Application of polysaccharide from long-stem grape fern algae in preparing drugs for treating osteoporosis

Abstract

The present invention relates to the field of marine biotechnology, and in particular to the use of long-stem grape fern algae polysaccharide in the preparation of drugs for treating osteoporosis. The present invention finds that the long-stem grape fern algae polysaccharide can improve the bone density of ovariectomized osteoporosis model rats. Therefore, the use of the long-stem grape fern algae polysaccharide in the preparation of drugs for treating osteoporosis is provided, and a preparation method of the long-stem grape fern algae polysaccharide is further provided.

Description

Technical Field

[0001] The invention relates to the field of marine biotechnology, and in particular to application of long-stem grape fern algae polysaccharide in preparing medicine for treating osteoporosis. Background Art

[0002] Caulerpa lentillifera belongs to the genus Caulerpa, order Bryopsidales, family Caulerpaceae, Chlorophyta, and is distributed in tropical and subtropical waters such as the South China Sea, Taiwan, Southeast Asia, Okinawa, Japan, and Oceania. It is rich in nutrients, delicious, and has health benefits. It is known as the "longevity vegetable" and is favored by coastal residents. It is an economic seaweed with research and development prospects.

[0003] According to relevant research, the water content of long-stem grape fern algae is rich, accounting for 97% of the wet weight, dietary fiber accounts for 24-26% of the dry weight, protein accounts for 16% of the dry weight, and fat accounts for only 0.03%. It does not contain cholesterol and is a high-protein, low-fat health food. It also contains a large amount of amino acids, minerals and unsaturated fatty acids, as well as trace elements such as calcium, phosphorus, magnesium, iodine, and copper. Other related studies have also found that long-stem grape fern algae has pharmacological effects such as antibacterial, treatment of diabetes, blood pressure reduction, anti-rheumatic and beauty and health care.

[0004] Osteoporosis is a group of systemic bone diseases characterized by decreased bone mass and destruction of bone microstructure, which leads to decreased bone strength, increased bone brittleness, and high susceptibility to fractures. With the aging of my country's population, osteoporosis has become an increasingly serious public health problem. The prevalence of osteoporosis in people over 65 years old in my country has reached 32%. The molecular mechanism of osteoporosis has not yet been clarified, the clinical manifestations and prognosis are relatively complex, and the course of the disease is prolonged. There is no effective treatment yet.

[0005] Marine plants have some characteristics that terrestrial plants do not have due to their unique living environment. Marine plants are widely distributed and large in number. Obtaining active ingredients with therapeutic activity for osteoporosis from marine plants is a novel and promising idea. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a new marine plant extract for the deficiencies of the prior art, and the extract can be used for the treatment and prevention of osteoporosis.

[0007] In order to achieve the purpose of the present invention, the following technical means are specifically adopted:

[0008] Use of long-stem grape fern algae polysaccharide in preparing medicine for treating osteoporosis.

[0009] The aforementioned method for preparing the long-stem grape fern algae polysaccharide is prepared by the following method:

[0010] (1) Drying and powdering: drying, crushing and sieving the cleaned long-stem grape fern algae to obtain long-stem grape fern algae powder;

[0011] (2) mixing feed and liquid: grinding and sieving the long-stem grape fern algae powder, adding water, and the feed-liquid ratio g / ml is 1:40-60 to obtain a feed-liquid mixture;

[0012] (3) Extracting crude polysaccharides with water: extracting crude polysaccharides from the mixture of feed and liquid obtained in step (2) at an extraction temperature of 70 to 90° C. for 1.5 to 2.5 hours, performing extraction 1 to 3 times, mixing the extracts, and concentrating to obtain a crude polysaccharide extract a equivalent to 0.8 to 1.3 g / ml of the original medicinal material;

[0013] (4) Alcohol precipitation of crude polysaccharides: the crude polysaccharide extract a obtained in step (3) is mixed with 95% ethanol at a volume ratio of 1:3-5, and allowed to stand at 3-5°C for alcohol precipitation, and then centrifuged at a speed of 4500-5500 r / min to obtain a precipitate, and the precipitate is prepared into a crude polysaccharide extract aqueous solution b equivalent to 0.8-1.3 g / ml of the original medicinal material;

[0014] (5) Sevage method for protein removal: The Sevage method is used to remove proteins to obtain polysaccharide extract c;

[0015] (6) Dialysis: The polysaccharide extract c obtained in step (5) is dialyzed in a dialysis bag with a molecular weight of 2000-3000 Da at 3-5° C. for 24-48 hours to remove the organic solvent, thereby obtaining a crude polysaccharide extract d;

[0016] (7) Freeze-drying and storage: The crude polysaccharide extract d obtained in step (6) is freeze-dried to obtain a seaweed polysaccharide extract.

