios, latencies of mice are significantly prolonged, error frequency is reduced, and cognitive ability of aging mice is significantly improved; the passive avoidance response ability of aging mice and the maximum load of the femur are significantly increased. At the same time, the present disclosure selects the pilose antler polypeptides from a plurality of APIs of the pilose antler, screens and obtains the total ginsenoside from a plurality of APIs of the ginseng, and uses both in combination. This not only clarifies the APIs in the prescription and improves the utilization rate of traditional Chinese medicine. Moreover, the preparation process can be partly simplified, and the production cost can be reduced.

The present disclosure provides a ginseng-antler energy peptide microcapsule having anti-aging effect, including a core material and a wall material, where the core material and the wall material have a mass ratio of 1:(6-10); the core material is the ginseng-antler energy peptide. In the present disclosure, preferably, the core material and the wall material may have a mass ratio of 1:8. The core material and wall material in a prescribed ratio may effectively improve the stability of the APIs of the drug, increase the slow-release efficiency and mask unpleasant odors. The present disclosure has no special restriction on the raw material type of the wall material, as long as the raw material type of the wall material of the microcapsule well known in the art may be used. Raw materials of the wall material may preferably include one or two of p-cyclodextrin, gelatin, chitosan, arabic gum, and carboxymethyl cellulose. The present disclosure provides a method for preparing the ginseng-antler energy peptide microcapsule, including the following steps: 1) under stirring, adding and dissolving fine powders of the ginseng-antler energy peptide in a saturated solution of a wall material 1 to obtain an intermediate solution; 2) sonicating the intermediate solution at 80-240 W for 40-100 min to obtain an encapsulation solution; and 3) adding a saturated solution of a wall material 2 to the encapsulation solution, stirring for 30 min, and freeze-drying to obtain ginseng-antler energy peptide microcapsules. In the present disclosure, the fine powders of the ginseng-antler energy peptide are added to the saturated solution of the wall material 1 under stirring and dissolved to obtain an intermediate solution. In the present disclosure, the fine powders of the ginseng-antler energy peptide may preferably pass through a 150-mesh sieve. When the wall material is preferably p-cyclodextrin and gelatin, the wall material 1 may preferably be p-cyclodextrin, and the saturated solution of the p-cyclodextrin may preferably have a concentration of 10 wt% (g/ml), which may preferably be dissolved at 60°C; the wall material 2 may preferably be gelatin, and the saturated solution of the gelatin may preferably have a concentration of 12 wt% (g/ml). The saturated solution of the p-cyclodextrin and the saturated solution of the gelatin may preferably have a volume ratio of 1:1. After the intermediate solution is obtained, the present disclosure sonicates the intermediate solution at 80-240 W for 40-100 min. to obtain an encapsulation solution. In the present disclosure, sonication is used to aggregate and deposit the wall material around the capsule core to form a capsule. In an embodiment of the present disclosure, a plurality of sets of sonication solutions are provided: sonication time 40 min, power 80 W; sonication time 70 min, power 150 W; sonication time 100 min, power 240 W; sonication time 40 min, power 150 W; sonication time 70 min, power 240 W; sonication time 100 min, power 80 W; sonication time 40 min, power 240 W; sonication time 70 min, power 80 W; sonication time 100 min, power 150 W; among them, the most preferred sonication power is 150W, and the sonication time is 70 min. After the encapsulation solution is obtained, the present disclosure adds the saturated solution of the wall material 2 to the encapsulation solution, stirs for 30 min, and freeze-dries to obtain ginseng antler energy peptide microcapsules. The present disclosure has no particular restriction on stirring methods, as long as manual or mechanical stirring well known in the art may be used, for example, a magnetic stirrer is used. The stirring causes the wall material to condense and deposit around the capsule core to form a capsule again, increasing the embedding rate. An objective of adding the wall material in twice in the present disclosure is to subject primarily encapsulated drugs and uncapsulated drugs to secondary encapsulation to increase the encapsulation rate of the drugs. If the two wall materials are sonicated once, similar results may not be obtained. In the present disclosure, the freeze-drying may preferably be to refrigerate for 48 h at 4°C, and pre-freeze overnight at -80°C. After freeze-drying, grinding a dried product may be further included to obtain microcapsules. A specific process flow of microcapsule preparation is shown in FIG. 1. The morphology of the microcapsules prepared is shown in FIG. 2. The microcapsules are regularly spherical or quasi-spherical in morphology. In the present disclosure, liquid chromatograph is used to verify the encapsulation efficiency and quality standards of the microcapsules prepared, according to the results, RSD is 1.29% for ginsenoside Rgl and 1.07% for ginsenoside Rd, indicating that the instrument has good precision; the method has good reproducibility, and the sample is stable within 24 h. The spike-and-recovery test results show that average recovery of ginsenoside is 97.1%, and that of ginsenoside Rd is 95.8%, indicating that the method has good accuracy. The present disclosure provides use of the ginseng-antler energy peptide or the ginseng-antler energy peptide microcapsule in the preparation of anti-aging health products. In the present disclosure, the ginseng-antler energy peptide product may be added with commonly used excipients to prepare various forms of acceptable medicaments or health products, including tablets, granules, capsules, pills, oral liquids, liquid beverages, and solid beverages; moreover, the oral decoctions, tablets, capsules or granules may all be prepared by conventional preparation methods of corresponding kinds of preparations. The present disclosure provides a ginseng-antler energy peptide dripping pill having anti-aging effect, including the ginseng-antler energy peptide microcapsule or the ginseng-antler energy peptide microcapsule prepared by the preparation method. In the present disclosure, the ginseng-antler energy peptide microcapsules and excipients of the dropping pill may preferably have a mass ratio of 1:(1-4), and more preferably 1:4. A process flow of dropping pill preparation is shown in FIG. 3. A method for preparing the ginseng-antler energy peptide dropping pill is preferably as follows:

