Preparation method and application of isodon amethystoides polysaccharide

Abstract

The invention provides a preparation method and application of isodon amethystoides polysaccharide. The invention provides an extraction and separation process of isodon amethystoides polysaccharide, high-content polysaccharide is obtained, and the polysaccharide has the functions of reducing blood fat, reducing body fat, local fat or fat-body ratio, preventing, treating, improving or relieving liver injury and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food processing, and particularly relates to a preparation method of Isodon amethystoides polysaccharide and application thereof. BACKGROUND

[0002] Isodon amethystoides (Benth) Cy Wu et Hsuan is a plant of Isodon in Labiatae, mainly grows in stone mountain shade in Huaibei area of Anhui, has the effects of antibacterial, anti-inflammatory and antitumor, and the main chemical components of Isodon amethystoides include diterpenoids, triterpenoids and flavonoids. Isodon amethystoides is commonly used for treating various diseases such as ulcer, tumor, snake bite, cold, prostatitis, acute infectious hepatitis and gynecological inflammation.

[0003] However, the method for separating polysaccharide from Isodon amethystoides and the physiological effects of Isodon amethystoides polysaccharide have not been reported. The deep processing method of Isodon amethystoides needs to be further developed, and the medicinal value or health value of Isodon amethystoides extract also needs to be further explored.

[0004] Plant polysaccharides are wrapped in tough plant cell walls, and it is difficult to extract polysaccharides completely by using conventional extraction methods (mechanical disruption, heat treatment, acid / alkali treatment, enzymatic hydrolysis). Plant often contains a large amount of impurities coexisting with polysaccharides, such as proteins, lipids, pigments, inorganic salts, monosaccharides / oligosaccharides, nucleic acids, small molecule metabolites, etc., which are difficult to remove completely, especially the proteins or pigments combined closely with polysaccharides, resulting in low purity of the final product. At the same time, the traditional separation methods (precipitation, dialysis, ion exchange, gel filtration) have limited resolution, small processing capacity and long time consumption. Therefore, it is necessary to efficiently and selectively destroy these physical barriers to release polysaccharides, while avoiding excessive damage leading to polysaccharide degradation or a large amount of impurities elution. Novel separation and purification methods can be selected to obtain high-purity plant polysaccharides.

[0005] The prevalence of metabolic diseases such as obesity, hyperlipidemia, fatty liver and the like is increasing. Hyperlipidemia refers to a condition in which one or more lipids in the blood is higher than normal due to abnormal metabolism or transport of lipids. Lipids are not soluble or slightly soluble in water and must be combined with proteins to exist in the form of lipoproteins. Therefore, hyperlipidemia is often hyperlipoproteinemia, which manifests as hypercholesterolemia, hypertriglyceridemia, or both. The incidence of hyperlipidemia is an important public health problem, and its incidence has been increasing at an alarming rate. It can significantly increase the risk of developing cardiovascular diseases such as atherosclerosis, which is one of the most common causes of morbidity and mortality worldwide. The use of lipid-lowering drugs such as statins is the most common and widely used method for treating hyperlipidemia at present. However, due to the high price and side effects of these drugs, people have begun to research and develop more effective and safer alternatives or adjuncts for lowering blood lipids. SUMMARY

[0006] To solve at least one of the above problems, the present disclosure provides a preparation method of a polysaccharide from Crataegus pinnatifida Bunge and applications thereof.

[0007] According to one aspect of the present disclosure, a preparation method of a polysaccharide from Crataegus pinnatifida Bunge is provided, comprising the following steps:

[0008] The present disclosure provides a preparation method of a polysaccharide from Crataegus pinnatifida Bunge, comprising the following steps:

[0009] 1) mixing dry powder obtained by crushing Crataegus pinnatifida Bunge leaves with water, and extracting using an ultrasonic extraction method to obtain an extract;

[0010] 2) mixing the extract with anhydrous ethanol after concentration, and centrifuging after standing to obtain a precipitate;

[0011] 3) dissolving the precipitate with water and filtering through a ceramic membrane to collect the permeate;

[0012] 4) filtering the permeate through an organic membrane, collecting the liquid, and drying to obtain a polysaccharide from Crataegus pinnatifida Bunge.

[0013] In some embodiments, the fresh Crataegus pinnatifida Bunge leaves are dried before crushing in step 1) to obtain dried Crataegus pinnatifida Bunge leaves.

[0014] In some embodiments, the drying is drying to a constant weight.

[0015] As understood by those skilled in the art, any known crushing method can be used for crushing, and non-limiting examples of crushing include mechanical impact crushing, air flow crushing, ball milling or vibration milling crushing, high pressure crushing, low temperature crushing, ultra-micro crushing, nano crushing, etc., all of which are routine choices for those skilled in the art.

[0016] In some embodiments, the dry powder after the pulverization in step 1) has a particle size of 5-100 μm, preferably 5-50 μm, more preferably 5-10 μm.

[0017] In some embodiments, the dry powder after the pulverization in step 1) has a particle size of 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm, 30 μm, 31 μm, 32 μm, 33 μm, 34 μm, 35 μm, 36 μm, 37 μm, 38 μm, 39 μm, 40 μm, 41 μm, 42 μm, 43 μm, 44 μm, 45 μm, 46 μm, 47 μm, 48 μm, 49 μm, 50 μm, 51 μm, 52 μm, 53 μm, 54 μm, 55 μm, 56 μm, 57 μm, 58 μm, 59 μm, 60 μm, 61 μm, 62 μm, 63 μm, 64 μm, 65 μm, 66 μm, 67 μm, 68 μm, 69 μm, 70 μm, 71 μm, 72 μm, 73 μm, 74 μm, 75 μm, 76 μm, 77 μm, 78 μm, 79 μm, 80 μm, 81 μm, 82 μm, 83 μm, 84 μm, 85 μm, 86 μm, 87 μm, 88 μm, 89 μm, 90 μm, 91 μm, 92 μm, 93 μm, 94 μm, 95 μm, 96 μm, 97 μm, 98 μm, 99 μm, or 100 μm.

[0018] In some embodiments, the mass to volume ratio of the dry powder after the pulverization in step 1) and water is 1 g : (5-200) mL, preferably 1 g : (10-200) mL, more preferably 1 g : (125-200) mL, most preferably 1 g : 150 mL.

[0019] In some embodiments, the mass to volume ratio of the dry powder after pulverization in step 1) to water is 1 g : (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200) mL,

[0020] In some embodiments, the ultrasonic power of the ultrasonic extraction method in step 1) is 200-1500 W, preferably 900-1500 W, most preferably 900 W.

[0021] In some embodiments, the ultrasonic power of the ultrasonic extraction method in step 1) is 200 W, 300 W, 400 W, 500 W, 600 W, 700 W, 800 W, 900 W, 1000 W, 1100 W, 1200 W, 1300 W, 1400 W, or 1500 W.

[0022] In some embodiments, the ultrasonic extraction method in step 1) has an ultrasonic time of 30 min to 120 min, preferably 45 min to 105 min, most preferably 45 min.

[0023] In some embodiments, the ultrasonic extraction method in step 1) has an ultrasonic time of 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min, 60 min, 61 min, 62 min, 63 min, 64 min, 65 min, 66 min, 67 min, 68 min, 69 min, 70 min, 71 min, 72 min, 73 min, 74 min, 75 min, 76 min, 77 min, 78 min, 79 min, 80 min, 81 min, 82 min, 83 min, 84 min, 85 min, 86 min, 87 min, 88 min, 89 min, 90 min, 91 min, 92 min, 93 min, 94 min, 95 min, 96 min, 97 min, 98 min, 99 min, 100 min, 101 min, 102 min, 103 min, 104 min, 105 min, 106 min, 107 min, 108 min, 109 min, 110 min, 111 min, 112 min, 113 min, 114 min, 115 min, 116 min, 117 min, 118 min, 119 min, or 120 min.

