CN112807416B

CN112807416B - Traditional Chinese medicine bone peptide composition with function of improving bone joint health and preparation method thereof

Info

Publication number: CN112807416B
Application number: CN202110059317.2A
Authority: CN
Inventors: 陶刚; 熊菲菲; 王俊; 涂宏建; 雷蕾; 王彩霞; 袁媛; 宴永球; 贾福怀
Original assignee: Ningbo Yufangtang Biotechnology Co ltd
Current assignee: Ningbo Yufangtang Biotechnology Co ltd
Priority date: 2021-01-18
Filing date: 2021-01-18
Publication date: 2023-05-05
Anticipated expiration: 2041-01-18
Also published as: CN112807416A

Abstract

The invention discloses a traditional Chinese medicine bone peptide composition with a function of improving bone joint health, which comprises the following components in parts by mass: 26-42 parts of marine fish bone collagen oligopeptide, 4-11 parts of alfalfa extract, 2.5-7.5 parts of pre-flatcar extract, 3-8 parts of acacia extract, 2-5 parts of mallow extract, 3-7 parts of rabdosia rubescens extract, 3-7 parts of herba patriniae extract, 0.5-1.5 parts of sodium hyaluronate, 3-7 parts of salmon nasal cartilage extract, 8-16 parts of shark cartilage extract, 2-5 parts of curcumin and 8-18 parts of chitosan oligosaccharide. The invention also discloses a preparation method of the traditional Chinese medicine bone peptide composition. Compared with the prior art, the traditional Chinese medicine bone peptide composition disclosed by the invention achieves the effects of improving the health of bone joints, does not damage viscera, gently reduces inflammation and pain, repairs cartilage and other bone tissues, promotes joint synovial fluid secretion, reduces swelling and discharges pus, and furthest reduces or avoids adverse reactions and side effects.

Description

Traditional Chinese medicine bone peptide composition with function of improving bone joint health and preparation method thereof

Technical Field

The invention relates to the technical field of traditional Chinese medicine compositions, in particular to a traditional Chinese medicine bone peptide composition with a function of improving bone joint health and a preparation method thereof.

Background

When the human joint ages to a certain extent, osteoarthritis is caused, namely, the degenerative change condition occurs at the joint cartilage of the human body, so that the joint cartilage of the human body is destroyed and lost, and simultaneously, the joint is accompanied with hyperosteogeny reaction diseases around the joint, thereby not only causing pain to patients, but also causing heavy economic and manpower and material resource burden to families of the patients. The protective layer of the bone joint is articular cartilage, the degeneration and abrasion of the articular cartilage are the main pathogenesis cause of osteoarthritis, and the proliferation spur, namely hyperosteogeny, is the osteophyte formed at the attachment position of the diseased joint ligament due to inflammatory stimulus, and cannot disappear by oneself.

Cartilage generally includes hyaline cartilage, fibrocartilage, and elastic cartilage. Hyaline cartilage generally covers the articular surface, and as the injury deteriorates, the cartilage wears away, breaks, etc., and is generally no longer good, although the tissue may be partially repaired. Fibrocartilage, commonly referred to as meniscal structures, including those of the knee joint, also partially repairs and regenerates, requiring surgical removal if damage and wear are particularly severe. Elastic cartilage includes ear, laryngeal cartilage, etc., and is generally not particularly treated.

Osteoarthritis is mainly classified into gouty, rheumatic and rheumatoid diseases, and its main causes are purine metabolic disorder hyperuricemia, systemic allergic connective tissue disease caused by bacteria infection, chronic diseases with unknown etiology, and systemic diseases mainly including inflammatory synovitis. These diseases often do not occur alone, often accompanied by hypertension, hyperlipidemia, arteriosclerosis, diabetes mellitus, often accompanied by uroacidosis nephropathy and kidney stones, acute fever and joint pain, heart, pneumonia, urinary system infection, rheumatic vasculitis, pericarditis, pleurisy, and other symptoms, which makes patients overwhelm. Joint health is associated with gout, inflammation, cartilage damage and rupture, bone tissue degradation, and the like.

Common bone joint treatment approaches are mainly divided into the following four types according to treatment modes: reconstruction treatment (joint replacement); prosthetic treatment (arthroscopic surgery, cartilage repair surgery, force line corrective surgery); drug treatment (analgesic drugs, joint cavity injection drugs, slow-acting drugs to relieve symptoms, traditional Chinese medicine); basic therapy (patient education, exercise therapy, physical therapy, mobility support therapy). Lifestyles that contribute to bone joint health include: maintaining optimistic emotion, reasonable life and working modes, avoiding overload, selecting proper shoes, performing physiotherapy massage by using auxiliary facilities, performing moderated aerobic exercises, proper health care food and natural health food intake to assist in improving the health of the bone joints, which are all proper bone joint health preserving means verified for a long period of time.

Along with the rapid development of social economy, the life rhythm is faster and faster, and the dietary structure and life habit of people are changed over the sky, and the symptoms such as bone joint damage, inflammation, chronic pain and the like can be caused due to the reasons of irregular sitting postures, sleeping postures, strenuous exercises, unprotected actions, too busy life work and rest and the like. Bone joint diseases become main diseases which endanger the health of people in China, and even can be regarded as large killers which afflict the health of human bodies.

The data of 2014 "national urban resident joint health survey" shows that more than 1 million people with arthritis in China are counted, and the number of people with osteoarthritis in the population over 50 years is continuously increased, and 90% of women and 80% of men in the population over 65 years suffer from osteoarthritis. Arthritis can be classified into osteoarthritis, rheumatoid disease, and the like according to the cause tonic, reactive, gouty, rheumatic, suppurative, etc. 52.5% of people are afflicted with joint disease. The patients suffering from bone joint diseases such as joint strain, lumbar disc herniation, compulsive spondylitis, rheumatic arthritis and rheumatoid arthritis are particularly more ill, and the development of products with smaller side effects and higher cost performance which can truly improve the health of bone joints is urgent to develop so as to improve the clinical symptoms of patients and promote the quality of life, thereby bringing good news to the vast bone joint health patients.

The traditional bone joint clinical medicine mainly uses analgesic and nonsteroidal anti-inflammatory medicines in combination, and common medicines are western medicines such as flurbiprofen, parecoxib, celecoxib, sodium hyaluronate, loxoprofen sodium, tetrandrine, ibuprofen, etoricoxib and the like. However, the medicine is three-component toxin, and a series of side effects exist in excessive intake of western medicines, so that the liver and kidney metabolism burden is increased, serious gastrointestinal adverse reaction is generated, the medicine dependence effect is also generated, and the medicine has a great potential safety hazard to the health of people, and is limited to a certain extent for applicable people; joint replacement belongs to a large-scale operation, is expensive and the process is irreversible; the reparative treatment treats both symptoms and root causes, and is easy to relapse; the curative effects of basic treatment, physical treatment, exercise support and the like are not clear, and improper operation can adversely affect.

Therefore, the ocean-source food raw materials, traditional Chinese medicines, medicinal and edible materials and other resource treasury are fully excavated and utilized, products with the function of improving the health of the bone joints are systematically and perfectly researched, utilized and developed, and the ocean-source food treasury is popularized, popularized and enjoyed by the masses, has extremely profound practical significance for improving the health level of people in China, and brings great social benefit and economic benefit.

In addition, the traditional marine fish bones and other raw materials are mostly directly pulverized, so that the consumption is large and the effect is not obvious; the traditional Chinese medicine decoction method is time-consuming and labor-consuming, the drug effect is reduced by long-time high-temperature decoction, the bioavailability is not high, and the method is a technical problem which needs to be solved urgently, namely, how to reduce the consumption of the drug, reduce the aversion mind of consumers to the drinking, keep the main efficacy components of the drug to the greatest extent, improve the drug bioavailability and improve the drug effect.

Disclosure of Invention

The first technical problem to be solved by the invention is to provide a traditional Chinese medicine bone peptide composition with the function of improving the health of bone joints, which not only achieves the effect of improving the health of bone joints, but also does not damage organs, gently diminishing inflammation, easing pain, repairing cartilage and other bone tissues, promoting joint synovial fluid secretion, relieving swelling and expelling pus, and furthest reducing or avoiding adverse reactions and side effects.

The second technical problem to be solved by the invention is to provide a preparation method of the traditional Chinese medicine bone peptide composition, which can keep main functional components of the medicine to the greatest extent, improve the bioavailability of the medicine and improve the efficacy.