A. melting and mixing the excipients well at 70°C, adding ground ginseng-antler energy peptide microcapsule powders while stirring, and after complete dissolution, letting the powders stand for 30 min to remove bubbles and obtain a molten liquid; B. dropping the molten liquid into 4°C liquid paraffin, cooling to mold, taking out, and wiping out excess liquid paraffin with filter paper; and C. drying to obtain ginseng-antler energy peptide dropping pills. The present disclosure has no particular restrictions on the types of the excipients, as long as the excipients well known in the art may be used. In an embodiment of the present disclosure, the excipients may be a mixture of PEG4000 and PEG6000. The mass ratio of the PEG4000 to the PEG6000 may be preferably 1:3. The ratio of the mass of ginseng-antler energy peptide microcapsule powders to the volume mass of excipients may be 1 g: 4 g. Dropping pills may be more suitable to carry around. In addition, the dripping pills may be given sublingually. The APIs of the medicament may be directly absorbed into the blood through the human mucosa, quickly participate in the human body's circulation, and act quickly in a short time, completely avoiding the shortcomings of low bioavailability, inconvenient administration, and slow acting of traditional Chinese medicine. The ginseng-antler energy peptide with anti-aging effect, the microcapsule, and the preparation method and use thereof provided by the present disclosure will be described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present disclosure. Embodiment 1 Extraction method of crude extract of pilose antler polypeptides 1. Pre-processing of pilose antler A fresh pilose antler was taken from a sika deer, sliced into small pieces of about 1 cm under cryogenic conditions, and stored at -20°C for later use. 2. Crude extraction of pilose antler polypeptides (1) One hundred grams (100 g) of pilose antler was quickly washed with distilled water at about 4°C until the blood was removed, and stored in the fresh-keeping layer of a refrigerator at 4°C for later use. (2) Acetic acid-sodium acetate buffer (pH 4.0) was prepared respectively and stored in the refrigerator at 4°C for later use. (3) Well-washed pilose antler slices were taken out and homogenized with a colloid (washed with acetate buffer) to obtain a homogenate; the homogenate was stored in the refrigerator at 4°C, stirred intermittently for 6 h every 20 min, and left to stand overnight. (4) After standing overnight, a colloidal pilose antler was taken out and centrifuged in a high speed refrigerated centrifuge (at 8,500 r-min' for 20 min). (5) After centrifugation, supernatant was collected and diluted with 90% ethanol aqueous solution to make a final concentration of the supernatant reach 65%. The supernatant was left to stand at 4°C, stirred for 6 h (for 20 min each), and left to stand overnight. (6) After a second centrifugation (at 8,500 r-min-' for 20 min), the lower layer of precipitates was collected and freeze-dried to obtain a crude extract of pilose antler polypeptides, which was stored at -80 0 C. 3. Protein assay Protein concentration of pilose antler polypeptides was assayed by Coomassie Brilliant Blue G 250 method: 1.00 g of pilose antler polypeptides powder was accurately weighed, dissolved in 1 mL of distilled water, and then detected by a microplate reader. The protein concentration was determined by the Easy Protein Quantitative Kit. The specific method was as follows: after Coomassie Brilliant Blue was left to stand at room temperature for 20 min, the microplate reader was preheated; 0, 1, 5, 7, 9, and 10 L of bovine serum albumin standard solutions (0.22 mg/mL) were placed in a 96-well plate, and water was added to make up 10 pL; 100 L of Coomassie Brilliant Blue stain was added to each well and shaken in a shaker for 10 min. Optical density (OD) values of different concentrations of protein samples were measured at 595 nm. A protein standard curve was calculated as follows: y=0.7511x+0.1668, R2=0.9992 (y-axis is OD value, and x-axis is concentration C). The protein standard curve is shown in FIG. 4. The detected protein concentration was 11.928 mg/ml. Embodiment 2 Preparation method of total ginsenosides: A ginseng was sliced into thick slices and decocted with water twice (the first time is 2 h, and the second time is 1.5 h); decoctions were filtered, and filtrates were combined; the combined filtrate was eluted to colorless through D101 macroporous adsorption resin columns and eluted with 60% ethanol aqueous solution; 60% ethanol eluent was collected and the filtrate concentrated to a clear paste with a relative density of 1.06 to 1.08 (800 C); the clear paste was dried and pulverized. The process and process parameters are shown in Table 1.