[0024] In some embodiments, the ultrasonic extraction method in step 1) has an extraction temperature of 30 to 60 °C, preferably 45 to 60 °C, most preferably 45 °C.

[0025] In some embodiments, the ultrasonic extraction method in step 1) has an extraction temperature of 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 51 °C, 52 °C, 53 °C, 54 °C, 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, or 60 °C.

[0026] In some embodiments, the ultrasonic power in the ultrasonic extraction method in step 1) is 200-1500 W, the ultrasonic time is 30 min-120 min, and the extraction temperature is 30-60 °C.

[0027] In some embodiments, the ultrasonic power in the ultrasonic extraction method in step 1) is 900-1500 W, the ultrasonic time is 45 min-120 min, and the extraction temperature is 45-60 °C.

[0028] In some embodiments, the ultrasonic power in the ultrasonic extraction method in step 1) is 900 W, the ultrasonic time is 45 min, and the extraction temperature is 45 °C.

[0029] In some embodiments, the concentrating in step 2) comprises concentrating to a dehydrated extract.

[0030] As understood by those skilled in the art, any known concentrating method can be used for the concentrating, non-limiting examples of which include evaporation concentration, freeze concentration, membrane separation concentration, adsorption concentration, extraction concentration, electrodialysis concentration, centrifugal concentration, etc., wherein the evaporation concentration includes atmospheric evaporation concentration, reduced pressure evaporation concentration (referred to as reduced pressure concentration) and thin film evaporation concentration, etc., all of which are routine choices for those skilled in the art.

[0031] In some embodiments, the volume ratio of the extract and anhydrous ethanol in step 2) is 1:(3-10), preferably 1:(3-5).

[0032] In some embodiments, the volume ratio of the extract and anhydrous ethanol in step 2) is 1:(3, 4, 5, 6, 7, 8, 9 or 10).

[0033] In some embodiments, the standing time in step 2) is 12-48 h, for example 24 h.

[0034] In some embodiments, the standing time in step 2) is 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h, 41 h, 42 h, 43 h, 44 h, 45 h, 46 h, 47 or 48 h.

[0035] In some embodiments, the centrifugal time in step 2) is 5-30 min, preferably 10-20 min, more preferably 15 min.

[0036] In some embodiments, the centrifugation time in step 2) is 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, or 30 min.

[0037] In some embodiments, the centrifugation speed in step 2) is 10000-20000 rpm.

[0038] In some embodiments, the centrifugation speed in step 2) is 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, or 20000 rpm.

[0039] In some embodiments, the mass ratio of the precipitate to water in step 3) is 1:(1-20), preferably 1:(1-10), more preferably 1:(1-5).

[0040] In some embodiments, the mass ratio of the precipitate to water in step 3) is 1:(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).

[0041] In some embodiments, the pore size of the ceramic membrane in step 3) is 0.1-1 μm, preferably 0.2-0.5 μm, most preferably 0.2 μm.

[0042] In some embodiments, the pore size of the ceramic membrane in step 3) is 0.1, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, or 0.1 μm.

[0043] In some embodiments, the flow rate of the ceramic membrane in step 3) is 50-200 mL / min, preferably 150-200 mL / min, most preferably 150 mL / min.

[0044] 3) The flow rate of the ceramic membrane is 50mL / min, 51mL / min, 52mL / min, 53mL / min, 54mL / min, 55mL / min, 56mL / min, 57mL / min, 58mL / min, 59mL / min, 60mL / min, 61mL / min, 62mL / min, 63mL / min, 64mL / min, 65mL / min, 66mL / min, 67mL / min, 68mL / min, 69mL / min, 70mL / min, 71mL / min, 72mL / min, 73mL / min, 74mL / mi n, 75mL / min, 76mL / min, 77mL / min, 78mL / min, 79mL / min, 80mL / min, 81mL / min, 82mL / min, 83mL / min, 84mL / min, 85mL / min, 86mL / min, 87mL / min, 88mL / min, 89mL / min, 90mL / min, 91mL / min, 92mL / min, 93mL / min, 94mL / min, 95mL / min, 96mL / min, 97mL / min, 98mL / min, 99mL / min, 100mL / min, 101mL / min, 10 2mL / min, 103mL / min, 104mL / min, 105mL / min, 106mL / min, 107mL / min, 108mL / min, 109mL / min, 110mL / min, 111mL / min, 112mL / min, 113mL / min, 114mL / min, 115mL / min, 116mL / min, 117mL / min, 118mL / min, 119mL / min, 120mL / min, 121mL / min, 122mL / min, 123mL / min, 124mL / min, 125mL / min, 126mL / min, 1 27mL / min, 128mL / min, 129mL / min, 130mL / min, 131mL / min, 132mL / min, 133mL / min, 134mL / min, 135mL / min, 136mL / min, 137mL / min, 138mL / min, 139mL / min, 140mL / min, 141mL / min, 142mL / min, 143mL / min, 144mL / min, 145mL / min, 146mL / min, 147mL / min, 148mL / min, 149mL / min, 150mL / min, 151mL / min,152 mL / min, 153 mL / min, 154 mL / min, 155 mL / min, 156 mL / min, 157 mL / min, 158 mL / min, 159 mL / min, 160 mL / min, 161 mL / min, 162 mL / min, 163 mL / min, 164 mL / min, 165 mL / min, 166 mL / min, 167 mL / min, 168 mL / min, 169 mL / min, 170 mL / min, 171 mL / min, 172 mL / min, 173 mL / min, 174 mL / min, 175 mL / min, 176 mL / min, 177 mL / min, 178 mL / min, 179 mL / min, 180 mL / min, 181 mL / min, 182 mL / min, 183 mL / min, 184 mL / min, 185 mL / min, 186 mL / min, 187 mL / min, 188 mL / min, 189 mL / min, 190 mL / min, 191 mL / min, 192 mL / min, 193 mL / min, 194 mL / min, 195 mL / min, 196 mL / min, 197 mL / min, 198 mL / min, 199 mL / min, or 200 mL / min.

[0045] In some embodiments, the pore size of the ceramic membrane in step 3) is 0.1-1 μm and the flow rate is 50-200 mL / min.

[0046] In some embodiments, the pore size of the ceramic membrane in step 3) is 0.2-0.5 μm and the flow rate is 150-200 mL / min.

[0047] In some embodiments, the pore size of the ceramic membrane in step 3) is 0.1 μm, 0.2 μm or 0.5 μm and the flow rate is 150 mL / min.

[0048] In some embodiments, the pore size of the ceramic membrane in step 3) is 0.2 μm and the flow rate is 150 mL / min.

[0049] In some embodiments, the rejection capacity of the organic membrane in step 4) is 5000-50000 Dalton, preferably 10000-30000 Dalton, most preferably 10000 Dalton.

[0050] In some embodiments, the organic membrane in step 4) has a rejection capacity of 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, 11000, 11500, 12000, 12500, 13000, 13500, 14000, 14500, 15000, 15500, 16000, 16500, 17000, 17500, 18000, 18500, 19000, 19500, 20000, 20500, 21000, 21500, 22000, 22500, 23000, 23500, 24000, 24500, 25000, 25500, 26000, 26500, 27000, 27500, 28000, 28500, 29000, 29500, 30000, 30500, 31000, 31500, 32000, 32500, 33000, 33500, 34000, 34500, 35000, 35500, 36000, 36500, 37000, 37500, 38000, 38500, 39000, 39500, 40000, 40500, 41000, 41500, 42000, 42500, 43000, 43500, 44000, 44500, 45000, 45500, 46000, 46500, 47000, 47500, 48000, 48500, 49000, 49500, or 50000 Daltons.