The invention solves the first technical problem by adopting the technical scheme that: a traditional Chinese medicine bone peptide composition with a function of improving bone joint health is characterized by comprising the following components in parts by mass:

the formula of the composition comprises the following components:

the marine fish bone collagen oligopeptide belongs to a small molecular protein peptide with the molecular weight of 100-1000 Da, is derived from fish bones of deep sea fish, and has the effects of keeping skin moisture, improving osteoporosis, promoting calcium absorption, locking bone calcium, promoting bone tissue regeneration and enhancing immunity;

the salmon nasal cartilage extract is derived from high-purity proteoglycan and short peptide extracted from deep sea salmon nasal cartilage, and has effects of recovering cartilage cell function, promoting proliferation of precursor cells, promoting cartilage differentiation, inhibiting cartilage calcification, reducing inflammatory reaction, maintaining cartilage structure, promoting joint health, and improving motor function;

the shark cartilage extract is prepared from the cartilage tissue of the mackerel, is rich in chondroitin, proteoglycan and short peptide, and has the natural effects of promoting the regeneration of articular cartilage, promoting the secretion of synovial fluid of joint cavities, diminishing inflammation, easing pain, inhibiting joint deformation and inhibiting new blood vessels;

Sodium hyaluronate is a glucuronic acid which is widely present in placenta, amniotic fluid, lens, articular cartilage, dermis layer of skin and other tissues; the composition is one of main components of joint cavity synovial fluid and cartilage matrix, plays a role in lubricating and moisturizing joints, reduces friction among tissues, can obviously improve inflammatory response of synovial fluid tissues, enhances the viscosity and lubricating function of joint fluid, protects and promotes healing and regeneration of joint cartilage, relieves pain and increases the mobility of joints;

curcumin is derived from diketone compound extracted from Curcuma longa of Zingiberaceae, and has wide pharmacological activities such as antiinflammatory, antioxidant, lipid regulating, antiviral, antiinfectious, antitumor, anticoagulation, liver fibrosis resisting, atherosclerosis resisting, etc.; aiming at the bone joint, the effects of improving inflammation, relaxing pressure, easing pain, improving immunity, resisting oxidation, inhibiting bacteria and the like can be achieved;

the chitosan oligosaccharide is a cationic alkaline amino oligosaccharide with positive charge and a polymerization degree of 2-20, which is obtained from chitosan derived from shrimp and crab shells through biological enzymolysis or chemical degradation technology, and has a molecular weight of less than or equal to 3200Da, good water solubility and high bioavailability; has antibacterial, antiinflammatory, intestinal microecology regulating, macrophage and T-lymphocyte activating, pathogenic bacteria growth and reproduction inhibiting, protein synthesis promoting, active oxygen content increasing, and bone tissue growth promoting, immunity enhancing effects;

The alfalfa is a perennial herb of alfalfa genus of leguminous family, is rich in nutrients such as protein, vitamin, mineral and the like, and has the efficacy components such as alfalfa saponin, flavone, isoflavone, coumarin and the like, and has the effects of reducing cholesterol and blood lipid content, regulating immunity, resisting oxidation, delaying aging and improving bone joint health through diminishing inflammation;

the dried whole plant of plantain herb which is derived from the herb plant before flatcar has sweet and cold taste and has the effects of diminishing inflammation, promoting urination, clearing heat, improving eyesight and eliminating phlegm; the effects of eliminating inflammation and relieving pain symptoms are achieved by removing oxygen free radicals;

acacia is Leguminosae of Rosales, is rich in volatile oil and flavonoid substances, has slightly sour and astringent taste, has effects of resolving carbuncle, expelling pus, astringing to stop bleeding, relieving pain, resisting bacteria, diminishing inflammation and resisting virus;

the herba Abelmoschi Manihot is leaf or whole herb of herba Abelmoschi Manihot of Abelmoschus, has sweet and sour taste, is cold in nature, and has effects of nourishing heart, liver, spleen, large intestine and small intestine channel, relieving constipation, clearing heat and promoting diuresis, cooling blood and removing toxic substance, promoting blood circulation for removing blood stasis, and promoting bone fracture and relieving pain;

rabdosia rubescens is dried branches and leaves of Rabdosia rubescens (Hemsl.) Hara of Labiatae, and has effects of clearing heat, reducing dryness, reducing blood lipid and blood pressure, treating inflammation, diminishing inflammation and relieving pain;

Herba Patriniae is dry whole herb with root of herba Patriniae of Patrinia of Patriniaceae, has effects of detumescence, expelling pus and relieving inflammation, and has strong medicinal value.

Preferably, the marine fish bone collagen oligopeptide is obtained by crushing marine fish bones, pulping, performing enzymolysis, performing centrifugal filtration, concentrating and performing spray drying treatment.

Further, the oligopeptide component with the molecular weight smaller than 1000D in the marine fish bone collagen oligopeptide accounts for more than 90%, so that the absorption and utilization rate of the marine fish bone collagen oligopeptide by an organism can be effectively improved, and the effect of being capable of being absorbed without digestion or slightly digested is achieved.

Preferably, the salmon nasal cartilage extract and the shark cartilage extract are obtained by respectively treating corresponding salmon nasal cartilage and shark cartilage by crushing, ultrasonic extraction, acidolysis and enzymolysis, centrifugation, membrane filtration and column filtration, and vacuum freeze drying technology, and the aggregation degree of the functional components is greatly improved.

Further, the content of proteoglycan in the salmon nasal cartilage extract is more than 60wt%, the content of short peptide in the shark cartilage extract is more than 50wt%, and the content of chondroitin is more than 20 wt%.

Preferably, the alfalfa extract, the alloy-happy extract and the front-flatcar extract are respectively prepared from corresponding alfalfa, alloy-happy and front-flatcar coarse powder and supercritical CO ₂ Extracting, refining by column, concentrating under reduced pressure, vacuum freeze drying, pulverizing to obtain the final product, wherein the effective components have high content of crude polysaccharide, protein and total flavone, good effect, high stability, and high bioavailability (8-15 times higher than crude drug).

The invention solves the second technical problem by adopting the technical proposal that: the preparation method of the medicinal bone peptide composition is characterized by comprising the following steps of:

(1) Crushing ocean fish bones, pulping, enzymolysis, centrifugal filtration, decompression concentration and spray drying to obtain the needed ocean fish bone collagen oligopeptide, wherein the oligopeptide component with the molecular weight of less than 1000D in the ocean fish bone collagen oligopeptide accounts for more than 90 percent;

(2) Respectively carrying out crushing ultrasonic extraction, acidolysis enzymolysis, centrifugal filtration, reduced pressure concentration, vacuum freeze drying and crushing on salmon nasal cartilage and shark cartilage to obtain a required salmon nasal cartilage extract and a required shark cartilage extract, wherein the content of proteoglycan in the salmon nasal cartilage extract is more than 60wt%, the content of short peptide in the shark cartilage extract is more than 50wt% and the content of chondroitin is more than 20 wt%;

(3) Respectively passing herba Medicaginis, radix Albiziae, and herba Artemisiae Annuae through coarse powder and supercritical CO ₂ Extracting, refining, concentrating under reduced pressure, vacuum freeze drying, pulverizing, and making into herba Medicaginis extract, radix Arisaema Albae extract, and herba Ipomoeae extract;

(4) Mixing the prepared marine fish bone collagen oligopeptide, salmon nasal cartilage extract and shark cartilage extract uniformly according to the mass portion ratio to obtain a mixture, and adding the prepared alfalfa extract, the front-flatcar extract, the acacia extract, the herba Abutili extract, the rabdosia rubescentis extract, the herba patriniae extract, the sodium hyaluronate, the curcumin and the chitosan oligosaccharide into the mixture, and mixing uniformly to obtain the required traditional Chinese medicine bone peptide composition.

Preferably, the marine ocean fish bone collagen oligopeptide in the step (1) is obtained through the following steps:

(a) Crushing and size mixing: pulverizing marine fishbone, sieving with 60-100 mesh sieve, adding purified water 10-20 times of the weight of the material, dispersing in water solution to obtain marine fishbone powder dispersion, and adjusting pH of the marine fishbone powder dispersion to 6.5-8.5;

(b) Enzymolysis: heating the marine fishbone powder dispersion liquid and keeping the temperature at 37+/-1 ℃, adding trypsin and neutral protease, uniformly stirring and carrying out enzymolysis for 1.5-2.5 hours, heating to 55+/-3 ℃, and continuing stirring and carrying out enzymolysis for 1-2.5 hours to obtain an enzymolysis liquid, wherein the adding amount of the trypsin is 0.2-0.35 wt% of the substrate content, the adding amount of the neutral protease is 0.25-0.4 wt% of the substrate content, and carrying out enzyme deactivation by a high-temperature instantaneous enzyme deactivation method after the enzymolysis is finished;

(c) And (3) centrifugal filtration: centrifugally separating enzyme-deactivated enzymolysis liquid at 12000-14000 rpm to eliminate impurity, collecting clear liquid, starting membrane filtering equipment, filtering clear liquid with microporous membrane of thickness 40-90 microns, pore size 2-5 microns and operating pressure 0.05-0.2 MPa to ensure clear and transparent filtrate and filter residue; desalting and decolorizing the filtered filtrate with ion exchange column and active carbon column, and nanofiltration to remove free amino acid;

(d) Concentrating under reduced pressure: concentrating the ion-exchanged solution under vacuum and reduced pressure at 65-85 ℃ and a vacuum degree of-0.075 to-0.09 Mpa to obtain oligopeptide concentrated solution, wherein the relative density is controlled between 1.04 and 1.06;

(e) Spray drying: spray drying the oligopeptide concentrated solution, wherein the technological parameters are that the nozzle temperature is 155-175 ℃, the outlet temperature is 80-85 ℃, and the feeding speed is 1.0-1.8L/min, so that the required marine fish bone collagen oligopeptide is obtained.

