Angelica sinensis blood replenishing decoction compound polysaccharide for preventing and treating atherosclerosis as well as preparation method and application thereof

Abstract

The invention discloses a Chinese angelica blood replenishing decoction compound polysaccharide for preventing and treating atherosclerosis as well as a preparation method and application of the Chinese angelica blood replenishing decoction compound polysaccharide. The total sugar content in the Chinese angelica blood replenishing decoction compound polysaccharide is greater than or equal to 85%, the sulfate group content is greater than or equal to 3%, the uronic acid content is greater than or equal to 6%, and the average molecular weight is 150-165 kDa. Under the assistance of ultrasonic waves, raw medicinal materials of the angelica sinensis blood replenishing decoction and chlorine type anion exchange resin are subjected to blending extraction, and polysaccharide is synchronously analyzed and precipitated from the resin in a sodium chloride ethanol solution high-pressure leaching manner, so that the preparation of the compound polysaccharide of the angelica sinensis blood replenishing decoction realizes synchronous coupling of ultrasonic-assisted extraction of the polysaccharide and static exchange adsorption separation of the resin; moreover, the polysaccharide is analyzed from the resin and cooperates with ethanol precipitation. The preparation method disclosed by the invention has the advantages of simple steps, no need of using acid and alkali reagents in the operation process, mild operation conditions, low equipment requirements, easiness in industrialization and the like, and accords with the traditional medication habit of traditional Chinese medicine compounds obtained by co-decocting multiple medicines. The obtained Chinese angelica blood replenishing decoction compound polysaccharide has remarkable activity of preventing and treating atherosclerosis.

Description

Technical Field

[0001] The present invention relates to an Angelica sinensis decoction compound polysaccharide for preventing and treating atherosclerosis, its preparation method and application, and belongs to the technical field of preparation of bioactive compound polysaccharides. Background Art

[0002] According to statistics, cardiovascular and cerebrovascular diseases rank first among the major diseases threatening human health. Atherosclerosis is the common pathological basis and main inducement of ischemic cardiovascular and cerebrovascular diseases. With the improvement of living standards, the high prevalence, high disability rate and high mortality rate of cardiovascular and cerebrovascular diseases induced by atherosclerosis have brought a heavy social and economic burden, making it an important global public health problem that cannot be ignored at present. Therefore, the research and development of pharmacodynamic components with significant activity against atherosclerosis have important social and economic value for the prevention and treatment of this disease.

[0003] Polysaccharides are high-molecular carbohydrates formed by polymerization of more than 10 monosaccharides through glycosidic bonds in a sugar chain manner. In recent years, studies have found that some polysaccharides containing sulfate groups and uronic acids derived from traditional Chinese medicines exhibit significant biological activities in preventing and treating atherosclerosis due to their obvious lipid-lowering, antioxidant, anti-inflammatory and cholesterol-regulating effects, and no adverse reactions such as rhabdomyolysis occur during the process of preventing and treating atherosclerosis, making them highly effective and low-toxic natural active compounds for preventing and treating atherosclerosis. However, current research and development of polysaccharides are mostly based on the polysaccharide components of single traditional Chinese medicines, and there are some traditional Chinese medicines used for polysaccharide preparation whose indications have nothing to do with the syndromes and principles of traditional Chinese medicine clinical treatment of diseases. There are few research reports on traditional Chinese medicine compound polysaccharides developed based on classical prescriptions for clinical treatment of diseases aggregated according to the theory of traditional Chinese medicine syndrome differentiation treatment, resulting in the inability of related polysaccharides to reflect the characteristics of traditional Chinese medicine syndrome differentiation and compatibility. At the same time, a large number of studies have also revealed that polysaccharides happen to be the main active components of compound decoctions, which are the important material basis for various decoctions to exert their therapeutic effects. Therefore, developing a preparation process of compound polysaccharides corresponding to the efficacy of its mother prescription based on the classical famous prescriptions for preventing and treating atherosclerosis in traditional Chinese medicine clinical practice can greatly promote the research and development of compound polysaccharide preparations for preventing and treating atherosclerosis, and better create compound polysaccharide-based atherosclerosis prevention and treatment preparations that conform to the characteristics of traditional Chinese medicine theory, with broad application prospects.

