CN114522183A - Sea cucumber extract for increasing bone mineral density - Google Patents
Sea cucumber extract for increasing bone mineral density Download PDFInfo
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- CN114522183A CN114522183A CN202210085751.2A CN202210085751A CN114522183A CN 114522183 A CN114522183 A CN 114522183A CN 202210085751 A CN202210085751 A CN 202210085751A CN 114522183 A CN114522183 A CN 114522183A
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- Prior art keywords
- sea cucumber
- bone
- cucumber extract
- calcium
- enzymolysis
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Abstract
The invention discloses a sea cucumber extract for increasing bone mineral density, and belongs to the technical field of health-care food. The method comprises the steps of crushing the sea cucumber, carrying out enzymolysis, and carrying out enzyme deactivation, centrifugation, filtration and drying on enzymolysis liquid after enzymolysis to obtain the sea cucumber extract. The sea cucumber extract with the function of increasing the bone mineral density provided by the invention contains a plurality of beneficial components such as sea cucumber polypeptide and sea cucumber polysaccharide, has various action mechanisms, can balance the bone metabolism condition from multiple aspects, promotes the body to absorb substances such as calcium and the like, and has a synergistic effect. It has effects in promoting mineral element absorption and deposition, inhibiting bone inflammation, balancing bone metabolism, repairing and promoting bone and bone joint, and maintaining bone health. In addition, after the sea cucumber extract and the calcium ions are compounded, the two have a synergistic effect on the aspect of improving the bone mineral density in a rat body.
Description
Technical Field
The invention relates to a sea cucumber extract for increasing bone mineral density, belonging to the technical field of health-care food.
Background
Osteoporosis is a disease induced by a variety of causes that can lead to decreased bone density and quality, destruction of bone microarchitecture, and increased risk of fracture. Osteoporosis patients are mostly old people and menopausal women, the life of the patients is very inconvenient, the brittleness of bones is increased, the fracture risk is increased, even slight trauma can cause serious consequences such as fracture, and the life quality and the life span are seriously influenced. Osteoporosis is not easily treated because of its many causes. With the aging of society, the incidence of osteoporosis is getting higher and higher, not only the patients suffer from pain, but also the requirements of treatment and nursing related to the patients are increased, which is a problem needing important attention in China and all over the world.
Sea cucumber contains various active substances, such as collagen, polysaccharide, etc. Sea cucumbers can be used as medicines since ancient times, and have various efficacies such as anti-inflammation and the like, which have potential benefits for bone health.
At present, health-care food aiming at osteoporosis in the market is mainly a calcium supplement product, such as calcium-containing capsules or granules, and the like, the main aim of the health-care food is to increase bone mineral density by supplementing calcium in a body, and the action mechanism of the product is single. Due to complicated pathogenesis of osteoporosis, such as bone metabolism and calcium balance disorder, the expected effect cannot be achieved by simply supplementing calcium, and the calcium in the osteoporosis is low in absorption and utilization rate due to defects of product characteristics or body function decline and the like. Therefore, it is very interesting to develop some natural sources as a therapeutic approach.
Disclosure of Invention
[ problem ] to provide a method for producing a semiconductor device
At present, health-care food aiming at osteoporosis in the market is mainly calcium supplement products such as calcium-containing capsules or granules, and the main aim of the health-care food is to increase bone density by supplementing calcium in a body. The product has a single action mechanism, the expected effect cannot be achieved by simply supplementing calcium due to the complicated pathogenesis of osteoporosis, and the calcium cannot be well absorbed and utilized due to the defects of the product characteristics or the decline of the body function and the like.
Therefore, the technical problems to be solved by the invention are as follows: provided is a sea cucumber extract capable of increasing bone density, which is effective in preventing and treating osteoporosis by improving bone metabolism and promoting calcium absorption.
[ technical solution ] A
The sea cucumber extract rich in sea cucumber polypeptide and sea cucumber polysaccharide is obtained by enzymolysis of sea cucumber, and has the effects of increasing bone mineral density, improving bone metabolism, inhibiting inflammation in bones and treating osteoporosis symptoms. In addition, the sea cucumber extract can enable calcium to be better absorbed and utilized by organisms, thereby achieving the effects of balancing calcium metabolism and further increasing bone density.
The first purpose of the invention is to provide a method for preparing a sea cucumber extract, which comprises the steps of carrying out enzymolysis on ground sea cucumbers, and carrying out enzyme deactivation, centrifugation, filtration and drying on enzymolysis liquid after enzymolysis to obtain the sea cucumber extract; the enzymolysis is carried out by adopting compound protease firstly and then carrying out enzymolysis by adopting flavourzyme, the adding amount of the compound protease is 15-75U/g of the sea cucumber, the enzymolysis temperature is 40-60 ℃, the pH value is 6.5-7.5, and the time is 0.5-1 h; the addition amount of the flavourzyme is 100-500U/g sea cucumber, the enzymolysis temperature is 50-60 ℃, the pH value is 6.5-7.5, and the time is 1-5 h. Preferably, the enzymolysis time of the flavourzyme is 3 to 5 hours, and further, the enzymolysis time is 3.5 to 4.5 hours.
In one embodiment of the present invention, the preparation method of the sea cucumber powder comprises the following steps:
(1) pretreatment: cutting open fresh sea cucumber from abdomen, removing viscera and gonad, keeping body wall, and washing.
(2) Blanching: blanching the sea cucumber body wall treated in the step (1) in water with the temperature of more than 90 ℃ for 15min, then cooling to 15-20 ℃, and cutting into small pieces.
(3) Desalting: and (3) soaking the sea cucumber treated in the step (2) in water at 0-5 ℃ for desalting until the conductivity of the soaking solution is lower than 1000 us/cm.
(4) Homogenizing: and (4) crushing the sea cucumber blocks treated in the step (3) by using a colloid mill to obtain homogenate, and then mixing the homogenate with water.
In one embodiment of the present invention, the sea cucumber block size in step (2) is (1-1.5 cm) × (1-1.5 cm).
In one embodiment of the present invention, the ratio of the sea cucumber homogenate to the water in the step (4) is 1: (5-10) (w/w).