Preferably, the salmon nasal cartilage extract and the shark cartilage extract in the step (2) are obtained by the following steps:

(a) Crushing and ultrasonic extraction: pulverizing salmon nasal cartilage or shark cartilage into coarse particles, sieving with a 10-mesh sieve, adding purified water with the weight 8-15 times of the material weight into an extraction kettle, starting steam heating to 90-105 ℃ and maintaining, starting ultrasonic auxiliary extraction equipment, wherein the ultrasonic power is 5-20 KW, the extraction times are 1-2 times, and the extraction time is 0.5-1.5 h;

(b) Acidolysis enzymolysis: transferring the extracting solution into an enzymolysis tank, regulating the pH to 3.0-5.0 by using dilute sulfuric acid, and keeping the temperature at 85-95 ℃ for 0.5-1 h; cooling to 45-55 ℃ after acidolysis is finished, maintaining, regulating the pH to 10-11, adding bacillus licheniformis protease with the concentration of 0.3-0.6wt% of salmon nasal cartilage or shark cartilage substrate, uniformly stirring and carrying out enzymolysis for 1.0-2.0 h, regulating the pH of an enzymolysis solution to be neutral, and carrying out enzyme deactivation by a high-temperature instantaneous enzyme deactivation method after the enzymolysis is finished;

(c) And (3) centrifugal filtration: centrifuging the enzyme-deactivated enzymolysis liquid at 15000-18000 rpm to remove impurities, collecting clear liquid, starting a membrane filtration device, filtering the clear liquid with a microporous membrane with a thickness of 80-120 μm and a pore diameter of 6-10 μm, and operating at 0.15-0.35 MPa to ensure that the filtrate is clear and transparent, and removing filter residues; the filtered filtrate is subjected to decolorization and deodorization treatment through an activated carbon column;

(d) Concentrating under reduced pressure: concentrating the extractive solution under reduced pressure at 65-75deg.C and vacuum degree of-0.06 to-0.09 Mpa and relative density of 1.12-1.18 to obtain concentrated extract;

(e) Vacuum freeze drying: putting salmon nasal cartilage or concentrated extract of shark cartilage into vacuum freeze drying equipment for drying, wherein parameters are set as follows: pre-freezing initial temperature of minus 40 ℃ to minus 50 ℃, pre-freezing speed of 0.35 to 0.55 ℃/min, pre-freezing end temperature of minus 68 ℃, drying chamber pressure of 30 to 50pa, heating plate temperature of 32 to 38 ℃ and drying time of 28 to 36 hours;

(f) Crushing: pulverizing the dried extract blocks, and sieving with 100-200 mesh sieve to obtain the required salmon nasal cartilage extract or shark cartilage extract.

Preferably, the alfalfa extract, the acacia extract and the pre-flatcar extract in the step (3) are obtained through the following steps:

(a) Coarse powder: pulverizing alfalfa or alloy or flatcar, and sieving with 8-12 mesh sieve;

(b) Supercritical CO ₂ Extraction: coarse powder of herba Medicaginis or alloy, CO ₂ Extracting by a supercritical extraction method, wherein parameters are set as follows: the extraction temperature is 32-38 ℃, the extraction pressure is 38-42 mpa, and CO ₂ The flow is 10-12 kg/h, the extraction time is 120-150 min, the separation pressure is 5-6MPa, the separation temperature is 38-45 ℃, entrainer with the crude drug amount of 5-10 wt% is added, the entrainer is ethanol solution with the concentration of 40-80% V/V, and the alfalfa or the alloy or the front-flat-car extract is obtained through separation;

(c) Refining by column: refining the extract by a macroporous resin separation column, wherein the column passing speed is 1-3L/h, the column passing time is 3-6 h, and discarding ineffective fractions;

(d) Concentrating under reduced pressure: vacuum concentrating the refined effective fraction at 60-70 deg.c and vacuum degree of-0.085 to-0.095 MPa to obtain concentrated Chinese medicinal liquid with relative density of 1.17-1.22;

(e) Vacuum freeze drying: drying the concentrated Chinese medicinal liquid in vacuum freeze drying equipment, with parameters set as follows: pre-freezing initial temperature-45 to-52 ℃, pre-freezing speed 0.25-0.35 ℃/min, pre-freezing end temperature-60 ℃, drying chamber pressure 35-40 pa, heating plate temperature 40-45 ℃ and drying time 24-32 h;

(f) Crushing: pulverizing the dried extract blocks, and sieving with 100-200 mesh sieve to obtain the required herba Medicaginis extract or radix Arisaema extract or herba Pachyrhizi Erosi extract.

Preferably, in the step (4), the sea ocean fish bone collagen oligopeptide, the salmon nasal cartilage extract and the shark cartilage extract are mixed by a small three-dimensional mixer for 5-15 min; finally, the total mixing time of all the raw materials by using a groove type mixer is 15-30 min.

Compared with the prior art, the invention has the advantages that:

(1) The traditional Chinese medicine bone peptide composition disclosed by the invention screens traditional Chinese medicine raw materials with extremely small toxic and side effects, carefully researches the traditional Chinese medicine raw materials according to clear traditional Chinese medicine proportion and quantitative relation thereof, ensures the edible safety, has no toxic or side effects, achieves the effect of improving the health function of bone joints, does not damage viscera, gently reduces inflammation, relieves pain, repairs cartilage and other bone tissues, promotes joint synovial fluid secretion, reduces swelling and discharges pus, and furthest reduces or avoids adverse reactions and side effects; most of the raw materials of the formula are from natural food materials and tonifying traditional Chinese medicines, so that the safety is high, the action principle of the functional components is mild, and no stimulation is generated;

(2) According to the invention, efficacy evaluation and research are carried out through efficacy animal model verification experiments, so that the functions of improving the health of bone joints and the health promoting mechanism of the traditional Chinese medicine bone peptide composition are clarified; the invention solves the technical problems of steady state maintenance of various bioactive substances such as short peptides, functional polysaccharides, total flavonoids and the like in natural raw materials, steady state delivery control of various functional factors and functional maintenance criticality through technical innovation of technology and process, has less consumption, faster dissolution, digestion and absorption speeds of effective components and higher bioavailability, ensures the safety and simultaneously ensures the remarkable efficacy to a great extent;

(3) The traditional Chinese medicine bone peptide composition has the unique function of improving the health of bone joints:

the main raw materials of the traditional Chinese medicine bone peptide composition of the invention are respectively two main types: (1) food raw materials with remarkable efficacy: such as marine fish bone collagen oligopeptide, sodium hyaluronate, curcumin, chitosan oligosaccharide, salmon nasal cartilage extract, shark cartilage extract, etc., and has the main effects of promoting joint synovial fluid secretion, promoting cartilage differentiation, and moistening and relieving pain; (2) mild traditional Chinese medicine extract: such as alfalfa extract, flatcar front extract, alloy-flower extract, herba Abutili extract, rabdosia rubescens extract, herba Patriniae extract, etc., which have the main effects of diminishing inflammation, relieving pain, astringing, stopping bleeding, detumescence and expelling pus;

Most of the traditional Chinese medicine preparations of alfalfa, alloy glaucescent, plantain herb, herba et Gemma Agrimoniae, rabdosia rubescens and herba patriniae pay attention to the effects of regulating blood fat, enhancing immunity, resisting oxidation, promoting urination and detoxification, clearing heat and eliminating phlegm, astringing and stopping bleeding, relaxing bowels, reducing dryness and expelling pus and the like, but the positive effects of improving the health of bone joints, inhibiting inflammation and relieving pain diseases and the like are not fully developed; according to the invention, the six traditional Chinese medicines are prepared into the extract through different technological processes according to different medicinal material properties, and the extract is compatible with the marine fish bone collagen oligopeptide, the salmon nasal cartilage extract and the shark cartilage extract which have the functions of promoting cartilage regeneration, eliminating inflammation and promoting joint health, so that the synergistic effect can be achieved, and the function of improving bone joint health is remarkably improved;

most of the prior curcumin and chitosan oligosaccharide preparations are concerned with the effects of resisting oxidation, regulating fat, enhancing immunity and the like, but the unique effects of curcumin and chitosan oligosaccharide in the aspects of protecting and improving inflammation, relaxing pressure and easing pain, promoting bone tissue growth and the like are not fully developed; the sodium hyaluronate in the formula has the effects of promoting joint synovial fluid secretion, lubricating, moisturizing and relieving pain; the curcumin, the chitosan oligosaccharide, the raw materials and the sodium hyaluronate are compounded for use, so that the curcumin, the chitosan oligosaccharide and the sodium hyaluronate have small stimulation on liver and kidney functions of a human body, and can achieve the effects of mildly nourishing, diminishing inflammation, easing pain and promoting bone tissue growth;

