A kidney-tonifying and anti-fatigue polypeptide composition and a preparation method thereof

Abstract

The application provides a kidney-tonifying and anti-fatigue polypeptide composition and a preparation method thereof, belongs to the technical field of medicinal biological peptide complexes, and the composition comprises oyster polypeptide, holothurian polypeptide, spider silk and blue ears grass-pentamerous fruit composite extract, cistanche-dendrobium officinale composite extract, ginseng extract and deer penis extract; the oyster polypeptide is a common enzymolysis product of oyster meat by trypsin and bovine chymotrypsin; the holothurian polypeptide is a common enzymolysis product of holothurian body wall by alkaline protease, collagenase type II and subtilisin; the spider silk and blue ears grass-pentamerous fruit composite extract is a product obtained by the following steps: subjecting spider silk and blue ears grass and pentamerous fruit to enzymolysis, extracting with an ethanol aqueous solution, and purifying with a SA sulfonic acid silica gel strong cation exchange column; the cistanche-dendrobium officinale composite extract is a product obtained by the following steps: subjecting cistanche and dendrobium officinale to enzymolysis, extracting with an ethanol aqueous solution, and purifying with a phenylboronic acid affinity column-polyamide column series connection column; and the composition realizes the synergistic effect of kidney tonification and anti-fatigue.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicinal biopeptide complexes, and particularly relates to a kidney-tonifying and fatigue-resisting polypeptide composition and a preparation method thereof. BACKGROUND

[0002] Kidney-tonifying and fatigue-resisting drugs are a cross field of traditional medical theory and modern biological medicine technology, and the core goal is to improve the fatigue syndrome caused by kidney essence deficiency, qi and blood deficiency or metabolic imbalance by regulating physiological functions of the body, while enhancing the functional activity of organs such as the kidney.

[0003] Traditional Chinese medicine believes that "the kidney is the root of preheaven", and fatigue is closely related to kidney essence deficiency and poor blood flow. Therefore, the development of kidney-tonifying and fatigue-resisting drugs based on traditional Chinese medicine is an important branch. Modern technology promotes the modern application of traditional prescriptions through the extraction of effective components of traditional Chinese medicine, the optimization of compound prescriptions, and the innovation of dosage forms. For example, the active ingredients in ginseng, deer antler and medlar are extracted by supercritical extraction and membrane separation technology to improve the bioavailability of drugs; the classic prescription is made into capsules, oral solutions and other dosage forms, taking into account the efficacy and convenience of taking.

[0004] Among them, biopeptides are small molecular compounds composed of amino acids connected by peptide bonds. Due to their small molecular weight, easy absorption and high biological activity, they have unique advantages in the field of kidney-tonifying and fatigue-resisting. For example, collagen peptides extracted from animal kidneys, bones and other tissues contain rich glycine, proline and other amino acids, which can promote kidney tissue repair and improve kidney function; they can regulate kidney microcirculation, enhance glomerular filtration function, and relieve symptoms such as soreness of the waist and knees caused by kidney essence deficiency. For example, small molecular peptides prepared by enzymatic hydrolysis of soybean protein have plant estrogen-like effects, which can regulate endocrine and improve symptoms of kidney deficiency caused by hormone imbalance; at the same time, they can promote protein synthesis and provide nutritional support for the kidney. Small molecular peptides produced by hydrolysis of whey protein can be quickly absorbed by the human body, providing energy for muscles and tissues, reducing lactic acid accumulation after exercise, and relieving physical fatigue; in addition, branched chain amino acids (leucine, isoleucine, etc.) contained in them can promote protein synthesis and enhance the body's endurance. Active peptides extracted from marine organisms such as sea cucumbers and abalones have antioxidant and energy metabolism regulating effects. For example, sea cucumber peptides can scavenge free radicals, protect cells from oxidative damage, promote glycolysis and aerobic respiration, improve the body's energy supply efficiency, and relieve mental fatigue and physical overexertion.

[0005] The application of peptide components in the field of kidney-tonifying and fatigue-resisting breaks through the limitations of traditional drugs with poor absorption and obvious side effects. Its advantages are: small molecular weight, can be directly absorbed by the intestinal tract, high bioavailability; strong specificity, can accurately act on target organs or metabolic pathways; high safety, peptides derived from natural substances usually have low toxicity, suitable for long-term use.

[0006] At present, the related technical research is mainly concentrated in the efficient extraction of peptides (such as optimization of enzymatic process), structural modification (enhancing stability), compound compatibility (synergistic effect with Chinese medicine ingredients) and the like. For example, the ginseng peptide is compounded with the marine biological peptide, which can not only play the kidney tonifying effect, but also enhance the anti-fatigue effect, and form the synergistic effect of "treating both the symptoms and the root cause". However, at present, the peptide components on the market are various, the bioavailability is low, the improvement effect is uneven, and the extraction technology and compound compatibility of the product still need to be continuously developed and improved to achieve more efficient treatment effect. SUMMARY

[0007] In view of the problems of low bioavailability, low bioactivity of peptide extraction, unreasonable component compatibility and slow improvement process of the existing kidney tonifying and anti-fatigue polypeptide products, the present application provides a kidney tonifying and anti-fatigue polypeptide composition and a preparation method thereof. The oyster polypeptide, the holothurian polypeptide, the spider silk and blue eared grass-pentamerous fruit composite extract, the cistanche-dendrobium officinale composite extract are prepared by a special method, and then reasonably compounded with ginseng extract and deer penis extract to form synergy from the aspects of "energy supply" and "damage repair". The plant source extract and the animal peptide form the bidirectional support of "kidney tonifying-anti-fatigue", and together realize the synergistic effect of "fast-acting anti-fatigue + long-acting kidney tonifying", effectively improve the treatment effect, and have high safety. The specific technical scheme is as follows:

[0008] A kidney tonifying and anti-fatigue polypeptide composition comprises the following raw materials in mass fraction: 25-30 parts of oyster polypeptide, 20-25 parts of holothurian polypeptide, 15-20 parts of spider silk and blue eared grass-pentamerous fruit composite extract, 18-22 parts of cistanche-dendrobium officinale composite extract, 8-10 parts of ginseng extract and 5-8 parts of deer penis extract. The oyster polypeptide is a product obtained by jointly enzymolysis of oyster meat by trypsin and bovine chymotrypsin; the holothurian polypeptide is a product below 5kDa obtained by jointly enzymolysis of holothurian body wall by alkaline protease, then by collagenase type II and subtilisin; the spider silk and blue eared grass-pentamerous fruit composite extract is a product obtained by jointly enzymolysis of the mixture of spider silk and blue eared grass and pentamerous fruit in a mass ratio of (3-5):(1-3) by cellulase, pectinase and beta-glucosidase, then by ultrasonic extraction by ethanol aqueous solution, and purification by SA sulfonic acid silica gel strong cation exchange column; and the cistanche-dendrobium officinale composite extract is a product obtained by jointly enzymolysis of the mixture of cistanche and dendrobium officinale in a mass ratio of (2-3):(0.5-1) by hemicellulase and beta-glucosidase, then by ultrasonic extraction by ethanol aqueous solution, and purification by benzene boronic acid affinity column-polyamide column series connection column.

[0009] The preparation method of the oyster polypeptide comprises the following steps: crushing oyster meat into paste, adding deionized water, adding trypsin and chymotrypsin, adjusting pH to 7.8-8.5, and enzymolysis at 36-40℃ for 3-4h; deactivating the enzyme, reducing the temperature to room temperature, centrifuging, taking the supernatant, and freeze-drying to obtain the oyster polypeptide.

[0010] The preparation method of the oyster polypeptide comprises the following steps: crushing oyster meat into paste, adding deionized water, adding trypsin and chymotrypsin, adjusting pH to 7.8-8.5, and enzymolysis at 36-40℃ for 3-4h; deactivating the enzyme, reducing the temperature to room temperature, centrifuging, taking the supernatant, and freeze-drying to obtain the oyster polypeptide.

[0011] The preparation method of the oyster polypeptide comprises the following steps: crushing oyster meat into paste, adding deionized water, adding trypsin and chymotrypsin, adjusting pH to 7.8-8.5, and enzymolysis at 36-40℃ for 3-4h; deactivating the enzyme, reducing the temperature to room temperature, centrifuging, taking the supernatant, and freeze-drying to obtain the oyster polypeptide.