In one embodiment of the invention, the enzyme deactivation is to raise the temperature of the enzymatic hydrolysate to above 90 ℃, keep the temperature for 10-30min, and then cool the enzymatic hydrolysate to 15-20 ℃.
In one embodiment of the invention, the filtration is to centrifuge the enzyme-deactivated enzymolysis liquid, and then take the supernatant to perform nanofiltration concentration until the solid content is 2-20%.
In one embodiment of the invention, the amount of the compound protease is 0.1-0.5% of that of the sea cucumber, and the enzyme activity is 15000U/g.
In one embodiment of the invention, the amount of the flavourzyme is 0.2-1.0% of the sea cucumber, and the enzyme activity is 50000U/g.
In one embodiment of the present invention, the flavourzyme enzymolysis conditions are: the enzymolysis temperature is 50-60 ℃, the pH is 6.5-7.5, and the enzymolysis time is 1-5 h; more preferably, the enzymolysis time is 3.5 h.
In one embodiment of the present invention, the centrifugal force is 1000-5000g, and the centrifugal time is 10-20 min.
In one embodiment of the invention, the nanofiltration device has a molecular weight cut-off of 100-300 Da.
In one embodiment of the invention, the sea cucumber extract is spray dried to obtain a powdered product.
The second purpose of the invention is to provide the sea cucumber extract prepared by the method.
In one embodiment of the present invention, the sea cucumber extract has a molecular weight of less than 1500 or more and 70% or more, and an average molecular weight of 400-1700 Da. Preferably, the average molecular weight is 400-500 Da.
The third purpose of the invention is to provide the application of the sea cucumber extract in preparing medicines for improving bone density or medicines for reducing high bone turnover rate, reducing osteoclast proliferation, inhibiting inflammation in bones and balancing bone metabolism.
In one embodiment of the present invention, the sea cucumber extract is added in an amount of 500-2000 mg/kg. Preferably, the sea cucumber extract is added in an amount of 1000 mg/kg.
In one embodiment of the present invention, the significant effects of the sea cucumber extract in increasing bone density and improving bone metabolism benefit from the synergistic effect of the sea cucumber polypeptide and the sea cucumber polysaccharide.
The fourth object of the present invention is to provide a composition comprising the above sea cucumber extract and calcium ions. The sea cucumber extract and the calcium ions are main active ingredients, the sea cucumber extract can promote the absorption of the calcium ions, and the sea cucumber extract and the calcium ions have a synergistic effect on the aspect of improving the bone mineral density.
In one embodiment of the present invention, the ratio of the sea cucumber extract to the calcium ion in the composition is (500- & ltSUB & gt 2000- & ltSUB & gt) & ltSUB & gt 166 (w/w). Preferably, the ratio of the sea cucumber extract to the calcium ions is 1000:166 (w/w).
In one embodiment of the present invention, the amount of the Stichopus japonicus extract added in the composition is 500-2000mg/kg, and the amount of the calcium ion added is 80-400 mg/kg. Preferably, the addition amount of the sea cucumber extract is 1000-2000mg/kg, and the addition amount of the calcium ion is 166-332 mg/kg.
In one embodiment of the invention, the calcium ions comprise calcium carbonate or calcium chloride.
In one embodiment of the invention, the composition is a medicament or nutraceutical.
In one embodiment of the present invention, the composition further comprises a pharmaceutical carrier and/or a pharmaceutical excipient.
In one embodiment of the present invention, the dosage form of the pharmaceutical composition is any pharmaceutically acceptable dosage form.
In one embodiment of the invention, the dosage form comprises powder, injection, capsule, tablet, oral liquid, soft candy.
In one embodiment of the invention, the animal experimental design is primarily in reference to health food testing and evaluation specifications. The experiment for balanced bone metabolism adopts a de-ovarian model. The method comprises the following specific steps:
(1) rats were anesthetized with pentobarbital sodium intraperitoneal injection at a dose of 25mg/kg body weight.
(2) After thorough anesthesia (body can not be overturned after abdomen is upwards), the rat with abdomen upwards can be fixed on a rat surgical board by cotton threads on four limbs and the mouth if necessary, and the center of the rat abdomen is about 100cm lower2After the mouse hair is carefully removed from the area with the size of the area by a shaver, the cut mouse hair is wiped off by 75% alcohol, so that the mouse hair is prevented from entering the wound to cause infection in the operation.
(3) Lightly scratching the skin and muscle layer of the rat along the midline of the abdomen of the shaved part by using a No. 11 scalpel, and exposing the intestinal tract and the internal organs; for convenient suture, the wound is as small as possible, preferably 1-2 cm. The rat skins on the two sides are respectively clamped by hemostatic forceps, the handle parts of the hemostatic forceps are placed on a table top, and the rat skins are always kept in a separated state by means of the gravity of the hemostatic forceps.
(4) Gently poking the intestinal tract aside by using forceps to find an oviduct which is obviously different from the intestinal tract, finding an ovary (pink, flat oval and convex surface) at the tail end of the oviduct along the oviduct, clamping the oviduct connected below the ovary by using hemostatic forceps, and completely cutting off the ovary and the oviduct on the upper part of the hemostatic forceps by using ophthalmic surgical scissors. The hemostatic forceps is kept in a clamping state for 1-2min to prevent hemorrhage. The other ovary was treated the same way. In sham groups, the ovaries were not removed, and only a portion of the fat surrounding both ovaries was removed.
(5) After the operation is finished, the oviduct and the intestinal tract are restored to the original positions, 2 ten thousand units of gentamicin is lightly sprayed in the abdominal cavity, and the needle head is prevented from touching the organs and tissues in the abdomen.
(6) Firstly suturing the muscle layer of the abdomen of the rat (continuous suturing method, knotting every two needles), then taking down the hemostats on the skin of the rat and suturing the outer skin of the rat (continuous suturing method, each needle needs to be knotted, the suturing is firm, the wound is prevented from being opened), spraying 2 ten thousand units of penicillin on the wound after the suturing is completed, placing the single abdomen of the single rat cage in the stainless steel rat cage upwards, and paying attention to the fact that the temperature of an air conditioner in an animal house is slightly increased, and the most suitable temperature is about 27-28 ℃.