The marine fish bone collagen oligopeptide, salmon nasal cartilage extract and shark cartilage extract in the composition are rich in substances beneficial to bone joints such as short peptide, proteoglycan, aminosugar, chondroitin and the like, and can promote bone tissue regeneration, diminish inflammation and relieve pain; the sodium hyaluronate can promote joint synovial fluid secretion, lubricate and moisturize, and relieve pain; curcumin and chitosan can improve immunity, inhibit bacteria, relieve pain and eliminate inflammation; the six traditional Chinese medicine extracts mainly give full play to the magic effects of bone reunion, detumescence and pus discharge, anti-inflammation and analgesia, and the six traditional Chinese medicines follow the traditional Chinese medicine theory of monarch, minister, assistant, ascending, descending, floating, nature, and returning to the warp, carefully select the medicinal materials for the road and place for compatibility, wherein the acacia is a monarch drug, carbuncle and pus elimination, convergence and hemostasis, pain and bacteria resistance, the herba semiaquilegiae and the herba plantaginis are ministerial drugs, the heat clearing and diuresis promoting, blood circulation and stasis removing, bone setting and pain relieving, the alfalfa and the herba patriniae are adjuvant drugs for regulating immunity, detumescence and pus discharge, the rabdosia is a drug, the heat clearing and dryness reducing, anti-inflammation and pain relieving, and the six medicinal materials respectively play different roles in the composition of the invention, are mutually dependent, complementary advantages and toxicity and drug property removing; the six medicinal materials are used for warming and cold, relieving exterior and interior, nourishing yin and yang, exerting the medicinal effects to the greatest extent, and the six medicinal materials are matched according to the formula proportion to play a role in synergy;

(4) The invention breaks through the bottleneck of the prior art, innovatively improves and optimizes through a large number of orthogonal experimental designs and single-factor experimental grops, and designs a brand-new preparation process flow:

respectively carrying out crushing ultrasonic extraction, acidolysis enzymolysis, centrifugal filtration, reduced pressure concentration, vacuum freeze drying and crushing on salmon nasal cartilage and shark cartilage to obtain a required salmon nasal cartilage extract and shark cartilage extract, wherein the proteoglycan content in the salmon nasal cartilage extract is up to more than 60wt%, the short peptide content in the shark cartilage extract is up to more than 50wt% and the chondroitin content is up to more than 20 wt%; the salmon nasal cartilage extract (or the shark cartilage extract) prepared by the above extraction acidolysis enzymolysis process has greatly reduced amount compared with the raw materials, greatly enriched effective components such as proteoglycan and short peptide, and the like, is more beneficial to improving bioavailability, and plays the roles of promoting cartilage regeneration, increasing joint synovial fluid and improving bone joint health; the method adopts the vacuum freeze drying technology to treat the extract innovatively, overcomes the defects of high temperature, long time and high energy consumption of the conventional vacuum drying, has lower drying temperature, and has the advantages of good drying quality, low processing cost, intelligent and accurate control of moisture and the like; in addition, the oxygen content is extremely low when the material is dried in a vacuum environment, so that the oxidation reaction of the dried material is greatly reduced, the flavor, appearance and color of the material are ensured, and the material has the unique advantage;

The marine fish bone collagen oligopeptide is prepared from marine fish bones as raw materials through crushing, pulping, enzymolysis, centrifugal filtration, reduced pressure concentration and spray drying, wherein the oligopeptide component with the molecular weight of less than 1000D accounts for more than 90 percent, so that the absorption and utilization rate of the marine fish bone collagen oligopeptide by an organism is effectively improved, and the effect of absorption without digestion or slight digestion is achieved;

the invention combines the characteristics of main efficacy components of the medicine, such as category, polarity and the like, adopts the pretreatment processing technology of coarse powder before the alfalfa, the alloy, and the flatcar, and passes through supercritical CO ₂ The preparation method comprises the steps of extraction, column refining, vacuum concentration, vacuum freeze drying, crushing and other process technologies to prepare the required alfalfa extract, the alloy extract and the front-of-flatcar extract, the effective components are fully released and extracted, the effective components in the medicinal materials are furthest reserved and are not damaged, meanwhile, the concentration reduces the edible amount of the medicinal materials, the dispersion and the dissolution are uniform, the absorption is quick, the effects can be rapidly exerted, the content of the effective components of crude polysaccharide, protein and total flavone is high, the effect is good, the stability is high, the bioavailability is greatly improved by 8-15 times compared with the crude drugs, and the medicinal material utilization rate and the medicinal effect are also greatly improved;

The invention creatively combines the advanced technologies together, and searches out corresponding technological processes, operation steps and technological operation parameters, thereby greatly improving the efficacy and bioavailability and reducing the consumption;

(5) The traditional Chinese medicine bone peptide composition with the function of improving the health of bone joints has strong general applicability, is suitable for the production and processing of various dosage forms such as hard capsules, granules, powder or tablets, has the advantages of easy operation, stable process, controllable quality, high production efficiency and high economic added value, and is suitable for large-scale and industrialized mass production.

Detailed Description

The present invention is described in further detail below with reference to examples.

Example 1:

(1) The marine fish bone collagen oligopeptide is prepared by crushing and pulping marine fish bone, performing enzymolysis, performing centrifugal filtration, concentrating under reduced pressure and spray drying, and the specific process is as follows:

(a) Crushing and size mixing: pulverizing marine fishbone, sieving with 80 mesh sieve, adding 12 times of purified water, mixing marine fishbone powder uniformly, dispersing in water solution to obtain marine fishbone powder dispersion, and adjusting pH of the marine fishbone powder dispersion to 8.0;

(b) Enzymolysis: heating and keeping the temperature at 37 ℃, adding trypsin and neutral protease into the marine fish bone powder dispersion liquid, uniformly stirring and carrying out enzymolysis for 1.5 hours, heating to 56 ℃, continuously stirring and carrying out enzymolysis for 2 hours, wherein the adding amount of the trypsin is 0.3wt% of the substrate content, the adding amount of the neutral protease is 0.4wt% of the substrate content, and carrying out enzyme deactivation by a high-temperature instantaneous enzyme deactivation method after the enzymolysis is finished;

(c) And (3) centrifugal filtration: centrifugally separating enzyme-deactivated enzymolysis liquid at 13000rpm to remove impurities, collecting clear liquid, starting a membrane filtering device, filtering the clear liquid with a microporous membrane with a thickness of 80 μm and a pore diameter of 3 μm and an operating pressure of 0.1MPa, ensuring that the filtrate is clear and transparent, and removing filter residues; desalting and decolorizing the filtered filtrate with ion exchange column and active carbon column, and nanofiltration to remove free amino acid;

(d) Concentrating under reduced pressure: concentrating the ion-exchanged solution under reduced pressure at 75deg.C under vacuum degree of-0.085 Mpa to obtain oligopeptide concentrated solution with relative density controlled at 1.06;

(e) Spray drying: and (3) spray-drying the oligopeptide concentrated solution, wherein the technological parameters are that the nozzle temperature is 165 ℃, the outlet temperature is 82 ℃, and the feeding speed is 1.4L/min, so that the marine fish bone collagen oligopeptide meeting the required quality requirement is obtained.

Table 1 shows the GPC relative molecular weight distribution and content of the main components of the marine fish collagen oligopeptide prepared in this example, and it can be seen from Table 1 that the molecular weight of the marine fish collagen oligopeptide prepared in this example is up to 91.77% of 1000D, which is to the extent that the marine fish collagen oligopeptide can be absorbed without digestion or with little digestion, and has high absorption efficiency.