[0051] In some embodiments, the organic membrane in step 4) has a flow rate of 50 mL / min to 250 mL / min, preferably 100 to 150 mL / min, most preferably 100 mL / min.

[0052] In some embodiments, the organic membrane in step 4) has a flow rate of 50 mL / min, 60 mL / min, 70 mL / min, 80 mL / min, 90 mL / min, 100 mL / min, 110 mL / min, 120 mL / min, 130 mL / min, 140 mL / min, 150 mL / min, 160 mL / min, 170 mL / min, 180 mL / min, 190 mL / min, 200 mL / min, 210 mL / min, 220 mL / min, 230 mL / min, 240 mL / min, or 250 mL / min.

[0053] In some embodiments, the organic membrane in step 4) has a retention capacity of 5000-50000 daltons and a flow rate of 50-250 mL / min.

[0054] In some embodiments, the organic membrane in step 4) has a retention capacity of 10000-30000 daltons and a flow rate of 100-250 mL / min.

[0055] In some embodiments, the organic membrane in step 4) has a retention capacity of 10000 daltons and a flow rate of 100 mL / min.

[0056] In some embodiments, the drying includes, but is not limited to, freeze-drying, hot air drying, spray drying, steam drying, vacuum drying, microwave drying, or vacuum microwave drying, etc.

[0057] In some embodiments, the cold trap temperature of the freeze-drying is -60 to -50°C, more preferably -55°C.

[0058] In some embodiments, the cold trap temperature of the freeze-drying is -60°C, -59°C, -58°C, -57°C, -56°C, -55°C, -54°C, -53°C, -52°C, -51°C, or -50°C,

[0059] In some embodiments, the vacuum degree is 0.1-1 Mpa, more preferably 0.2 Mpa.

[0060] In some embodiments, the vacuum degree is 0.1 Mpa, 0.2 Mpa, 0.3 Mpa, 0.4 Mpa, 0.5 Mpa, 0.6 Mpa, 0.7 Mpa, 0.8 Mpa, 0.9 Mpa, or 1 Mpa.

[0061] In some embodiments, the temperature of the hot air drying is 40-60°C, preferably 50°C.

[0062] In some embodiments, the temperature of the hot air drying is 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, or 60°C.

[0063] In some embodiments, the temperature of the vacuum microwave drying is 40-50°C, preferably 45°C.

[0064] In some embodiments, the temperature of the vacuum microwave drying is 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, or 50°C.

[0065] In some embodiments, the microwave power is 800-1200 W, preferably 1000 W.

[0066] In some embodiments, the microwave power is 800 W, 900 W, 1000 W, 1100 W or 1200 W.

[0067] In some embodiments, the method comprises the following steps:

[0068] 1) Dry powder of crushed Paliurus ramosissimus leaf with a particle size of 5-100 μm is mixed with water in a ratio of 1 g:(5-200) mL, and extracted using ultrasonic extraction method to obtain an extract, the ultrasonic power being 200-1500 W, the ultrasonic time being 30-120 min, and the extraction temperature being 30-60°C;

[0069] 2) The extract is concentrated and dehydrated to a extractum, mixed with anhydrous ethanol in a volume ratio of 1:(3-10), and centrifuged for 5-30 min at a speed of 10000-20000 rpm after standing for 12-48 h to obtain a precipitate;

[0070] 3) The precipitate is dissolved with water in a ratio of precipitate to water of 1:(1-20) by mass, filtered through a ceramic membrane with a pore size of 0.1-1 μm at a flow rate of 50-200 mL / min, and the permeate is collected;

[0071] 4) The permeate is filtered using an organic membrane with a cutoff capacity of 5000-50000 daltons at a flow rate of 50-250 mL / min, and the liquid is collected and dried to obtain Paliurus ramosissimus polysaccharide.

[0072] In some embodiments, the method comprises the following steps:

[0073] 1) Dry powder of crushed Paliurus ramosissimus leaf with a particle size of 5-50 μm is mixed with water in a ratio of 1 g:(10-200) mL, and extracted using ultrasonic extraction method to obtain an extract, the ultrasonic power being 900-1500 W, the ultrasonic time being 45-105 min, and the extraction temperature being 45-60°C;

[0074] 2) The extract is concentrated and dehydrated to a extractum, mixed with anhydrous ethanol in a volume ratio of 1:(3-5), and centrifuged for 10-20 min at a speed of 10000-20000 rpm after standing for 12-48 h to obtain a precipitate;

[0075] 3) dissolving the precipitate with water in a ratio of precipitate to water by mass of 1:(1-10), and filtering through a ceramic membrane with a pore size of 0.2-0.5, at a flow rate of 150-200 mL / min, and collecting the permeate;

[0076] 4) filtering the permeate through an organic membrane with a cutoff of 10,000-30,000 daltons, at a flow rate of 100-150 mL / min, and collecting the liquid, and drying to obtain the polysaccharide from Fructus Psidii.

[0077] In some embodiments, the method comprises the following steps:

[0078] 1) mixing dry powder of Fructus Psidii leaves with a particle size of 10 μm with water in a ratio of 1 g:150 mL, and extracting using ultrasonic extraction, to obtain an extract, at an ultrasonic power of 900 W, for 45 min, and at an extraction temperature of 45°C;

[0079] 2) mixing the extract with anhydrous ethanol in a ratio of 1:5 by volume, and centrifuging for 15 min at a speed of 10,000-20,000 rpm after standing for 24 h, to obtain a precipitate;

[0080] 3) dissolving the precipitate with water in a ratio of precipitate to water by mass of 1:1 or 1:2, and filtering through a 0.2 μm ceramic membrane, at a flow rate of 150 mL / min, and collecting the permeate;

[0081] 4) filtering the permeate through an organic membrane with a cutoff of 10,000 daltons, at a flow rate of 100 mL / min, and collecting the liquid, and drying to obtain the polysaccharide from Fructus Psidii.

[0082] According to yet another aspect of the present disclosure, there is provided a polysaccharide from Fructus Psidii prepared according to the method.

[0083] According to yet another aspect of the present disclosure, there is provided a medicament comprising the polysaccharide from Fructus Psidii.

[0084] According to yet another aspect of the present disclosure, there is provided use of the preparation method or the polysaccharide from Fructus Psidii in the preparation of a medicament for preventing, treating, ameliorating, controlling or alleviating hyperlipidemia, dyslipidemia, obesity, and / or improving lipid metabolism.

[0085] According to yet another aspect of the present disclosure, there is provided use of the preparation method or the polysaccharide from Fructus Psidii in preventing, treating, ameliorating, controlling or alleviating hyperlipidemia, dyslipidemia, obesity, and / or improving lipid metabolism.

[0086] In some embodiments, the use comprises one or more of the following:

[0087] (1) weight loss or weight control;

[0088] (2) prevention, treatment, improvement, control or alleviation of hyperlipidemia;

[0089] (3) improvement of lipid metabolism;

[0090] (4) control or reduction of triglyceride, total cholesterol and low-density lipoprotein cholesterol levels;

[0091] (5) increase of high-density lipoprotein cholesterol levels;

[0092] (6) control or reduction of body fat, local fat or fat body ratio;

[0093] (7) prevention, treatment, improvement, control or alleviation of liver function impairment;

[0094] (8) prevention, treatment, improvement, control or alleviation of atherosclerosis.

[0095] In some embodiments, the liver function impairment includes at least one of fatty liver, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, liver cirrhosis, liver cancer or liver cell damage.

[0096] In some embodiments, the cause of the liver function impairment includes, but is not limited to, at least one of high-fat diet, high-cholesterol diet, hyperlipidemia or high cholesterol.