(7) The body state of the rats after awakening is observed within 24 hours, the rats with good health condition are transferred to a common rat cage, and each rat is injected with 2 ten thousand units of gentamicin per day in 3 days. If the wound suture of the rat is bitten, the rat should be anesthetized in time and then sutured firmly.
The calcium absorption promotion experiment mainly uses rats in low calcium environment. The specific conditions are that the rats in the experimental group are fed with low-calcium feed (100mg/kg), the rats in the blank group are fed with common feed, and all rats can only drink deionized water.
The invention has the beneficial effects that:
the sea cucumber extract with the function of increasing the bone mineral density provided by the invention contains a plurality of beneficial components such as sea cucumber polypeptide and sea cucumber polysaccharide, has various action mechanisms, can balance the bone metabolism condition from multiple aspects, promotes the body to absorb substances such as calcium and the like, and has a synergistic effect. It has effects in promoting mineral element absorption and deposition, inhibiting bone inflammation, balancing bone metabolism, repairing and promoting bone and bone joint, and maintaining bone health. On the other hand, the designed substance has small molecular weight, is easy to digest and absorb by human bodies, has natural and healthy sources, and has no toxic or side effect on organisms. In addition, after the sea cucumber extract and the carbon ions are compounded, the sea cucumber extract and the carbon ions have a synergistic effect on the aspect of improving the bone density in the rat body.
Drawings
FIG. 1 is a graph showing the effect of sea cucumber extracts on bone density of ovariectomized rats at different enzymatic hydrolysis times in example 2. SHAM stands for SHAM group, OVX stands for anovulatory group，E2The positive group is shown, and the rest are the extract groups prepared by each enzymolysis time. Wherein "+" indicates that the two groups of data have significant difference, and P is less than 0.05; ". indicates that there was a very significant difference between the two sets of data, P < 0.01. The letters and the legends are the same.
FIG. 2 shows the effect of different doses of Stichopus japonicus extract on bone density in ovariectomized rats in example 3. L, M, H the content of Stichopus japonicus extract in low, medium, and high dosage groups is shown.
FIG. 3 shows the serum estradiol levels in the rats of each group after ovariectomy in example 3.
Figure 4 shows the change in the levels of bone formation markers in the rats of each group in example 3.
FIG. 5 shows the change in the level of bone resorption markers in the rats of each group in example 3.
FIG. 6 shows the change of the level of inflammatory factors in rats of each group in example 3.
FIG. 7 shows the effect of different proportions of Stichopus japonicus extract and calcium on bone density in low calcium rats in example 4. Control represents blank Control group, Model represents low calcium Model group, CaCo3Indicating calcium carbonate positive group, L, M, H indicating low, medium and high proportion groups of sea cucumber extract and calcium ion, respectively. The letters and the legends are the same.
FIG. 8 shows the effect of different doses on bone density in low calcium rats in example 5 at the same ratio of extract to calcium ions. Wherein L, M, H represent the low, medium and high dose groups, respectively.
FIG. 9 shows the effect of Stichopus japonicus extract, Stichopus japonicus polypeptide, and Stichopus japonicus polysaccharide on bone density of ovariectomized rats in comparative example 1.
FIG. 10 is a graph showing the effect of collagen peptides of different sources on bone density in rats with low calcium in comparative example 2.
Detailed Description
The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.
1. Detection of protein content of sea cucumber extract
The protein content of the sea cucumber extract is detected by adopting a Kjeldahl method. The method is carried out according to the first method of GB 5009.5-2016. Here, an SKD-200 semi-automatic kjeldahl apparatus was used.
2. Detection of polysaccharide content in sea cucumber extract
The polysaccharide content of the sea cucumber extract is determined by the phenol-sulfuric acid method.
Accurately weighing 0.02g of anhydrous fucose, dissolving, and diluting to 100mL to obtain fucose standard solution. 50mL of concentrated sulfuric acid is added into 10mL of water, and after cooling, 0.6g of phenol is added to prepare a phenol-sulfuric acid reagent. Taking 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL and 0.5mL of standard solution, respectively supplementing the standard solution to 1mL by using distilled water, then adding 5mL of phenol-sulfuric acid reagent, uniformly mixing, heating in a boiling water bath for 30min, taking out, immediately cooling, uniformly mixing again, standing for 10min, and measuring the absorbance at 490nm to obtain a standard curve.
Accurately weighing 0.02g of the sea cucumber extract, dissolving in water, fixing the volume to 100mL, and taking 1mL of solution to be detected to perform the operation to obtain the polysaccharide content of the sea cucumber extract.
3. Amino acid composition and content detection of sea cucumber extract
About 0.1g of sea cucumber extract is accurately weighed, added with 8mL of 6M HCl and then filled with nitrogen, and then hydrolyzed at 120 ℃ for 22 h. After hydrolysis, 4.8mL of 10M NaOH was added to neutralize and the volume was adjusted to 25 mL. After filtration through a double-layer filter paper, 1mL of the filtrate was centrifuged at 15000rpm for 30 min. Aspirate 400 μ L of supernatant into the liquid phase vial. The detection instrument is Agilent1100HPLC (high performance liquid chromatography) and is provided with an ultraviolet detector.
The chromatographic setting parameters are as follows: chromatography column Agilent Hypersil ODS (5 μm, 4.0 mm. times.250 mm); mobile phase: 27.6mmol/L sodium acetate-triethylamine-tetrahydrofuran (volume ratio 500: 0.11:2.5) +80.9mmol/L sodium acetate-methanol-acetonitrile (volume ratio 1:2: 2); the detection wavelength was 338nm (proline 262 nm); the flow rate was 1.0mL/min, and the column temperature was 40 ℃.
4. Molecular weight detection of sea cucumber extract polypeptide
The sea cucumber extract is fully dissolved by ultrapure water to prepare a solution with the protein concentration of 1mg/mL, and the molecular weight of the polypeptide is measured by Waters1525HPLC (high performance liquid chromatograph) after passing through a 0.22 mu m water system film. The instrument was equipped with a 2487 uv detector and GPC analysis software in an Em-power workstation.