[0012] The preparation method of the oyster polypeptide comprises the following steps: crushing oyster meat into paste, adding deionized water, adding trypsin and chymotrypsin, adjusting pH to 7.8-8.5, and enzymolysis at 36-40℃ for 3-4h; deactivating the enzyme, reducing the temperature to room temperature, centrifuging, taking the supernatant, and freeze-drying to obtain the oyster polypeptide.

[0013] The preparation method of the above-mentioned spider silk blue-ear grass-pentacene composite extract includes the following steps: mixing spider silk blue-ear grass powder and pentacene pulp at a mass ratio of (3-5):(1-3) to obtain a mixture, adding deionized water, adding cellulase, pectinase and β-glucosidase, adjusting pH to 4.8-5.0, enzymolysis at 40-45°C for 3-4h, enzyme inactivation, cooling to room temperature, and obtaining an enzymolysis liquid; adding an ethanol aqueous solution, ultrasonic treatment, centrifugation, taking the supernatant, vacuum concentration, obtaining a concentrated liquid, loading the concentrated liquid onto a SA sulfonic silica gel strong cation exchange column, first washing away impurities with 20-25% volume concentration ethanol aqueous solution for 1.5-2BV, then eluting with 50-60% volume concentration ethanol aqueous solution for 2-3BV, collecting the eluate, vacuum concentration, and drying to obtain the spider silk blue-ear grass-pentacene composite extract.

[0014] The preparation method of the above-mentioned spider silk blue-ear grass-pentacene composite extract includes the following steps: taking dried whole plants of spider silk blue-ear grass, crushing through a 40-60 mesh sieve to obtain spider silk blue-ear grass powder; taking the pulp of mature pentacene, beating, adding 0.1-0.3% ascorbic acid by mass of the pulp, and stirring uniformly to obtain pentacene pulp; mixing the spider silk blue-ear grass powder and the pentacene pulp at a mass ratio of (3-5):(1-3) to obtain a mixture, adding deionized water to the mixture at a solid-liquid mass ratio of 1:(5-6), adding 0.5-1.5wt% cellulase, 0.5-1.5wt% pectinase and 0.5-1.5wt% β-glucosidase based on the mass of the mixture, adjusting pH to 4.8-5.0, enzymolysis at 40-45°C for 3-4h, enzyme inactivation at 80-90°C for 15-20min, cooling to room temperature, and obtaining an enzymolysis liquid; adding an ethanol aqueous solution with a volume concentration of 60-70% to the enzymolysis liquid at 2-3 times the volume of the enzymolysis liquid, ultrasonic treatment at 40-45kHz for 30-60min, centrifugation at 8000-10000r / min for 10-20min, taking the supernatant, vacuum concentration at 50-60°C to 10-20% of the volume, obtaining a concentrated liquid, loading the concentrated liquid onto a SA sulfonic silica gel strong cation exchange column, first washing away impurities with 20-25% volume concentration ethanol aqueous solution for 1.5-2BV, then eluting with 50-60% volume concentration ethanol aqueous solution for 2-3BV, collecting the eluate, vacuum concentration at 50-60°C, and drying at 50-60°C to a water content of less than 5wt% to obtain the spider silk blue-ear grass-pentacene composite extract.

[0015] The preparation method of the Cistanche- Dendrobium compound extract includes the following steps: mixing Cistanche powder and Dendrobium powder according to the mass ratio (2-3) :(0.5-1), obtaining a compound, adding deionized water, adding hemicellulase and β-glucosidase, adjusting pH 5.0-5.5, enzymolysis at 50-55℃ for 3-5h, enzyme inactivation, and cooling to room temperature to obtain an enzymolysis solution; adding an ethanol aqueous solution, ultrasonic, centrifugation, taking the supernatant, and reducing pressure concentration to obtain a concentrated solution; loading the concentrated solution onto a phenylboronic acid affinity column-polyamide column series column; first washing with deionized water 1.5-2BV to remove impurities; then eluting with 0.1-0.15mol / L NH4HCO3 aqueous solution 2-3BV, and then eluting with 0.1-0.15mol / L acetic acid aqueous solution 2-3BV; combining the eluate, reducing pressure concentration, and drying to obtain the Cistanche-Dendrobium compound extract.

[0016] The preparation method of the Cistanche-Dendrobium compound extract includes the following steps: crushing Cistanche dry product to pass through a 60-80 mesh sieve to obtain Cistanche powder; crushing Dendrobium dry product to pass through a 60-80 mesh sieve to obtain Dendrobium powder; mixing the Cistanche powder and the Dendrobium powder according to the mass ratio (2-3) :(0.5-1) to obtain a compound; adding deionized water to the compound according to the material-liquid mass ratio 1 :(6-8), adding 1-2wt% hemicellulase and 1-2wt% β-glucosidase based on the mass of the compound, adjusting pH 5.0-5.5, enzymolysis at 50-55℃ for 3-5h, enzyme inactivation at 80-90℃ for 15-20min, cooling to room temperature, and obtaining an enzymolysis solution; adding an ethanol aqueous solution with a concentration of 60-70% by volume in an amount of 2-3 times the volume of the enzymolysis solution, ultrasonic at 40-45kHz for 30-60min, centrifugation at 8000-10000r / min for 10-20min, taking the supernatant, reducing pressure concentration at 50-60℃ to 20-30% of the volume, and obtaining a concentrated solution; loading the concentrated solution onto a phenylboronic acid affinity column-polyamide column series column; first washing with deionized water 1.5-2BV to remove impurities; then eluting with 0.1-0.15mol / L NH4HCO3 aqueous solution 2-3BV, and then eluting with 0.1-0.15mol / L acetic acid aqueous solution 2-3BV; combining the eluate, reducing pressure concentration at 50-60℃, and drying at 50-60℃ until the water content is less than 5wt% to obtain the Cistanche-Dendrobium compound extract.

[0017] The preparation method of the one kind of kidney-tonifying and anti-fatigue polypeptide composition includes: uniformly mixing oyster polypeptide, sea cucumber polypeptide, spider silk hair bluegrass-pentamerous fruit compound extract, Cistanche-Dendrobium compound extract, ginseng extract, and deer penis extract according to mass parts to obtain a composition; mixing the composition with a pharmaceutically acceptable excipient to prepare an oral preparation, and the oral preparation includes capsules, granules, and tablets.

[0018] The present invention provides a kidney-tonifying and fatigue-relieving polypeptide composition and its preparation method, the beneficial effects of which include:

[0019] I. Complementary Functions of Core Components: Oyster peptides and sea cucumber peptides, as animal-derived bioactive peptides, function by regulating energy metabolism and providing antioxidant effects, respectively. Oyster peptides are rich in amino acids such as glycine and arginine, which can activate the AMPK energy-sensing pathway and promote ATP production in muscle cells; the hydrophobic bioactive peptides in sea cucumber peptides can scavenge free radicals and reduce cellular oxidative damage. The two work synergistically from the perspectives of "energy supply" and "damage repair." Plant-derived extracts and animal peptides provide dual support for "kidney tonification and anti-fatigue." The active ingredients in the *Solanum nigrum*-*Dendrobium nobile* compound extract can scavenge free radicals generated during exercise, and its organic acids can regulate intestinal pH to promote peptide absorption; the echinacoside and *Dendrobium nobile* polysaccharides in the *Cistanche deserticola*-*Dendrobium officinale* compound extract can repair kidney function by activating the renal SIRT1 pathway and inhibiting the release of inflammatory factors, working together with animal peptides to achieve a synergistic effect of "rapid anti-fatigue + long-lasting kidney tonification."

[0020] II. Scientific Design of Ingredient Ratios: The optimized ratios of each ingredient avoid "component antagonism." For example, an imbalance in the ratio of *Symplocos buergeriana*-*Dalbergia odorifera* compound extract to *Cistanche deserticola*-*Dendrobium officinale* compound extract can lead to decreased antioxidant capacity or hindered peptide absorption. A reasonable ratio ensures that the former's absorption-promoting effect complements the latter's kidney-tonifying activity. The addition of ginseng extract and deer antler extract further enhances the synergistic effect of animal peptides and plant extracts by strengthening endocrine regulation.

[0021] III. Synergistic Effect of Enzymatic Hydrolysis: Oyster polypeptides are hydrolyzed by a combination of trypsin and bovine chymotrypsin. The former cleaves the carboxyl-terminal peptide bonds of lysine and arginine, while the latter targets the amino-terminal peptide bonds of aromatic amino acids, breaking down large protein molecules into specific active small peptide molecules and enhancing their kidney-tonifying efficacy. Sea cucumber polypeptides are initially hydrolyzed by alkaline protease, then synergistically treated with collagenase type II and subtilisin. The former disrupts the triple helix structure of collagen, while the latter releases hydrophobic active peptides, ensuring the full utilization of their antioxidant and energy metabolism regulating activities.