[0097] In some embodiments, the liver function impairment includes high-fat diet-induced, high-cholesterol diet-induced and / or high-fat high-cholesterol-induced.

[0098] According to yet another aspect of the present disclosure, there is provided use of the preparation method or the polysaccharide of Fructus Crataegi in the preparation of health products for helping to maintain a healthy level of blood lipids.

[0099] According to yet another aspect of the present disclosure, there is provided use of the preparation method or the polysaccharide of Fructus Crataegi in maintaining a healthy level of blood lipids.

[0100] Beneficial effects:

[0101] Currently, there is little research on polysaccharides of Fructus Crataegi. The present disclosure provides an extraction and separation process of polysaccharides of Fructus Crataegi, and a high-content polysaccharide is obtained. The polysaccharide has the functions of lowering blood lipids, reducing body fat, local fat or fat body ratio, preventing, treating, improving or alleviating liver damage, etc. BRIEF DESCRIPTION OF DRAWINGS

[0102] Figure 1 A Fourier transform infrared spectrum of the polysaccharide of Fructus Crataegi in Example 4 is shown.

[0103] Figure 2 The blood lipid test results after gavage administration in Example 6 are shown, including triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). The ordinate unit is mmol / L.

[0104] Figure 3 The tissue section observation of mouse liver after gavage administration in Example 6 is shown. DETAILED DESCRIPTION

[0105] DEFINITIONS

[0106] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following definitions are applied, for the purposes of interpreting this description, and shall have the meanings set forth below. Additionally, unless otherwise clear from context, singular articles, including "a," "an," and "the," are intended to include the plural forms of the same.

[0107] As used herein, the terms "a" and "one" include plural references unless the context clearly indicates otherwise.

[0108] The expression "about" as used herein is as understood by one of ordinary skill in the art and varies in its scope depending on the context in which it is used. If one of ordinary skill in the art is not reasonably aware of the context in which the term is used, "about" will mean up to plus or minus 10% of the particular value.

[0109] The term "nonalcoholic fatty liver disease (NAFLD)" used in the present disclosure is a liver disease characterized by hepatocyte lipid deposition induced by glucose and lipid metabolism disorders, which has become the most common chronic liver disease worldwide. NAFLD includes simple liver steatosis (NAFL) and nonalcoholic steato-hepatitis (NASH). NASH is a key stage of the progression of NAFLD and is also closely related to the high incidence of metabolic diseases such as hyperlipidemia and arteriosclerosis.

[0110] The term "polysaccharide" as used in the present disclosure means a polymeric carbohydrate molecule composed of long chains of monosaccharide units joined together through glycosidic bonds and which upon hydrolysis yields constituent monosaccharides or oligosaccharides. Included are glycan or oligosaccharides, as well as glycoconjugate carbohydrate moieties. Oligosaccharides are carbohydrate polymers containing a small number (typically 3-10) of saccharide components (monosaccharides). Polysaccharides are generally composed of monosaccharides linked through O- or N-glycosidic bonds to the side chains of amino acids or lipid moieties in appropriate proteins. The glycoconjugates include, but are not limited to, glycoproteins, glycolipids, proteoglycans, and glycophosphosphingolipids, or other known or unknown glycoconjugates.

[0111] The term "liver coefficient" as used in the present disclosure refers to the ratio of the liver weight of an animal to its body weight, which is a commonly used index in pharmacology. The greater the liver coefficient, the more serious the edema and hyperplasia of the liver of the body.

[0112] The term "fat body ratio" as used in the present disclosure refers to the ratio of fat weight to body weight. The greater the ratio, the more fat content and the more obese the body. In some embodiments, the fat body ratio can be calculated using the ratio of perirenal fat content to body weight.

[0113] To make the objectives, technical solutions and advantages of the present application clearer, further detailed description of the present application is made below in combination with examples. The specific examples described herein are only used to explain the present application and do not constitute any limitation to the present application. The actual protection scope of the present application is set forth in the claims. In the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications. The equipment, instruments, reagents and / or kits used in the following examples are not mentioned for their sources, and are commercially available or obtained by conventional methods known to those skilled in the art.

[0114] Examples

[0115] Polysaccharide content determination method:

[0116] Preparation of standard stock solution: accurately weigh the glucose standard (molecular weight 180, purity greater than 95%, Shanghai Yuan Ye Biological Technology Co., Ltd.) dried to constant weight at 105°C into a 100 mL volumetric flask, add water to dissolve, dilute to the mark, and shake well for use.

[0117] Preparation of standard curve: precisely pipette 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL, 3.0 mL, 3.5 mL, 4.0 mL of the above standard stock solution into a 50 mL volumetric flask, add water to the mark, shake well, as the standard solution, respectively, precisely pipette 2.0 mL of the standard solution into a test tube, add 1 mL of 5% phenol solution, mix well, quickly add 7.0 mL of concentrated sulfuric acid, shake well. Incubate in a 40°C water bath for 30 min, then place in an ice water bath for 5 min, take out. The first one is blank. Measure the absorbance at 490 nm wavelength. The standard curve is prepared with the absorbance as the ordinate and the concentration (mg / mL) as the abscissa.

[0118] Preparation of sample solution: accurately weigh 0.1 g of the sample to be tested into a 50 mL core-shaped flask, add nearly 30 mL of water, heat reflux for 2 hours, filter, and dilute to 50 mL. Shake well, then take 1 mL of the solution into a 10 mL volumetric flask, add water to the mark, mix well, as the test solution.

[0119] Determination of sample solution: accurately pipette 2.0 mL of the prepared test solution into a test tube, then find the "Preparation of standard curve" item from "add 1 mL of 5% phenol solution" operation, measure the absorbance at 490 nm wavelength, detect the polysaccharide concentration (mg / mL) by external standard method, and calculate the polysaccharide content.

[0120] Polysaccharide content = C x V x dilution factor / m,

[0121] Where C is the concentration value (mg / mL) calculated according to the standard curve, V is the constant volume (50 mL), m is the sample weight (0.1 g), and the dilution factor is 10.

[0122] Example 1 Extraction method 1 of polysaccharides from Wangzaozi

[0123] Method: (1) Dry the fresh Wangzaozi leaves to constant weight to obtain dried Wangzaozi leaves;

[0124] (2) The dried Wangzaozi leaves are pulverized into dry powder with a particle size of 5 μm by using a super micro grinder;

[0125] (3) Add water to the dry powder according to the liquid-solid ratio of 10 mL: 1 g, then extract by using ultrasonic extraction method, the power of the ultrasonic extraction method is 200 W, the temperature of the ultrasonic extraction method is 30°C, and the time of the ultrasonic extraction method is 30 min, to obtain the extraction liquid;

[0126] (4) The extraction liquid is concentrated under reduced pressure (50-90°C, vacuum pressure 0.07 MPa) to dehydrate into extract, to obtain the concentrated liquid;

[0127] (5) The concentrated solution is added with 3 times volume of anhydrous ethanol to precipitate, and after 24 hours of static state, centrifugation is performed for 10 minutes at 10000 rpm, and the precipitate A is collected, to obtain the crude polysaccharide of the leaves of Evonymus alata Benth;

[0128] (6) The crude polysaccharide of the leaves of Evonymus alata Benth is dissolved in 1 times mass of pure water, and then ceramic membrane (Hefei Shijie Membrane Equipment) is used for impurity removal, with a pore size of 0.1 μm and a flow rate of 50 mL / min, and the permeate is collected.

[0129] (7) The permeate is subjected to organic membrane filtration, with an organic membrane (Hefei Shijie Membrane Equipment) having a pore size of 10000 Dalton and a flow rate of 50 mL / min, and the permeate is collected.

[0130] (8) The permeate is subjected to freeze-drying, with a cold trap temperature of -55℃ and a vacuum degree of 0.2 Mpa, and after drying, the high-content polysaccharide is obtained.