[0017] Preferably, the specific operation of step (5) is as follows: add 1 / 4 to 1 / 2 times the volume of sevage reagent: chloroform: n-butanol = 4:1 to the crude polysaccharide extract b obtained in step (4), shake at room temperature for 20 minutes, and after the reaction is completed, high-speed centrifuge at a speed of 4500 to 5500 r / min to take the supernatant; repeat the above steps for about 5 to 8 times until there is no precipitation in the solution.

[0018] Preferably, the extraction temperature of step (3) is 80° C., the extraction time is 2 hours, the number of extractions is 2 times, and the solid-liquid ratio of step (2) is 1:50.

[0019] Beneficial Effects

[0020] (1) The present invention provides a method for preparing high-purity long-stem grape fern algae polysaccharide.

[0021] (2) The polysaccharide extracted from the long-stem grape fern algae using this method showed the effect of treating osteoporosis in animal models. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Example 1

[0024] The method for determining the content of the long-stem grape fern algae polysaccharide of the present invention is to use the sulfuric acid-anthrone method to determine the content of the long-stem grape fern algae polysaccharide.

[0025] The operation method of sulfuric acid-anthrone method for determining polysaccharide content is as follows:

[0026] A. Preparation of standard solution: (1) Anthrone reagent: Accurately weigh 0.1g of anthrone, add 80% concentrated sulfuric acid to dissolve, transfer to a brown volumetric flask, dilute to 100ml with 80% concentrated sulfuric acid, shake well; use immediately after preparation (no more than 2 hours). (2) Dextran standard solution: Place dextran in a phosphorus pentoxide desiccator, accurately weigh 0.1g after 12 hours, dilute to 100ml with distilled water, and prepare a 1.0mg / ml dextran standard solution.

[0027] B. Drawing of standard curve: Accurately pipette 0mL, 0.20mL, 0.40mL, 0.60mL, 0.80mL and 1.00mL of dextran standard solution into 10mL stoppered test tubes, and fill to 1.0mL with distilled water. Prepare 3 groups for each concentration. Add 4.0mL of anthrone-sulfuric acid solution respectively, use a vortex oscillator to mix the reaction solution, and then place the test tube in a boiling water bath to react for 10min. Cool to room temperature in a cold water bath for 10min, and immediately measure the maximum required wavelength at 626nm in a UV-visible spectrophotometer. Use the mass of dextran as the horizontal axis and the absorbance as the vertical axis to develop a standard curve.

[0028] C. Determination of sample content: Take 1.0 ml of sample solution and measure the absorbance of the polysaccharide sample of long-stem grape fern algae at 626 nm according to the above method. The measurement is repeated three times in parallel to calculate the polysaccharide content.

[0029] Example 2

[0030] The preparation method of long-stem grape fern algae polysaccharide is as follows:

[0031] (1) Selecting algae: Select fresh long-stem grape fern algae as raw material, remove weeds, mud and other impurities, and wash them for later use;

[0032] (2) Drying and powdering: drying the long-stem grape fern algae selected in step (1) at 50° C. for 36 to 72 hours, taking out the dried seaweed, crushing it, and passing it through a 60-mesh sieve to obtain long-stem grape fern algae powder;

[0033] (3) Mixing the feed and liquid: Weigh the crushed and sieved long-stem grape fern algae powder, add distilled water, and the feed-liquid ratio (g / ml) is 1:50, and stir evenly to obtain a feed-liquid mixture;

[0034] (4) Extracting crude polysaccharides with water: extracting crude polysaccharides from the mixture of feed and liquid obtained in step (3) at an extraction temperature of 80° C. and an extraction time of 2 h, performing extraction twice, mixing the extracts, and rotary evaporating to a crude polysaccharide extract a of 1 ml / g dry seaweed;

[0035] (5) Alcohol precipitation of crude polysaccharides: the crude polysaccharide extract a obtained in step (4) was uniformly mixed with 95% ethanol at a volume ratio of 1:4, and allowed to stand for alcohol precipitation at 4°C for 12 hours. The mixture was centrifuged at a speed of 5000 r / min for 10 minutes to obtain a precipitate, which was dissolved into a crude polysaccharide extract b at a concentration of 1 ml / g dry seaweed;

