CN109453188B - Application of sandworm polysaccharide in preparation of medicine for preventing and treating osteoporosis - Google Patents
Application of sandworm polysaccharide in preparation of medicine for preventing and treating osteoporosis Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention discloses an application of sandworm polysaccharide in preparing a medicine or a health-care product for preventing and treating osteoporosis. Pharmacological experimental research proves that the sandworm polysaccharide can obviously improve the proliferation activity of the preosteoblasts MC3T3-E1 cultured in vitro and promote the differentiation of the preosteoblasts, and can obviously increase the bone density of a mouse with the age of 10 months. The above results indicate that sandworm polysaccharide has the potential to treat or prevent osteoporosis. The new application of the sandworm polysaccharide disclosed by the invention enlarges the indication or application range of the sandworm polysaccharide, and is expected to be developed into a new medicine or health-care product for preventing and treating osteoporosis.
Description
Technical Field
The invention relates to the technical field of application of natural products, in particular to application of marine product polysaccharide in medicines or health-care products.
Background
Osteoporosis (OP) is a systemic, systemic skeletal disease characterized by decreased bone mass, increased bone fragility due to destruction of the bone tissue microstructure and increased risk of fracture, and is a degenerative disease caused by aging of bones. Epidemiological investigations show that: women are obviously higher than men, and the incidence rate tends to increase along with the acceleration of the aging process of China's society. By 2013, the old population in China breaks through 2 hundred million, and by 2020, osteoporosis patients in China are expected to reach 2.866 hundred million. It is expected that by the 50 s of this century, hip fracture patients due to osteoporosis will exceed 300 million/year. Thus, osteoporosis has become a significant health problem that is not negligible.
OP is classified into primary osteoporosis and secondary osteoporosis, wherein the primary osteoporosis accounts for 90% of osteoporosis, and 2 subtypes, i.e. type I and type II (type I is also called postmenopausal osteoporosis, and type II is senile osteoporosis). Among them, type I osteoporosis accounts for the majority of primary osteoporosis, and the onset of type I osteoporosis is mainly due to decreased ability of postmenopausal women to synthesize and secrete estrogen, decreased osteoblast formation, decreased osteogenic function, increased osteoclast formation and recruitment, and enhanced osteoclastic action (bone resorption). A negative balance between bone formation and bone resorption occurs, resulting in a decrease in bone mass and thus osteoporosis. The main theoretical bases of the current clinical osteoporosis prevention and treatment medicines comprise: 1. inhibit excessive bone resorption by osteoclast, and inhibit high bone turnover rate caused by postmenopausal osteoporosis patients and other bone metabolism disorder; 2. Stimulates osteoblast bone formation and mineralization maturation, and simultaneously inhibits osteoclast proliferation and differentiation, so that new bone formation is over absorption, and finally bone mass is increased. The target of its action is mainly directed to osteoblast bone formation and osteoclast bone resorption.
Traditionally, the occurrence of primary osteoporosis has been associated with endocrine factors, particularly estrogen deficiency. However, primary osteoporosis is also considered to be a bone disease associated with chronic inflammation (e.g., rheumatoid, viral infection). Primary and secondary osteoporosis caused by various reasons is mostly related to immune dysfunction, inflammatory reaction can lead to the increase of differentiation and formation of osteoclasts and the increase of absorption capacity of mature osteoclasts, thereby breaking the steady bone reconstruction where bone absorption and bone formation are located, leading to the excessive absorption and destruction of bone to cause bone diseases, and finally developing into osteoporosis from the initial decrease of bone mass, so that the research of anti-osteoporosis through anti-inflammatory action becomes a new hotspot. The polysaccharide can play a regulating role on the immune system in multiple ways and multiple layers, such as promoting the phagocytic function of a reticuloendothelial system, inducing the generation of antibodies, activating phagocytes, regulating T, B the functions of lymphocytes and NK cells, inducing the expression of immune regulatory factors and the like. Research proves that the divaricate saposhnikovia root polysaccharide for treating osteoporosis rats can effectively reduce the concentration of calcium and magnesium ions and ALP in blood serum, regulate cell factors, relieve the bone high decomposition state of castrated rats and improve bone density; the research also shows that the polygonatum polysaccharide can reduce the bone loss and the bone density reduction of ovariectomized rats, improve the bone microstructure damage, promote the osteogenesis related genes and inhibit the expression of the osteoclastogenesis related gene mRNA, and can promote the fracture healing. In addition, many polysaccharides such as cynomorium songaricum polysaccharide, lycium barbarum polysaccharide, morinda officinalis polysaccharide, persimmon leaf polysaccharide, tea polysaccharide, achyranthes bidentata polysaccharide and the like also have obvious osteoporosis resisting effect.