Table 1 Process and process parameters Process Process parameter Raw material quality Certification Pretreatment Sorting Remove foreign bodies Drug moistening and Moisten thoroughly and slice into 2-4 m thick slices slicing In the first extraction, add water having an amount Water addition of 15 times as much as that of ginseng Extraction In the second extraction, add water having an amount of 12 times as much as that of ginseng Decocting time in water The first decoction lasts for 2 h The second decoction lasts for 1.5 h Sample loading and Resin type D101 macroporous adsorption resin adsorption Flow rate of sample 2-4 BV/h loading Ethanol (analytical grade) 60% concentration Elution Addition amount of 60% 5,00010 kg ethanol Flow rate of analysis 2-4 BV/h Filtration 0.22 m microporous membrane Filtration and Vacuum degree -0.02 to -0.06 MPa concentration Temperature 75-85 0 C Specific gravity of the 1.06-1.08 (80°C) concentrated solution Drying temperature 95-105 0 C Vacuum drying Vacuum degree -0.06 to 0.08 MPa Pulverization Particle size If >95%, pass through a 120 mesh sieve Sieving Sieving No impurity Batch mixing Batch mixing Homogeneous and uniform in color Inner packaging Weight 1 kg/bag or 25 kg/bag Embodiment 3 1 Method for preparing ginseng-antler energy peptide (SN) microcapsules Step A: Pilose antler polypeptides and total ginsenoside were ground into fine powders by parts by weight, passed through a 150-mesh sieve, and mixed evenly; Step B: A given amount of p-cyclodextrin was prepared into a 10% (1 g: 10 ml) solution, stirred and dissolved into a transparent saturated solution in a 60°C water bath; Step C: While stirring, a mixed powder of pilose antler polypeptides and total ginsenoside was added thereto until completely dissolved; Step D: The mixture was transferred to an ultrasonic cleaner and sonicated at a given power and a given temperature, so as to make wall materials condense and deposit around the capsule core to form a capsule; Step E: The mixture was transferred to a magnetic stirrer and mixed with a 12% (1 g: 12 ml) gelatin saturated solution equal in volume to p-cyclodextrin, and stirred at a given temperature, so as to make the wall material condense again and deposit around the capsule core to form a capsule, increasing the embedding rate; Step F: The mixture was refrigerated for 48 h at 4°C, pre-frozen overnight at -80°C, and freeze driedinafreezedryer;dried product was ground to obtain ginseng-antler energy peptide microcapsules. 2 Orthogonal experimental design of ginseng-antler energy peptide (SN) microcapsule According to the nature of SN and the results of preliminary screening, factors to be investigated in the orthogonal experimental design are determined as the core-wall weight ratio (A), sonication time (B), ultrasonic power (C), and frequency of sonication (D); with encapsulation rates of ginsenosides Rg l and Rd as indicators, an L9 (3 x3) orthogonal array was used. The factors and levels are shown in Table 2.