[0022] IV. Precision of Extraction and Purification Processes: Complex enzymatic hydrolysis of plant extracts (such as using cellulase and pectinase in *Dendrobium nobile* and *Dendrobium truncata*) breaks down cell walls, releasing active ingredients such as polyphenols and flavonoids, avoiding the encapsulation and absorption barriers caused by un-hydrolyzed components. The use of dedicated purification columns enhances the purity of active ingredients: SA sulfonic acid-based silica gel strong cation exchange columns specifically capture positively charged active ingredients, while the tandem combination of phenylboronic acid affinity columns and polyamide columns specifically retains key components such as *Dendrobium nobile* polysaccharides and polyphenols, reducing the loss of active ingredients due to non-specific adsorption.

[0023] In summary, the composition of the present application performs complex enzymolysis + complex extraction + exclusive purification process, can guarantee the yield of small molecule active peptides and glycosides, realize better therapeutic effect, and has less impurities and high safety. The oyster is cut by trypsin + bovine pancreatic chymotrypsin to cut hydrophobic and aromatic amino acids; the sea cucumber is cut by collagenase II + subtilisin to release effective sequence peptides; the plant extraction is cut by complex enzymolysis to release more effective active substances, and a special purification column is used to capture effective active ingredients; the compatibility is more reasonable, can synergistically play a better role, and can provide quick-acting anti-fatigue and long-acting kidney tonifying effect. DETAILED DESCRIPTION

[0024] The present application will be further described below in combination with specific implementation examples, but the present application is not limited to these examples.

[0025] Example 1

[0026] A kidney tonifying and anti-fatigue polypeptide composition, comprising the following raw materials in mass fraction: 28 parts of oyster polypeptide, 22 parts of sea cucumber polypeptide, 18 parts of spider silk hair blue ears grass-pentamerous fruit complex extract, 20 parts of cistanche-dendrobium officinale complex extract, 9 parts of ginseng extract and 7 parts of deer penis extract. The particle size of the ginseng extract and the deer penis extract is all below 150 mesh.

[0027] The preparation method of the oyster polypeptide comprises the following steps: the fresh and washed oyster meat is crushed into a slurry, deionized water is added at a material-liquid mass ratio of 1:7, 1.0wt% trypsin and 1.0wt% bovine pancreatic chymotrypsin are added based on the mass of the oyster meat, the pH is adjusted to 8.2, and the enzymolysis is carried out at 37℃ for 3.5h; the enzyme is inactivated at 85℃ for 18min, the temperature is reduced to room temperature, centrifugation is carried out at 4500r / min for 15min, the supernatant is taken, and freeze-drying is carried out until the water content is 4.2wt%, to obtain the oyster polypeptide.

[0028] The preparation method of the sea cucumber polypeptide comprises the following steps: the sea cucumber body wall is cut into small pieces below 1cm, deionized water is added at a material-liquid mass ratio of 1:9, the solid is taken after filtration after ultrasonic treatment at 40kHz for 25min; the solid is added with deionized water at a material-liquid mass ratio of 1:7, 1.0wt% alkaline protease is added based on the mass of the sea cucumber body wall, the pH is adjusted to 9.2, and the enzymolysis is carried out at 42℃ for 1.5h; the enzyme is inactivated at 85℃ for 18min, the temperature is reduced to room temperature, the pH is adjusted to 6.8, 1.0wt% collagenase type II and 0.8wt% subtilisin are added based on the mass of the sea cucumber body wall, the enzymolysis is carried out at 48℃ for 2.5h, the enzyme is inactivated at 85℃ for 18min, the temperature is reduced to room temperature, centrifugation is carried out at 4500r / min for 15min, the supernatant is taken, ultrafiltration is carried out on the supernatant through a 5kDa ultrafiltration membrane, the permeate is collected, and freeze-drying is carried out until the water content is 4.5wt%, to obtain the sea cucumber polypeptide.

[0029] The preparation method of the C. filiseta- P. multiflorum composite extract comprises the following steps: taking dry whole plants of C. filiseta, crushing and passing through a 40-mesh sieve to obtain C. filiseta powder; taking pulp of mature P. multiflorum, beating, adding 0.2% ascorbic acid to the pulp, and stirring to obtain P. multiflorum pulp; mixing the C. filiseta powder and the P. multiflorum pulp according to a mass ratio of 4:1.5 to obtain a mixture; adding deionized water to the mixture according to a solid-liquid mass ratio of 1:5.5, adding 1.0 wt% cellulase, 1.0 wt% pectinase and 1.0 wt% β-glucosidase to the mixture, adjusting pH to 4.9, and performing enzymolysis at 42°C for 3.5 h; inactivating the enzyme at 85°C for 18 min, cooling to room temperature, and obtaining an enzymolysis solution; adding 65% volume concentration ethanol aqueous solution to the enzymolysis solution in a volume ratio of 2.5:1, performing ultrasonic treatment at 40 kHz for 45 min, centrifuging at 9000 r / min for 15 min, taking the supernatant, and concentrating at 55°C under reduced pressure to a volume of 18%; loading the concentrated solution onto a SA sulfonic silica gel strong cation exchange column, first washing impurities with 22% volume concentration ethanol aqueous solution for 1.5 BV, and then eluting with 55% volume concentration ethanol aqueous solution for 2.5 BV; collecting the eluate, concentrating at 55°C under reduced pressure, and drying at 55°C until the water content is 3.8 wt% to obtain the C. filiseta-P. multiflorum composite extract.

[0030] The preparation method of the C. filiseta- P. multiflorum composite extract comprises the following steps: taking dry whole plants of C. filiseta, crushing and passing through a 40-mesh sieve to obtain C. filiseta powder; taking pulp of mature P. multiflorum, beating, adding 0.2% ascorbic acid to the pulp, and stirring to obtain P. multiflorum pulp; mixing the C. filiseta powder and the P. multiflorum pulp according to a mass ratio of 4:1.5 to obtain a mixture; adding deionized water to the mixture according to a solid-liquid mass ratio of 1:5.5, adding 1.0 wt% cellulase, 1.0 wt% pectinase and 1.0 wt% β-glucosidase to the mixture, adjusting pH to 4.9, and performing enzymolysis at 42°C for 3.5 h; inactivating the enzyme at 85°C for 18 min, cooling to room temperature, and obtaining an enzymolysis solution; adding 65% volume concentration ethanol aqueous solution to the enzymolysis solution in a volume ratio of 2.5:1, performing ultrasonic treatment at 40 kHz for 45 min, centrifuging at 9000 r / min for 15 min, taking the supernatant, and concentrating at 55°C under reduced pressure to a volume of 18%; loading the concentrated solution onto a SA sulfonic silica gel strong cation exchange column, first washing impurities with 22% volume concentration ethanol aqueous solution for 1.5 BV, and then eluting with 55% volume concentration ethanol aqueous solution for 2.5 BV; collecting the eluate, concentrating at 55°C under reduced pressure, and drying at 55°C until the water content is 3.8 wt% to obtain the C. filiseta-P. multiflorum composite extract.

[0031] The preparation method of the above-mentioned kidney-tonifying and anti-fatigue polypeptide composition comprises: uniformly mixing, in mass parts, oyster polypeptide, sea cucumber polypeptide, spider silk and blue-eared grass-pentamerous fruit composite extract, cistanche-dendrobium officinale composite extract, ginseng extract, and deer penis extract to obtain a composition; and mixing the composition with pharmaceutically acceptable excipients to prepare an oral preparation, which comprises capsules, granules, and tablets.

[0032] Embodiment 2

[0033] A kidney-tonifying and anti-fatigue polypeptide composition comprises the following raw materials in mass parts: oyster polypeptide 25 parts, sea cucumber polypeptide 25 parts, spider silk and blue-eared grass-pentamerous fruit composite extract 15 parts, cistanche-dendrobium officinale composite extract 22 parts, ginseng extract 8 parts, and deer penis extract 8 parts. The particle size of the ginseng extract and the deer penis extract is both below 150 mesh.

[0034] The preparation method of the oyster polypeptide comprises the following steps: crushing fresh and washed oyster meat into a slurry, adding deionized water at a material-to-liquid mass ratio of 1:8, adding 0.5 wt% trypsin and 1.5 wt% chymotrypsin based on the mass of the oyster meat, adjusting the pH to 7.8, and performing enzymatic hydrolysis at 40°C for 3 h; inactivating the enzyme at 90°C for 15 min, reducing the temperature to room temperature, centrifuging at 5000 r / min for 10 min, taking the supernatant, and freeze-drying to a water content of 4.3 wt% to obtain the oyster polypeptide.