Table 1:

(2) The salmon nasal cartilage (or shark cartilage) is prepared by pulverizing, ultrasonic extracting, acidolysis and enzymolysis, centrifuging, concentrating under reduced pressure, vacuum freeze drying, and pulverizing to obtain salmon nasal cartilage extract (or shark cartilage extract), which comprises the following steps:

(a) Crushing and ultrasonic extraction: pulverizing salmon nasal cartilage or shark cartilage into coarse particles, sieving with 10 mesh sieve, adding purified water 12 times the weight of the materials into an extraction kettle, heating with steam to 102 deg.C, maintaining, and turning on ultrasonic auxiliary extraction equipment with ultrasonic power of 8KW for 2 times and extraction time of 1 hr;

(b) Acidolysis enzymolysis: transferring the extract into an enzymolysis tank, regulating the pH to be between 4.0 by using dilute sulfuric acid, and keeping the temperature at 95 ℃ for 1h; cooling to 50 ℃ after acidolysis is finished, maintaining, adjusting the pH to 11, adding bacillus licheniformis protease with the substrate content of salmon nasal cartilage or shark cartilage of 0.4wt%, uniformly stirring and carrying out enzymolysis for 1.5 hours, adjusting the pH of an enzymolysis solution to be neutral, and carrying out enzyme deactivation by a high-temperature instantaneous enzyme deactivation method after the enzymolysis is finished;

(c) And (3) centrifugal filtration: centrifuging the enzyme-deactivated enzymolysis liquid at 16000rpm by a centrifuge to remove impurities, collecting clear liquid, starting a membrane filtering device, filtering the clear liquid by a microporous membrane with a thickness of 110 μm and a pore diameter of 8 μm, and operating at 0.25MPa to ensure that the filtrate is clear and transparent, and removing filter residues; the filtered filtrate is subjected to decolorization and deodorization treatment through an activated carbon column;

(d) Concentrating under reduced pressure: concentrating under reduced pressure at 70deg.C under vacuum of-0.09 Mpa and relative density of 1.16 to obtain concentrated extract;

(e) Vacuum freeze drying: putting salmon nasal cartilage or concentrated extract of shark cartilage into vacuum freeze drying equipment for drying, wherein parameters are set as follows: pre-freezing initial temperature-45 ℃, pre-freezing speed 0.45 ℃/min, pre-freezing end temperature-68 ℃, drying chamber pressure 45pa, heating plate temperature 36 ℃ and drying time 28h;

(f) Crushing: pulverizing the dried extract, and sieving with 150 mesh sieve to obtain salmon nasal cartilage extract or shark cartilage extract.

The salmon nasal cartilage extract (or the shark cartilage extract) prepared by the above extraction acidolysis enzymolysis process has greatly reduced amount compared with the raw materials, greatly enriched effective components such as proteoglycan and short peptide, and the like, is more beneficial to improving bioavailability, plays roles of promoting cartilage regeneration, increasing joint synovial fluid and improving bone joint health, and has the proteoglycan content of 65wt%, the short peptide content of 55wt% and the chondroitin content of 25wt% in the shark cartilage extract.

(3) Pulverizing herba Medicaginis or herba Albiziae or herba Arenariae Albiziae, and supercritical CO ₂ Extracting, refining by column, concentrating under reduced pressure, vacuum freeze drying, and pulverizing to obtain herba Medicaginis extract or radix Arisaema extract or herba Arisaema extract, which is prepared by the following steps:

(a) Coarse powder: pulverizing herba Medicaginis or herba Albiziae or herba Blumeae Balsamiferae, and sieving with 8 mesh sieve;

(b) Supercritical CO ₂ Extraction: coarse powder of herba Medicaginis or alloy, CO ₂ Supercritical extraction (extraction temperature 36 deg.C, extraction pressure 38Mpa, CO) ₂ Flow rate is 10kg/h, extraction time is 120min, separation pressure is 5MPa, separation temperature is 42 ℃, entrainer with 5wt% of crude drug is added, the entrainer is ethanol solution with 50% V/V concentration), and the process is carried outSeparating to obtain herba Medicaginis or radix Arisaema or herba Arisaema;

(c) Refining by column: refining the extract with macroporous resin separation column at column passing rate of 2L/h for 5 hr, and discarding ineffective fraction;

(d) Concentrating under reduced pressure: concentrating the refined effective fraction under reduced pressure at 65deg.C and vacuum degree of-0.095 Mpa to obtain Chinese medicinal concentrated solution with relative density of 1.18;

(e) Vacuum freeze drying: drying the concentrated Chinese medicinal materials in vacuum freeze drying equipment, with parameters set as follows: pre-freezing initial temperature-45 ℃, pre-freezing speed 0.35 ℃/min, pre-freezing end temperature-60 ℃, drying chamber pressure 40pa, heating plate temperature 45 ℃ and drying time 24h;

(f) Crushing: pulverizing the dried extract, and sieving with 200 mesh sieve to obtain herba Medicaginis extract or radix Arisaema extract or herba Ipomoeae extract.

Due to the adoption of supercritical CO ₂ The preparation method comprises the steps of extraction, column refining, vacuum freeze drying and other processing technologies, and the obtained alfalfa extract, the alloy-leaved sweetgum extract and the pre-flatcar extract have the advantages of high content of crude polysaccharide, protein and total flavone, good effect, high stability and high bioavailability which are greatly improved by 12 times compared with crude drugs.

(4) Mixing 34 parts of marine fish bone collagen oligopeptide, 5 parts of salmon nasal cartilage extract and 12 parts of shark cartilage extract by using a small three-dimensional mixer for 10min to obtain a mixture, and adding 7.5 parts of alfalfa extract, 5 parts of front-flatcar extract, 5.5 parts of acacia extract, 3.5 parts of Abelmoschus manihot extract, 5 parts of rabdosia rubescens extract, 5 parts of patrinia extract, 1 part of sodium hyaluronate, 3.5 parts of curcumin and 13 parts of chitosan oligosaccharide into the mixture, and continuously stirring for 20min to be completely uniform by using a groove-type mixer to obtain the required traditional Chinese medicine bone peptide composition.

Example 2:

(1) Pulverizing marine fish bones, sieving with 100 mesh sieve, adding purified water with the weight of 20 times of the material weight to prepare dispersion, adjusting pH to 8.5, heating at 37deg.C for heat preservation, adding trypsin and neutral protease, stirring for enzymolysis for 2.5h, heating to 52deg.C, continuing enzymolysis for 2h to obtain enzymolysis solution, wherein the addition amount of trypsin is 0.35wt% of the substrate amount, the addition amount of neutral protease is 0.25wt% of the substrate amount, and inactivating enzyme after enzymolysis is completed; centrifuging the enzymolysis solution at 12000rpm, collecting clear liquid, filtering with microporous membrane with thickness of 60 μm and pore diameter of 3 μm and pressure of 0.1MPa, and removing residue; desalting and decolorizing the filtrate with ion exchange column and active carbon column, and nanofiltration to remove free amino acid; concentrating the filtrate under vacuum at 65deg.C and vacuum degree of-0.075 Mpa to obtain oligopeptide concentrated solution, and controlling the relative density at 1.04; spray drying the concentrated solution, wherein the nozzle temperature is 175 ℃, the outlet temperature is 85 ℃, and the feeding speed is 1.2L/min, so that the required marine fish bone collagen oligopeptide is obtained.

(2) Pulverizing salmon nasal cartilage or shark cartilage into coarse particles, sieving with 10 mesh sieve, adding purified water 8 times the weight of the materials into an extraction kettle, heating to 95deg.C, maintaining, and turning on ultrasonic assistance with power of 5KW for 2 times for 1 hr; transferring the extract into an enzymolysis tank, regulating pH to 3.5 with dilute sulfuric acid, and maintaining at 85deg.C for 0.5 hr; cooling to 55deg.C after acidolysis, maintaining, adjusting pH to 10, adding Bacillus licheniformis protease 0.3wt% of salmon nasal cartilage or shark cartilage substrate, stirring uniformly, performing enzymolysis for 1.0 hr, adjusting pH to neutrality, and inactivating enzyme after enzymolysis is completed; centrifuging the enzymolysis solution at 15000rpm, removing impurities, collecting clear liquid, filtering the clear liquid with microporous membrane with thickness of 80 μm and pore diameter of 10 μm and pressure of 0.15MPa, and removing residue; decolorizing and deodorizing the filtrate by an activated carbon column; concentrating the extractive solution under reduced pressure at 75deg.C under vacuum degree of-0.09 Mpa with relative density of 1.15 to obtain concentrated extract; vacuum freeze-drying the extract, wherein the pre-freezing initial temperature is-50 ℃, the pre-freezing speed is 0.35 ℃/min, the pre-freezing end temperature is-68 ℃, the drying chamber pressure is 35pa, the heating plate temperature is 32 ℃, and the drying time is 28h; pulverizing the extract, and sieving with 200 mesh sieve to obtain the desired salmon nasal cartilage extract or shark cartilage extract.

(3) Pulverizing herba Medicaginis or herba Albiziae or herba Blumeae Balsamiferae, and sieving with 12 mesh sieve; CO ₂ Supercritical extraction at 38deg.C under 42mpa and CO ₂ Flow rate is 12kg/h, extraction time is 120min, and separation pressure is 5MPa, adding entrainer (60% V/V concentration ethanol solution) with crude drug content of 5wt% at a separation temperature of 38 ℃ to obtain an extract by separation; refining the extract by using a macroporous resin separation column at a column passing rate of 3L/h for 4h, and discarding invalid fractions; vacuum concentrating the effective fraction at 60deg.C under vacuum degree of-0.085 Mpa to obtain Chinese medicinal concentrated solution with relative density of 1.18; vacuum freeze drying of the concentrated Chinese medicinal liquid, pre-freezing initial temperature of-45deg.C, pre-freezing rate of 0.25deg.C/min, pre-freezing final temperature of-60deg.C, drying chamber pressure of 40pa, heating plate temperature of 45deg.C, and drying time of 24 hr; pulverizing the extract blocks, and sieving with 100 mesh sieve to obtain herba Medicaginis extract, alloy flower extract or front-of-flatcar extract.