[0036] (6) Sevage method for protein removal: add 1 / 3 volume of sevage reagent (chloroform: n-butanol = 4:1) to the crude polysaccharide extract b obtained in step (5), shake at room temperature for 20 min, centrifuge at 5000 r / min for 10 min after the reaction, and collect the supernatant; repeat the above steps for about 5 to 8 times until there is no precipitation in the solution, and concentrate by rotary evaporation to a crude polysaccharide extract c of 1 ml / g dry seaweed;

[0037] (7) Dialysis: The polysaccharide extract c obtained in step (6) is dialyzed in a dialysis bag with a molecular weight of 2500 Da at 4° C. for 48 h to remove the organic solvent, thereby obtaining a crude polysaccharide extract d;

[0038] (8) Freeze-drying and storage: The crude polysaccharide extract d obtained in step (7) is freeze-dried to obtain a seaweed polysaccharide extract, and the obtained freeze-dried powder is stored at -20°C.

[0039] Example 3

[0040] The above method was used to extract the polysaccharide from the long-stem grape fern algae for the anti-osteoporosis experiment.

[0041] Modeling:

[0042] Female SD rats were adaptively fed for two weeks and randomly divided into a sham operation group of 10 rats and an ovariectomized model group. 2% sodium pentobarbital was used for intraperitoneal anesthesia. The ovariectomized group had their bilateral ovaries completely removed, and the sham operation group had a small piece of fat removed (except that the ovaries were not removed, the rest of the operations were the same as those of the ovariectomized group rats), and the incisions were sutured. Penicillin was injected intramuscularly for 7 consecutive days after surgery. Ovariectomized osteoporosis model rats were formed 4 weeks after modeling.

[0043] Grouping and dosing:

[0044] Four weeks after modeling, the rats were divided into sham operation group, model group, Xianlinggubao group, low-dose long-stem grape fern algae polysaccharide group, medium-dose long-stem grape fern algae polysaccharide group, and high-dose long-stem grape fern algae polysaccharide group.

[0045] Sham operation group: intragastric administration of 0.5% CMC-Na solution, the intragastric volume was 10 ml / kg;

[0046] Model group: intragastric administration of 0.5% CMC-Na solution, the intragastric volume was 10 ml / kg;

[0047] Xianlinggubao (positive drug) group: intragastrically administered Xianlinggubao 0.5% CMC-Na solution, the dosage was 1.5g / kg / d, and the administration volume was 10ml / kg; Long-stem grape fern algae polysaccharide low-dose group: intragastrically administered Long-stem grape fern algae polysaccharide 0.5% CMC-Na solution, the dosage was 0.1g / kg / d, and the administration volume was 10ml / kg;

[0048] The medium-dose group of long-stem grape fern algae polysaccharide was intragastrically administered with 0.5% CMC-Na solution of long-stem grape fern algae polysaccharide, with a dosage of 0.2 g / kg / d and a volume of 10 ml / kg.

[0049] The high-dose group of long-stem grape fern algae polysaccharide was intragastrically administered with 0.5% CMC-Na solution of long-stem grape fern algae polysaccharide, with a dosage of 0.3 g / kg / d and a volume of 10 ml / kg.

[0050] The above groups were administered drugs continuously for 12 weeks.

[0051] Index determination:

[0052] (1) Body weight was measured once a week before and after administration. (2) After 12 weeks of administration, the femur bone density of the rats was measured.

[0053] The experimental results of the effects of positive drugs and long-stem grape fern algae polysaccharide on the body weight and bone density of rats in the model group are as follows:

[0054] Table 1 Effects of positive drugs and long-stem grape fern polysaccharide on body weight of rats in the model group

[0055] Group Before administration (g) After administration (g) Sham operation group 282.6±10.09 <![CDATA[376.2±10.88 ### ]]> Model Group 273.9±11.38 429.0±12.90 Xianlinggubao Group 275.1±12.06 <![CDATA[391.9±9.30 ### ]]> Low-dose group of long-stem grape fern polysaccharide 276.9±10.12 <![CDATA[398.3±9.23 ### ]]> Medium dose group of long stem grape fern algae polysaccharide 276.1±11.51 <![CDATA[396.3±8.52 ### ]]> High-dose group of long-stem grape fern polysaccharide 274.4±10.61 <![CDATA[388.8±11.16 ### ]]>