The chromatographic setting parameters were as follows: column TSK GEL 2000SWXL (300 mm. times.7.8 mm); mobile phase: 40% acetonitrile (containing 0.1% trichloroacetic acid) + 60% ultrapure water (v/v); the detection wavelength is 220 nm; the flow rate is 0.5 mL/min; the column temperature was 30 ℃.
The sample injection volume is 10 mu L; the sample concentration is 1 mg/mL; and (4) standard product: cytochrome C (Mw ═ 12384Da), aprotinin (Mw ═ 6500Da), bacillase (Mw ═ 1422Da), ethionine-tyrosine-arginine (Mw ═ 451Da), and ethionine-ethionine (Mw ═ 189 Da).
5. Animal experiment design for improving bone mineral density by using sea cucumber extracts with different molecular weights
After the animals are parked, the animals are adaptively raised for one week, the animals are weighed on the 7 th day and then are randomly grouped according to the weight, wherein a blank group is treated by a fake operation, the rest experimental groups are subjected to bilateral ovariectomy, each rat needs to be injected with 20000 units of gentamicin to prevent infection after the operation is finished, the administration is continuously carried out for 90 days, the rats are weighed every week, the weight change condition of the rats is observed, and the intragastric administration dosage is adjusted at any time. Rats in each group are fasted for 12 hours after the last gastric lavage, then are anesthetized by intraperitoneal injection of 3% sodium pentobarbital solution, blood and serum are separated from heart, and the femur and tibia on two sides are taken, and connective tissues and muscles are carefully removed to measure various indexes. Specific groupings can be seen in the following table:
TABLE 1
6. Animal experiment design for improving bone mineral density by using sea cucumber extracts with different dosages
Animal experimental design is as above, and the specific grouping can be seen in the following table:
TABLE 2
7. Animal experimental design for increasing bone mineral density after compounding sea cucumber extracts and calcium in different proportions
Animals were acclimatized for one week after dwelling and were randomly assigned to 6 groups on day 7 after weighing. The specific administration scheme is as follows:
TABLE 3
Except for the normal calcium control group, each group ingested only low calcium feed. The source of the exogenous ingested calcium is calcium carbonate.
The administration is continued for 90 days according to the above scheme, and the rats need to be fed with low-calcium feed in the whole process. Rats were weighed weekly, observed for weight changes, and gavage was adjusted at any time. If the rats are too big to be filled with the drug, the drug should be mixed into the feed at a fixed ratio and the intake of the drug should be recorded daily (generally, the intake of the drug per time of the rats is about 3-5% of the body weight of the rats, and the mixing ratio can be roughly calculated according to the body weight).
8. Animal experiment design for increasing bone mineral density after compounding sea cucumber extracts with different dosages and calcium
The animal experiment design is as above, and the specific grouping is as follows:
TABLE 4
9. Biological material
The compound protease is purchased from Novoxil, and has the model of Protomax 500MG and the enzyme activity of 15000U/g; the Flavourzyme is purchased from Novoxin company, the model is Flavourzyme 500MG, and the enzyme activity is 50000U/g.
Example 1: method for preparing sea cucumber extract
A method of preparing a sea cucumber extract, the method comprising the steps of:
(1) cutting open fresh sea cucumber from abdomen, removing viscera and gonad, keeping body wall, and washing.
(2) Blanching the sea cucumber body wall in water with the temperature of more than 95 ℃ for 15min, then cooling to 20 ℃, and cutting into small blocks (1-1.5 cm) by 1-1.5 cm.
(3) Soaking the sea cucumber blocks in water at 0-5 deg.C for desalting until the electric conductivity of the soaking solution is lower than 500 us/cm.
(4) The sea cucumber blocks are crushed by a colloid mill and then put into an enzymolysis tank together with water which is 8 times of the weight of the sea cucumber. Heating the liquid to 95 ℃, keeping the temperature for 30min, cooling to 50 ℃, adding compound protease for enzymolysis, wherein the addition of the compound protease is 0.3 percent of the weight of the sea cucumber, adding flavourzyme for enzymolysis after the enzymolysis for 30min, the addition of the flavourzyme is 0.6 percent of the weight of the sea cucumber, and the enzymolysis conditions are as follows: the enzymolysis temperature is 55 deg.C, and pH is 7.0.
(5) Raising the temperature of the enzymolysis liquid to 95 ℃ to inactivate enzyme, preserving the heat for 30min, and then cooling the enzymolysis liquid to 15-20 ℃. Then centrifuging, taking supernatant, and carrying out nanofiltration concentration until the solid content is 10%. And finally, spray drying to obtain the sea cucumber extract.
Wherein, the enzymolysis time of the stroke flavor protease in the step (4) is 1.5h, 2.5h, 3.5h and 4.5 h. The molecular weight distribution of the enzymatic hydrolysate in example 1 was measured according to the above-mentioned molecular weight distribution measuring method, and the results are shown in Table 5, which shows that more small-molecule peptides can be obtained as the time for enzymatic hydrolysis is longer. In addition, the inventor also tries to carry out enzymolysis by separately adopting compound protease (the addition amount is 0.3 percent of the weight of the sea cucumber) or flavourzyme (the addition amount is 0.6 percent of the weight of the sea cucumber), the enzymolysis time is 5 hours, the molecular weight is found to be less than 1500Da and less than 60 percent, the average molecular weight is more than 3000Da, and the molecular weight of the sea cucumber extract obtained by single enzyme hydrolysis is larger.