(4) Mixing 40 parts of marine fish bone collagen oligopeptide, 6 parts of salmon nasal cartilage extract and 9.5 parts of shark cartilage extract by using a small three-dimensional mixer for 5min to obtain a mixture, and adding 8.5 parts of alfalfa extract, 4.5 parts of front-flatcar extract, 4.5 parts of acacia extract, 2.5 parts of herba Abutili extract, 6 parts of rabdosia rubescens extract, 4 parts of herba patriniae extract, 0.5 part of sodium hyaluronate, 4 parts of curcumin and 10 parts of chitosan oligosaccharide into the mixture, and continuously stirring for 15min to be completely uniform by using a groove-type mixer to obtain the required traditional Chinese medicine bone peptide composition.

Example 3:

(1) Pulverizing marine fish bones, sieving with a 60-mesh sieve, adding purified water 10 times the weight of the materials to prepare dispersion, adjusting the pH to 6.5, heating and preserving heat at 38 ℃, adding trypsin and neutral protease, stirring for enzymolysis for 1.5h, heating to 58 ℃, continuing to carry out enzymolysis for 2.5h to obtain enzymolysis liquid, wherein the adding amount of trypsin is 0.35wt% of the substrate amount, the adding amount of neutral protease is 0.3wt% of the substrate amount, and inactivating enzyme after the enzymolysis is finished; centrifugally separating the enzymolysis liquid at 14000rpm, collecting clear liquid, filtering with microporous membrane with thickness of 90 μm and pore diameter of 5 μm and pressure of 0.1MPa, and removing filter residue; desalting and decolorizing the filtrate with ion exchange column and active carbon column, and nanofiltration to remove free amino acid; concentrating the filtrate under vacuum and reduced pressure at 65deg.C and vacuum degree of-0.09 Mpa to obtain oligopeptide concentrated solution with relative density controlled at 1.04; spray drying the concentrated solution, wherein the nozzle temperature is 155 ℃, the outlet temperature is 80 ℃, and the feeding speed is 1.8L/min, so that the required marine fish bone collagen oligopeptide is obtained.

(2) Pulverizing salmon nasal cartilage or shark cartilage into coarse particles, sieving with 10 mesh sieve, adding purified water 15 times the weight of the materials into an extraction kettle, heating to 102 deg.C, maintaining, and turning on ultrasonic auxiliary power 20KW for 2 times for 1.5 hr; transferring the extract into an enzymolysis tank, regulating pH to 4.0 with dilute sulfuric acid, and maintaining at 95deg.C for 0.5 hr; cooling to 55deg.C after acidolysis, maintaining, adjusting pH to 11, adding Bacillus licheniformis protease 0.4wt% of salmon nasal cartilage or shark cartilage substrate, stirring uniformly, performing enzymolysis for 1.5 hr, adjusting pH to neutrality, and inactivating enzyme after enzymolysis is completed; centrifuging the enzymolysis solution at 18000rpm, removing impurities, collecting clear liquid, filtering the clear liquid with microporous membrane with thickness of 120 μm and pore diameter of 10 μm and pressure of 0.35MPa, and removing residue; decolorizing and deodorizing the filtrate by an activated carbon column; concentrating the extractive solution under reduced pressure at 75deg.C under vacuum degree of-0.07 Mpa with relative density of 1.18 to obtain concentrated extract; vacuum freeze-drying the extract, wherein the pre-freezing initial temperature is-40 ℃, the pre-freezing speed is 0.35 ℃/min, the pre-freezing end temperature is-68 ℃, the drying chamber pressure is 50pa, the heating plate temperature is 32 ℃, and the drying time is 36h; pulverizing the extract, and sieving with 150 mesh sieve to obtain the desired salmon nasal cartilage extract or shark cartilage extract.

(3) Pulverizing herba Medicaginis or herba Albiziae or herba Blumeae Balsamiferae, and sieving with 12 mesh sieve; CO ₂ Supercritical extraction at 32deg.C under 42mpa and CO ₂ The flow is 10kg/h, the extraction time is 130min, the separation pressure is 5MPa, the separation temperature is 42 ℃, the entrainer (80% V/V concentration ethanol solution) with the crude drug content of 5wt% is added, and the extract is obtained by separation; refining the extract by using a macroporous resin separation column, wherein the column passing speed is 3L/h, the time is 3h, and discarding invalid fractions; vacuum concentrating the effective fraction at 60deg.C under vacuum degree of-0.095 Mpa to obtain Chinese medicinal concentrated solution with relative density of 1.17; vacuum freeze drying of the concentrated Chinese medicinal liquid, pre-freezing initial temperature of-52deg.C, pre-freezing rate of 0.35 deg.C/min, pre-freezing final temperature of-60deg.C, drying chamber pressure of 35pa, heating plate temperature of 40deg.C, and drying time of 32 hr; pulverizing the extract, sieving with 120 mesh sieve to obtain herba Medicaginis extract, alloy flower extract or front-of-flatcar extract。

(4) Mixing 29 parts of marine fish bone collagen oligopeptide, 4 parts of salmon nasal cartilage extract and 10 parts of shark cartilage extract by using a small three-dimensional mixer for 15min to obtain a mixture, adding 11 parts of alfalfa extract, 3.5 parts of front-flatcar extract, 7 parts of acacia extract, 2 parts of herba Abutili extract, 4 parts of rabdosia rubescens extract, 7 parts of herba patriniae extract, 1.5 parts of sodium hyaluronate, 3 parts of curcumin and 18 parts of chitosan oligosaccharide into the mixture, and continuously stirring for 30min to be completely uniform by using a trough-type mixer to obtain the required traditional Chinese medicine bone peptide composition.

Example 4:

(1) Pulverizing marine fish bones, sieving with 100 mesh sieve, adding purified water 15 times the weight of the materials to prepare dispersion, adjusting pH to 7.5, heating at 36.5deg.C, maintaining temperature, adding trypsin and neutral protease, stirring for enzymolysis for 2.5h, heating to 53deg.C, continuing enzymolysis for 2.0h to obtain enzymolysis solution, wherein the adding amount of trypsin is 0.25wt% of the substrate amount, the adding amount of neutral protease is 0.3wt% of the substrate amount, and inactivating enzyme after enzymolysis is completed; centrifugally separating the enzymolysis liquid at 13000rpm, collecting clear liquid, filtering with microporous membrane with thickness of 60 μm and pore diameter of 2 μm and pressure of 0.05MPa, and removing residue; desalting and decolorizing the filtrate with ion exchange column and active carbon column, and nanofiltration to remove free amino acid; concentrating the filtrate under reduced pressure at 65deg.C and vacuum degree of-0.085 Mpa to obtain oligopeptide concentrated solution with relative density controlled at 1.05; spray drying the concentrated solution, wherein the nozzle temperature is 175 ℃, the outlet temperature is 80 ℃, and the feeding speed is 1.0L/min, so that the required marine fish bone collagen oligopeptide is obtained.

(2) Pulverizing salmon nasal cartilage or shark cartilage into coarse particles, sieving with 10 mesh sieve, adding purified water 8 times the weight of the materials into an extraction kettle, heating to 90deg.C, maintaining, and turning on ultrasonic assistance with power of 5KW for 0.5 hr for 1 time; transferring the extracting solution into an enzymolysis tank, regulating the pH to 3.0 by dilute sulfuric acid, and keeping the temperature at 85 ℃ for 1h; cooling to 55deg.C after acidolysis, maintaining, adjusting pH to 11, adding Bacillus licheniformis protease 0.35wt% of salmon nasal cartilage or shark cartilage substrate, stirring uniformly, performing enzymolysis for 1.5 hr, adjusting pH to neutrality, and inactivating enzyme after enzymolysis is completed; centrifuging the enzymolysis solution at 18000rpm, removing impurities, collecting clear liquid, filtering the clear liquid with microporous membrane with thickness of 110 μm and pore diameter of 8 μm and pressure of 0.15MPa, and removing residue; decolorizing and deodorizing the filtrate by an activated carbon column; concentrating the extractive solution under reduced pressure at 70deg.C under vacuum degree-0.075 Mpa with relative density of 1.15 to obtain concentrated extract; vacuum freeze-drying the extract, wherein the pre-freezing initial temperature is-45 ℃, the pre-freezing speed is 0.45 ℃/min, the pre-freezing end temperature is-68 ℃, the drying chamber pressure is 40pa, the heating plate temperature is 36 ℃, and the drying time is 32h; pulverizing the extract, and sieving with 150 mesh sieve to obtain the desired salmon nasal cartilage extract or shark cartilage extract.