[0131] (9) The mass of the high-content polysaccharide obtained in step (8) is weighed, and the ratio of the mass to the mass of the fresh leaves of Evonymus alata Benth in step (1) is calculated as the yield, which is 25%, the polysaccharide concentration is determined, and the polysaccharide content is calculated as 78%.

[0132] Example 2: Extraction method 2 of the polysaccharide of the leaves of Evonymus alata Benth

[0133] Method: (1) The fresh leaves of Evonymus alata Benth are dried to constant weight to obtain dried leaves of Evonymus alata Benth;

[0134] (2) The dried leaves of Evonymus alata Benth are pulverized into dry powder with a particle size of 10 μm by using a super micro pulverizer;

[0135] (3) Water is added to the dry powder at a liquid-solid ratio of 30 mL:1 g, and then ultrasonic extraction is performed, with a power of 400 W, a temperature of 40℃, and a time of 90 min, to obtain an extraction solution;

[0136] (4) The extraction solution is subjected to vacuum concentration (50-90℃, vacuum pressure 0.07 MPa) to dehydrate into extract residue, to obtain a concentrated solution;

[0137] (5) The concentrated solution is added with 4 times volume of anhydrous ethanol to precipitate, and after 24 hours of static state, centrifugation is performed for 20 minutes at 20000 rpm, and the precipitate A is collected, to obtain the crude polysaccharide of the leaves of Evonymus alata Benth;

[0138] (6) The crude polysaccharide of the leaves of Evonymus alata Benth is dissolved in 2 times mass of pure water, and then ceramic membrane is used for impurity removal, with a pore size of 0.2 μm and a flow rate of 200 mL / min, and the permeate is collected.

[0139] (7) The permeate is filtered by organic membrane, the pore size of the organic membrane is 50000 Dalton, and the flow rate is 100 mL / min to collect the permeate.

[0140] (8) The permeate is dried by hot air, and the drying temperature is 50℃, and the high content polysaccharide is obtained after drying.

[0141] (9) The yield is 23% and the polysaccharide content is 82% according to the method of Example 1.

[0142] Example 3 Extraction method 3 of Wangzaozi polysaccharide

[0143] Method: (1) The fresh Wangzaozi leaves are dried to constant weight to obtain dried Wangzaozi leaves;

[0144] (2) The dried Wangzaozi leaves are crushed into dry powder with a particle size of 100 μm by using a super micro grinder;

[0145] (3) The dry powder is added with water at a liquid-solid ratio of 200 mL: 1 g, and then extracted by ultrasonic extraction method, the power of the ultrasonic extraction method is 1000 W, the temperature of the ultrasonic extraction method is 50℃, and the time of the ultrasonic extraction method is 120 min to obtain an extraction solution;

[0146] (4) The extraction solution is concentrated under reduced pressure (50-90℃, vacuum pressure 0.07 MPa) and dehydrated to extract paste to obtain a concentrated solution;

[0147] (5) The concentrated solution is added with anhydrous ethanol with a volume of 4 times that of the concentrated solution for precipitation, and after standing for 24 h, centrifugation is performed at a speed of 20000 r / min for 15 min, and the precipitate A is collected to obtain Wangzaozi crude polysaccharide;

[0148] (6) The Wangzaozi crude polysaccharide is dissolved in pure water with a mass of 1 times that of the Wangzaozi crude polysaccharide, and then ceramic membrane is used for impurity removal, the pore size of the ceramic membrane is 0.5 μm, and the flow rate is 100 mL / min to collect the permeate.

[0149] (7) The permeate is filtered by organic membrane, the pore size of the organic membrane is 20000 Dalton, and the flow rate is 150 mL / min to collect the permeate.

[0150] (8) The permeate is dried by vacuum microwave, the drying temperature is 45℃, and the microwave power is 1000 W, and the high content polysaccharide is obtained after drying.

[0151] (9) The yield is 22% and the polysaccharide content is 78% according to the method of Example 1.

[0152] Comparative Example 1

[0153] A method similar to example 1 was used to prepare the polysaccharide from the fruit of the plant Malus hupehensis, except that step 1 was not performed, and the polysaccharide was obtained by hot reflux extraction at a temperature of 80°C and a solid-to-liquid ratio of 1:20. The yield was 19% and the polysaccharide content was 46%.

[0154] Comparative Example 2

[0155] A method similar to example 1 was used to prepare the polysaccharide from the fruit of the plant Malus hupehensis, except that step 1 was not performed, and the polysaccharide was obtained by ceramic membrane purification. The yield was 22% and the polysaccharide content was 38%.

[0156] Comparative Example 3

[0157] A method similar to example 1 was used to prepare the polysaccharide from the fruit of the plant Malus hupehensis, except that step 1 was not performed, and the polysaccharide was obtained by organic membrane purification. The yield was 23% and the polysaccharide content was 45%.

[0158] Comparative Example 4

[0159] Method: (1) Fresh leaves of the plant Malus hupehensis were dried to a constant weight to obtain dried leaves of the plant Malus hupehensis;

[0160] (2) The dried leaves of the plant Malus hupehensis were ground to a powder having a particle size of 10 μm using a super-micro grinder;

[0161] (3) Water was added to the powder at a liquid-to-solid ratio of 10 mL:1 g, and then the mixture was extracted using an ultrasonic extraction method at a power of 900 W, a temperature of 45°C, and for a time of 45 min to obtain an extract;

[0162] (4) The extract was then purified using a ceramic membrane having a pore size of 0.2 μm at a flow rate of 1500 mL / min to collect a permeate;

[0163] (5) The extract was concentrated under reduced pressure (50-90°C, vacuum pressure 0.07 MPa) and then dehydrated to obtain a concentrated extract; the volume ratio of the concentrated extract to the extract was 1:6;

[0164] (6) The concentrated extract was precipitated by adding anhydrous ethanol to a volume of 3 times that of the concentrated extract, and then the precipitate was collected by centrifugation at a speed of 20000 rpm for 15 min after standing for 24 h;

[0165] (7) The precipitate was dissolved in water at a volume of 1 times that of the precipitate, and then the solution was filtered using an organic membrane having a molecular weight cut-off of 10000 Da at a flow rate of 100 mL / min to obtain a filtrate;

[0166] (8) The filtrate was concentrated and then freeze-dried at a cold trap temperature of -55°C and a vacuum degree of 0.2 MPa to obtain the polysaccharide from the fruit of the plant Malus hupehensis.

[0167] (9) Yield 25%, polysaccharide content 43%.

[0168] Example 4 Polysaccharide infrared spectrum determination

[0169] The polysaccharide prepared in Example 2 was mixed with KBr in a marvellous mortar and then pressed into a thin sheet, and Fourier transform infrared spectroscopy was used to determine it, the scanning range being 4000-400 cm -1 . The infrared spectrum is shown in Figure 1 . It can be seen that there is a strong hydroxyl stretching vibration peak at wave number 3297.16 cm -1 ; 2928.39 cm -1 is the C-H stretching vibration peak; 1641.7 cm -1 is a clear absorption peak, which is likely to be due to the amide bond I >C=O stretching vibration or the peptide amide bond C-N bending; in addition, the absorption peak at 1400.9 cm -1 is the >C=O stretching of the carboxyl group. The presence of characteristic hydroxyl groups preliminarily indicates that the extracted sample is polysaccharide. In addition, in the fingerprint region of the spectrum, the main absorption peak is at 1034.3 cm -1 , which is the absorption peak of the alcohol hydroxyl C-O, and the weak peak at 1231.6 cm -1 is the absorption peak of the C-O-C group. The infrared spectrum shows that the extracted polysaccharide sample of Wangjiazi contains most of the characteristic absorption peaks related to polysaccharide and can be identified as polysaccharide.