[0004] Danggui Buxue Decoction is a famous prescription for replenishing qi, nourishing blood, and promoting blood production created by Li Dongyuan in the Jin Dynasty. It is composed of Astragalus membranaceus and Angelica sinensis in a ratio of 5:1. In the formula, Astragalus membranaceus is used as the monarch drug to greatly replenish the spleen qi and provide the source for blood production; Angelica sinensis is used as the assistant drug to promote blood circulation, nourish blood, nourish the yang qi in the body, and dispel stasis; together, they achieve the effects of replenishing qi and blood, and removing stasis to promote new blood production. Danggui Buxue Decoction is an important basic and classic prescription for treating atherosclerosis by physicians of past dynasties. However, there has been no research report on preparing compound polysaccharides with the effect of preventing and treating atherosclerosis based on Danggui Buxue Decoction, which not only hinders the in-depth development of Danggui Buxue Decoction itself, but also causes the loss of an important class of candidate active ingredients that conform to the characteristics of traditional Chinese medicine in the prevention and treatment of atherosclerosis.

[0005] The current mainstream process for polysaccharide preparation is: hot water extraction - ethanol precipitation - decolorization and protein removal - DEAE-52 cellulose ion exchange column chromatography - molecular sieve column chromatography. Although this process can obtain purified polysaccharide components with different properties, however, it not only has cumbersome and time-consuming process steps, harsh operating conditions, but also has a small single-treatment volume, and there is also the drawback of requiring a large amount of acid and base reagents, resulting in the inability of this process to meet the requirements of industrial production. Therefore, there is an urgent need to establish a new method for polysaccharide preparation with simple process steps, no need to use acid and base reagents during the operation process, mild operating conditions, low equipment requirements, and easy industrialization. Summary of the Invention

[0006] Object of the Invention: Aiming at the deficiencies of the prior art, the first object of the present invention is to provide a Danggui Buxue Decoction compound polysaccharide for preventing and treating atherosclerosis, the second object of the present invention is to provide a preparation method of the Danggui Buxue Decoction compound polysaccharide for preventing and treating atherosclerosis, and the third object of the present invention is to provide the application of the Danggui Buxue Decoction compound polysaccharide for preventing and treating atherosclerosis in the preparation of drugs for preventing and treating atherosclerosis.

[0007] Technical Solution: The Danggui Buxue Decoction compound polysaccharide for preventing and treating atherosclerosis described in the present invention has a total sugar content ≥ 85%, a sulfate group content ≥ 3%, a uronic acid content ≥ 6%, and an average molecular weight of 150 - 165 kDa in the Danggui Buxue Decoction compound polysaccharide for preventing and treating atherosclerosis.

[0008] The preparation method of the Danggui Buxue Decoction compound polysaccharide for preventing and treating atherosclerosis described in the present invention includes the following steps:

[0009] (1) Under ultrasonic assistance, the original medicinal materials of Danggui Buxue Decoction and anion exchange resin are mixed and extracted in an extraction solvent;

[0010] (2) Wash the anion exchange resin to obtain the anion exchange resin containing the Danggui Buxue Decoction compound polysaccharide;

[0011] (3) Synchronously resolve and precipitate the polysaccharides of Danggui Buxue Decoction from the anion exchange resin containing the polysaccharides of Danggui Buxue Decoction with a sodium chloride ethanol solution, centrifuge to precipitate, and obtain the product by freeze-drying.

[0012] Further, in step (1), the original medicinal materials of Danggui Buxue Decoction include Astragalus membranaceus and Angelica sinensis.

[0013] Further, in step (1), the mass ratio of Astragalus membranaceus to Angelica sinensis is 5:1.

[0014] Further, in step (1), the original medicinal materials of Danggui Buxue Decoction are pulverized to pass through a 40 - 80 mesh sieve.

[0015] Further, in step (1), the anion exchange resin is chloride-form D301 ion exchange resin or chloride-form 717 ion exchange resin.

[0016] Further, in step (1), the extraction solvent is deionized water.

[0017] Further, in step (1), the mass ratio of the original medicinal materials of Danggui Buxue Decoction, the anion exchange resin to the extraction solvent is 1:(0.5 - 1.5):(15 - 25).

[0018] Further, in step (1), the ultrasonic power is 100 - 300 W, the extraction temperature is 50 - 70 °C, and the extraction time is 40 - 60 min.

[0019] Further, in step (1), during extraction, the original medicinal materials of Danggui Buxue Decoction and the anion exchange resin are separately packed into different filter bags for ultrasonic extraction, and the aperture of the filter bag is 100 - 150 mesh.

[0020] Further, in step (2), the method for washing the anion exchange resin includes: taking out the filter bag containing the anion exchange resin in the co - mixed extraction, and rinsing the filter bag with deionized water until the outflow from the filter bag has no original medicinal materials of Danggui Buxue Decoction.