TABLE 5 molecular weight of the polypeptides in sea cucumber extracts
Example 2: application of sea cucumber extracts with different molecular weights in increasing bone mineral density
The sea cucumber extract prepared in example 1 is adopted to intervene in ovariectomized rats to carry out animal experiments, the daily gavage amount of the sea cucumber extract with different molecular weights is 1000mg/kg, and the experimental results and conclusions are as follows:
the data in fig. 1 show that the bone density of the model group is significantly reduced after the operation is finished, and the bone density of the positive group is significantly increased after the treatment. The bone density of the ovariectomized rat is also increased by each experimental group, wherein the sea cucumber extract obtained after 3.5h and 4.5h of enzymolysis has a good effect on the bone density, and the bone density in the rat body can be remarkably increased. Therefore, as shown in figure 1, the sea cucumber extract has the effect of improving the bone density, and the extracts subjected to enzymolysis for 3.5h and 4.5h have significance and better effect. The sea cucumber extract prepared in example 1 with the enzymolysis time of 3.5h and 4.5h has a similar effect of improving the bone density, so the sea cucumber extract with the enzymolysis time of 3.5h is more preferable. Preferably, the average molecular weight of the sea cucumber extract is controlled within the range of 400-500Da, so that the effect of increasing the bone density is better.
Example 3: application of sea cucumber extracts with different dosages in increasing bone mineral density
Based on the results of example 2, the sea cucumber extract of example 1, which had an enzymatic hydrolysis time of 3.5 hours, was used for the experiment.
(1) Results of animal experiments
The 90-day feeding experiment shows that the experimental animal has good growth condition in the whole growth period, and the apparent physiological indexes, serum biochemical indexes, organ index indexes, pathological sections and the like of the experimental group have no obvious difference with those of the control group except the related indexes of bone density, thereby proving that the invention is safe and nontoxic to organisms.
The beneficial effect of the invention on increasing the bone density is further detailed through animal experiments, and the experiments are carried out according to authoritative documents at home and abroad, an evaluation method for increasing the bone density function in health food inspection and evaluation technical specifications, and an evaluation method for increasing the bone density function (for obtaining comments).
The experimental scheme and the results of the function of increasing the bone mineral density provided by the invention are as follows:
1 materials and methods
1.1 Experimental animals and Environment
60 healthy SPF-grade female SD rats with the weight of 300 +/-20 g and the age of 3 months are purchased from Witongli laboratory animals, Limited liability company. During the experiment, SD rats are fed in the experimental animal center of the university in south of the Yangtze river. The ethical approval serial number is JN.No2020930S0600117[231], and the license number of the experimental animal is SCXK (Zhe) 2019-.
1.2 animal groups and dosing
The estrogen-free feed base formula was referenced to AIN-93G feed, but with the soybean oil component replaced with the corn oil component. The concrete components are as follows (%): casein 20, L-cystine 0.3, corn starch 39.7, maltodextrin 13.2, sucrose 10, cellulose 5, corn oil 7, t-butylhydroquinone 0.0014, mineral 3.5, vitamin 1, choline tartrate 0.25.
The mineral reference contents were as follows (mg/kg feed): MnSO4 110、CuSO4 0.8、FeSO4 1.2、ZnSO42960、CaHPO4 2890、MgSO412500. The vitamin reference content was as follows (/ kg feed): VitA 14000IU, VitD 1500IU, VitE 120mg, VitK 3mg, VitKB1 12mg、VitKB2 20mg、VitKB6 12mg、VitKB120.03mg, 60mg of nicotinic acid, 24mg of pantothenic acid, 6mg of folic acid and 0.54mg of biotin.
The experimental animals are adaptively fed for one week and then are randomly grouped according to body weight, and then are subjected to an ovarian surgery for osteoporosis making model. And 5 days after the operation, blood is taken from the tail tip to measure the content of estradiol in the serum of the tail tip, and whether the molding is successful is judged. After grouping, each group contains 10, six groups are respectively: sham group + saline; ovariectomized model group + normal saline; ovariectomized + estradiol group; ovariectomized + sea cucumber extract low dose group; ovariectomized and sea cucumber extract medium dosage group; ovariectomized + sea cucumber extract high dose group. The positive group has estradiol content of 0.1mg/kg, and the sea cucumber extract low-medium content group has low-medium content of 500mg/kg, 1000mg/kg and 2000mg/kg per day. The daily intake of the human body is 5 times, 10 times and 20 times respectively. The whole gavage period is 90 days.
1.3 Experimental methods
The weight of the rat is measured every week, the rat is killed by breaking the neck after the gastric lavage period is finished, and the relevant indexes are detected by taking samples of thighbone, blood and the like. The proximal femur bone density and the remaining bone related data of the rat are measured using Perkin Elmer Quantum GX micro-CT after the left femur is isolated.
2 results of the experiment
2.2 bone Density data
The influence of the sea cucumber extract provided by the invention on the change condition of the bone density of rats can be shown in figure 2. From the figure, it can be seen that the bone density of the rats in the model group is significantly reduced compared to that in the sham operation group. The positive drug group has obvious improvement effect. The sea cucumber extract dosage groups have obvious therapeutic action on osteoporosis rats, especially middle dosage groups, compared with model groups, and the bone density of the rat is remarkably increased.
2.3 serum Biochemical indicators
2.3.1 estradiol content
The serum estradiol content is shown in figure 3. From the figure, it can be seen that the serum estradiol content of the rats in the experimental group is significantly reduced compared with that in the experimental group, which indicates that the molding operation is successful, and the animals can be used for the next series of operations.
2.3.2 bone formation indicator
The influence of the sea cucumber extract provided by the invention on the change condition of the contents of PINP and CTX-1 in the serum of a rat can be shown in figure 4. PINP is produced by osteoblasts and can specifically reflect the rate of bone formation. From the figure, the PINP content in the model group is obviously increased compared with that in the sham operation group, and the dosage group of the sea cucumber extract can prevent the increase to different degrees and is dose-dependent, wherein the effect of the high dosage group is most obvious. CTX-1 is an important product of bone resorption. From the figure, the CTX-1 content of the rats in the model group is obviously increased, which indicates that the bone metabolism rate of the rats in the group is abnormal, and the phenomenon can be relieved after the sea cucumber extract is treated. The above results indicate that the sea cucumber extract of the present invention can balance the unbalanced bone metabolism in the osteoporotic rat and reduce the abnormally increased bone metabolism rate due to menopause.