[0170] Example 5 Single factor comparison experiment of process parameters in the extraction method of Wangjiazi leaf polysaccharide

[0171] 1. Selection of solid-liquid ratio:

[0172] Take several portions of Wang Zaizi, crushed to a particle size of 10 μm of dry powder, respectively, add different volume of water, the ratio of solid to liquid is 1g:5mL, 1g:25mL, 1g:50mL, 1g:75mL, 1g:100mL, 1g:125mL, 1g:150mL, 1g:175mL, 1g:200mL, 1g:225mL, 1g:250mL, respectively, then ultrasonic extraction, ultrasonic power is 900W, ultrasonic time is 60min, extraction temperature is 50℃ to obtain the extract; The extract is concentrated and mixed with anhydrous ethanol in a ratio of 1:5, and then centrifuged at 10000 rpm for 15 min after standing for 24 h to obtain the precipitate; The precipitate is dissolved in water with a mass of 1 times the mass of the precipitate, and then filtered through a ceramic membrane with a pore size of 0.2 μm and a flow rate of 100 mL / min to collect the permeate; The permeate is filtered through an organic membrane with a pore size of 20000 daltons and a flow rate of 150 mL / min to collect the liquid, and then freeze-dried to obtain Wang Zaizi polysaccharide. The yield is calculated according to the method described in Example 1, and the polysaccharide content is determined. The specific solid-liquid ratio test results are shown in Table 1.

[0173] Table 1. Single factor comparison test results of solid-liquid ratio

[0174]

[0175] The results are shown in Table 1. When the solid-liquid ratio is 1g:5mL-1g:200mL, the polysaccharide content is above 65%, and the yield is above 17%; when the solid-liquid ratio is 1g:125mL-1g:200mL, the polysaccharide content is above 72%, and the yield is above 21%; when the solid-liquid ratio is 1g:150mL, the polysaccharide content is the highest, reaching 75%, and the yield is the highest, reaching 25%.

[0176] 2. Extraction power selection:

[0177] Take several portions of Wang Zaizi, crushed to a particle size of 10 μm of dry powder, respectively, add different volume of water, the ratio of solid to liquid is 1:150, respectively, ultrasonic extraction, ultrasonic power is 200W, 500W, 700W, 900W, 1200W, 1500W, ultrasonic time is 45min, extraction temperature is 45℃ to obtain the extract; The extract is concentrated and mixed with anhydrous ethanol in a ratio of 1:5, and then centrifuged at 10000 rpm for 15 min after standing for 24 h to obtain the precipitate; The precipitate is dissolved in water with a mass of 1 times the mass of the precipitate, and then filtered through an inorganic membrane with a pore size of 0.2 μm and a flow rate of 150 mL / min to collect the permeate; The permeate is filtered through an organic membrane with a pore size of 10000 daltons and a flow rate of 100 mL / min to collect the liquid, and then freeze-dried to obtain Wang Zaizi polysaccharide. The yield is calculated according to the method described in Example 1, and the polysaccharide content is determined. The specific solid-liquid ratio test results are shown in Table 2.

[0178] Table 2. Results of single-factor comparison experiment of extraction power

[0179]

[0180] The results are shown in Table 2. When the ultrasonic power is 200W-1500W, the polysaccharide content is above 67%; when the ultrasonic power is 900W-1500W, the polysaccharide content is above 72% and the yield is above 22%; when the ultrasonic power is 900W, the polysaccharide content is the highest, which is 75%, and the yield is 22%.

[0181] 3. Ultrasonic time selection:

[0182] A number of Wangzaozi were weighed, crushed into dry powder with a particle size of 10μm, and different volumes of water were added, with a solid-liquid ratio of 1:150. Ultrasonic extraction was performed, the ultrasonic power was 900W, the ultrasonic time was 15min, 30min, 45min, 60min, 75min, 90min, 105min, and 120min, and the extraction temperature was 45℃ to obtain the extraction liquid. After the extraction liquid was concentrated, it was mixed with anhydrous ethanol at a ratio of 1:5, and after standing for 24h, centrifugation was performed at 10000r / min for 15min to obtain the precipitate. The precipitate was dissolved with water in an amount of 1 times the mass of the precipitate, and then filtered through an inorganic membrane with a pore size of 0.2μm and a flow rate of 150mL / min to collect the permeate. The permeate was filtered through an organic membrane with a pore size of 10000Daltons and a flow rate of 100mL / min to collect the liquid. After freeze-drying, Wangzaozi polysaccharide was obtained. The yield was calculated according to the method described in Example 1, and the polysaccharide content was determined. The specific solid-liquid ratio test results are shown in Table 3.

[0183] Table 3. Results of single-factor comparison experiment of ultrasonic extraction time

[0184]

[0185] The results are shown in Table 3. When the ultrasonic time is 45min-105min, the polysaccharide content is above 63% and the yield is above 20%; when the ultrasonic time is 45min, the polysaccharide content is the highest, which is 73%, and the yield is 22%.

[0186] 4. Ultrasonic temperature selection:

[0187] Take several portions of Wang Zaizi, crushed to a particle size of 10 μm dry powder, respectively, add different volume of water, the ratio of solid to liquid is 1:150, respectively, using ultrasonic extraction, ultrasonic power is 900W, ultrasonic time is 45min, extraction temperature is 30℃, 45℃, 60℃, 75℃, 90℃ to obtain the extract; the extract is concentrated and mixed with anhydrous ethanol in a ratio of 1:5, after standing for 24h, centrifuged for 15min, 10000r / min, to obtain the precipitate; the precipitate is dissolved with 1 times its mass of pure water, filtered through an inorganic membrane, the pore size of the inorganic membrane is 0.2 μm, the flow rate is 150mL / min, collect the permeate; the permeate is filtered through an organic membrane, the pore size of the organic membrane is 10000 daltons, the flow rate is 100mL / min, collect the liquid, freeze-dried to obtain Wang Zaizi polysaccharide, calculate the yield and determine the polysaccharide content according to the method described in Example 1, the specific solid-liquid ratio test results are shown in Table 4.

[0188] Table 4. Single factor comparison test results of ultrasonic extraction temperature

[0189]

[0190] The results are shown in Table 4, when the ultrasonic temperature is 45℃-60℃, the polysaccharide content is more than 50%, and the yield is more than 23%; when the ultrasonic temperature is 45℃, the polysaccharide content is the highest, which is 65%, and the yield is 23%.

[0191] 5. Ceramic membrane pore size selection:

[0192] Take several portions of Wang Zaizi, crushed to a particle size of 10 μm dry powder, respectively, add different volume of water, the ratio of solid to liquid is 1:150, respectively, using ultrasonic extraction, ultrasonic power is 900W, ultrasonic time is 45min, extraction temperature is 45℃ to obtain the extract; the extract is concentrated and mixed with anhydrous ethanol in a ratio of 1:5, after standing for 24h, centrifuged for 15min, 10000r / min, to obtain the precipitate; the precipitate is dissolved with 1 times its mass of pure water, filtered through an inorganic membrane, the pore size of the inorganic membrane is 0.1 μm, 0.2 μm, 0.5 μm, 1 μm, the flow rate is 100mL / min, collect the permeate; the permeate is filtered through an organic membrane, the pore size of the organic membrane is 10000 daltons, the flow rate is 100mL / min, collect the liquid, freeze-dried to obtain Wang Zaizi polysaccharide, calculate the yield and determine the polysaccharide content, the specific solid-liquid ratio test results are shown in Table 5.