Table 2 Factor level table Core-wall weight Sonication time Ultrasonic power Frequency of ratio (min) (W) sonication (D) 1 1:6 40 80 1 2 1:8 70 150 2 3 1:10 100 240 3 A total of nine parts of pilose antler polypeptides and total ginsenoside powder were taken according to the ratio and tested according to the orthogonal array. The specific tests were arranged as follows: Sample 1: p-cyclodextrin 3-fold, sonication time 40 min, power 80 W, sonicating once, gelatin 3-fold, magnetic stirring for 30 min. Sample 2: p-cyclodextrin 3-fold, sonication time 70 min, power 150 W, sonicating twice, gelatin 3-fold, magnetic stirring for 30 min. Sample 3: p-cyclodextrin 3-fold, sonication time 100 min, power 240 W, sonicating thrice, gelatin 3-fold, magnetic stirring for 30 min. Sample 4: p-cyclodextrin 4-fold, sonication time 40 min, power 150 W, sonicating thrice, gelatin 4-fold, magnetic stirring for 30 min. Sample 5: p-cyclodextrin 4-fold, sonication time 70 min, power 240 W, sonicating once, gelatin 4-fold, magnetic stirring for 30 min. Sample 6: p-cyclodextrin 4-fold, sonication time 100 min, power 80 W, sonicating twice, gelatin 4-fold, magnetic stirring for 30 min. Sample 7: p-cyclodextrin 5-fold, sonication time 40 min, power 240 W, sonicating twice, gelatin 5-fold, magnetic stirring for 30 min. Sample 8: p-cyclodextrin 5-fold, sonication time 70 min, power 80 W, sonicating thrice, gelatin 5-fold, magnetic stirring for 30 min. Sample 9: p-cyclodextrin 5-fold, sonication time 100 min, power 150 W, sonicating once, gelatin 5-fold, magnetic stirring for 30 min. The orthogonal results are shown in Table 3.

Table 3 Orthogonal results A B C D Frequency of sonication Encapsulation efficiency (Fold) Time (h) Power (W) (time) (%) 1 6 40 80 1 51.02342969 2 6 70 150 2 58.45238399 3 6 100 240 3 76.09083191 4 8 40 150 3 94.80914534 5 8 70 240 1 92.00146876 6 8 100 80 2 60.73825827 7 10 40 240 2 61.941992

8 10 70 80 3 79.15656324 9 10 100 150 1 85.16628101 185.56664 207.77456 190.91825 228.1911795 I 56 7 12 247.54887 229.61041 238.42781 181.1326343 II 24 6 03 226.26483 221.99537 230.03429 250.0565405 III 62 12 27 I 61.856 69.258 63.639 76.064 82.516 76.537 79.476 60.378 III 75.422 73.998 76.678 83.352 R 20.661 7.279 15.837 22.975 34434.979 43170.270 36449.778 95 71 64 52071.21438 61280.444 52720.943 56847.820 21 13 74 32809.03119 51195.776 49281.944 52915.775 12 82 8 62528.27344 48970.400 48391.052 48737.791 1 89 73 49136.17301 Sum of squares of deviations 661.239 81.892 428.630 827.012 Variance 330.619 40.946 214.315 413.506 F 8.075 1.000 5.234 10.099

Conclusion According to the orthogonal experimental results, i.e., D>A>C>B, A2>A3>A1, B2>B3>B1, C2>C3>C1, and D3>D1>D2, the best process is: 1:8 core-wall weight ratio, sonication time 70 min, ultrasonic power 150 W, magnetic stirring for 30 min. For saving the time and cost, sonication is considered to conduct once. 3. Process verification test Three batches of SN microcapsules were prepared according to the best plan, and the encapsulation efficiency of ginsenosides was detected by high performance liquid chromatography (HPLC). Formula I was substituted to obtain drug loading, and Formula II was substituted to obtain encapsulation efficiency (based on ginsenoside Rgl and ginsenoside Rd). Drug loading = the amount of drug contained in the microcapsule/total amount of the microcapsules * 100% Formula I Encapsulation efficiency = the amount of encapsulated drugs in the system/the total amount of encapsulated and unencapsulated drugs in the system * 100% Formula II The results showed that the average drug loading of the three batches of samples was (86.440.001)%, and the average encapsulation efficiency was (91.09+0.49)%. This shows that this preparation method is stable and feasible. 4. Determination of the potential and morphology of SN