[0035] The preparation method of the sea cucumber polypeptide comprises the following steps: cutting the sea cucumber body wall into small pieces below 1 cm, adding deionized water at a material-to-liquid mass ratio of 1:10, and filtering to obtain the solid; adding deionized water to the solid at a material-to-liquid mass ratio of 1:6, adding 1.5 wt% alkaline protease based on the mass of the sea cucumber body wall, adjusting the pH to 9.0, and performing enzymatic hydrolysis at 45°C for 1.5 h; inactivating the enzyme at 90°C for 15 min, reducing the temperature to room temperature, adjusting the pH to 7.0, adding 0.5 wt% collagenase type II and 1.0 wt% subtilisin based on the mass of the sea cucumber body wall, and performing enzymatic hydrolysis at 45°C for 3 h; inactivating the enzyme at 80°C for 20 min, reducing the temperature to room temperature, centrifuging at 4000 r / min for 20 min, taking the supernatant, performing ultrafiltration through a 5 kDa ultrafiltration membrane, collecting the permeate, and freeze-drying to a water content of 4.9 wt% to obtain the sea cucumber polypeptide.

[0036] The preparation method of the C. filiseta- P. lucidum composite extract comprises the following steps: taking dry whole plants of C. filiseta, crushing and passing through a 40-mesh sieve to obtain C. filiseta powder; taking pulp of mature P. lucidum, beating, adding 0.3% ascorbic acid by mass of the pulp, and stirring uniformly to obtain P. lucidum pulp; mixing the C. filiseta powder and the P. lucidum pulp according to a mass ratio of 3:1 to obtain a mixture; adding deionized water to the mixture according to a solid-liquid mass ratio of 1:5, adding 1.5 wt% cellulase, 0.5 wt% pectinase and 1.5 wt% β-glucosidase based on the mass of the mixture, adjusting pH to 4.8, and performing enzymolysis at 45°C for 3 h; inactivating the enzyme at 90°C for 15 min, and cooling to room temperature to obtain an enzymolysis liquid; adding 3 times the volume of 60% (by volume) ethanol aqueous solution to the enzymolysis liquid, performing ultrasonic treatment at 45 kHz for 30 min, centrifuging at 10 000 r / min for 10 min, taking the supernatant, and concentrating at 60°C under reduced pressure to 10% of the volume to obtain a concentrated liquid; loading the concentrated liquid onto a SA sulfonic silica gel strong cation exchange column, first washing impurities with 25% (by volume) ethanol aqueous solution for 1.5 BV, and then eluting with 60% (by volume) ethanol aqueous solution for 2 BV; collecting the eluate, concentrating at 60°C under reduced pressure, and drying at 50°C until the water content is 4.7 wt% to obtain the C. filiseta-P. lucidum composite extract.

[0037] The preparation method of the C. filiseta- P. lucidum composite extract comprises the following steps: taking dry whole plants of C. filiseta, crushing and passing through a 40-mesh sieve to obtain C. filiseta powder; taking pulp of mature P. lucidum, beating, adding 0.3% ascorbic acid by mass of the pulp, and stirring uniformly to obtain P. lucidum pulp; mixing the C. filiseta powder and the P. lucidum pulp according to a mass ratio of 3:1 to obtain a mixture; adding deionized water to the mixture according to a solid-liquid mass ratio of 1:5, adding 1.5 wt% cellulase, 0.5 wt% pectinase and 1.5 wt% β-glucosidase based on the mass of the mixture, adjusting pH to 4.8, and performing enzymolysis at 45°C for 3 h; inactivating the enzyme at 90°C for 15 min, and cooling to room temperature to obtain an enzymolysis liquid; adding 3 times the volume of 60% (by volume) ethanol aqueous solution to the enzymolysis liquid, performing ultrasonic treatment at 45 kHz for 30 min, centrifuging at 10 000 r / min for 10 min, taking the supernatant, and concentrating at 60°C under reduced pressure to 10% of the volume to obtain a concentrated liquid; loading the concentrated liquid onto a SA sulfonic silica gel strong cation exchange column, first washing impurities with 25% (by volume) ethanol aqueous solution for 1.5 BV, and then eluting with 60% (by volume) ethanol aqueous solution for 2 BV; collecting the eluate, concentrating at 60°C under reduced pressure, and drying at 50°C until the water content is 4.7 wt% to obtain the C. filiseta-P. lucidum composite extract.

[0038] The preparation method of the above-mentioned kidney-tonifying and anti-fatigue polypeptide composition comprises: uniformly mixing, in mass parts, oyster polypeptide, sea cucumber polypeptide, spider silk and blue-eared grass-pentamerous fruit composite extract, cistanche-dendrobium officinale composite extract, ginseng extract and deer penis extract to obtain a composition; and mixing the composition with pharmaceutically acceptable excipients to prepare oral preparations, including capsules, granules and tablets.

[0039] Example 3

[0040] A kidney-tonifying and anti-fatigue polypeptide composition comprises the following raw materials in mass parts: oyster polypeptide 30 parts, sea cucumber polypeptide 20 parts, spider silk and blue-eared grass-pentamerous fruit composite extract 20 parts, cistanche-dendrobium officinale composite extract 18 parts, ginseng extract 10 parts and deer penis extract 5 parts. The particle size of the ginseng extract and the deer penis extract is both below 200 mesh.

[0041] The preparation method of the oyster polypeptide comprises the following steps: crushing fresh and washed oyster meat into a slurry, adding deionized water at a material-to-liquid mass ratio of 1:6, adding 1.5 wt% trypsin and 0.5 wt% chymotrypsin based on the mass of the oyster meat, adjusting the pH to 8.5, and performing enzymatic hydrolysis at 36℃ for 4 h; inactivating the enzyme at 80℃ for 20 min, reducing the temperature to room temperature, centrifuging at 4000 r / min for 20 min, taking the supernatant, and freeze-drying to a water content of 3.9 wt% to obtain the oyster polypeptide.

[0042] The preparation method of the sea cucumber polypeptide comprises the following steps: cutting the sea cucumber body wall into small pieces below 1 cm, adding deionized water at a material-to-liquid mass ratio of 1:8, and filtering to obtain the solid after ultrasonic treatment at 45 kHz for 20 min; adding deionized water at a material-to-liquid mass ratio of 1:8, adding 0.5 wt% alkaline protease based on the mass of the sea cucumber body wall, adjusting the pH to 9.5, and performing enzymatic hydrolysis at 40℃ for 2 h; inactivating the enzyme at 80℃ for 20 min, reducing the temperature to room temperature, adjusting the pH to 6.5, adding 1.5 wt% collagenase type II and 0.5 wt% subtilisin based on the mass of the sea cucumber body wall, and performing enzymatic hydrolysis at 50℃ for 2 h; inactivating the enzyme at 90℃ for 15 min, reducing the temperature to room temperature, centrifuging at 5000 r / min for 10 min, taking the supernatant, performing ultrafiltration through a 5 kDa ultrafiltration membrane, collecting the permeate, and freeze-drying to a water content of 4.6 wt% to obtain the sea cucumber polypeptide.

[0043] The preparation method of the C. filiseta- P. multiflora compound extract comprises the following steps: taking dry whole plants of C. filiseta, crushing and passing through a 60-mesh sieve to obtain C. filiseta powder; taking pulp of mature P. multiflora, beating, adding 0.1% ascorbic acid to the pulp, and stirring to obtain P. multiflora pulp; mixing the C. filiseta powder and the P. multiflora pulp according to a mass ratio of 5:3 to obtain a mixture; adding deionized water to the mixture according to a solid-liquid mass ratio of 1:6, adding 0.5 wt% cellulase, 1.5 wt% pectinase and 0.5 wt% β-glucosidase based on the mass of the mixture, adjusting pH to 5.0, and performing enzymatic hydrolysis at 40°C for 4 h; inactivating the enzyme at 80°C for 20 min; cooling to room temperature to obtain an enzymatic hydrolysate; adding 2 times the volume of 70% volume concentration ethanol aqueous solution to the enzymatic hydrolysate, performing ultrasonic treatment at 40 kHz for 60 min, centrifuging at 8000 r / min for 20 min, taking the supernatant, and reducing the volume to 20% at 50°C under reduced pressure to obtain a concentrated solution; loading the concentrated solution onto a SA sulfonic silica gel strong cation exchange column, first washing with 20% volume concentration ethanol aqueous solution to remove impurities, and then eluting with 50% volume concentration ethanol aqueous solution to collect the eluate; reducing the volume at 50°C under reduced pressure, and drying at 60°C until the water content is 4.1 wt% to obtain the C. filiseta- P. multiflora compound extract.