[0193] Table 5. Single factor comparison test results of ceramic membrane pore size

[0194]

[0195] The results are shown in Table 5. When the ceramic membrane pore size is 0.1 μm-1 μm, the polysaccharide content is more than 65%, and the yield is more than 20%; when the ceramic membrane pore size is 0.2 μm-0.5 μm, the polysaccharide content is more than 72%, and the yield is more than 23%; when the ceramic membrane pore size is 0.2 μm, the polysaccharide content is the highest, being 73%, and the yield is the highest, being 25%.

[0196] 6. Ceramic membrane flow rate selection:

[0197] A number of Wangzaozi were weighed, crushed into dry powder with a particle size of 10 μm, and added with different volumes of water, with a solid-liquid ratio of 1:150, respectively. Ultrasonic extraction was performed, with an ultrasonic power of 900 W, an ultrasonic time of 45 min, and an extraction temperature of 45°C to obtain an extraction liquid. After the extraction liquid was concentrated and mixed with anhydrous ethanol at a ratio of 1:5, it was allowed to stand for 24 h and then centrifuged at 10,000 r / min for 15 min to obtain a precipitate. The precipitate was dissolved with water in an amount of 1 times the mass of the precipitate, and then filtered through an inorganic membrane with a pore size of 0.2 μm and a flow rate of 50 mL / min, 100 mL / min, 150 mL / min, 200 mL / min, 250 mL / min, and 300 mL / min to collect the permeate. The permeate was filtered through an organic membrane with a pore size of 10,000 Dalton and a flow rate of 100 mL / min to collect the liquid. After freeze-drying, Wangzaozi polysaccharide was obtained. The yield was calculated according to the method described in Example 1, and the polysaccharide content was determined. The specific solid-liquid ratio test results are shown in Table 6.

[0198] Table 6. Single-factor comparison test results of ceramic membrane flow rate

[0199]

[0200] The results are shown in Table 6. When the ceramic membrane flow rate is 150 mL / min-200 mL / min, the polysaccharide content is more than 67%, and the yield is more than 25%; when the ceramic membrane flow rate is 150 mL / min, the polysaccharide content is the highest, being 78%, and the yield is 28%.

[0201] 7. Organic membrane pore size selection:

[0202] A number of Wangzaozi were weighed, crushed to a dry powder with a particle size of 10 μm, different volumes of water were added, the solid-liquid ratio was 1:150, respectively, and ultrasonic extraction was used, the ultrasonic power was 900 W, the ultrasonic time was 45 min, and the extraction temperature was 45°C to obtain an extract; the extract was concentrated and mixed with anhydrous ethanol at a ratio of 1:5, and then centrifuged at 10,000 rpm for 15 min after standing for 24 h to obtain a precipitate; the precipitate was dissolved in 1 times its mass of pure water, filtered through an inorganic membrane with a pore size of 0.2 μm and a flow rate of 150 mL / min, and the permeate was collected; the permeate was filtered through an organic membrane with a pore size of 5000, 10,000, 30,000, 50,000 and 100,000 daltons and a flow rate of 100 mL / min, and the liquid was collected, and Wangzaozi polysaccharides were obtained after freeze-drying; the yield was calculated according to the method described in Example 1, and the polysaccharide content was determined, and the specific solid-liquid ratio test results are shown in Table 7.

[0203] Table 7. Results of single-factor comparison experiment of organic membrane pore size

[0204]

[0205]

[0206] The results are shown in Table 7. When the organic membrane pore size is 5000-50,000 daltons, the polysaccharide content is above 63%, and the yield is above 25%; when the organic membrane pore size is 10,000-30,000 daltons, the polysaccharide content is above 76%, and the yield is above 27%; when the organic membrane pore size is 10,000 daltons, the polysaccharide content is the highest, reaching 78%, and the yield is the highest, reaching 28%.

[0207] 8 Organic membrane flow rate selection:

[0208] A number of Wangzaozi were weighed, crushed to a dry powder with a particle size of 10 μm, different volumes of water were added, the solid-liquid ratio was 1:150, respectively, and ultrasonic extraction was used, the ultrasonic power was 900 W, the ultrasonic time was 45 min, and the extraction temperature was 45°C to obtain an extract; the extract was concentrated and mixed with anhydrous ethanol at a ratio of 1:5, and then centrifuged at 10,000 rpm for 15 min after standing for 24 h to obtain a precipitate; the precipitate was dissolved in 1 times its mass of pure water, filtered through an inorganic membrane with a pore size of 0.2 μm and a flow rate of 150 mL / min, and the permeate was collected; the permeate was filtered through an organic membrane with a pore size of 5000, 10,000, 30,000, 50,000 and 100,000 daltons and a flow rate of 100 mL / min, and the liquid was collected, and Wangzaozi polysaccharides were obtained after freeze-drying; the yield was calculated according to the method described in Example 1, and the polysaccharide content was determined, and the specific solid-liquid ratio test results are shown in Table 7.

[0209] Table 8. Single factor comparison experiment results of organic membrane flow rate

[0210]

[0211] As shown in Table 8, when the organic membrane flow rate is 50 mL / min to 250 mL / min, the polysaccharide content is more than 70%, and the yield is more than 23%; when the organic membrane flow rate is 100 mL / min to 150 mL / min, the polysaccharide content is more than 78%, and the yield is more than 27%; when the organic membrane flow rate is 100 mL / min, the polysaccharide content is the highest, which is 82%, and the yield is the highest, which is 31%.

[0212] Based on the single factor comparison experiments 1-8, the optimal parameter of the extraction method of the polysaccharide of the leaves of Paliurus ramosissimus is obtained.

[0213] (1) The fresh leaves of Paliurus ramosissimus are dried to constant weight to obtain dried leaves of Paliurus ramosissimus;

[0214] (2) The dried leaves of Paliurus ramosissimus are crushed to a dry powder with a particle size of 10 μm by using a super micro grinder;

[0215] (3) Water is added to the dry powder at a liquid-to-material ratio of 150 mL: 1 g, and then ultrasonic extraction is used for extraction, the power of the ultrasonic extraction is 900 W, the temperature of the ultrasonic extraction is 45°C, and the time of the ultrasonic extraction is 45 min, to obtain an extraction liquid;

[0216] (4) The extraction liquid is concentrated under reduced pressure (50-90°C, vacuum pressure 0.07 MPa) and then dehydrated to a paste to obtain a concentrated liquid;

[0217] (5) Anhydrous ethanol is added to the concentrated liquid in an amount of 5 times the volume of the concentrated liquid for precipitation, and after standing for 24 h, centrifugation is performed at a speed of 10,000 rpm for 15 min, and the precipitate A is collected, to obtain crude polysaccharide of Paliurus ramosissimus;

[0218] (6) The crude polysaccharide of Paliurus ramosissimus is dissolved in water in an amount of 1 times the mass of the crude polysaccharide, and then ceramic membrane is used for impurity removal, the pore size of the ceramic membrane is 0.2 μm, and the flow rate is 150 mL / min, and the permeate is collected.

[0219] (7) The permeate is subjected to organic membrane filtration, the pore size of the organic membrane is 10,000 daltons, and the flow rate is 100 mL / min, and the permeate is collected.

[0220] (8) The permeate is subjected to freeze-drying, and after drying, high-content polysaccharide is obtained.

[0221] Example 6: The polysaccharide of Paliurus ramosissimus has the effects of lowering blood lipids and inhibiting and improving obesity

[0222] Experimental method

[0223] Determination of blood lipid: After the rats were fasted for 12 h, the rats were anesthetized by inhaling anhydrous ether, about 1.2 mL of blood was collected by capillary glass, and then transferred to a sterile centrifuge tube and stood for 1 h. After the blood coagulated, it was centrifuged at 10,000 r / min for 15 min to separate the serum. The triglyceride (TG) determination kit (single reagent GPO-PAP method), total cholesterol (TCH / T-CHO) determination kit (single reagent GPO-PAP method), low-density lipoprotein cholesterol (LDL-C) determination kit (double reagent direct method), and high-density lipoprotein cholesterol (HDL-C) determination kit (double reagent direct method) (Nanjing Jiancheng Biological Institute) were used to determine the levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) according to the instructions.