Two hundred milligrams (200 mg) of SN microcapsules weighed, dissolved in 1 mL of distilled water solution for vortex mixing, and centrifuged to collect a supernatant; afield emission scanning electron microscope was used to investigate the surface morphology of micelles. The sample was mounted on the surface of short columns and sputter-coated with gold-palladium in an argon atmosphere. Accelerating voltage used was 3 kV, and the shape was regularly spherical or quasi spherical, as shown in FIG. 2. Embodiment 4 Method for preparing ginseng-antler energy peptide dropping pills Separately, 3.6 g of pilose antler polypeptides and 6.4 g of total ginsenoside weighed and ground into fine powders, passed through a 150-mesh sieve, and mixed well; 40 g of p-cyclodextrin was prepared into a 10% (1 g: 10 ml) solution, stirred and dissolved into a transparent saturated solution in a 60°C water bath; while stirring, a mixed powder of pilose antler polypeptides and total ginsenoside was added thereto until completely dissolved. The mixture was transferred to an ultrasonic cleaner and sonicated at 150 W for 70 min. The mixture was transferred to a magnetic stirrer and mixed with 40 g of 12% (1 g: 12 ml) gelatin saturated solution, stirred for 30 min, and taken out. The mixture was refrigerated for 48 h at 4°C, pre-frozen overnight at -80°C, and freeze driedinafreezedryer;dried product was ground to obtain ginseng-antler energy peptide microcapsules. Ten grams (10 g) of PEG4000 and 30 g of PEG6000 were weighed, molten and mixed in a 70°C water bath; the ginseng-antler energy peptide microcapsules prepared above were added while stirring; after complete dissolution, the mixture was left to stand for 30 min to remove bubbles; a dropper was used to aspirate the molten liquid and drip to liquid paraffin. The distance between the dropper and the liquid paraffin was 10 cm; condensing temperature was 5°C, and drip rate was 50 drops/min. After cooling and molding, droplets were taken out, and excess liquid paraffin was wiped out with filter paper, following by drying in an oven at 50°C to obtain ginseng-antler energy peptide dropping pills. Embodiment 5 The determination method and quality standard of the encapsulation efficiency of the SN microcapsules prepared in Embodiment 4 1. Chromatographic conditions and system suitability test Chromatographic column was Zorbax SB-C18 (4.6*250 mm, 5 m); flow rate was 0.4 mL-min 1; column temperature was 30°C; detection wavelength was 203 nm; injection volume was 5 L; mobile phase was acetonitrile: water was used for gradient elution. Elution program is shown in Table 4.

Table 4 Elution program Time (min) Acetonitrile (%)

0 15 5 19 10 19 13 25 15 28 18 28 22 30 25 32 27 33 30 35 35 55 38 80 40 100 45 100 2. Preparation of reference solution Appropriate amounts of ginsenoside Rgl reference standard and ginsenoside Rd reference standard were accurately weighed, and mixed with methanol to prepare a mixed solution containing 0.026 mg of ginsenoside Rgl and 0.026 mg of ginsenoside Rd in 1 ml. 3. Preparation of test solution One hundred and fifty milligrams (150 mg) of this product was accurately weighed, put in a 10 ml volumetric flask, dissolved in methanol ultrasonically to and diluted to volume; after filtration, subsequent filtrate was collected. Negative sample solutions were prepared in the same way. 4. Assay Precisely, 5 L each of the reference solution and the test solution was pipetted and injected into a liquid chromatograph for measurement. 5. Methodological study 5.1 Establishment of standard curves Precisely, 0, 1, 3, 4, 5, 70 L of SN reference solutions were pipetted and measured according to the above chromatographic conditions. Regression equations were obtained using peak area (y) as the ordinate and SN concentration (x) as the abscissa: y=105535x+0.02 for ginsenoside Rgl and y=92020x-0.0938 for ginsenoside Rd. The results showed that ginsenoside Rgl of SN had a good linear relationship in the range between 0 and 0.00183 mg-mL-', as shown in FIG. 5; ginsenoside Rd had a good linear relationship in the range between 0 and 0.0039 mg-mL-1, as shown in FIG. 6. 5.2 Precision test An SN sample was prepared into a test solution and determined according to the above chromatographic conditions, with each injection of 5 L; six injections were repeated. Measurement results showed that: RSD was 1.29% for ginsenoside Rgl and 1.07% for ginsenoside Rd, indicating that the instrument has good precision. 5.3 Reproducibility test