Shachong is also called sea ginseng, a known Sipunulus nudus (Sipunculus nudus), also known as Sipunculus nudus, and is produced in the North sea, Qinzhou, etc., of the northern gulf of Guangdong, Zhanjiang and Guangxi. As a famous seafood, the sandworms are delicious, crisp and tender in taste, rich in various active ingredients, and need to develop more subsequent products to prolong the industrial chain of the sandworm products. Current studies show that: the crude polysaccharide of the sandworm can recover thymus atrophy and spleen atrophy of mice caused by cyclophosphamide, obviously antagonize leukopenia, and has no obvious weight gaining effect on immune organs of normal mice; the phagocytic capacity of normal mouse abdominal cavity macrophages can be obviously enhanced; the sandworm polysaccharide crude product and the refined polysaccharide can promote the mouse spleen lymphocyte proliferation and can act synergistically with the sword bean protein A; can obviously improve the cellular immunity and the humoral immunity of the mouse, promote the carbon clearance rate of the mouse and the phagocytosis function of the liver and the spleen of the mouse to foreign matters, and improve the lung specificity immunity of the mouse. In conclusion, the sandworm polysaccharide is a biological activity regulator which can obviously enhance the immune function of the organism. However, no report about the anti-osteoporosis activity of sandworm polysaccharide exists at present.
Disclosure of Invention
The invention aims to provide application of sandworm polysaccharide in preparation of a medicine or health-care product for preventing and treating osteoporosis.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
an application of sandworm polysaccharide in preparing medicine or health-care product for preventing and treating osteoporosis is disclosed.
As a further scheme, the sandworm polysaccharide is a bioactive substance obtained by means of an enzyme method, a solvent extraction method, an ultrasonic-assisted extraction method, a microwave-assisted extraction method, a supercritical fluid extraction method and the like, and is a natural macromolecule consisting of aldose or ketose linked by glycoside bonds.
As a further scheme, the medicine or health-care product is a capsule, a tablet, a pill, a granule or an oral liquid.
As a further scheme, the medicine or health care product is prepared from sandworm polysaccharide and auxiliary materials allowed by a preparation or other optional active ingredients.
With the further development of marine biological resources and the increasing importance of the research on saccharides and drugs, the research and development of marine animal polysaccharides will be more and more concerned and become one of the hot spots. The research on the physiological activity of the sandworm active substance shows that the sandworm polysaccharide has various biological functions of resisting radiation, fatigue, oxidation, virus, immunity and the like. Experimental results show that the sandworm polysaccharide can remarkably promote the proliferation and differentiation of MC3T3-E1 osteogenic precursor cells cultured in vitro, and can remarkably improve the bone density of a mouse with the age of 10 months. The results show that the sandworm polysaccharide has a better function of treating or preventing osteoporosis.
The invention has the following beneficial effects:
the sandworm polysaccharide can promote the proliferation and differentiation of MC3T3-E1 osteogenic precursor cells in vitro and can increase the bone density of a 10-month-old mouse in vivo. The sandworm is used as a food material and has no toxic or side effect, so that the polysaccharide of the sandworm has better application potential in preparing medicaments or health-care products for treating or preventing osteoporosis.
Drawings
FIG. 1 is a graph showing the effect of sandworm polysaccharide on ALP activity of MC3T3-E1 preosteoblasts, compared with a blank controlaP＜0.05；
FIG. 2 is a graph showing the effect of sandworm polysaccharide on bone Density (BMD) in mice, compared to a placebo groupaP＜0.05。
Detailed Description
The present invention will be further described with reference to the following embodiments.
