CN115844920A - Application of caulerpa lentillifera polysaccharide in preparation of medicine for treating osteoporosis - Google Patents
Application of caulerpa lentillifera polysaccharide in preparation of medicine for treating osteoporosis Download PDFInfo
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Abstract
The invention relates to the technical field of marine organisms, in particular to application of long-stem grape caulerpan polysaccharide in preparation of a medicine for treating osteoporosis. Therefore, the application of the polysaccharide in preparing the medicine for treating osteoporosis is provided, and a preparation method of the botryococcus longissimus polysaccharide is further provided.
Description
Technical Field
The invention relates to the technical field of marine organisms, in particular to application of caulerpa lentillifera polysaccharide in preparation of a medicine for treating osteoporosis.
Background
Caulerpa lentillifera (Caulerpa lentillifera) belongs to the phylum Chlorophyta (Chlorophyta) order of Lupeophytales (Bryopsidales) and belongs to the genus Caulerpa (Caulerpa) of the family Caulerpaceae, and is distributed in tropical and subtropical areas such as south China sea, taiwan China, southeast Asia, okinawa Japan, and oceania. The caulerpa lentillifera is rich in nutrition, delicious in taste and has a health-care effect, is called as a 'longevity vegetable', is deeply favored by vast coastal residents, and is an economic seaweed with research and development prospects.
According to related researches, the caulerpa lentillifera is rich in moisture content which accounts for 97% of wet weight, dietary fiber accounts for 24-26% of dry weight, protein accounts for 16% of dry weight, fat only accounts for 0.03%, cholesterol is not contained, and the caulerpa lentillifera is a high-protein low-fat health-care food. Also contains a large amount of amino acids, minerals, unsaturated fatty acids, and trace elements such as calcium, phosphorus, magnesium, iodine, and copper. Other related researches also find that the caulerpa lentillifera has the pharmacological effects of resisting bacteria, treating diabetes, reducing blood pressure, dispelling wind-damp, beautifying, protecting health and the like.
Osteoporosis is a systemic skeletal disease characterized by a decrease in bone mass and destruction of the microstructure of bone tissue, resulting in a decrease in bone strength, an increase in bone fragility, and a high risk of fracture. With the aging of the population in China, osteoporosis becomes an increasingly serious public health problem, and the prevalence rate of osteoporosis in the population over 65 years old in China reaches 32%. The molecular mechanism related to the onset of osteoporosis is not clear, the clinical manifestation and prognosis are complex, the course of disease is prolonged, and an effective treatment method is not available.
The marine plants have the characteristics which are not possessed by some terrestrial plants due to the unique living environment, and the marine plants are widely distributed and have large quantity, so that the method for obtaining the active ingredients with the activity of treating the osteoporosis from the marine plants is a novel thought with development prospect.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a novel marine plant extract aiming at the defects of the prior art, and the extract can be used for treating and preventing osteoporosis.
In order to realize the purpose of the invention, the following technical means are specifically adopted:
use of Botrytis longipedicularis polysaccharide in preparing medicine for treating osteoporosis is provided.
According to the preparation method of the vitis vinifera dunaliella polysaccharide, the vitis vinifera dunaliella polysaccharide is prepared by adopting the following method:
(1) Drying and powdering: drying cleaned caulerpa lentillifera, pulverizing, and sieving to obtain caulerpa lentillifera powder;
(2) Mixing the material liquid: crushing the sieved caulerpa lentillifera powder, adding water, wherein the g/ml ratio of the material to the liquid is 1: 40-60, and obtaining a material-liquid mixture;
(3) Water extraction of crude polysaccharide: extracting the crude polysaccharide from the feed liquid mixture obtained in the step (2) at the extraction temperature of 70-90 ℃ for 1.5-2.5 h for 1-3 times, mixing the extracting solutions, and concentrating to obtain a crude polysaccharide extracting solution a equivalent to 0.8-1.3 g/ml of the original medicinal material;
(4) Alcohol precipitation of crude polysaccharide: mixing the crude polysaccharide extract a obtained in the step (3) with 95% ethanol in a volume ratio of 1: 3-5, standing at 3-5 ℃ for alcohol precipitation, centrifuging at a high speed of 4500-5500 r/min to obtain a precipitate, and preparing the precipitate into a crude polysaccharide extract water solution b equivalent to 0.8-1.3 g/ml of a raw medicinal material;
(5) Removing protein by Sevage method: removing protein by adopting a Sevage method to obtain a polysaccharide extracting solution c;
(6) And (3) dialysis: dialyzing the polysaccharide extracting solution c obtained in the step (5) for 24-48h at 3-5 ℃ in a dialysis bag with the molecular weight of 2000-3000Da, and removing the organic solvent to obtain a crude polysaccharide extracting solution d;
(7) Freeze-drying and storing: and (4) carrying out freeze drying on the crude polysaccharide extracting solution d prepared in the step (6) to obtain the algal polysaccharide extract.