2.3.3 bone resorption index
The influence of the sea cucumber extract on the content change of BALP, TGF-beta 1 and TRACP-5b in rat serum can be shown in figure 5. BALP (bone specific alkaline phosphatase) is an extracellular enzyme of osteoblasts, derived only from osteoblasts. TGF-. beta.1 (transforming growth factor-. beta.1) has a powerful regulatory effect on bone and is synthesized in the skeletal system primarily by osteoblasts. Osteoclasts secrete TRACP-5b (serum tartrate acid-fast phosphatase) into the blood, and thus it can characterize the strength of bone resorption. The metabolism in ovariectomized rats is disordered, so that the osteoporosis symptoms are mainly caused by overactive bone metabolism. From the figure, the 3 indexes in the model group are obviously increased compared with those in the pseudo-operation group, and the contents of the 3 substances in the blood serum are reduced after the treatment of the sea cucumber extract with different doses. The sea cucumber extract provided by the invention has a good balance effect on bone metabolism, and has a remarkable promotion effect on the improvement of the bone mass and the bone density of an organism.
2.3.3 inflammation index
2.3.3.1 inflammatory factors
The influence of the sea cucumber extract on the content change conditions of IL-6, IL-1 beta and TNF-alpha in rat serum can be shown in figure 6. The change conditions of the three are similar, the content of the three in the model group is obviously increased compared with that in the sham operation group, and the inflammatory phenomenon in the osteoporosis rat is relatively serious. The prognosis of the sea cucumber extract is reduced to different degrees and is dose-dependent, wherein the curative effect of a high-dose group is obvious. The results show that the sea cucumber extract provided by the invention can obviously relieve the inflammation condition caused by osteoporosis, and the inflammatory factor is an important cytokine in the downstream of an OPG/RANKL passage, so that the sea cucumber extract can prevent osteoclast differentiation through the OPG/RANKL passage, thereby promoting bone formation and maintaining bone metabolic balance.
2.3.3.2 anti-inflammatory factor
The influence of the sea cucumber extract provided by the invention on the content change condition of IL-10 in rat serum can be seen in FIG. 6D. Rats in the model group were severely inflamed and the IL-10 content was significantly reduced due to inflammatory conditions caused by associated pathways and by osteoporosis of fractures. These inflammatory conditions were alleviated and the anti-inflammatory factor IL-10 was increased to varying degrees, especially in the medium dose group, when treated with sea cucumber extract. Therefore, the sea cucumber extract provided by the invention can relieve inflammation and increase the bone density of rats.
3 small knot
The sea cucumber extract prepared by the embodiment contains polypeptide and polysaccharide, wherein the content of the polypeptide is 63.7 +/-1.0%, and the content of the polysaccharide is 18.9 +/-0.0%.
After the rat is perfused with the three sea cucumber extracts with different dosages (500mg/kg, 1000mg/kg and 2000mg/kg), the bone density of the osteoporosis rat can be obviously increased; improving the dysregulated bone metabolic balance; can obviously reduce the content of inflammatory factors in the osteoporosis rat, thereby relieving inflammatory symptoms, preventing the over-differentiation of osteoclast, promoting bone formation and maintaining the metabolic balance of bones. Wherein the sea cucumber extract has the best effect at medium dosage. In conclusion, the sea cucumber extract provided by the invention has the effects of increasing the bone density of rats and improving the bone metabolism.
Example 4: application of compound of sea cucumber extracts with different proportions and calcium in increasing bone mineral density
The sea cucumber extract of example 1 with an enzymolysis time of 3.5h was used for the experiment.
Animals were acclimatized for one week after dwelling, weighed on day 7 and then randomly assigned to 6 groups of 8 animals per group by weight. The specific dosing regimen and bone density data were as follows:
TABLE 6
Except the normal calcium control group, the other groups only took in low calcium feed. The source of exogenous ingested calcium is uniformly calcium carbonate. The dosage of calcium ion is converted into the recommended dosage of human body.
The administration is continued for 90 days according to the above scheme, and the rats need to be fed with low-calcium feed in the whole process. Rats were weighed weekly, observed for weight changes, and gavage was adjusted at any time. If the rats are too big to be filled with the drug, the drug should be mixed into the feed at a fixed ratio and the intake of the drug should be recorded daily (generally, the intake of the drug per time of the rats is about 3-5% of the body weight of the rats, and the mixing ratio can be roughly calculated according to the body weight).
Fig. 7 shows that after the holothurian extract and calcium are compounded, the holothurian extract and calcium have synergistic effect on improving the bone density in rats, and the medium proportion group is significantly different from the high proportion group and the low calcium model group, but the medium proportion group is not greatly different from the high proportion group. In addition, the bone density of the medium proportion group is remarkably improved compared with that of the calcium carbonate group with the same calcium intake, so that the proportion of the medium proportion group can be used as a more preferable proportion, namely the proportion of the sea cucumber extract and the calcium ions is 1000:166, and the effect of increasing the bone density is better.
Example 5: application of different dosages of sea cucumber extract and calcium compounded in increasing bone mineral density
The sea cucumber extract of example 1 with an enzymolysis time of 3.5h was used for the experiment. The proportions of the sea cucumber extract and calcium were based on the proportion group in example 4.
Animals were acclimatized for one week after dwelling, weighed on day 7 and then randomly assigned to 6 groups of 8 animals per group by weight. The specific dosing regimen and bone density data were as follows:
TABLE 7
Except the normal calcium control group, the other groups only took in low calcium feed. In the low, medium and high dose groups, the proportions of the sea cucumber extract and calcium in each group were kept the same.