[0044] The preparation method of the C. filiseta- P. multiflora compound extract comprises the following steps: taking dry whole plants of C. filiseta, crushing and passing through a 60-mesh sieve to obtain C. filiseta powder; taking pulp of mature P. multiflora, beating, adding 0.1% ascorbic acid to the pulp, and stirring to obtain P. multiflora pulp; mixing the C. filiseta powder and the P. multiflora pulp according to a mass ratio of 5:3 to obtain a mixture; adding deionized water to the mixture according to a solid-liquid mass ratio of 1:6, adding 0.5 wt% cellulase, 1.5 wt% pectinase and 0.5 wt% β-glucosidase based on the mass of the mixture, adjusting pH to 5.0, and performing enzymatic hydrolysis at 40°C for 4 h; inactivating the enzyme at 80°C for 20 min; cooling to room temperature to obtain an enzymatic hydrolysate; adding 2 times the volume of 70% volume concentration ethanol aqueous solution to the enzymatic hydrolysate, performing ultrasonic treatment at 40 kHz for 60 min, centrifuging at 8000 r / min for 20 min, taking the supernatant, and reducing the volume to 20% at 50°C under reduced pressure to obtain a concentrated solution; loading the concentrated solution onto a SA sulfonic silica gel strong cation exchange column, first washing with 20% volume concentration ethanol aqueous solution to remove impurities, and then eluting with 50% volume concentration ethanol aqueous solution to collect the eluate; reducing the volume at 50°C under reduced pressure, and drying at 60°C until the water content is 4.1 wt% to obtain the C. filiseta- P. multiflora compound extract.

[0045] The preparation method of the above-mentioned kidney-tonifying and anti-fatigue polypeptide composition comprises: uniformly mixing, by mass fraction, oyster polypeptide, sea cucumber polypeptide, spider silk and bluegrass-pentamerous fruit composite extract, cistanche-dendrobium candidum composite extract, ginseng extract, and deer penis extract to obtain a composition; and mixing the composition with a pharmaceutically acceptable excipient to prepare an oral preparation, wherein the oral preparation comprises capsules, granules, and tablets.

[0046] The raw materials involved in each of the above embodiments are as follows: the oysters are selected from Mactra veneriformis. The sea cucumbers are selected from Apostichopus japonicus. The ginseng extract is water-soluble and is obtained from Xinyang He Biological Technology Co., Ltd. in Shaanxi, with model number XYHRS-001. The deer penis extract is water-soluble and is obtained from Shanxi Haocheng Biological Technology Co., Ltd. The trypsin has an enzyme activity of 4000 U / g and is obtained from Anhui Weimao Biological Technology Co., Ltd. The chymotrypsin is obtained from Shanghai Liandai Biological Engineering Co., Ltd., with model number LM1002. The alkaline protease has an enzyme activity of 200,000 U / g and is obtained from Zhengzhou Chaofan Chemical Co., Ltd. The collagenase type II is obtained from Hunan Woke Biological Technology Co., Ltd., with BR, type II, and >125 CDU / mg. The subtilisin has an enzyme activity of 400,000 U / g and is obtained from Hubei Jianmo Biological Technology Co., Ltd. The cellulase has an enzyme activity of 10,000 U / g and is obtained from Nanning Dongheng Huadao Biological Technology Co., Ltd. The pectinase has an enzyme activity of 30,000 U / g and is obtained from Nanning Pangbo Biological Engineering Co., Ltd. The β-glucosidase has an enzyme activity of 20,000 U / g and is obtained from Beijing Solabio Technology Co., Ltd. The hemicellulase has an enzyme activity of 50,000 U / g and is obtained from Ningxia Xiasen Industry Group Co., Ltd. The SA sulfonic acid-based silica gel strong cation exchange column is obtained from Xi'an Qiyue Biological Technology Co., Ltd. The medium of the phenylboronic acid affinity column is phenylboronic acid affinity medium, which is obtained from Suzhou Namicro Technology Co., Ltd., with model number UniPB-80L. The medium of the polyamide column is 80-100 mesh polyamide resin, which is obtained from Shanghai Liandai Biological Engineering Co., Ltd., with model number LM1233.

[0047] Comparative Example 1

[0048] The difference from Example 1 is that the spider silk and bluegrass-pentamerous fruit composite extract is changed to 8 parts, and the cistanche-dendrobium candidum composite extract is changed to 30 parts.

[0049] Comparative Example 2

[0050] The difference from Example 1 is that the spider silk and bluegrass-pentamerous fruit composite extract is changed to 30 parts, and the cistanche-dendrobium candidum composite extract is changed to 8 parts.

[0051] Comparative Example 3

[0052] The difference from Example 1 is that no chymotrypsin is added in the preparation method of the oyster polypeptide.

[0053] Comparative Example 4

[0054] The difference from Example 1 is that in the preparation method of oyster polypeptide, trypsin is replaced by bromelain.

[0055] Comparative Example 5

[0056] The difference from Example 1 is that in the preparation method of sea cucumber polypeptide, no protease subtilisin is added.

[0057] Comparative Example 6

[0058] The difference from Example 1 is that in the preparation method of sea cucumber polypeptide, collagenase type II is replaced by collagenase type I.

[0059] Comparative Example 7

[0060] The difference from Example 1 is that in the preparation method of Gahnia trichopus- Pentaclethra glaucoides complex extract, Gahnia trichopus powder and Pentaclethra glaucoides paste are mixed at a mass ratio of 1:5.

[0061] Comparative Example 8

[0062] The difference from Example 1 is that in the preparation method of Gahnia trichopus- Pentaclethra glaucoides complex extract, no enzymatic hydrolysis is performed, and ethanol aqueous solution is directly added for ultrasonic extraction.

[0063] Comparative Example 9

[0064] The difference from Example 1 is that in the preparation method of Gahnia trichopus- Pentaclethra glaucoides complex extract, SA sulfonic acid silica gel strong cation exchange column is replaced by D001-CC type cation exchange resin column.

[0065] Comparative Example 10

[0066] The difference from Example 1 is that in the preparation method of Cistanche deserticola- Dendrobium officinale complex extract, Cistanche deserticola powder and Dendrobium officinale powder are mixed at a mass ratio of 0.8:2.5.

[0067] Comparative Example 11

[0068] The difference from Example 1 is that in the preparation method of Cistanche deserticola- Dendrobium officinale complex extract, no enzymatic hydrolysis is performed, and ethanol aqueous solution is directly added for ultrasonic extraction.

[0069] Comparative Example 12

[0070] The difference from Example 1 is that in the preparation method of Cistanche deserticola- Dendrobium officinale complex extract, phenylboronic acid affinity column is replaced by carboxymethyl sepharose gel FF column.

[0071] Comparative Example 13

[0072] The difference from Example 1 is that, in the preparation method of the Cynomorium songaricum-Dendrobium officinale composite extract, the polyamide column is replaced by a macroporous adsorption resin AB-8 column.

[0073] Comparative Example 14

[0074] The difference from Example 1 is that, in the preparation method of the Cynomorium songaricum-Dendrobium officinale composite extract, NH4HCO3 aqueous solution is not used for elution.

[0075] Comparative Example 15

[0076] The difference from Example 1 is that, in the preparation method of the Cynomorium songaricum-Dendrobium officinale composite extract, acetic acid aqueous solution is not used for elution.

[0077] The sources of the above comparative examples are as follows: bromelain is from Shaanxi Jinrun Biological Technology Co., Ltd., with an enzyme activity of 200,000 U / g. D001-CC type cation exchange resin medium, carboxymethyl sepharose gel FF, and macroporous adsorption resin AB-8 medium are all from Shanghai Yuanye Biological Technology Co., Ltd.

[0078] I. Safety detection (cytotoxicity)

[0079] Sample preparation: 10.0 mg of the composition was dissolved in 10 mL of DMEM medium containing 10% fetal bovine serum, ultrasonically dissolved for 30 min, sterilized with a 0.22 μm filter membrane, and 1 mg / mL of the mother liquor was obtained; and the complete culture medium was used to dilute to 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, and 0.25 mg / mL working solutions.