Six SB samples were prepared into test solutions and measured according to the above chromatographic conditions. Each injection volume was 5 L. Measurement results showed that: RSD was 1.51% for ginsenoside Rgl and 1.03% for ginsenoside Rd, indicating that the method has good reproducibility. 5.4 Stability test An SN sample was prepare into a test solution and measured according to the above chromatographic conditions. Each injection volume was 5 L. Assays were conducted at 0, 2, 4, 8, 12, and 24 h, respectively. Results showed that: RSD was 3.02% for ginsenoside Rgl and 3.25% for ginsenoside Rd, indicating that the sample is stable within 24 h. 5.5 Spike-and-recovery test An appropriate amount of reference substance was precisely added to an SN sample with a known content to prepare a test solution, and determination was performed according to the chromatographic conditions under item 1 of Embodiment 3. Each injection volume was 5 L. Results showed that the average recovery was 97.1% for ginsenosides and 95.8% for ginsenosides Rd, indicating that the method has good accuracy. 5.6 Determination results of ginsenoside Rgl and ginsenoside Rd in SN Three SN samples were prepared; 20 mg of the sample was accurately weighed, dissolved in 10 mL of methanol ultrasonically, and filtered with a 0.22 m microporous membrane. After the sample preparation was completed, HPLC was carried out. The average total content of ginsenoside Rgl and ginsenoside Rd in SN was measured to be 0.44 mg/g. Embodiment 6 Determination of the protein concentration in the SN prepared in Embodiment 4 1. Plotting of standard curve Protein concentration was determined by the Easy Protein Quantitative Kit. The specific determination method was as follows: a microplate reader was preheated for 30 min, and Coomassie Brilliant Blue was left to stand for 20 min at room temperature; 0, 1, 5, 7, 9, and 10 L of bovine serum albumin standard solutions (0.22 mg/mL) were added to a 96-well plate, respectively, and distilled water was added to each well to make the volume up to 10 L; 100 L of Coomassie Brilliant Blue stain was added to each well and shaken on a shaker for 10 min. OD values of protein samples of difference groups were measured at 595 nm, and a protein standard curve was calculated as: y=0.919x+0.2346, R2=0.9907 (the y-axis is the concentration C, and the x-axis is the OD). The protein standard curve is shown in FIG. 7. 2. Preparation of test solution One hundred milligrams (100 mg) of SN sample was mixed with 1 mL of lysis buffer, lysed on ice for 1 h, and then centrifuged for 15 min at 14,000 rpm and 4°C; supernatant was collected, diluted 10-fold, and mixed well with 10 L of test solution and 100 L of Coomassie Brilliant Blue on a shaker for 5 min; OD values were measured at 595 nm by using a microplate reader and substituted into the standard curve to calculate the content. The protein content in SN was determined to be 16 pg/mg. 3. SDS-PAGE SDS-PAGE was used to observe the protein distribution in SN microcapsules. 3.1 Protein sample processing The protein loading volume per well was 20 g, Buffer and protein were mixed in a volume ratio of 4:1; the processed sample was placed in a water bath at 100°C and boiled for 10 min, centrifuged and then loaded. 3.2. SDS-PAGE Glass plates for making gel were washed and blown dry with a hair dryer, and a slab for making gel was assembled; 12% running gel was prepared and gelatinized for 30 min; after that, 5% stacking gel was prepared and gelatinized for 30 min. A gel slab was assembled in an electrophoresis tank, where running buffer was poured up; protein samples were loaded successively. Blank wells were filled up with the buffer. The upper gel was electrophoresed at a constant voltage of 90 V for 40 min, and the lower gel was electrophoresed at a constant voltage of 110 V for 120 min. 3.3 Staining and destaining After the electrophoresis is terminated, the gel was placed in a well-prepared Coomassie Brilliant Blue R250 stain solution, soaked in not less than 5 times the volume of the stain, placed on a slowly shaking shaker, and stained for 50 min at room temperature. After staining, the gel was immersed in a destaining solution (methanol: glacial acetic acid: water = 4:1:5, in a volume ratio) and shaken gently; the destaining solution was replaced at intervals to destain until clear bands were visible to the naked eye. After that, the gel was immersed in distilled water to stop destaining. 3.4 Gel imaging The gel was placed in a gel imager to observe test results and take pictures. The results are shown in FIG. 8. The results showed that the protein molecular weight of SN microcapsules ranged between 25 and 70 kDa. Embodiment 7 1. Experimental animals and grouping Sixty ICR mice (30 males and 30 females, provided by Changchun Yisi Laboratory Animal Technology Co. Ltd.) weighing (20 2) g were selected, and all animals were housed at (25 1)°C and relative humidity of 45%-50%. The mice were randomly divided into a blank group (K), a model group (M), a positive group (Y), and a ginseng-antler energy peptide group: the mass ratio of pilose antler polypeptides to total ginsenoside was 1:1 (SN1), 1:1.5 (SN2), 1:2.3 (SN3), and 1:2.8 (SN4), respectively. After the ICR mice were acclimatized for three days, the positive group and the ginseng-antler energy peptide group (SNI, SN2, SN3, and SN4 groups) were on prophylactic vitamin E (150 mg/kg); the ginseng-antler energy peptide group (SN1, SN2, SN3, and SN4 groups) was on 150 mg/kg pilose antler polypeptides for eight days. The blank group was given an equal volume of normal saline by gavage. After eight days, mice in the other groups, except for the blank group, were subcutaneously injected with D-galactose (1,000 mg/kg) at the scruff; the blank group was subcutaneously injected with normal saline for 60 days. The mice were re-weighed every 3 to 5 days, and the dosage was adjusted according to the body weight. 2. Behavioral experiments 2.1 Mouse step-through test Acclimation phase: On day 1, each mouse was placed into a brightly lit compartment in numerical order; a switch connecting a dark compartment was turned on, and the mouse was allowed to move freely in the brightly lit and dark compartments for about 2-3 min. After complete acclimation of apparatus, the mouse was put back into the cage and was ready for the next day's training. Learning phase: On day 2, the switch connecting the brightly lit and dark compartments were turned off and the mice was placed in the brightly lit compartment. After the mouse was placed well, an electric shock device was switched on while turning on the switch connecting the brightly lit and dark compartments. Because of skototaxis, the mouse scurried into the dark compartment, and the mouse was shocked by the electric shock device once it entered the dark compartment. Step-through latency of a mouse was defined as the time from the start of the test to the first electric shock, which was no more than 5 min. After the test, the total number of electric shocks received by each mouse in 5 min was recorded, i.e., error frequency in the step-through test. 2.2 Morris water maze test Place navigation task: A hidden platform was randomly placed in the middle of one of the four quadrants of the Morris water maze, which must not be above the water surface and be disposed 2 cm underwater. An appropriate amount of titanium dioxide was added into the Morris water maze and stirred well so that a mouse could be marked successfully. The mouse was placed in the Morris water maze from each of the four quadrants. When placed, the mouse's head faced the wall of the pool. After the start of the task, had the mouse found the platform, instrument would automatically record the time when the mouse found the platform, that is, latency to reach the platform, and timing would be stopped to end the task. During the first five days of learning and training, had the mouse not found the platform within 2 min, the mouse would be placed on the platform for 15-30 s, and the latency of the mouse that did not find the platform would be 120 s. After the task, the mouse was blow-dried and returned to the animal room. 2.3 Step-down test A step-down test was carried out 40 days after drug administration. A mouse step-down chamber was opened, a mouse was put into the step-down apparatus, and 32 V alternating current was applied.