The administration is continued for 90 days according to the above scheme, and the rats need to be fed with low-calcium feed in the whole process. Rats were weighed weekly, observed for weight changes, and gavage was adjusted at any time. If the rats are too big to be filled with the drug, the drug should be mixed into the feed at a fixed ratio and the intake of the drug should be recorded daily (generally, the intake of the drug per time of the rats is about 3-5% of the body weight of the rats, and the mixing ratio can be roughly calculated according to the body weight). Calcium metabolism experiments were performed after the growth experiments were completed. Moving the animals into a metabolism cage, respectively collecting excrement and urine of each tested animal for 72 hours, and accurately recording the feed intake of each tested animal; and (4) measuring the calcium content in the feed, the excrement and the urine. The apparent absorption and retention of calcium were calculated as follows:
the calcium in the feed plus the calcium by oral gavage
Calcium absorption-ingestion of calcium-faecal calcium
Calcium absorption (%) as ═ (calcium absorbed/calcium ingested) × 100%
The stored calcium is ingested calcium-faecal calcium-urinary calcium
The calcium storage rate (%) was (stored calcium/ingested calcium) × 100%
Rats in each group are fasted for 12 hours after the last gastric lavage, then are anesthetized by intraperitoneal injection of 3% sodium pentobarbital solution, blood and serum are separated from heart, and the femur and tibia on two sides are taken, and connective tissues and muscles are carefully removed to measure various indexes.
As can be further seen from the data in FIG. 8 and Table 7, bone density increased by 0.08g/mm after gavage calcium carbonate alone relative to the low calcium control group3(ii) a After the sea cucumber extract is separately infused into the stomach, the bone density is increased by 0.02g/mm3(ii) a After the intragastric dose of the extract calcium compound, the bone density is increased by 0.14g/mm3(ii) a Is far more than the sum of the effects of the calcium carbonate and the sea cucumber extract (0.1 g/mm)3) This shows that the compound of the sea cucumber extract and calcium has synergistic effect in increasing bone density in rats. The bone density of the experimental group was significantly increased compared to the model group, and the middle-dose group and the high-dose group had the effect of significantly increasing the bone density compared to the calcium carbonate group and the extract group, and thus the middle-dose and the high-dose groups were preferable.
The calcium apparent absorption and retention can be seen in the table below.
TABLE 8
From the above table, it can be seen that the compound dry prognosis of the sea cucumber extract and calcium can greatly improve the calcium intake and storage of the body compared with the pure calcium group with the same dosage, so that the sea cucumber extract has the capability of improving the calcium absorption of the body.
Comparative example 1
The sea cucumber extract with enzymolysis time of 3.5h in example 1 is used for carrying out experiments, and after being redissolved in water, 95% ethanol with 2 times of volume is added, and the sea cucumber polypeptide and the sea cucumber polysaccharide are obtained through precipitation separation. Referring to example 3, the ovarian removal animal intervention experiment was performed, the gavage dose of the sea cucumber extract was a medium dose, the gavage dose of the sea cucumber polypeptide and the sea cucumber polysaccharide was an equivalent gavage dose of the sea cucumber extract, other conditions were the same as those in example 3, and the bone density results are shown in fig. 9. From the figure, the effect of the sea cucumber extract on improving the bone density is obvious for the ovariectomized rat, while the effect of single sea cucumber polypeptide or sea cucumber polysaccharide is not obvious and only shows the trend of improvement, so that the effect of single substance on improving the bone metabolism is not good, and the remarkable effect of the sea cucumber extract is benefited from the synergistic effect of the components aiming at the complex mechanism of osteoporosis.
Comparative example 2
Referring to example 5, a low-calcium animal model was used for experiments, and a medium dose (peptide/calcium) was used for gastric lavage, and compared with the calcium absorption promoting effects of sea cucumber polypeptide (3.5h enzymolysis), yak collagen polypeptide, and tilapia skin collagen polypeptide, the other conditions were the same as in example 5, and the bone density results are shown in fig. 10. From the figure, it can be seen that the effect of sea cucumber polypeptide intervention is better than that of collagen polypeptides from two other sources under the condition of the same calcium intake in calcium-deficient rats. Therefore, the sea cucumber polypeptide has better calcium absorption promoting effect compared with the collagen polypeptides from two other sources.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method for preparing a sea cucumber extract is characterized in that the sea cucumber is ground and then subjected to enzymolysis, and after the enzymolysis, the enzymolysis liquid is subjected to enzyme deactivation, centrifugation, filtration and drying to obtain the sea cucumber extract; the enzymolysis is carried out by adopting compound protease firstly and then carrying out enzymolysis by adopting flavourzyme, the adding amount of the compound protease is 15-75U/g of the sea cucumber, the enzymolysis temperature is 40-60 ℃, the pH value is 6.5-7.5, and the time is 0.5-1 h; the addition amount of the flavourzyme is 100-500U/g sea cucumber, the enzymolysis temperature is 50-60 ℃, the pH value is 6.5-7.5, and the time is 1-5 h.
2. The method as claimed in claim 1, wherein the filtration is carried out with a membrane having a molecular weight cut-off of 100-300 Da.
3. A sea cucumber extract prepared according to the method of claim 1 or 2.
4. The sea cucumber extract as claimed in claim 3, wherein the sea cucumber extract has a molecular weight of less than 1500Da and more than 70%, and an average molecular weight of 400-1700 Da.
5. Use of a sea cucumber extract as claimed in claim 3 or 4 for the preparation of a medicament for increasing bone density or lowering high bone turnover rate, reducing osteoclast proliferation, inhibiting inflammation in bone, and balancing bone metabolism.
6. The use as claimed in claim 5, wherein the amount of Stichopus japonicus extract added in the use is 500-2000 mg/kg.
7. A composition comprising the sea cucumber extract of claim 3 or 4 and calcium ions.
8. The composition as claimed in claim 7, wherein the amount of the Stichopus japonicus extract added in the composition is 500-2000mg/kg, and the amount of the calcium ion added is 80-400 mg/kg.
9. The composition according to claim 7 or 8, wherein the composition is a pharmaceutical or nutraceutical.
10. The composition according to any one of claims 7 to 9, wherein the composition further comprises a pharmaceutical carrier and/or a pharmaceutical excipient.