Preferably, the specific operation of step (5) is: adding a sevage reagent with the volume of 1/4-1/2 times of the crude polysaccharide extracting solution b obtained in the step (4): chloroform and n-butanol are = 4: 1, the mixture is oscillated for 20min at room temperature, and after the reaction is finished, the mixture is centrifuged at high speed at the rotating speed of 4500-5500 r/min, and supernatant is taken; the above procedure is repeated about 5 to 8 times until the solution is not precipitated.
Preferably, the extraction temperature in the step (3) is 80 ℃, the extraction time is 2 hours, the extraction times are 2 times, and the feed-liquid ratio in the step (2) is 1: 50.
Advantageous effects
(1) The invention provides a preparation method of high-purity caulerpa lentillifera polysaccharide.
(2) The caulerpa lentillifera polysaccharide extracted by the method shows the effect of treating osteoporosis on an animal model.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The method for measuring the content of the polysaccharide in the vitis amurensis dunn of the invention adopts a sulfuric acid-anthrone method to measure
The operation method for measuring the polysaccharide content by the sulfuric acid-anthrone method comprises the following steps:
A. preparation of a standard solution: (1) anthrone reagent: accurately weighing 0.1g of anthrone, adding 80% concentrated sulfuric acid to dissolve, transferring to a brown volumetric flask, adding 80% concentrated sulfuric acid to a constant volume of 100ml, and shaking up; it is ready for use (not more than 2 hours). (2) dextran standard solution: placing the glucan in a phosphorus pentoxide dryer, precisely weighing 0.1g after 12h, and fixing the volume to 100ml with distilled water to prepare a glucan standard solution of 1.0 mg/ml.
B. Drawing a standard curve: 0mL, 0.20mL, 0.40mL, 0.60mL, 0.80mL and 1.00mL of the dextran standard solution were precisely pipetted into 10mL stoppered tubes, and 1.0mL was made up with distilled water to prepare 3 groups per concentration. 4.0ml of the anthrone-sulfuric acid solution was added, respectively, and the reaction solutions were mixed using a vortex shaker, and then the tube was placed in a boiling water bath to react for 10min. Cooling to room temperature in cold water bath for 10min, and immediately measuring the maximum required wavelength at 626nm of an ultraviolet-visible spectrophotometer. And (3) establishing a standard curve by taking the mass of the glucan as an abscissa and the absorbance as an ordinate.
C. And (3) determination of sample content: taking 1.0ml of sample solution, measuring the absorbance of the polysaccharide sample of the Caulerpa lentillifera at 626nm according to the method, measuring for 3 times in parallel, and calculating the polysaccharide content.
Example 2
The preparation method of the caulerpa lentillifera polysaccharide comprises the following steps:
(1) Selecting algae: selecting fresh caulerpa lentillifera as a raw material, removing impurities such as weeds, silt and the like, and cleaning for later use;
(2) Drying and powdering: drying the caulerpa lentillifera selected in the step (1) at 50 ℃ for 36-72 hours, taking out the dried seaweed, crushing, and sieving with a 60-mesh sieve to obtain caulerpa lentillifera powder;
(3) Mixing the material liquid: weighing crushed and sieved caulerpa lentillifera powder, adding distilled water, and properly and uniformly stirring to obtain a material liquid mixture, wherein the material liquid ratio g/ml is 1: 50;
(4) Water extraction of crude polysaccharide: extracting the crude polysaccharide from the feed liquid mixture obtained in the step (3) at the extraction temperature of 80 ℃ for 2 hours for 2 times, mixing the extracting solutions, and performing rotary evaporation to obtain a crude polysaccharide extracting solution a of 1ml/g dry seaweed;
(5) Alcohol precipitation of crude polysaccharide: uniformly mixing the crude polysaccharide extracting solution a obtained in the step (4) with 95% ethanol in a volume ratio of 1: 4, standing at 4 ℃ for alcohol precipitation for 12h, centrifuging at a high speed of 5000r/min for 10min to obtain a precipitate, and dissolving the precipitate into a crude polysaccharide extracting solution b of 1ml/g dry seaweed;
(6) Removing protein by Sevage method: adding a sevage reagent (chloroform: n-butanol = 4: 1) with the volume of 1/3 times of the crude polysaccharide extracting solution b obtained in the step (5), oscillating at room temperature for 20min, centrifuging at a high speed of 5000r/min for 10min after the reaction is finished, and taking supernatant; repeating the above steps for about 5-8 times until the solution is not precipitated, and concentrating by rotary evaporation to obtain 1ml/g crude polysaccharide extract c of dry seaweed;
(7) And (3) dialysis: dialyzing the polysaccharide extract c obtained in the step (6) at 4 ℃ for 48h in a dialysis bag with the molecular weight of 2500Da to remove an organic solvent to obtain a crude polysaccharide extract d;
(8) Freeze-drying and storing: and (4) freeze-drying the crude polysaccharide extracting solution d prepared in the step (7) to obtain a seaweed polysaccharide extract, and storing the obtained freeze-dried powder at the temperature of-20 ℃.
Example 3
The anti-osteoporosis experiment of the long-stem grape fern polysaccharide extracted by the method.