The animal received an electric shock, and its avoidance response was to jump on the platform. A majority of animals would jump to a copper grid again or repeatedly and stepped down from the platform quickly after receiving electric shocks. The mice were trained for 5 min, and the number of electric shocks received by each mouse was recorded as learning performance (EN1). On day 41, the mice were directly placed on the platform in an energized state, and the time from the mouse being placed on the platform to the first step-down was recorded as the latency (LT); the mice would jump on the platform evasively after receiving an electric shock, and the frequency of the mouse stepping down again within 5 min (EN2) was recorded as the error frequency; both LT and EN2 were used as memory performance. 2.4 Determination of bone biomechanical properties A bone biomechanics tester was used to perform a three-point bending test at the Changchun University of Chinese Medicine R&D Center. The test femur was placed on the support of the biomechanics tester; gauze was soaked with normal saline and wrapped around the femur to make the convex surface downward, ensuring that the midpoint of the femur and the center of bilateral fulcrums, along with pressure points, were all in a straight line. The span between the two fulcrums (L) was 27 mm, the descending speed was 5.0 mm/min; while descending at even speed, a force was applied on the midpoint of the femur until the femur fractured. Maximum load born by the femur was determined according to the pressure sensor data. 3. Experimental results 3.1 Step-through test results The step-through test results showed that in the memory test on day 2, as shown in FIG. 9a: compared with the blank control group, the latencies of the mice in the model group were significantly shortened (P<0.01); compared with the model control group and the control group, the latencies of the mice in the positive group and the ginseng-antler energy peptide group were significantly prolonged (P<0.01), and the SN2 and SN3 groups were significantly better than the SNI and SN4 groups (P<0.05, P<0.01). As shown in FIG. 9b: the mice in the model group showed significantly higher error frequency within 5 min compared with the blank control group (P<0.01); the mice in the positive group and the ginseng-antler energy peptide group showed lower error frequency within 5 min compared with the model group and the control group (P<0.05). 3.2 Morris water maze The results of the place navigation task are shown in FIG. 10. As the training time increased, the swim distance to reach the platform gradually decreased for each group of mice. Compared with the blank control group, the mice in the model group increased the swim distance to reach the platform on day 5 of training (P<0.05). Compared with the model group, on day 5 of training, the mice in the ginseng-antler energy peptide group significantly shortened the latency to reach the platform (P<0.01). This suggests that the ginseng-antler energy peptide can improve the cognitive ability of