[0056] Compared with the model group, #P<0.05, ##P<0.01, ###P<0.001

[0057] Table 2 Effects of positive drugs and long-stem grape fern polysaccharide on bone density of rats in the model group

[0058] Group <![CDATA[Bone density (g / cm 2 )]]> Sham operation group <![CDATA[0.202±0.0036 ## ]]> Model Group 0.175±0.0045 Xianlinggubao Group <![CDATA[0.194±0.0046 ## ]]> Low-dose group of long-stem grape fern polysaccharide <![CDATA[0.186±0.0043 # ]]> Medium dose group of long stem grape fern algae polysaccharide 0.191±0.0051# High-dose group of long-stem grape fern polysaccharide <![CDATA[0.201±0.0046 ## ]]>

[0059] Compared with the model group, #P<0.05, ##P<0.01, ###P<0.001

[0060] As shown in Table 1, the body weight of the rats in the model group after administration was significantly higher than that in the sham operation group, indicating that the body weight of the rats increased significantly after osteoporosis modeling. Compared with the model group, the low, medium and high dose groups of Xianlinggubao and long stem grape fern algae polysaccharide can significantly reduce the body weight of rats, indicating that Xianlinggubao and long stem grape fern algae polysaccharide have a significant effect on reducing the body weight of ovariectomized osteoporotic rats. As shown in Table 2, the bone density of rats in the model group was significantly lower than that in the sham operation group, while the low, medium and high dose groups of Xianlinggubao and long stem grape fern algae polysaccharide can increase bone density to varying degrees, indicating that Xianlinggubao and long stem grape fern algae polysaccharide significantly increase the bone density value of ovariectomized osteoporotic rats and can effectively improve bone density. In summary, long stem grape fern algae polysaccharide can show a therapeutic effect on the ovariectomized osteoporotic rat model.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of long-stem grape fern algae polysaccharide in the preparation of drugs for treating osteoporosis, It is characterized in that The polysaccharide is prepared by the following method: (1) Selecting algae: Select fresh long-stem grape fern algae as raw material, remove weeds, mud and other impurities, and wash them for later use; (2) Drying and powdering: drying the long-stem grape fern algae selected in step (1) at 50° C., taking out the dried seaweed, crushing it, and passing it through a 60-mesh sieve to obtain long-stem grape fern algae powder; (3) Mixing the feed and liquid: weigh the crushed and sieved long-stem grape fern algae powder, add distilled water, and the feed-liquid ratio is 1:50 g / ml, and stir evenly to obtain a feed-liquid mixture; (4) Extracting crude polysaccharides with water: extracting crude polysaccharides from the mixture of feed and liquid obtained in step (3) at an extraction temperature of 80° C. and an extraction time of 2 h, performing extraction twice, mixing the extracts, and rotary evaporating to a crude polysaccharide extract a of 1 ml / g dry seaweed; (5) Alcohol precipitation of crude polysaccharides: the crude polysaccharide extract a obtained in step (4) was uniformly mixed with 95% ethanol at a volume ratio of 1:4, and allowed to stand for alcohol precipitation at 4°C for 12 h. The mixture was centrifuged at a speed of 5000 r / min for 10 min to obtain a precipitate, which was dissolved into a crude polysaccharide extract b at a concentration of 1 ml / g dry seaweed; (6) Sevage method for protein removal: add 1 / 3 volume of sevage reagent (chloroform: n-butanol = 4:1) to the crude polysaccharide extract b obtained in step (5), shake at room temperature for 20 min, centrifuge at 5000 r / min for 10 min after the reaction, and take the supernatant; repeat the above steps for about 5 to 8 times until there is no precipitation in the solution, and concentrate by rotary evaporation to a crude polysaccharide extract c of 1 ml / g dry seaweed; (7) Dialysis: The polysaccharide extract c obtained in step (6) is dialyzed in a dialysis bag with a molecular weight of 2500 Da at 4° C. for 48 h to remove the organic solvent, thereby obtaining a crude polysaccharide extract d; (8) Freeze-drying and storage: The crude polysaccharide extract d obtained in step (7) is freeze-dried to obtain a seaweed polysaccharide extract, and the obtained freeze-dried powder is stored at -20°C.