The sandworm polysaccharide may be obtained by any suitable extraction method, such as: hot water extraction, acid extraction, alkaline extraction, enzymatic methods, and the like. The sandworm polysaccharide used in the examples of the present invention can be obtained by the following method: fresh sandworms (purchased in Zhanjiang Dongfeng aquatic product market) are cut open longitudinally by a dissecting scissors, and the body wall is washed by distilled water and cut into small sections. 250g of the above small fragment was taken, 1000mL of distilled water (ultrapure water, Milli-Q) was added, and the mixture was homogenized at high speed for 5min (tissue triturator, Shanghai Biao national model Co., Ltd.) and allowed to stand at 4 ℃ for 24 hours. The sample is put into a water bath at 90 ℃ for leaching for 2h, and is centrifuged at 4900r/min for 15min (a floor type low-speed centrifuge, Hunan Hexi apparatus and Equipment Co., Ltd.), and the supernatant and the precipitate are collected respectively. To the supernatant was added 3% trichloroacetic acid (Nanjing chemical Co., Ltd.) until no turbidity appeared, and the mixture was left overnight to remove the protein by centrifugation. Adding 95% ethanol into the supernatant after protein removal to a final concentration of 70%, centrifuging at 4900r/min, and precipitating to obtain water-extracted polysaccharide.
The following describes in detail preferred embodiments of the present invention, and experimental methods in which specific conditions are not specified in the examples are generally conventional conditions.
Example 1 Effect of sandworm polysaccharide on proliferation of MC3T3-E1 preosteoblasts cultured in vitro
1. Cells
Mouse preosteoblasts MC3T3-E1 (purchased from Shanghai cell Bank, Chinese academy of sciences). The cells were routinely cultured in alpha-MEM (Hyclone, USA) complete medium containing 10% fetal bovine serum (GIBCO, USA) and 5% CO2Incubated at 37 ℃ in an incubator (Thermo), and when the cell fusion degree reaches 80%, 0.25% of pancreatin (sigma in USA) is digested and subcultured.
MTT method for detecting proliferation of MC3T3-E1 preosteoblasts
Taking cells in logarithmic growth phase, and adopting alpha-MEM culture solution containing 10% fetal calf serum to resuspend the cells, wherein the cell density is 5 multiplied by 104one/mL. Respectively inoculating to 3 96-well cell culture plates, wherein each well is 100 mu L, cells adhere to the wall after 24h, removing the culture solution, washing with Phosphate Buffer Solution (PBS) for 1 time, and adding culture solution containing sargassum polysaccharide (0, 6.25, 12.5, 25, 50 and 100mg/mL) with different concentrations. The experiment is provided with 6 groups according to the different concentrations of sandworm polysaccharide, and each group has 8 multiple holes. At 24, 36 and 48 hours of culture, 1 plate was removed, 20. mu.L of MTT solution (5g/L) was added to each well, and culture was continued at 37 ℃ for 4 hours, and the culture medium was discarded. Adding DMSO (100 mu L) into each hole, oscillating at room temperature for 10min, detecting the absorbance (OD value) of each hole at 490nm by using an enzyme-linked immunosorbent assay (ELx800 enzyme-linked immunosorbent assay, BIO-TEK), calculating the cell proliferation activity, and performing statistical analysis by using SPSS 12 software to perform group t test.
3. Results
As can be seen from Table 1, the sandworm polysaccharide with 5 concentrations can remarkably promote the proliferation of MC3T3-E1 cells (P < 0.05) at 3 different time points, and the sandworm polysaccharide has the proliferation promoting effect on MC3T3-E1 cells. Comparison with blank control groupaP＜0.05。
Table 1 effect of sandworm polysaccharide on pro-osteoblastic MC3T3-E1 proliferative activity (n ═ 8)
Example 2: effect of sandworm polysaccharide on osteogenic differentiation of MC3T3-E1 cells cultured in vitro
1. Alkaline phosphatase (ALP) Activity assay
The preosteoblasts MC3T3-E1 are inoculated to a 12-well cell plate, the original culture solution is removed after 24h of cell adherence, and then alpha-MEM culture solutions containing different sandworm polysaccharide concentrations (0, 6.25, 12.5, 25, 50 and 100mg/mL) are respectively added, the total is 6 groups, and each group has 3 duplicate wells. Cell culture broth, 2.5X 10, was aspirated on day 63Centrifuging at r/min for 10min, collecting supernatant, and determining ALP activity with ALP activity detection kit (Nanjing kit). Blank wells (50. mu.L of buffer, 50. mu.L of matrix solution, 30. mu.L of double distilled water), standard wells (50. mu.L of buffer, 50. mu.L of matrix solution, 30. mu.L of 0.02mg/mL phenol standard application solution), assay wells (50. mu.L of buffer, 50. mu.L of matrix solution, 30. mu.L of test supernatant) were prepared. Mixing well, and water bathing at 37 deg.C for 15 min. 150 mu L of color developing agent is added into each hole, the hole plate is shaken gently and mixed evenly, and the OD value of each hole is measured at the wavelength of 520 nm. Statistical analysis was performed using the SPSS 12 software for the set t-test.