D-galactose-induced aging mice, and the SN2 and SN3 groups are significantly better than the SNI and SN4 groups (P<0.05, P<0.01). 3.3 Step-down test As shown in FIG. 11, the error frequency significantly increased and the latency significantly shortened in the model group compared with the blank control group (P<0.05). After learning for 5 min, the error frequency significantly decreased (P<0.05, P<0.01) and the latency increased in the positive group and the ginseng-antler energy peptide group compared with the model group. The results show that the ginseng-antler energy peptide group can improve the passive avoidance response ability of aging mice, and the SN2 and SN3 groups are significantly better than the SNI and SN4 groups (P<0.05, P<0.01). 3.4 The result of the effect of ginseng-antler energy peptide on bone biomechanics is shown in FIG. 12. Compared with the blank group, the maximum femoral load significantly decreased in the model group (P<O.01). Compared with the model group, the maximum femoral load increased in the ginseng-antler energy peptide group (P<0.05), and the SN2 and SN3 groups were significantly superior to the SNI and SN4 groups (P<0.05, P<0.01). This shows that the ginseng-antler energy peptide can improve the biomechanical properties of aging mice and delay bone aging. Compared with the blank control group, "P<0.05, ""P<0.01; compared with the model group, *P<0.05, **P<0.01; compared with the SN2 group, ^P<0.01, AAP<0.01; compared with the SN3 group, AP<0.01, "P<0.01. As can be seen from the above-mentioned embodiments, in the present disclosure, D-galactose is used to establish a subacute aging mouse model to observe effects of the ginseng-antler energy peptide on learning and memory ability in aging mice and bone biomechanical efficacy. Thus, anti aging effect of the ginseng-antler energy peptide and the microcapsule and dropping pill thereof is demonstrated. The foregoing descriptions are merely preferred embodiments of the present disclosure; it should be noted that several improvements and modifications can also be made by one of ordinary skill in the art without departing from the principles of the present disclosure, and these improvements and modifications should also be regarded as the protection scope of the present disclosure.

Claims (5)

1. What is claimed is: 1. A ginseng-antler energy peptide having an anti-aging effect, comprising pilose antler polypeptides and total ginsenoside; wherein the pilose antler polypeptides and the total ginsenoside have a mass ratio of (3-4): (6-7).
2. 2. The ginseng-antler energy peptide according to claim 1, wherein the pilose antler polypeptides and the total ginsenoside have a mass ratio of 1:1.8.
3. 3. A ginseng-antler energy peptide microcapsule having an anti-aging effect, comprising a core material and a wall material, wherein the core material and the wall material have a mass ratio of 1:(6-10); the core material is the ginseng-antler energy peptide according to claim 1 or 2; wherein the core material and the wall material have a mass ratio of 1:8; wherein raw materials of the wall material comprise one or two of p-cyclodextrin, gelatin, chitosan, arabic gum and carboxymethyl cellulose.
4. 4. A method for preparing the ginseng-antler energy peptide microcapsule according to claim 3, comprising the following steps: 1) under stirring, adding and dissolving fine powders of the ginseng-antler energy peptide according to claim 1 or 2 in a saturated solution of a wall material I to obtain an intermediate solution; 2) sonicating the intermediate solution at 80-240 W for 40-100 min to obtain an encapsulation solution; and 3) adding a saturated solution of a wall material 2 to the encapsulation solution, stirring for 30 min, and freeze-drying to obtain ginseng-antler energy peptide microcapsules; wherein when the wall material comprises p-cyclodextrin and gelatin, the wall material 1 is p cyclodextrin, and the saturated solution of p-cyclodextrin has a concentration of 10 wt%; the wall material 2 is gelatin, and the saturated solution of gelatin has a concentration of 12 wt%; the saturated solution of p-cyclodextrin and the saturated solution of gelatin have a volume ratio of 1:1; wherein the sonication is conducted once at 150 W for 70 min.
5. 5. A ginseng-antler energy peptide dropping pill having an anti-aging effect, comprising the ginseng-antler energy peptide microcapsule according to claim 3 or a ginseng-antler energy peptide microcapsule prepared by the preparation method according to claim 4.