[0021] Further, in step (3), the sodium chloride ethanol solution is a 60% ethanol solution containing 0.4 - 0.6 mol / L sodium chloride.

[0022] Further, in step (3), when synchronously resolving and precipitating the polysaccharides of the original medicinal materials of Danggui Buxue Decoction from the anion exchange resin with the sodium chloride ethanol solution, the sodium chloride ethanol solution is pumped in with a ceramic high - pressure pump, the pressure is 1.5 - 2.5 Mpa, and the flow rate is 0.5 - 1.0 times the volume of the anion exchange resin per minute.

[0023] Further, in step (3), the centrifugal separation is carried out at 4500 rpm for more than 30 min.

[0024] Use of the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis in the preparation of drugs for preventing and treating atherosclerosis.

[0025] The preparation method of the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis. In this method, the original medicinal materials of Danggui Buxue Decoction are mixed and extracted with chloride-type anion exchange resin under ultrasonic assistance. The ultrasonic-assisted extraction of polysaccharide and ion exchange adsorption separation are combined into one, realizing the synchronous coupling of ultrasonic-assisted extraction of polysaccharide and static exchange adsorption separation of resin. This not only simplifies the process steps, reduces energy consumption, saves production time, and improves separation efficiency, but also does not require the use of acid-base reagents during the operation process, and the operation conditions are mild. The polysaccharide is simultaneously desorbed and precipitated from the resin by high-pressure elution with sodium chloride ethanol solution. Compared with the traditional process, it not only realizes the coordination of polysaccharide desorption from the resin and ethanol precipitation, but also omits the processes of dialysis for desalting and protein removal. The process steps are simple, the equipment requirements are low, and it is easy to industrialize. The extraction of the compound polysaccharide by this preparation method is based on the co-decoction of all the medicinal materials of the classic formula Danggui Buxue Decoction for preventing and treating atherosclerosis, and the obtained compound polysaccharide extraction process conforms to the traditional medication habit of multi-herb co-decoction in traditional Chinese medicine compound. At the same time, through the synergistic effect of the specific adsorption of chloride-type anion resin and the specific elution of sodium chloride ethanol solution with appropriate concentration, the obtained compound polysaccharide of Danggui Buxue Decoction is rich in sulfate groups and uronic acid. The biological activity endowed by the high sulfate group and uronic acid content of the compound polysaccharide of Danggui Buxue Decoction fully follows and continues the actual clinical application of the mother formula Danggui Buxue Decoction for preventing and treating atherosclerosis in traditional Chinese medicine, and has a significant effect on preventing and treating atherosclerosis.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0027] (1) The preparation process of the present invention has the advantages of simple process steps, no need to use acid-base reagents during the operation process, mild operation conditions, low equipment requirements, easy industrialization, etc., and conforms to the traditional medication habit of multi-herb co-decoction in traditional Chinese medicine compound.

[0028] (2) The total sugar content in the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis obtained by the present invention is ≥85%, the sulfate group content is ≥3%, and the uronic acid content is ≥6%. The biological activity endowed by its high sulfate group and uronic acid content fully follows and continues the actual clinical application of the mother formula Danggui Buxue Decoction for preventing and treating atherosclerosis in traditional Chinese medicine, and has a significant effect on preventing and treating atherosclerosis. Specific embodiments

[0029] The technical solutions of the present invention will be further described below.

[0030] The implementation modes of the present invention are not limited thereto. The parts mentioned in the examples are all in parts by mass.

[0031] Example 1

[0032] (1) Pretreatment of medicinal materials

[0033] Weigh 5 parts of commercially available Chinese medicinal material Astragalus membranaceus and 1 part of commercially available Chinese medicinal material Angelica sinensis by mass fraction. After mixing, pulverize them with a pulverizer until they pass through a 40-mesh sieve to obtain the original medicinal material powder of Danggui Buxue Decoction.

[0034] (2) Preparation of anion exchange resin

[0035] Weigh the chloride-form D301 ion exchange resin at 0.5 times the mass of the original medicinal materials of Danggui Buxue Decoction by mass ratio.

[0036] (3) Co-blending extraction of the original medicinal materials of Danggui Buxue Decoction and anion exchange resin assisted by ultrasonic

[0037] Put the pretreated original medicinal material powder of Danggui Buxue Decoction and the prepared chloride-form D301 ion exchange resin into different 100-mesh filter bags respectively. Tie the mouths of the filter bags tightly, place them in an extraction container, add deionized water at 15 times the mass of the original medicinal materials of Danggui Buxue Decoction, and perform ultrasonic-assisted extraction for 40 min under the conditions of an extraction temperature of 50 °C and an extraction power of 100 W.