2. Results
ALP is an enzyme protein secreted by osteoblasts, promotes cell maturation and calcification, and the quantitative detection of ALP can reflect the differentiation level of osteoblasts, and the higher the enzyme activity is, the more obvious the differentiation from preosteoblasts to mature osteoblasts is, so that the activity of ALP is a good index reflecting the differentiation degree and the functional state of osteoblasts.
As shown in FIG. 1, the sandworm polysaccharide can enhance ALP activity of the preosteoblasts MC3T3-E1 compared with the blank control group, and the sandworm polysaccharide can promote preosteoblasts of MC3T3-E1 to differentiate into osteogenesis.
Example 3: effect of sandworm polysaccharide on bone Density of 10-month-old mice
1. Grouping experimental animals:
after 40 KM mice (purchased from the center of medical laboratory animals in Guangdong province) at 8 months of age were acclimatized for 2 weeks, they were randomly divided into 4 groups of 10 mice each. Group A is blank Control (CON) group, and the mice in the group are gavaged with 10mL/(kg/d) of physiological saline every day; the group B is a sandworm polysaccharide low-dose group, and the mice in the group are administrated with sandworm polysaccharide 25mg/(kg/d) by intragastric administration every day; c, in the middle dose group of the group of sandworm polysaccharide, the group of mice is gavaged with 50mg/(kg/d) of sandworm polysaccharide every day; and D, a group of sandworm polysaccharide high-dose groups, wherein the group of mice is subjected to gavage administration of 100mg/(kg/D) of sandworm polysaccharide every day. All 4 groups of mice had free access to water and food. The experiment was administered for 10 weeks, and after completion of the experiment, the right femur was subjected to Micro-CT (viva CT 40; SCANCO Medical AG) scanning and three-dimensional reconstruction.
2. Micro-CT measurement:
placing the treated mouse femur into a Micro-CT (Micro-computed tomography) instrument, and carrying out X-ray scanning on the proximal metaphysis of the femur; the scanning conditions were: the image matrix is 2048 multiplied by 2048, the integration time is 200 ms, and the energy/intensity is 70kVp, 114 muA and 8W; scanning was performed with 0 ° rotation. After the scanning is finished, selecting bone tissues with the distal end of 1.0mm and the layer thickness of 2.0mm as cancellous bone interested areas (ROI) for three-dimensional recombination, and extracting image information with the lowest threshold value of 160. After the recombined image is obtained, the Micro-CT software is used for quantitative analysis. Statistical analysis was performed using the SPSS 12 software for the set t-test.
3. The experimental results are as follows:
as shown in FIG. 2, the bone density (BMD) of the mice in the high, medium and low dose groups of the sandworm polysaccharide is obviously increased compared with the blank control group.
Various other modifications and changes may occur to those skilled in the art, such as those described above, and other embodiments, and it is intended that all such modifications and changes fall within the scope of the appended claims.
Claims (3)
1. The application of sandworm polysaccharide in preparing the medicine for preventing and treating osteoporosis is characterized in that the sandworm polysaccharide is obtained by the following method: cutting fresh sandworms longitudinally by adopting dissecting scissors, washing the body wall of the fresh sandworms with distilled water, cutting the fresh sandworms into small sections, taking 250g of the small sections, adding 1000mL of distilled water, homogenizing at a high speed for 5min, standing at 4 ℃ for 24h, leaching the samples in a water bath at 90 ℃ for 2h, centrifuging at 4900r/min for 15min, respectively collecting supernatant and precipitates, adding 3% of trichloroacetic acid into the supernatant until turbidity does not appear, standing overnight, centrifuging to remove protein, adding 95% of ethanol into the supernatant after protein removal until the final concentration is 70%, and centrifuging at 4900r/min to obtain the precipitate, namely polysaccharide water extraction.
2. The use of claim 1, wherein the medicament is one of a capsule, a tablet, a pill, a granule, or an oral liquid.
3. The use of claim 2, wherein the medicament is prepared from sargassum polysaccharide and pharmaceutically acceptable adjuvant ingredients.
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