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Citations (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1951402A (en) * | 2006-10-13 | 2007-04-25 | 黄世宽 | Method for preparing active polypeptide and zinc protein of sea cucumber |
| CN101514354A (en) * | 2009-02-25 | 2009-08-26 | 山东大学 | Sea cucumber polypeptide, preparation method and application thereof |
| CN101589823A (en) * | 2008-05-26 | 2009-12-02 | 马正军 | A kind of production technology of sea cucumber health milk |
| CN102907558A (en) * | 2012-11-16 | 2013-02-06 | 山东省海洋水产研究所 | Processing method of sea cucumber polypeptide |
| CN104397764A (en) * | 2014-12-11 | 2015-03-11 | 山东省科学院生物研究所 | Method for desalting, removing silt and extracting active materials in sea cucumber intestine processing and utilizing procedure |
| CN104522728A (en) * | 2014-11-27 | 2015-04-22 | 山东好当家海洋发展股份有限公司 | Method for extracting phosphatide and sea cucumber oil by utilizing sea cucumber blanching liquid |
| RU2013147388A (en) * | 2013-10-23 | 2015-04-27 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Дальневосточный государственный технический рыбохозяйственный университет" | METHOD FOR PRODUCING PRODUCT POSSESSING BIOLOGICALLY ACTIVE PROPERTIES FROM HOLOTURIUM |
| CN104888192A (en) * | 2015-04-30 | 2015-09-09 | 威海特伦斯生物工程有限公司 | Sea cucumber glucosamine preparation for increasing bone mineral density, and production method thereof |
| CN107164441A (en) * | 2017-05-18 | 2017-09-15 | 华南理工大学 | A kind of method for improving dried sea-cucumber albumen and polysaccharide extract rate |
| CN107549792A (en) * | 2017-09-13 | 2018-01-09 | 山东圣洲海洋生物科技股份有限公司 | A kind of sea cucumber peptide composite nutrition powder and its preparation technology |
| CN107823634A (en) * | 2017-12-20 | 2018-03-23 | 大连深蓝肽科技研发有限公司 | Treatment medicine for treating osteoporosis based on marine polysaccharide |
| CN109007649A (en) * | 2018-06-29 | 2018-12-18 | 烟台源力德海洋生物有限公司 | A kind of preparation method and applications of sea cucumber oyster glycopeptide calcium complexing compound |
| CN112274536A (en) * | 2020-11-05 | 2021-01-29 | 江南大学 | Application of sea cucumber powder in preparation of medicine for promoting skin wound healing |
| CN112425737A (en) * | 2020-11-05 | 2021-03-02 | 江南大学 | Fishy smell-removed and decolored sea cucumber powder and preparation method thereof |
| CN113004384A (en) * | 2021-02-20 | 2021-06-22 | 中国海洋大学 | Preparation method and application of sea cucumber intestine bone-promoting peptide |
| CN113265437A (en) * | 2021-06-01 | 2021-08-17 | 大连工业大学 | Preparation method and application of sea cucumber protein peptide for promoting proliferation and differentiation of MC3T3-E1 cells |
-
2022
- 2022-01-24 CN CN202210085751.2A patent/CN114522183B/en active Active
Patent Citations (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1951402A (en) * | 2006-10-13 | 2007-04-25 | 黄世宽 | Method for preparing active polypeptide and zinc protein of sea cucumber |
| CN101589823A (en) * | 2008-05-26 | 2009-12-02 | 马正军 | A kind of production technology of sea cucumber health milk |
| CN101514354A (en) * | 2009-02-25 | 2009-08-26 | 山东大学 | Sea cucumber polypeptide, preparation method and application thereof |
| CN102907558A (en) * | 2012-11-16 | 2013-02-06 | 山东省海洋水产研究所 | Processing method of sea cucumber polypeptide |
| RU2013147388A (en) * | 2013-10-23 | 2015-04-27 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Дальневосточный государственный технический рыбохозяйственный университет" | METHOD FOR PRODUCING PRODUCT POSSESSING BIOLOGICALLY ACTIVE PROPERTIES FROM HOLOTURIUM |
| CN104522728A (en) * | 2014-11-27 | 2015-04-22 | 山东好当家海洋发展股份有限公司 | Method for extracting phosphatide and sea cucumber oil by utilizing sea cucumber blanching liquid |
| CN104397764A (en) * | 2014-12-11 | 2015-03-11 | 山东省科学院生物研究所 | Method for desalting, removing silt and extracting active materials in sea cucumber intestine processing and utilizing procedure |
| CN104888192A (en) * | 2015-04-30 | 2015-09-09 | 威海特伦斯生物工程有限公司 | Sea cucumber glucosamine preparation for increasing bone mineral density, and production method thereof |
| CN107164441A (en) * | 2017-05-18 | 2017-09-15 | 华南理工大学 | A kind of method for improving dried sea-cucumber albumen and polysaccharide extract rate |
| CN107549792A (en) * | 2017-09-13 | 2018-01-09 | 山东圣洲海洋生物科技股份有限公司 | A kind of sea cucumber peptide composite nutrition powder and its preparation technology |
| CN107823634A (en) * | 2017-12-20 | 2018-03-23 | 大连深蓝肽科技研发有限公司 | Treatment medicine for treating osteoporosis based on marine polysaccharide |
| CN109007649A (en) * | 2018-06-29 | 2018-12-18 | 烟台源力德海洋生物有限公司 | A kind of preparation method and applications of sea cucumber oyster glycopeptide calcium complexing compound |
| CN112274536A (en) * | 2020-11-05 | 2021-01-29 | 江南大学 | Application of sea cucumber powder in preparation of medicine for promoting skin wound healing |
| CN112425737A (en) * | 2020-11-05 | 2021-03-02 | 江南大学 | Fishy smell-removed and decolored sea cucumber powder and preparation method thereof |
| CN113004384A (en) * | 2021-02-20 | 2021-06-22 | 中国海洋大学 | Preparation method and application of sea cucumber intestine bone-promoting peptide |
| CN113265437A (en) * | 2021-06-01 | 2021-08-17 | 大连工业大学 | Preparation method and application of sea cucumber protein peptide for promoting proliferation and differentiation of MC3T3-E1 cells |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115770266A (en) * | 2023-01-30 | 2023-03-10 | 云南中医药大学 | Pharmaceutical composition for improving bone mineral density based on regulation of intestinal bacteria metabolism |
| CN115770266B (en) * | 2023-01-30 | 2023-05-09 | 云南中医药大学 | Pharmaceutical composition for treating postmenopausal osteoporosis |
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