[0038] (4) Collect and wash to remove the adhered residual medicinal residue powder on the resin

[0039] Take out the filter bag containing the chloride-form D301 ion exchange resin in the co-blending extraction, and rinse the filter bag with deionized water until there is no medicinal powder in the effluent from the filter bag, thus obtaining a clean resin filter bag adsorbed with the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis.

[0040] (5) Synchronously desorb and precipitate polysaccharides from the resin with sodium chloride ethanol solution

[0041] Keep the mouth of the filter bag facing upward, hang the clean resin filter bag adsorbed with the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis in a synchronous desorption and precipitation container, open the mouth of the filter bag, and use a ceramic high-pressure pump to pump the 60% ethanol solution containing 0.4 mol / L sodium chloride into the filter bag under the conditions of a pressure of 1.5 MPa and a flow rate of 0.5 times the resin volume per minute until there is no polysaccharide precipitation in the effluent from the filter bag.

[0042] (6) Formation of the product of the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis

[0043] Remove the resin filter bag, centrifuge the desorption and precipitation solution containing the compound polysaccharide at a rotation speed of 4500 rpm for 30 min, collect the centrifuged compound polysaccharide precipitate and freeze-dry it to obtain the product of the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis.

[0044] (7) Quality evaluation of the product of the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis

[0045] Weigh the compound polysaccharide of Danggui Buxue Decoction to calculate the product yield. Take the product of the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis, and determine its total sugar content by the phenol-sulfuric acid method; determine its sulfuric acid content by the barium chloride-gelatin colorimetric method; determine its uronic acid content by the sulfuric acid-meta-hydroxybiphenyl method; determine its molecular weight by high performance size exclusion chromatography. The results show that the yield of the product obtained in this example is 10.11±0.42%, the total sugar content is 88.32±0.48%; the sulfate group content is 3.49±0.25%, the uronic acid content is 6.99±0.39%, and the average molecular weight is 157.9 kDa.

[0046] Example 2

[0047] The experimental process is the same as that in Example 1:

[0048] (1) Pretreatment of medicinal materials

[0049] Weigh 5 parts of commercially available Chinese medicinal material Astragalus membranaceus and 1 part of commercially available Chinese medicinal material Angelica sinensis by mass, mix them and pulverize them with a pulverizer until they pass through an 80-mesh sieve to obtain the original medicinal material powder of Danggui Buxue Decoction.

[0050] (2) Preparation of anion exchange resin

[0051] Weigh 1.5 times the mass of the original medicinal material of Danggui Buxue Decoction of chloride-form 717 ion exchange resin by mass ratio.

[0052] (3) Co-mixing extraction of the original medicinal material of Danggui Buxue Decoction and anion exchange resin assisted by ultrasonic wave

[0053] Put the pretreated original medicinal material of Danggui Buxue Decoction and the prepared chloride-form 717 ion exchange resin into different 150-mesh filter bags respectively, tie the filter bag mouths tightly, place them in an extraction container, add 25 times the mass of deionized water of the original medicinal material of Danggui Buxue Decoction, and carry out ultrasonic-assisted extraction for 60 min under the conditions of an extraction temperature of 70 °C and an extraction power of 300 W.

[0054] (4) Collect and wash to remove the adhered residual medicinal residue powder on the resin

[0055] Take out the filter bag containing chloride-form 717 ion exchange resin in the co-mixing extraction, and rinse the filter bag with deionized water until there is no medicinal powder in the effluent of the filter bag, that is, obtain a clean resin filter bag adsorbed with the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis.

[0056] (5) Synchronously desorb and precipitate polysaccharides from the resin with sodium chloride ethanol solution

[0057] Keep the filter bag mouth facing upward, hang the clean resin filter bag adsorbed with the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis in the synchronous resolution and precipitation container, open the filter bag mouth, and under the conditions of a pressure of 2.5 MPa and a flow rate of 1.0 times the resin volume per minute, pump a 60% ethanol solution containing 0.6 mol / L sodium chloride into the filter bag with a ceramic high-pressure pump until no polysaccharide precipitation appears in the effluent of the filter bag.

[0058] (6) Formation of the product of the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis

[0059] Remove the resin filter bag, centrifuge the resolution and precipitation solution containing the compound polysaccharide at a rotational speed of 4500 rpm for 30 min, collect the precipitated compound polysaccharide after centrifugation and freeze-dry it to obtain the product of the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis.