[0080] Detection steps:

[0081] 1. Logarithmic growth phase HepG2 cells were inoculated in a 96-well plate at 1×10 4 cells / well (100 μL / well), and cultured at 37°C, 5% CO2 for 24 h.

[0082] 2. Discard the supernatant, and set groups:

[0083] Blank group: cell-free culture medium;

[0084] Negative control group: cells + complete culture medium;

[0085] Positive control group: cells + 5 μM cisplatin (cytotoxic drug);

[0086] Experimental group: different concentrations of samples (100 μL / well);

[0087] Each group has 6 replicate wells.

[0088] 3. After 48 h of culture, 10 μL of CCK-8 reagent was added to each well, incubated for 2 h, and the OD value was measured at 450 nm.

[0089] 4. Calculation:

[0090] Cell survival rate (%) = [(OD of experimental group - OD of blank) / (OD of negative control group - OD of blank)] x 100%.

[0091] The results are shown in Table 1 below:

[0092] Table 1 Results of cytotoxicity test (cell survival rate %, interval value of parallel samples)

[0093]

[0094]

[0095] II. Anti-fatigue function test and kidney physiological function test

[0096] 1. Experimental animals and grouping:

[0097] Animals: SPF grade SD male rats (21 groups x 6, body weight 180 ± 20 g); grouping (6 rats per group):

[0098] Blank control group: no modeling + intragastric solvent;

[0099] Fatigue model group: modeling + intragastric solvent;

[0100] Positive drug group: modeling + Jinkui Shenqi Pill (0.5 g / kg);

[0101] Example 1-3 group: modeling + test composition (100 mg / kg + intragastric solvent); Comparative example 1-15 group: modeling + test composition (100 mg / kg + intragastric solvent);

[0102] Stage 1, adaptive training (3 days):

[0103] All rats (except the blank group) were subjected to 10 min of weightless swimming per day in a 25°C water tank; Stage 2, modeling period (14 days):

[0104] Days 4-7: swimming to exhaustion (submerged for >10 s) with 5% body weight on the tail; Days 8-14: swimming to exhaustion twice a day (with a 4 h interval);

[0105] Stage 3, drug intervention (30 days):

[0106] From the 15th day of modeling, once daily intragastric administration was performed;

[0107] Drug administration group: composition suspension (0.5% CMC-Na preparation);

[0108] Model and blank group: equal volume of 0.5% CMC-Na solution;

[0109] 3. Sample collection timeline:

[0110] Completed within 24h after the last administration:

[0111]

[0112]

[0113] 4. Project index detection:

[0114] 4.1 Anti-fatigue function detection:

[0115]

[0116] The detection results are shown in Table 2 below:

[0117] Table 2 Anti-fatigue function detection results (parallel sample interval value)

[0118]

[0119]

[0120] 4.2 Kidney function and oxidative damage detection:

[0121]

[0122] The detection results are shown in Table 3 below:

[0123] Table 3 Kidney function and oxidative damage detection results (parallel sample interval value)

[0124]

[0125]

[0126] From the above detection results, it can be seen that the implementation of the combined enzyme hydrolysis + combined extraction + exclusive purification process of the compositions of Example 1 to Example 3 can guarantee the yield of small molecule active peptides and glycosides, achieve better therapeutic effect, and have less impurities and high safety. Oysters are cut by trypsin + bovine pancreatic chymotrypsin to cut hydrophobic and aromatic amino acids; sea cucumbers are released by collagenase II + subtilisin to release effective sequence peptides; plant extraction is broken by complex enzyme hydrolysis to release more effective active substances, and a special purification column is used to capture effective active ingredients; The compatibility is more reasonable, which can synergistically play a better role, provide rapid-acting anti-fatigue and long-acting kidney tonifying effect.

[0127] Comparative Example 1 (8 parts of spider-hair blue-ear grass and five-meric compound extract, 30 parts of cistanche and dendrobium compound extract): The imbalance of the proportion of the compound extract destroys the "synergistic effect" mechanism. The spider-hair blue-ear grass and five-meric compound extract is rich in active substances, which can scavenge free radicals generated during exercise, reduce oxidative damage to kidney cells, and at the same time, the organic acids contained therein can regulate intestinal pH, promote the absorption of polypeptides, and improve the stability of ingredient absorption. When the extract is insufficient, the antioxidant capacity decreases, and the polypeptide absorption efficiency decreases. The core components of the cistanche and dendrobium extract (such as echinacoside in cistanche and dendrobium polysaccharide in dendrobium) have the effects of tonifying kidney and essence, and can promote the repair of renal tubular epithelial cells, but in excess, the high concentration of polysaccharides increases the osmotic pressure of the intestinal tract, hindering the transmembrane transport of other components (especially oyster and holothurian polypeptides), resulting in a decrease in bioavailability.

[0128] Comparative Example 2 (30 parts of spider-hair blue-ear grass and five-meric compound extract, 8 parts of cistanche and dendrobium compound extract): The lack of cistanche and dendrobium extract leads to the absence of "kidney tonifying foundation", and the excessive spider-hair blue-ear grass and five-meric compound extract causes "component antagonism". The phenylethanoid glycoside components in cistanche can activate the SIRT1 signaling pathway in the kidney, promote mitochondrial function repair, and improve creatinine clearance rate; dendrobium polysaccharide can inhibit the release of inflammatory factors in the kidney. When it is insufficient, the kidney repair mechanism cannot be started, and the inflammatory response is intensified. When the spider-hair blue-ear grass and five-meric compound extract is excessive, the tannins contained therein will combine with the amino groups in the polypeptides to form insoluble complexes, reducing the absorption of oyster and holothurian polypeptides, and high-dose flavonoids interfere with the hypothalamic-pituitary-adrenal axis (HPA axis) regulation, affecting the normal secretion of anti-fatigue hormones (such as cortisol).

[0129] Comparative Example 3 (oyster polypeptide preparation without adding bovine chymotrypsin): The absence of "synergistic cutting" in the enzyme system leads to abnormal polypeptide structure. Oyster protein is rich in basic amino acids such as glycine and arginine, and trypsin mainly cuts the carboxylic acid end peptide bond of lysine and arginine, while bovine chymotrypsin specifically cuts the amino acid end peptide bond of phenylalanine and tyrosine. The two can synergistically decompose large molecular proteins into high-efficiency small molecular peptides. After the absence of bovine chymotrypsin, the enzyme product contains a large amount of medium molecular peptides, which have poor efficiency, and such peptides cannot be directly absorbed through the intestinal villus membrane and need to be fermented by intestinal flora for utilization, resulting in a decrease in bioavailability. At the same time, medium molecular peptides cannot effectively activate the AMPK energy sensing pathway, leading to a decrease in ATP generation in muscle cells and a decrease in exercise endurance; their nutritional support for the proximal tubule of the kidney is also significantly weakened.

[0130] Comparative Example 4 (Oyster polypeptide prepared by replacing trypsin with bromelain): The mismatch between the "pH adaptability" and "substrate specificity" of the enzyme leads to the loss of peptide activity. Different enzymes have different activities and protein cleavage sites. Bromelain mainly cleaves the amino-terminal peptide bond of arginine and lysine, which overlaps with the cleavage site of trypsin but is less efficient, and cannot generate the unique active sequence in oyster polypeptide, which is insufficient to more efficiently promote the synthesis of NO in the kidney and improve microcirculation.

[0131] Comparative Example 5 (Stichopus polypeptide preparation without adding subtilisin): Incomplete enzymolysis leads to the absence of "hydrophobic active peptides" in Stichopus polypeptide. Stichopus body wall protein is rich in glycine, proline, and hydroxyproline, forming a unique triple helix structure. Collagenase type II mainly cleaves the peptide bond between glycine and proline, while subtilisin can specifically hydrolyze the peptide bond formed by hydrophobic amino acids (such as leucine and isoleucine). The combination of the two can release peptide sequences with antioxidant activity. After the absence of subtilisin, this type of hydrophobic peptide remains in the macromolecular protein and cannot play a role in scavenging free radicals. At the same time, the lack of hydrophobic peptides can lead to a decrease in mitochondrial membrane potential and a decrease in cellular aerobic respiration efficiency, accelerating lactic acid accumulation.

[0132] Comparative Example 6 (Stichopus polypeptide prepared by replacing collagenase type II with type I): Differences in the "substrate specificity" of the enzyme lead to different kidney repair activities. In addition, the compatibility with subtilisin is also different, and the sequence of small molecule peptide products after combined enzymolysis is different, which directly affects the degree of efficacy.