(3) Pulverizing herba Medicaginis or herba Albiziae or herba Bl Albiziae, and sieving with 10 mesh sieve; CO ₂ Supercritical extraction at 32deg.C under 42mpa and CO ₂ The flow is 12kg/h, the extraction time is 140min, the separation pressure is 5.5MPa, the separation temperature is 42 ℃, the entrainer (40% V/V concentration ethanol solution) with the crude drug content of 7wt% is added, and the extract is obtained by separation; refining the extract by using a macroporous resin separation column, wherein the column passing speed is 2L/h, the time is 3h, and discarding invalid fractions; vacuum concentrating the effective fraction at 60deg.C under vacuum degree of-0.085 Mpa to obtain Chinese medicinal concentrated solution with relative density of 1.17; vacuum freeze drying of the concentrated Chinese medicinal liquid, pre-freezing initial temperature of-45deg.C, pre-freezing rate of 0.25deg.C/min, pre-freezing final temperature of-60deg.C, drying chamber pressure of 35pa, heating plate temperature of 40deg.C, and drying time of 24 hr; pulverizing the extract blocks, and sieving with 100 mesh sieve to obtain herba Medicaginis extract, alloy flower extract or front-of-flatcar extract.

(4) Mixing 42 parts of marine fish bone collagen oligopeptide, 5 parts of salmon nasal cartilage extract and 12 parts of shark cartilage extract by using a small three-dimensional mixer for 15min to obtain a mixture, adding 7 parts of alfalfa extract, 6.5 parts of front-flatcar extract, 4 parts of acacia extract, 3 parts of herba Abutili extract, 4 parts of rabdosia rubescentis extract, 4 parts of herba patriniae extract, 1 part of sodium hyaluronate, 3.5 parts of curcumin and 8 parts of chitosan oligosaccharide into the mixture, and continuously stirring for 15min to be completely uniform by using a trough-type mixer to obtain the required traditional Chinese medicine bone peptide composition.

Example 5:

(1) Pulverizing marine fish bones, sieving with 80 mesh sieve, adding purified water 15 times the weight of the materials to prepare dispersion, adjusting pH to 7.0, heating at 37deg.C for heat preservation, adding trypsin and neutral protease, stirring for enzymolysis for 2h, heating to 55deg.C, continuing enzymolysis for 2.0h to obtain enzymolysis solution, wherein the addition amount of trypsin is 0.25wt% of the substrate amount, the addition amount of neutral protease is 0.3wt% of the substrate amount, and inactivating enzyme after enzymolysis is completed; centrifugally separating the enzymolysis liquid at 14000rpm, collecting clear liquid, filtering with microporous membrane with thickness of 50 μm and pore diameter of 3 μm and pressure of 0.15MPa, and removing filter residue; desalting and decolorizing the filtrate with ion exchange column and active carbon column, and nanofiltration to remove free amino acid; concentrating the filtrate under vacuum and reduced pressure at 65deg.C and vacuum degree of-0.08 Mpa to obtain oligopeptide concentrated solution with relative density controlled at 1.05; spray drying the concentrated solution, wherein the nozzle temperature is 165 ℃, the outlet temperature is 82 ℃, and the feeding speed is 1.2L/min, so that the required marine fish bone collagen oligopeptide is obtained.

(2) Pulverizing salmon nasal cartilage or shark cartilage into coarse particles, sieving with 10 mesh sieve, adding purified water 12 times the weight of the materials into an extraction kettle, heating to 95deg.C, maintaining, and turning on ultrasonic assistance with power of 10KW for 2 times for 1.0 hr; transferring the extract into an enzymolysis tank, regulating pH to 4.0 with dilute sulfuric acid, and maintaining at 90deg.C for 1 hr; cooling to 45 ℃ after acidolysis, keeping the temperature, adjusting the pH to 10.5, adding bacillus licheniformis protease with the substrate content of salmon nasal cartilage or shark cartilage of 0.45wt%, uniformly stirring and carrying out enzymolysis for 1.5 hours, adjusting the pH to be neutral, and inactivating the enzyme after the enzymolysis is finished; centrifuging the enzymolysis liquid at 16000rpm, removing impurities, collecting clear liquid, filtering the clear liquid with microporous membrane with thickness of 100 μm and pore diameter of 8 μm and pressure of 0.20MPa, and removing residue; decolorizing and deodorizing the filtrate by an activated carbon column; concentrating the extractive solution under reduced pressure at 68deg.C under vacuum degree-0.075 Mpa with relative density of 1.15 to obtain concentrated extract; vacuum freeze-drying the extract, wherein the pre-freezing initial temperature is-45 ℃, the pre-freezing speed is 0.45 ℃/min, the pre-freezing end temperature is-68 ℃, the drying chamber pressure is 40pa, the heating plate temperature is 32 ℃, and the drying time is 30h; pulverizing the extract, and sieving with 100 mesh sieve to obtain the desired salmon nasal cartilage extract or shark cartilage extract.

(3) By mixing herba ViolaePulverizing herba Medicaginis or alloy, and sieving with 10 mesh sieve; CO ₂ Supercritical extraction at 32deg.C under 40Mpa under CO ₂ The flow is 12kg/h, the extraction time is 120min, the separation pressure is 5MPa, the separation temperature is 38 ℃, the entrainer (40% V/V concentration ethanol solution) with the crude drug content of 6wt% is added, and the extract is obtained by separation; refining the extract by using a macroporous resin separation column at a column passing rate of 1L/h for 3.5h, and discarding invalid fractions; vacuum concentrating the effective fraction at 65deg.C under vacuum degree of-0.085 Mpa to obtain Chinese medicinal concentrated solution with relative density of 1.18; vacuum freeze drying of the concentrated Chinese medicinal liquid, pre-freezing initial temperature of-45deg.C, pre-freezing rate of 0.30 deg.C/min, pre-freezing final temperature of-60deg.C, drying chamber pressure of 37pa, heating plate temperature of 42 deg.C, and drying time of 28 hr; pulverizing the extract blocks, and sieving with 100 mesh sieve to obtain herba Medicaginis extract, alloy flower extract or front-of-flatcar extract.

(4) Mixing 26 parts of marine fish bone collagen oligopeptide, 7 parts of salmon nasal cartilage extract and 11 parts of shark cartilage extract by using a small three-dimensional mixer for 12 minutes to obtain a mixture, adding 8 parts of alfalfa extract, 7.5 parts of front-flatcar extract, 6 parts of acacia extract, 5 parts of herba Abutili extract, 6 parts of rabdosia rubescentis extract, 5 parts of herba patriniae extract, 1.5 parts of sodium hyaluronate, 5 parts of curcumin and 12 parts of chitosan oligosaccharide into the mixture, and continuously stirring for 25 minutes by using a trough-type mixer until the mixture is completely uniform, thus obtaining the required traditional Chinese medicine bone peptide composition.

The foregoing description of the preferred embodiments of the invention should not be taken as limiting the scope of the invention, which is defined by the appended claims, or any modifications or adaptations of the invention using its general principles and without departing from the principles of the invention, or by direct or indirect application in other relevant fields.

The prepared traditional Chinese medicine bone peptide composition is subjected to human body test feeding test:

sample: the bone peptide composition of the traditional Chinese medicine prepared in the embodiment 1 of the invention is taken orally 2 times per day with 6g of warm boiled water as a test food sample, and the total daily dosage is 12g.

Inclusion of subject criteria: voluntary subjects who are in line with osteoarthritis or joint pain swelling.

Efficacy evaluation criteria: cure (complete disappearance of clinical symptoms, complete disappearance of inflammation, swelling and pain), obvious effect (obvious improvement of clinical symptoms, great reduction of occurrence probability of inflammation, swelling and pain), effective (improvement of clinical symptoms, even inflammation, swelling and pain but reduction of occurrence probability), ineffective (no change or even aggravation of clinical symptoms), and total effective rate=cure rate+obvious rate+effective rate.

1. General conditions:

the initial test population has 30 cases of test feeding group, 30 cases of negative control group and 30 cases of positive control group, and the test subjects have normal mental, sleeping, diet and urination and defecation states before and after test feeding, and no abnormal phenomenon occurs. The number, sex, number of people, age and course of disease are shown in Table 2. From the data in Table 2, the data of three groups of patients were found to have no significant differences, no statistical significance, comparability, and P > 0.05.

Table 2:

2. the test method comprises the following steps:

the test group takes the traditional Chinese medicine bone peptide composition prepared in the embodiment 1 of the invention, and the daily dosage is 12g; the ibuprofen slow-release capsule is taken by a positive control group, 2 capsules are taken daily, and the dosage of the ibuprofen slow-release capsule is 600mg daily; the negative control group took the same dose of placebo for a period of 15 days. After 15 days, the statistical diagnostic data were analyzed comprehensively by clinical diagnosis of the inflammatory and pain severity.