Molding:
female SD rats were acclimatized for two weeks, 10 sham operated groups were randomly assigned, and the remaining groups were ovariectomized. Intraperitoneal anesthesia is carried out by adopting 2% sodium pentobarbital, bilateral ovaries are completely removed from the castration group, small fat blocks are removed from the sham operation group (except for not removing the ovaries, the operation is the same as that of the castration group rats), and incisions are sutured. Post-operative intramuscular injection of penicillin was continued for 7 days. Ovariectomized osteoporosis model rats were formed 4 weeks after molding.
Grouping and administration:
4 weeks after molding, rats were divided into a sham operation group, a model group, a Xianlinggubao group, a long-stem grape caulerpan polysaccharide low dose group, a long-stem grape caulerpan polysaccharide medium dose group, and a long-stem grape caulerpan polysaccharide high dose group.
Sham group: gavage 0.5% CMC-Na solution, the gavage volume is 10ml/kg;
model group: gavage 0.5% CMC-Na solution, the gavage volume is 10ml/kg;
xianlinggubao (positive drug) group: gavage administration of 0.5% CMC-Na solution of Xianlinggubao, with administration dosage of 1.5g/kg/d and administration volume of 10ml/kg; low dose group of vitis vinifera dunaliella polysaccharide: 0.5% CMC-Na solution of periphyton, administered at a dose of 0.1g/kg/d and a volume of 10ml/kg;
dose groups for vitis vinifera dunaliella polysaccharide: 0.5% CMC-Na solution of periphyton, administered at a dose of 0.2g/kg/d and a volume of 10ml/kg;
long stem grape fern polysaccharide high dose group: 0.5% CMC-Na solution of periphyton, administered at a dose of 0.3g/kg/d and a volume of 10ml/kg;
each of the above groups was administered continuously for 12 weeks.
Index measurement:
(1) Body weight was measured 1 time per week before and after dosing; (2) bone density of rat femur was measured 12 weeks after administration.
The experimental results of the positive drug and the effect of the caulerpa lentinan on the weight and bone density of the rats in the model group are as follows:
TABLE 1 Positive drug and Caulerpa lentinan Effect on body weight of model group rats
| Group of | Before administration (g) | After administration (g) |
| Artificial operation group | 282.6±10.09 | 376.2±10.88 ### |
| Model set | 273.9±11.38 | 429.0±12.90 |
| Xianlinggubao group | 275.1±12.06 | 391.9±9.30 ### |
| Low dose group of Caulerpa lentillifera polysaccharides | 276.9±10.12 | 398.3±9.23 ### |
| Medium-dose group of Caulerpa lentillifera polysaccharide | 276.1±11.51 | 396.3±8.52 ### |
| High dose group of Caulerpa lentillifera polysaccharides | 274.4±10.61 | 388.8±11.16 ### |
Compared with the model group, # P < 0.05, # P < 0.01, # P < 0.001
TABLE 2 Effect of Positive drugs and Caulerpa lentinan on bone Density in model groups of rats
| Group of | Bone mineral density (g/cm) 2 ) |
| Artificial operation group | 0.202±0.0036 ## |
| Model set | 0.175±0.0045 |
| Xianlinggubao group | 0.194±0.0046 ## |
| Low dose of Caulerpa lentillifera polysaccharideGroup of | 0.186±0.0043 # |
| Long stem grape caulerpa polysaccharide medium dose group | 0.191±0.0051# |
| High dose group of Caulerpa lentillifera polysaccharides | 0.201±0.0046 ## |
Compared with the model group, # P < 0.05, # P < 0.01, # P < 0.001
As can be seen from Table 1, the body weight of the model group rats after administration was significantly higher than that of the sham operation group, indicating that the body weight of the rats was significantly increased after the osteoporosis molding. Compared with the model group, the Xianlinggubao and the long-stem grape fern algae polysaccharide in the low, medium and high dose groups can obviously reduce the weight of the rat, and the Xianlinggubao and the long-stem grape fern algae polysaccharide have obvious reducing effect on the weight of the rat with ovarian osteoporosis. As can be seen from table 2, the bone density of the rats in the model group is significantly lower than that of the sham operation group, and the bone density of the rats in the Xianlinggubao group and the Caulerpa lentillifera polysaccharide in the low, medium and high dose groups can be increased to different degrees, which indicates that the bone density of the rats with ovarian osteoporosis can be significantly improved by the Xianlinggubao group and the Caulerpa lentillifera polysaccharide, and the bone density can be effectively improved. In conclusion, the vitis amurensis dunaliella polysaccharide can show a treatment effect on the ovariectomized osteoporosis rat model.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (1)
1. Use of Botrytis longipedicularis polysaccharide in preparing medicine for treating osteoporosis is provided.
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| CN117323350A (en) * | 2023-10-27 | 2024-01-02 | 江苏海洋大学 | Application of pteridophyta longifolia polyphenol in preparation of medicines for treating thrombus |
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| CN117323350A (en) * | 2023-10-27 | 2024-01-02 | 江苏海洋大学 | Application of pteridophyta longifolia polyphenol in preparation of medicines for treating thrombus |
| CN117323350B (en) * | 2023-10-27 | 2024-03-05 | 江苏海洋大学 | Application of pteridophyta longifolia polyphenol in preparation of medicines for treating thrombus |
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