[0133] Comparative Example 7 (Aphrodite hair blue grass - five-core fruit mixed ratio 1:5): Imbalance in raw material ratio destroys the "enzymatic microenvironment" and "ingredient stability". High concentration of fruit acid can oxidize and destroy the phenolic hydroxyl structure of blue grass glycoside, leading to a decrease in its role in regulating central nervous system neurotransmitters (such as dopamine and 5-hydroxytryptamine), and a decrease in the effect of relieving mental fatigue.

[0134] Comparative Example 8 (Aphrodite hair blue grass - five-core fruit complex extract without enzymolysis): The absence of enzymolysis leads to "insufficient release of active ingredients" and "absorption barriers". The effective components of Aphrodite hair blue grass are wrapped in plant cell walls (containing cellulose and pectin). Without enzymolysis, they can only be extracted by ethanol ultrasonic extraction, with low extraction rate. Moreover, the released components are combined with macromolecular cellulose to form insoluble complexes, which cannot pass through the tight junctions of intestinal epithelial cells.

[0135] Comparative Example 9 (Aphrodite hair blue grass - five-core fruit extract prepared by replacing SA sulfonic acid silica gel column with D001-CC resin): The "selective adsorption difference" of the chromatographic medium leads to the loss of effective components. The SA sulfonic acid silica gel strong cation exchange column has high selectivity for positively charged active ingredients, while the D001-CC resin has strong adsorption for neutral components (such as polysaccharides) and low adsorption rate for cationic components.

[0136] Comparative Example 10 (Cistanche- Dendrobium mix ratio 0.8:2.5): Imbalance of ratio leads to "kidney- tonifying active ingredient ratio imbalance".

[0137] The optimal compatibility of Cistanche and Dendrobium is based on "yin and yang complementation": phenylethanoid glycosides in Cistanche (warm nature) can promote androgen synthesis, while polysaccharides in Dendrobium (cool nature) can inhibit excessive inflammatory response. When the ratio of Cistanche to Dendrobium is 2.5:0.8, a balance can be achieved. After the ratio is reversed, the excessive polysaccharides in Dendrobium can inhibit the appropriate inflammatory response (such as macrophage recruitment) required for kidney repair, leading to a slower regeneration rate of renal tubular epithelial cells. At the same time, the insufficient components in Cistanche can reduce androgen synthesis, hindering muscle protein synthesis.

[0138] Comparative Example 11 (Cistanche-Dendrobium extract without enzymatic hydrolysis): Enzymatic hydrolysis leads to "active ingredient encapsulation" and "absorption barrier". Echinacoside in Cistanche and dendrobium polysaccharide in Dendrobium are embedded in the cellulose-lignin network of plant cells. Without enzymatic hydrolysis, they can only be released by ethanol extraction, with low extraction efficiency. Moreover, the released components are mostly macromolecules (polysaccharide molecular weight > 500 kDa), which cannot be absorbed by intestinal villi (and need to be fermented by intestinal flora into small molecules, with low utilization rate). Insufficient echinacoside can weaken the activation of the SIRT1 pathway in the kidney, hindering mitochondrial function repair. Insufficient dendrobium polysaccharides cannot inhibit the TLR4 inflammatory pathway in the kidney, resulting in poor kidney- tonifying performance.

[0139] Comparative Example 12 (Cistanche-Dendrobium extract with carboxymethyl sepharose gel instead of phenylboronic acid affinity column): "Specific recognition difference" of affinity medium leads to loss of active ingredients. Phenylboronic acid affinity column specifically binds to cis-diol structures in polysaccharides (such as mannose residues in dendrobium polysaccharides), while carboxymethyl sepharose gel only adsorbs by charge, with low recovery rate for neutral polysaccharides (such as dendrobium polysaccharides). The absence of dendrobium polysaccharides leads to the inability to activate the AMPK pathway, reducing cellular energy sensing and hepatic glycogen synthesis. At the same time, phenylboronic acid column can remove phenolic acid impurities (such as caffeic acid) in the extract, while the replacement column cannot, affecting the absorption activity of the components.

[0140] Comparative Example 13 (Cistanche-Dendrobium extract with AB-8 macroporous resin instead of polyamide column): "Adsorption selectivity" difference of resin leads to loss of polyphenolic active ingredients. Polyamide column has high affinity (through hydrogen bonding) for polyphenolic components (such as chlorogenic acid in Cistanche and gallic acid in Dendrobium). AB-8 macroporous resin is non-polar adsorption, with low recovery rate for polyphenols. These polyphenolic components are important antioxidants, and their absence leads to the inability to scavenge ROS produced during exercise, resulting in oxidative damage to muscle cells. At the same time, insufficient chlorogenic acid can inhibit the reabsorption of urea by the renal tubule, leading to elevated serum BUN.

[0141] Comparative Example 14 (Cistanche- Dendrobium extract not eluted with NH4HCO3) and Comparative Example 15 (Cistanche- Dendrobium extract not eluted with acetic acid): The absence of the elution step results in the loss of target active ingredients and reduces efficacy.

Claims

1. A kidney-tonifying and anti-fatigue polypeptide composition, characterized in that, The composition comprises the following raw materials in parts by weight: 25-30 parts of oyster polypeptide, 20-25 parts of sea cucumber polypeptide, 15-20 parts of spider silk grass-five-leaf fruit compound extract, 18-22 parts of Cistanche deserticola-Dendrobium officinale compound extract, 8-10 parts of ginseng extract and 5-8 parts of deer antler extract. The oyster polypeptide is a product obtained by co-enzymatic hydrolysis of oyster meat with trypsin and bovine chymotrypsin. The sea cucumber polypeptide is a product with a value of less than 5 kDa obtained by alkaline protease hydrolysis of the sea cucumber body wall, followed by co-hydrolysis with collagenase type II and subtilisin. The compound extract of *Symplocos edulis* and *Dypsis chinensis* was obtained by mixing *Symplocos edulis* and *Dypsis chinensis* in a mass ratio of (3-5):(1-3), enzymatically hydrolyzing them with cellulase, pectinase and β-glucosidase, then extracting them with an ethanol aqueous solution by ultrasonication, and purifying them with an SA sulfonic acid silica gel strong cation exchange column. The Cistanche deserticola-Dendrobium officinale composite extract is obtained by mixing Cistanche deserticola and Dendrobium officinale at a mass ratio of (2-3):(0.5-1), enzymatically hydrolyzing with hemicellulase and β-glucosidase, then ultrasonically extracting with ethanol aqueous solution, and purifying with phenylboronic acid affinity column-polyamide column tandem column. The ginseng extract is a water-soluble ginseng extract, sourced from Shaanxi Xinyanghe Biotechnology Co., Ltd., model XYHRS-001; the deer antler extract is a water-soluble deer antler extract, sourced from Shanxi Haocheng Biotechnology Co., Ltd.

2. The kidney-tonifying and fatigue-resisting polypeptide composition according to claim 1, characterized in that, The preparation method of the oyster polypeptide includes the following steps: crushing oyster meat into a paste, adding deionized water, adding trypsin and bovine chymotrypsin, adjusting the pH to 7.8-8.5, enzymatically hydrolyzing at 36-40℃ for 3-4 hours; inactivating the enzyme, cooling to room temperature, centrifuging, taking the supernatant, and freeze-drying to obtain the oyster polypeptide.

3. The kidney-tonifying and fatigue-resisting polypeptide composition according to claim 2, characterized in that, The preparation method of the oyster polypeptide includes the following steps: freshly washed oyster meat is crushed into a paste, and deionized water is added at a material-to-liquid mass ratio of 1:(6-8). Based on the mass of oyster meat, 0.5-1.5 wt% trypsin and 0.5-1.5 wt% bovine chymotrypsin are added, the pH is adjusted to 7.8-8.5, and enzymatic hydrolysis is carried out at 36-40℃ for 3-4 hours; enzyme inactivation is carried out at 80-90℃ for 15-20 minutes, the temperature is lowered to room temperature, and centrifuged at 4000-5000 r / min for 10-20 minutes. The supernatant is collected and freeze-dried until the water content is below 5 wt% to obtain the oyster polypeptide.