[0060] (7) Quality evaluation of the product of the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis

[0061] Weigh the compound polysaccharide of Danggui Buxue Decoction to calculate the product yield. Take the product of the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis. Determine its total sugar content by the phenol-sulfuric acid method; determine its sulfuric acid content by the barium chloride-gelatin colorimetric method; determine its uronic acid content by the sulfuric acid-meta-hydroxybiphenyl method; determine its molecular weight by high performance size exclusion chromatography. The results show that the yield of the product obtained in this example is 11.44 ± 0.51%, the total sugar content is 86.03 ± 0.31%; the sulfate group content is 4.99 ± 0.18%, the uronic acid content is 8.14 ± 0.27%, and the average molecular weight is 162.7 kDa.

[0062] Example 3

[0063] The experimental process is the same as that in Example 1:

[0064] (1) Pretreatment of medicinal materials

[0065] Weigh 5 parts of commercially available Chinese medicinal material Astragalus membranaceus and 1 part of commercially available Chinese medicinal material Angelica sinensis by mass fraction, mix them and pulverize them with a pulverizer to pass through a 60-mesh sieve to obtain the original medicinal material powder of Danggui Buxue Decoction.

[0066] (2) Preparation of anion exchange resin

[0067] Weigh the chloride-form D301 ion exchange resin in an amount 1.0 times the mass of the original medicinal material of Danggui Buxue Decoction by mass ratio.

[0068] (3) Co-blending extraction of the original medicinal material of Danggui Buxue Decoction and anion exchange resin assisted by ultrasonic wave

[0069] The pretreated original medicinal materials of Danggui Buxue Decoction and the prepared chloride-form D301 ion exchange resin were respectively packed into different 120-mesh filter bags. The filter bag mouths were tied tightly and placed in an extraction container. Deionized water 20 times the mass of the original medicinal materials of Danggui Buxue Decoction was added, and ultrasonic-assisted extraction was carried out for 50 min under the conditions of an extraction temperature of 60 °C and an extraction power of 200 W.

[0070] (4) Collect and wash to remove the adhered residual medicinal dregs powder on the resin

[0071] Take out the filter bag containing the chloride-form D301 ion exchange resin in the co-mixed extraction, and rinse the filter bag with deionized water until there is no medicinal powder in the filtrate flowing out of the filter bag, thus obtaining a clean resin filter bag adsorbed with the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis.

[0072] (5) Synchronously desorb and precipitate polysaccharides from the resin with a sodium chloride ethanol solution

[0073] Keep the filter bag mouth facing upwards, hang the clean resin filter bag adsorbed with the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis in the synchronous desorption and precipitation container, open the filter bag mouth, and use a ceramic high-pressure pump to pump a 60% ethanol solution containing 0.5 mol / L sodium chloride into the filter bag under the conditions of a pressure of 2.0 MPa and a flow rate of 0.75 times the resin volume per minute until there is no polysaccharide precipitation in the filtrate flowing out of the filter bag.

[0074] (6) Formation of the product of the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis

[0075] Remove the resin filter bag, centrifuge the desorption and precipitation solution containing the compound polysaccharide at a rotation speed of 4500 rpm for 30 min, collect the compound polysaccharide precipitate after centrifugation and freeze-dry it to obtain the product of the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis.

[0076] (7) Quality evaluation of the product of the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis

[0077] Weigh the compound polysaccharide of Danggui Buxue Decoction to calculate the product yield. Take the product of the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis. Determine its total sugar content by the phenol-sulfuric acid method; determine its sulfuric acid content by the barium chloride-gelatin colorimetric method; determine its uronic acid content by the sulfuric acid-meta-hydroxybiphenyl method; determine its molecular weight by high performance size exclusion chromatography. The results show that the yield of the product obtained in this example is 10.52 ± 0.37%, the total sugar content is 87.11 ± 0.29%; the sulfate group content is 4.33 ± 0.21%, the uronic acid content is 7.71 ± 0.18%, and the average molecular weight is 159.4 kDa.

[0078] Example 4

[0079] The experimental process was the same as that in Example 1:

[0080] (1) Pretreatment of medicinal materials

[0081] Weigh 5 parts of commercially available Chinese medicinal material Astragalus membranaceus and 1 part of commercially available Chinese medicinal material Angelica sinensis by mass fraction. After mixing, pulverize them with a pulverizer until they pass through a 14-mesh sieve to obtain the original medicinal material powder of Danggui Buxue Decoction.

[0082] (2) Preparation of anion exchange resin

[0083] Weigh the hydroxide-form D301 ion exchange resin at 0.5 times the mass of the original medicinal materials of Danggui Buxue Decoction by mass ratio.