3. Test results:

the data pair of effects of the three groups of volunteers after the test food are shown in table 3:

table 3:

as can be seen from the results in Table 3, 7 cases are cured in the test feeding group, 13 cases are effectively treated, 8 cases are effectively treated, the total effective rate is 93.3%, and the total effective rate of the test feeding group is obviously higher than that of the negative control group (P < 0.01) and is also obviously higher than that of the positive control group. All three groups of patients have no obvious adverse reaction.

4. Summarizing:

after the traditional Chinese medicine bone peptide composition prepared in the embodiment 1 of the invention is taken according to a specified method and dosage by a test group, clinical symptoms are greatly improved until the patients are healed, the total effective rate is higher than that of a negative control group and a positive control group (the test results of other embodiments are similar to those of the embodiment 1), and the traditional Chinese medicine bone peptide composition has obvious and positive promotion effect on improving the health of bone joints, so that the composition can be considered to have the effect of improving the health function of bone joints and has unique effect.

Claims

1. A traditional Chinese medicine bone peptide composition with a function of improving bone joint health is characterized by comprising the following components in parts by mass:

26-42 parts of marine fish bone collagen oligopeptide

4-11 parts of alfalfa extract

2.5-7.5 parts of plantago asiatica extract

3-8 parts of acacia extract

2-5 parts of an extract of herba Abelmoschi Manihot

3-7 parts of rabdosia rubescens extract

3-7 parts of herba patriniae extract

0.5-1.5 parts of sodium hyaluronate

3-7 parts of salmon nasal cartilage extract

8-16 parts of shark cartilage extract

Curcumin 2-5 parts

8-18 parts of chitosan oligosaccharide;

the oligopeptide component with the molecular weight smaller than 1000D in the marine fish bone collagen oligopeptide accounts for more than 90 percent, so that the marine fish bone collagen oligopeptide can be absorbed without digestion or slightly digested;

The preparation method of the traditional Chinese medicine bone peptide composition comprises the following steps:

(4) Uniformly mixing the prepared marine fish bone collagen oligopeptide, salmon nasal cartilage extract and shark cartilage extract according to the mass part ratio to obtain a mixture, and then adding the prepared alfalfa extract, the pre-flatcar extract, the acacia extract, the mallow extract, the rabdosia rubescens extract, the patrinia herb extract, the sodium hyaluronate, the curcumin and the chitosan oligosaccharide into the mixture, and uniformly mixing to obtain the required traditional Chinese medicine bone peptide composition;

The ocean fish bone collagen oligopeptide in the step (1) is obtained through the following steps:

(a) Crushing and size mixing: crushing marine fishbone, sieving with a 60-100 mesh sieve, adding purified water with the weight of 10-20 times of the weight of the material, dispersing in an aqueous solution to prepare a marine fishbone powder dispersion liquid, and regulating the pH of the marine fishbone powder dispersion liquid to 6.5-8.5;

(b) Enzymolysis: heating the marine fishbone powder dispersion liquid, keeping the temperature at 37+/-1 ℃, adding trypsin and neutral protease, uniformly stirring and carrying out enzymolysis for 1.5-2.5 hours, heating to 55+/-3 ℃, and continuing stirring and carrying out enzymolysis for 1-2.5 hours to obtain an enzymolysis liquid, wherein the adding amount of the trypsin is 0.2-0.35 wt% of the substrate content, the adding amount of the neutral protease is 0.25-0.4 wt% of the substrate content, and carrying out enzyme deactivation by a high-temperature instantaneous enzyme deactivation method after the enzymolysis is finished;

(c) And (3) centrifugal filtration: centrifugally separating enzyme-deactivated enzymolysis liquid by a centrifugal machine 12000-14000 rpm to remove impurities, collecting clear liquid, starting membrane filtering equipment, filtering the clear liquid by a microporous membrane with the thickness of 40-90 mu m, the pore diameter of the membrane of 2-5 mu m and the operating pressure of 0.05-0.2 MPa, ensuring that the filtrate is clear and transparent, and removing filter residues; desalting and decolorizing the filtered filtrate with ion exchange column and active carbon column, and nanofiltration to remove free amino acid;

(e) Spray drying: spray drying the oligopeptide concentrated solution, wherein the technological parameters are that the nozzle temperature is 155-175 ℃, the outlet temperature is 80-85 ℃, and the feeding speed is 1.0-1.8L/min, so that the required marine fish bone collagen oligopeptide is obtained;

the salmon nasal cartilage extract and the shark cartilage extract in the step (2) are obtained by the following steps:

(a) Crushing and ultrasonic extraction: taking salmon nasal cartilage or shark cartilage, crushing into coarse particles, sieving with a 10-mesh sieve, adding purified water with the weight 8-15 times of the weight of the materials into an extraction kettle, starting steam heating to 90-105 ℃ and maintaining, starting ultrasonic auxiliary extraction equipment, wherein the ultrasonic power is 5-20 KW, the extraction times are 1-2 times, and the extraction time is 0.5-1.5 h;

(b) Acidolysis enzymolysis: transferring the extracting solution into an enzymolysis tank, regulating the pH to 3.0-5.0 by using dilute sulfuric acid, and maintaining the temperature at 85-95 ℃ for 0.5-1 h; cooling to 45-55 ℃ after acidolysis is finished, maintaining, regulating the pH to 10-11, adding bacillus licheniformis protease with the concentration of 0.3-0.6wt% of salmon nasal cartilage or shark cartilage substrate, uniformly stirring and carrying out enzymolysis for 1.0-2.0 h, regulating the pH of an enzymolysis solution to be neutral, and carrying out enzyme deactivation by a high-temperature instantaneous enzyme deactivation method after the enzymolysis is finished;

(c) And (3) centrifugal filtration: centrifuging the enzyme-deactivated enzymolysis liquid at 15000-18000 rpm by a centrifuge to remove impurities, collecting clear liquid, starting a membrane filtering device, filtering the clear liquid by a microporous membrane with the thickness of 80-120 μm and the pore diameter of 6-10 μm, and operating the pressure of 0.15-0.35 MPa to ensure that the filtrate is clear and transparent, and removing filter residues; the filtered filtrate is subjected to decolorization and deodorization treatment through an activated carbon column;

(d) Concentrating under reduced pressure: concentrating the extract under reduced pressure, controlling the concentration temperature at 65-75 ℃, controlling the vacuum degree at-0.06 to-0.09 Mpa, and controlling the relative density at 1.12-1.18 to obtain concentrated extract;

(e) Vacuum freeze drying: putting salmon nasal cartilage or concentrated extract of shark cartilage into vacuum freeze drying equipment for drying, wherein parameters are set as follows: pre-freezing initial temperature is-40 to-50 ℃, pre-freezing speed is 0.35 to 0.55 ℃/min, pre-freezing end temperature is-68 ℃, drying chamber pressure is 30 to 50pa, heating plate temperature is 32 to 38 ℃, and drying time is 28 to 36 hours;

(f) Crushing: crushing the dried extract blocks, and sieving the crushed extract blocks with a 100-200-mesh sieve to obtain a required salmon nasal cartilage extract or a required shark cartilage extract;

the alfalfa extract, the alloy-flower extract and the pre-flatcar extract in the step (3) are obtained through the following steps:

(b) Supercritical CO ₂ Extraction: coarse powder of herba Medicaginis or alloy, CO ₂ Extracting by a supercritical extraction method, wherein parameters are set as follows: the extraction temperature is 32-38 ℃, the extraction pressure is 38-42 mpa, and CO ₂ The flow is 10-12 kg/h, the extraction time is 120-150 min, the separation pressure is 5-6MPa, the separation temperature is 38-45 ℃, entrainer with the crude drug content of 5-10wt% is added, the entrainer is ethanol solution with the concentration of 40-80% V/V, and the alfalfa or the alloy or the front-flat-car extract is obtained through separation;

(d) Concentrating under reduced pressure: vacuum concentrating the effective fraction after column refining at 60-70deg.C under vacuum degree of-0.085 to-0.095 Mpa to obtain Chinese medicinal concentrated solution with relative density of 1.17-1.22;

(e) Vacuum freeze drying: drying the concentrated Chinese medicinal liquid in vacuum freeze drying equipment, with parameters set as follows: pre-freezing initial temperature is-45 to-52 ℃, pre-freezing speed is 0.25 to 0.35 ℃/min, pre-freezing end temperature is-60 ℃, drying chamber pressure is 35 to 40pa, heating plate temperature is 40 to 45 ℃, and drying time is 24 to 32 hours;

(f) Crushing: crushing the dried extract blocks, and sieving the crushed extract blocks with a 100-200-mesh sieve to obtain the required alfalfa extract or alloy-happy extract or front-flatcar extract.