4. The kidney-tonifying and fatigue-resisting polypeptide composition according to claim 1, characterized in that, The preparation method of the sea cucumber polypeptide includes the following steps: the sea cucumber body wall is cut into small pieces, deionized water is added, ultrasonication is performed, and the solid is filtered; the solid is added to deionized water, alkaline protease is added, the pH is adjusted to 9.0-9.5, enzymatic hydrolysis is performed at 40-45℃ for 1.5-2 hours, the enzyme is inactivated, the temperature is lowered to room temperature, the pH is adjusted to 6.5-7.0, collagenase type II and subtilisin are added, enzymatic hydrolysis is performed at 45-50℃ for 2-3 hours, the enzyme is inactivated, the temperature is lowered to room temperature, centrifugation is performed, the supernatant is collected, ultrafiltration is performed through a 5kDa ultrafiltration membrane, the permeate is collected, and freeze-dried to obtain the sea cucumber polypeptide.

5. The kidney-tonifying and fatigue-resisting polypeptide composition according to claim 4, characterized in that, The preparation method of the sea cucumber polypeptide comprises the following steps: cutting the sea cucumber body wall into small pieces with a size of less than 1 cm, adding deionized water in a material-liquid mass ratio of 1: (8-10), performing ultrasonic treatment at 40-45 kHz for 20-30 min, filtering to obtain a solid; adding deionized water in a material-liquid mass ratio of 1: (6-8) to the solid, adding 0.5-1.5 wt% alkaline protease based on the mass of the sea cucumber body wall, adjusting the pH to 9.0-9.5, performing enzymolysis at 40-45 DEG C for 1.5-2 h, inactivating the enzyme at 80-90 DEG C for 15-20 min, reducing the temperature to room temperature, adjusting the pH to 6.5-7.0, adding 0.5-1.5 wt% collagenase type II and 0.5-1.0 wt% subtilisin based on the mass of the sea cucumber body wall, performing enzymolysis at 45-50 DEG C for 2-3 h, inactivating the enzyme at 80-90 DEG C for 15-20 min, reducing the temperature to room temperature, centrifuging at 4000-5000 r / min for 10-20 min, taking the supernatant, performing ultrafiltration through a 5 kDa ultrafiltration membrane, collecting the permeate, and freeze-drying to a water content of less than 5 wt% to obtain the sea cucumber polypeptide.

6. The kidney-tonifying and fatigue-resisting polypeptide composition according to claim 1, characterized in that, The preparation method of the spider silk hair blue ears grass-pentamerous fruit composite extract comprises the following steps: mixing spider silk hair blue ears grass powder and pentamerous fruit pulp in a mass ratio of (3-5): (1-3) to obtain a mixture, adding deionized water, adding cellulase, pectinase and beta-glucosidase, adjusting the pH to 4.8-5.0, performing enzymolysis at 40-45 DEG C for 3-4 h, inactivating the enzyme, reducing the temperature to room temperature, and obtaining an enzymolysis liquid; adding an ethanol aqueous solution, performing ultrasonic treatment, centrifuging, taking the supernatant, performing vacuum concentration, obtaining a concentrated liquid, loading the concentrated liquid onto an SA sulfonic silica gel strong cation exchange column, first washing impurities with 20-25% volume concentration ethanol aqueous solution for 1.5-2 BV, then eluting with 50-60% volume concentration ethanol aqueous solution for 2-3 BV, collecting the eluate, performing vacuum concentration, drying, and obtaining the spider silk hair blue ears grass-pentamerous fruit composite extract.

7. The kidney-tonifying and fatigue-resisting polypeptide composition according to claim 6, characterized in that, The preparation method of the spiderweb blue-ear grass-pentamerous fruit composite extract includes the following steps: taking the dried whole plant of spiderweb blue-ear grass, crushing and passing through a 40-60 mesh sieve to obtain spiderweb blue-ear grass powder; taking the pulp of mature pentamerous fruit, beating, adding 0.1-0.3% ascorbic acid by mass of the pulp and stirring to obtain pentamerous fruit slurry; mixing the spiderweb blue-ear grass powder and the pentamerous fruit slurry according to a mass ratio of (3-5):(1-3) to obtain a mixture, adding deionized water according to a solid-liquid mass ratio of 1:(5-6), adding 0.5-1.5wt% cellulase, 0.5-1.5wt% pectinase and 0.5-1.5wt% beta-glucosidase based on the mass of the mixture, adjusting pH to 4.8-5.0, and enzymolysis at 40-45°C for 3-4h, inactivating the enzyme at 80-90°C for 15-20min, cooling to room temperature, and obtaining an enzymolysis liquid; adding an ethanol aqueous solution with a concentration of 60-70% by volume in an amount of 2-3 times the volume of the enzymolysis liquid, ultrasonicating at 40-45kHz for 30-60min, centrifuging at 8000-10000r / min for 10-20min, taking the supernatant, and vacuum concentrating at 50-60°C to 10-20% of the volume to obtain a concentrated liquid; loading the concentrated liquid onto a SA sulfonic silica gel strong cation exchange column, first washing with 20-25% ethanol aqueous solution to remove impurities, then eluting with 50-60% ethanol aqueous solution, collecting the eluate, vacuum concentrating at 50-60°C, and drying at 50-60°C until the water content is less than 5wt% to obtain the spiderweb blue-ear grass-pentamerous fruit composite extract.

8. The kidney-tonifying and fatigue-resisting polypeptide composition according to claim 1, characterized in that, The preparation method of the cistanche-dendrobium compound extract includes the following steps: mixing cistanche powder and dendrobium powder according to a mass ratio of (2-3):(0.5-1) to obtain a compound, adding deionized water, adding hemicellulase and beta-glucosidase, adjusting pH to 5.0-5.5, and enzymolysis at 50-55°C for 3-5h, inactivating the enzyme, cooling to room temperature, and obtaining an enzymolysis liquid; adding an ethanol aqueous solution, ultrasonicating, centrifuging, taking the supernatant, vacuum concentrating, and obtaining a concentrated liquid; loading the concentrated liquid onto a phenylboronic acid affinity column-polyamide column series column; first washing with deionized water to remove impurities; then eluting with 0.1-0.15mol / L NH4HCO3 aqueous solution for 2-3BV, and eluting with 0.1-0.15mol / L acetic acid aqueous solution for 2-3BV; combining the eluate, vacuum concentrating, and drying to obtain the cistanche-dendrobium compound extract.

9. The kidney-tonifying and fatigue-resisting polypeptide composition according to claim 8, characterized in that, The preparation method of the C. deserticola-D. candidum composite extract comprises the following steps: C. deserticola dry product is crushed to pass through a 60-80 mesh sieve to obtain C. deserticola powder; D. candidum dry product is crushed to pass through a 60-80 mesh sieve to obtain D. candidum powder; the C. deserticola powder and the D. candidum powder are mixed according to a mass ratio of (2-3):(0.5-1) to obtain a composite; deionized water is added to the composite according to a material-liquid mass ratio of 1:(6-8), 1-2 wt% hemicellulase and 1-2 wt% β-glucosidase are added according to the mass of the composite, the pH is adjusted to 5.0-5.5, and enzymolysis is carried out at 50-55°C for 3-5 h, and then the enzyme is inactivated at 80-90°C for 15-20 min, and then the temperature is reduced to room temperature to obtain an enzymolysis liquid; 2-3 times the volume of 60-70% volume concentration ethanol aqueous solution is added to the enzymolysis liquid, ultrasonic treatment is carried out at 40-45 kHz for 30-60 min, and then centrifugation is carried out at 8000-10000 r / min for 10-20 min, and then the supernatant is taken and concentrated at 50-60°C under reduced pressure to a volume of 20-30% to obtain a concentrated liquid; the concentrated liquid is loaded onto a phenylboronic acid affinity column-polyamide column series column; first, deionized water is used for washing 1.5-2 BV to remove impurities; then, 0.1-0.15 mol / L NH4HCO3 aqueous solution is used for elution for 2-3 BV, and then 0.1-0.15 mol / L acetic acid aqueous solution is used for elution for 2-3 BV; the eluate is combined, concentrated under reduced pressure at 50-60°C, and dried at 50-60°C until the water content is less than 5 wt% to obtain the C. deserticola-D. candidum composite extract.

10. The method for preparing the kidney-tonifying and anti-fatigue polypeptide composition according to claim 1, characterized in that, The preparation method comprises the following steps: the oyster polypeptide, the holothurian polypeptide, the spider silk hair blue larkspur-D. pentagyna composite extract, the C. deserticola-D. candidum composite extract, the ginseng extract and the deer penis extract are uniformly mixed according to mass parts to obtain a composition; and the composition is mixed with a pharmaceutically acceptable excipient to prepare an oral preparation, and the oral preparation comprises capsules, granules and tablets.

Images