[0084] (3) Co-blending extraction of the original medicinal materials of Danggui Buxue Decoction and anion exchange resin assisted by ultrasonic

[0085] Put the pretreated original medicinal materials of Danggui Buxue Decoction and the prepared hydroxide-form D301 ion exchange resin into different 100-mesh filter bags respectively. Tie the filter bag mouths tightly, place them in an extraction container, add deionized water at 15 times the mass of the original medicinal materials of Danggui Buxue Decoction, and perform ultrasonic-assisted extraction for 40 min under the conditions of an extraction temperature of 50 °C and an extraction power of 100 W.

[0086] (4) Collect and wash to remove the adhered residual medicinal residue powder on the resin

[0087] Take out the filter bag containing the hydroxide-form D301 ion exchange resin in the co-blending extraction, and rinse the filter bag with deionized water until there is no medicinal powder in the effluent from the filter bag, thus obtaining a clean resin filter bag adsorbed with the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis.

[0088] (5) Synchronously desorb and precipitate polysaccharides from the resin with sodium chloride ethanol solution

[0089] Keep the filter bag mouth facing upward, hang the clean resin filter bag adsorbed with the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis in the synchronous desorption and precipitation container, open the filter bag mouth, and use a ceramic high-pressure pump to pump 0.1 mol / L 60% ethanol solution containing sodium chloride into the filter bag under the conditions of a pressure of 1.5 MPa and a flow rate of 0.5 times the resin volume per minute until there is no polysaccharide precipitation in the effluent from the filter bag.

[0090] (6) Formation of the product of the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis

[0091] Remove the resin filter bag, centrifuge the desorption and precipitation solution containing the compound polysaccharide at a rotation speed of 4500 rpm for 30 min, collect the centrifuged compound polysaccharide precipitate and freeze-dry it to obtain the product of the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis.

[0092] (7) Quality evaluation of the product of the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis

[0093] Weigh the compound polysaccharide of Danggui Buxue Decoction to calculate the product yield. Take the product of the compound polysaccharide of Danggui Buxue Decoction for preventing and treating atherosclerosis. Use the phenol-sulfuric acid method to determine its total sugar content; use the barium chloride-gelatin colorimetric method to determine its sulfuric acid content; use the sulfuric acid-meta-hydroxybiphenyl method to determine its uronic acid content; use high performance size exclusion chromatography to determine its molecular weight. The results show that the yield of the product obtained in this example is 6.43 ± 0.51%, the total sugar content is 75.44 ± 0.74%; the sulfate group content is 2.15 ± 0.18%, the uronic acid content is 5.87 ± 0.61%, and the average molecular weight is 171.4 kDa.

[0094] Comparative Example 1

[0095] (1) Preparation by the traditional process (comparative process) of the polysaccharide of Danggui Buxue Decoction

[0096] Weigh 5 parts of commercially available Chinese medicinal material Astragalus membranaceus (the same batch as in Example 1) and 1 part of commercially available Chinese medicinal material Angelica sinensis (the same batch as in Example 1) by mass. After mixing, pulverize them with a pulverizer until they pass through a 14-mesh sieve to obtain the original medicinal material powder of Danggui Buxue Decoction. Place the original medicinal material powder of Danggui Buxue Decoction in an extraction device, add deionized water with a mass 30 times that of the raw materials, reflux and decoct for 2 h, then filter. Repeat the extraction of the filter residue 2 times, collect and combine the 3 filtrates, concentrate under reduced pressure to a relative density of 1.25, add anhydrous ethanol with a volume 3 times that of the concentrated solution, let it stand for precipitation for 1 h, centrifuge to obtain the precipitate and freeze-dry it to obtain the crude polysaccharide of Danggui Buxue Decoction. Load 100 mg of the crude polysaccharide of Danggui Buxue Decoction onto a DEAE-52 cellulose ion exchange column with a bed volume of 188 mL and a diameter-height ratio of 1:7.5, control the flow rate at 0.3 times the bed volume / min, after fully eluting with 400 mL of deionized water, elute with 400 mL of 0.4 mol / L sodium chloride, monitor with the phenol-sulfuric acid method and collect the eluate containing the crude polysaccharide of Danggui Buxue Decoction, and concentrate under reduced pressure to a relative density of 1.5. After fully desalting with a dialysis bag with a molecular weight of 4500 Da, concentrate under reduced pressure to a relative density of 1.25, add anhydrous ethanol with a volume 3 times that of the concentrated solution, let it stand for precipitation for 1 h, centrifuge to obtain the precipitate and freeze-dry it to obtain the polysaccharide of Danggui Buxue Decoction.

[0097] (2) Comparison of the preparation of the polysaccharide of Danggui Buxue Decoction between the traditional process and Example 1

[0098] Compare the efficiency, consumption and product quality of the process of Example 1 and the traditional process of Comparative Example 1 for the polysaccharide of Danggui Buxue Decoction. The results are shown in Table 1.

[0099] Table 1 Comparison of the process of Example 1 and the traditional process for the polysaccharide of Danggui Buxue Decoction

[0100]

[0101] Note: The superscript letter b indicates that Example 1 compared with Comparative Example 1, P < 0.01.

[0102] As can be seen from the experimental results in Table 1, compared with the traditional comparative process, the process of the present invention not only has simple process steps, saves time and energy, but also the product yield, sulfate group content, and uronic acid content are significantly better than those of the traditional process, and the average molecular weight is also significantly lower than that of the control process.

[0103] Effect verification of Example 5

[0104] Eighty-four SPF-grade ApoE - / - mice were fed with ordinary feed in the animal quarantine room for 1 week for adaptation. After 1 week, if there was no abnormality, they were transferred to the breeding room for grouped intervention. Eighty-four ApoE - / - mice were randomly divided into 7 groups, namely blank control group, model group, Example 1 group, Example 2 group, Example 3 group, Example 4 group, and Comparative Example 1 group. There were 12 mice in each group, with 6 males and 6 females. The blank control group was fed with ordinary feed (basic feed), and the remaining groups were fed with high-fat feed (21% fat, 0.25% cholesterol, 1% choline, 77.75% basic feed), with free access to food and water. At the same time, the Example 1 group - Example 4 group and the Comparative Example 1 group were intragastrically administered with a physiological saline solution of different example products at 0.1 mL / 10 g at a dose of 200 mg / kg / d (dissolve each example product in physiological saline according to the set dosing dose). The blank control group and the model group were given the same volume of physiological saline. The intervention was continuous for 12 weeks. 24 hours after the last administration, the mice were anesthetized, and blood was collected by eye socket after enucleation, and then sacrificed by cervical dislocation. The thoracic aorta was quickly dissected.

[0105] The contents of total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) in the serum were analyzed using the corresponding kits. On this basis, the arteriosclerosis index (AI) was calculated according to the formula "arteriosclerosis index (AI) = (TC - HDL) ÷ HDL"; Oil Red O staining was used to measure and evaluate the plaque size of the thoracic aorta sections, and the ratio of plaque to lumen (P / L%) was calculated to evaluate the effect of the prepared Angelica sinensis decoction compound polysaccharide product for preventing and treating atherosclerosis on improving atherosclerotic lesions. The results are shown in Table 2.

[0106] Table 2 Effects of the prepared Angelica sinensis decoction compound polysaccharide on atherosclerotic lesions

[0107]

[0108] Note: The superscript letter a indicates P < 0.05 compared with the model group, and b indicates P < 0.01 compared with the model group.

[0109] The experimental results in Table 2 show that, compared with the blank control group, the TC, TG, and LDL of the mice in the model group were significantly elevated. However, HDL was significantly decreased, suggesting that the blood lipid metabolism level of the mice in the model group had undergone severe disorders. At the same time, further calculation of the AI value and evaluation of P / L% also found that the AI value and P / L% of the model group were significantly higher than those of the blank control group, and the AI value of the model group was also much greater than the diagnostic value of 4.0 for atherosclerosis, indicating that the mice in the model group had developed severe atherosclerotic lesions while experiencing lipid metabolism disorders. However, compared with the model group, after simultaneously administering the prepared Angelica Sinensis Decoction compound polysaccharide product for preventing and treating atherosclerosis to the groups of Examples 1-3, their TC, TG, LDL, AI value, and P / L% were significantly decreased; at the same time, HDL was significantly increased, and the AI values of the groups of Examples 1-3 were significantly lower than the diagnostic value of 4.0 for atherosclerosis. These results confirm that the Angelica Sinensis Decoction compound polysaccharide products prepared by the groups of Examples 1-3 have a significant effect on regulating blood lipids and preventing and treating atherosclerosis. At the same time, the experimental results also found that, compared with the model group, in the groups of Example 4 and Comparative Example 1, although the TC, TG, LDL, AI value, and P / L% were significantly decreased; at the same time, HDL was also significantly increased, but the degree of reversing the abnormal pathological indicators of the model group was significantly weaker than that of the group of Example 1, and their AI values were all greater than the diagnostic value of 4.0 for atherosclerosis. These results imply that although the groups of Example 4 and Comparative Example 1 can improve the inducing factors of atherosclerosis such as TC, TG, and LDL, they cannot completely prevent atherosclerosis, and the mice in the relevant groups are still in a severe atherosclerotic state. This result also proves that the process of the present invention is the best for the preparation of the Angelica Sinensis Decoction compound polysaccharide for preventing and treating atherosclerosis.

[0110] The above embodiments are only for illustrating the technical concept and characteristics of the present invention, and their purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it accordingly, and cannot be used to limit the protection scope of the present invention. All equivalent transformations or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A Danggui Buxue Decoction compound polysaccharide for preventing and treating atherosclerosis, characterized in that: The Danggui Buxue Decoction compound polysaccharide for preventing and treating atherosclerosis has a total sugar content of ≥85%, a sulfate content of ≥3%, a uronic acid content of ≥6%, and an average molecular weight of 150-165 kDa.

2. The method for preparing the Danggui Buxue Tang compound polysaccharide for preventing and treating atherosclerosis according to claim 1, characterized in that: The following steps are involved: (1) Under the assistance of ultrasound, the original medicinal material of Danggui Buxue Decoction and anion exchange resin are mixed and extracted in an extraction solvent; (2) washing the anion exchange resin to obtain an anion exchange resin containing the Danggui Buxue Decoction compound polysaccharide; (3) Using sodium chloride ethanol solution, the Danggui Buxue Decoction compound polysaccharide is simultaneously resolved and precipitated from the anion exchange resin containing the Danggui Buxue Decoction compound polysaccharide, centrifuged and lyophilized to obtain the polysaccharide.

3. The method for preparing the Danggui Buxue Decoction compound polysaccharide for preventing and treating atherosclerosis according to claim 2, characterized in that: In step (1), the original medicinal materials of Danggui Buxue Decoction include Astragalus and Angelica, the mass ratio of Astragalus to Angelica is 5:1, and the original medicinal materials of Danggui Buxue Decoction are crushed to pass through a 40-80 mesh sieve.

4. The method for preparing the Danggui Buxue Decoction compound polysaccharide for preventing and treating atherosclerosis according to claim 2, characterized in that: In step (1), the anion exchange resin is a chlorine-type D301 ion exchange resin or a chlorine-type 717 ion exchange resin, and the extraction solvent is deionized water.

5. The method for preparing the Danggui Buxue Decoction compound polysaccharide for preventing and treating atherosclerosis according to claim 2, characterized in that: In step (1), the mass ratio of the Danggui Buxue Decoction raw material, the anion exchange resin and the extraction solvent is 1:(0.5-1.5):(15-25).

6. The method for preparing the Danggui Buxue Decoction compound polysaccharide for preventing and treating atherosclerosis according to claim 2, characterized in that: In step (1), the ultrasonic power is 100-300 W, the extraction temperature is 50-70° C., and the extraction time is 40-60 min.

7. The method for preparing the Danggui Buxue Decoction compound polysaccharide for preventing and treating atherosclerosis according to claim 2, characterized in that: In step (1), during extraction, the original medicinal materials of Danggui Buxue Decoction and the anion exchange resin are respectively placed in different filter bags for ultrasonic extraction, and the pore size of the filter bag is 100-150 meshes.

8. The method for preparing the Danggui Buxue Decoction compound polysaccharide for preventing and treating atherosclerosis according to claim 6, characterized in that: In step (2), the method for washing the anion exchange resin comprises: taking out the filter bag containing the anion exchange resin in the blended extraction, and washing the filter bag with deionized water until the effluent of the filter bag is free of the original medicinal materials of Danggui Buxue Decoction.

9. The method for preparing the Danggui Buxue Decoction compound polysaccharide for preventing and treating atherosclerosis according to claim 7, characterized in that: In step (3), the sodium chloride ethanol solution is a 60% ethanol solution containing 0.4-0.6 mol / L sodium chloride. When the sodium chloride ethanol solution is used to simultaneously resolve and precipitate the Danggui Buxue Decoction compound polysaccharide from the anion exchange resin, the sodium chloride ethanol solution is pumped in with a ceramic high-pressure pump at a pressure of 1.5-2.5 MPa and a flow rate of 0.5-1.0 times the volume of the anion exchange resin / min. The centrifugal separation is performed at 4500 rpm for more than 30 min.

10. Use of the Danggui Buxue Decoction compound polysaccharide for preventing and treating atherosclerosis according to claim 1 in the preparation of a drug for preventing and treating atherosclerosis.

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