[0224] Lee's index: After the last intragastric administration was completed, each mouse in the treatment group was fasted overnight, the body weight (g) of the mouse was measured, the body length (the distance from the nose tip to the anus, cm) was measured by a vernier caliper, and the Lee's index (an index for evaluating the degree of obesity) was calculated. Wherein: Lee's index = body weight (g)1 / 3×10 / body length (cm).

[0225] Liver coefficient and fat body ratio: The rats were quickly dissected, the abdominal skin tissue of the rats was stripped about 1 cm 2 ; the rat perirenal fat and liver were separated, quickly rinsed with physiological saline, and the water was absorbed with sterile filter paper. The mass of the liver and fat was measured, and the liver coefficient and fat body ratio were calculated. Wherein: liver coefficient = (liver mass / mouse body mass) × 100%; fat body ratio (%) = (perirenal fat mass / mouse body mass) × 100%.

[0226] The liver tissue and fat tissue were immersed and fixed in 10% paraformaldehyde, and were embedded, paraffin sectioned, and frozen sectioned according to the pathological tissue sampling and fixing. The sections were sequentially placed into environmental protection type dewaxing solution I, environmental protection type dewaxing solution II, anhydrous ethanol, and pure water for cleaning.

[0227] The frozen sections were taken out from the -20°C refrigerator and restored to room temperature, and then fixed in a tissue fixing solution for 15 min and rinsed with running water. The sections were placed into a hematoxylin dyeing solution for 5 min, washed with tap water, differentiated with a differentiation solution, washed with tap water, returned to blue with a return blue solution, and rinsed with running water. The sections were sequentially dehydrated in 85% and 95% gradient alcohol for 5 min each, dyed in an eosin dyeing solution for 5 min, and then sealed with a neutral balsam after being placed into anhydrous ethanol. The pathological changes of the liver tissue were observed under an optical microscope.

[0228] Experimental settings and detection indexes

[0229] SPF-grade male C57BL / 6J mice, weighing 15–20 g, were acclimatized for one week under natural light at a temperature of 25℃±2℃ and humidity of 50%±10%. Male mice were randomly divided into 6 groups of 7 mice each and numbered. These included: a normal control group, a model control group, a positive drug group (simvastatin, 10 mg / kg), a low-dose group (jujube polysaccharide, 100 mg / kg), a medium-dose group (jujube polysaccharide, 200 mg / kg), and a high-dose group (jujube polysaccharide, 500 mg / kg). The normal control group was fed a standard maintenance diet, while the other groups were fed a high-fat diet to induce obesity. One month after modeling, blood lipid levels in the mice were measured using the same method. Successful modeling was defined as a significant increase in TC, TG, and LDL-C levels in the model control group, positive drug group, and the low, medium, and high-dose groups compared to the normal control group, and a significant decrease in HDL-C levels compared to the normal control group.

[0230] After successful modeling, mice in the positive drug group were administered simvastatin (10 mg / kg) daily by gavage for 9 weeks (63 days); mice in the low, medium, and high dose groups were administered jujube polysaccharide prepared in Example 2 at doses of 100 mg / kg, 200 mg / kg, and 500 mg / kg daily by gavage, respectively, for 9 weeks (63 days). After the last gavage administration, blood lipids, Lee's index, liver coefficient, and fat body ratio were measured according to the above methods, and liver tissue samples were taken for pathological observation.

[0231] result

[0232] blood lipids

[0233] Blood lipid test results after polysaccharide gavage experiment are as follows Figure 2 As shown.

[0234] Depend on Figure 2 The results showed that the levels of TC, TG, and LDL-C in the model control group mice were significantly higher than those in the normal control group (P < 0.05); the level of HDL-C in the model control group mice was significantly lower than that in the normal control group (P < 0.05). Compared with the model group, the levels of TC, TG, and LDL-C in the high, medium, and low dose groups of jujube polysaccharide were significantly lower (P < 0.05), while the level of HDL-C in the high, medium, and low dose groups of jujube polysaccharide was significantly higher (P < 0.05). This indicates that jujube polysaccharide can improve lipid metabolism in obese mice, treat or improve hyperlipidemia, and has a lipid-lowering effect.

[0235] Lee's Index and Fat Body Ratio

[0236] The results of Lee's index and fat body ratio are shown in Table 9. The Lee's index and fat body ratio of the model control group mice were significantly higher than those of the normal control group (p<0.05), while the Lee's index and fat body ratio of the high-dose and medium-dose groups of *Ziziphus jujuba* extract were significantly lower than those of the model control group (p<0.05). This indicates that *Ziziphus jujuba* polysaccharide can treat or improve obesity symptoms in mice, improve lipid metabolism in obese mice, and reduce body fat.

[0237] Table 9. Results of Lee's Index and Fat Body Ratio

[0238]

[0239] In this context, # indicates that the value is significantly different from the normal control group, and * indicates that the value is significantly different from the model control group.

[0240] Liver coefficient

[0241] The liver coefficient results are shown in Table 10. The liver coefficient of the model control group mice showed an increasing trend compared to the normal control group, but the difference was not statistically significant. Compared to the model group, the liver coefficients of mice treated with high, medium, and low doses of *Ziziphus jujuba* polysaccharide and the positive control group were all lower than those of the model control group, but the differences were not statistically significant. These results indicate that a high-fat diet has a certain impact on the liver. The drugs and *Ziziphus jujuba* polysaccharide can cause a certain degree of decrease in the liver coefficient, and this decrease is dose-dependent. However, due to the short experimental period, the effect may not have been statistically significant.

[0242] Table 10. Liver coefficient results

[0243]

[0244] Pathological section observation results of liver tissue

[0245] The results are as follows Figure 3 As shown, HE staining of mouse liver tissue revealed that the liver tissue structure of the normal control group mice remained intact. Hepatic sinusoids were located between hepatocyte cords and connected to the central vein, exhibiting a radial distribution. Hepatocytes showed uniform morphology, with round nuclei located centrally, and evenly distributed cytoplasm. No fatty degeneration was observed.

[0246] Compared with the normal control group, the model control group showed obvious fatty degeneration and ballooning degeneration of liver tissue. With increasing dosage of jujube polysaccharide, the degree of liver damage in mice tended to decrease, the hepatocyte structure gradually became more complete and orderly, and the number of fat vacuoles also showed a decreasing trend, indicating a significant improvement in fat deposition and cell vacuolation. This demonstrates that jujube polysaccharide can treat or improve fatty liver in mice and reduce the degree of liver damage.

[0247] The technical solutions of the present application are not limited to the above specific embodiments, and any technical variations made according to the technical solutions of the present application fall within the protection scope of the present application.

Claims

1. A method for preparing polysaccharide of Malus prunifolia (Wild.) Britt, comprising the following steps: 1) mixing dry powder obtained by crushing leaves of Malus prunifolia (Wild.) Britt with water, and extracting by ultrasonic extraction to obtain an extract; 2) mixing the extract with anhydrous ethanol after concentration, and centrifuging after standing to obtain a precipitate; 3) dissolving the precipitate with water and filtering through a ceramic membrane to collect a permeate; 4) filtering the permeate through an organic membrane, and collecting a liquid after drying to obtain polysaccharide of Malus prunifolia (Wild.) Britt. 2.The method according to claim 1, wherein, in the step 1), the fresh leaves of Malus prunifolia (Wild.) Britt are dried before crushing to obtain dried leaves of Malus prunifolia (Wild.) Britt; and / or, the particle size of the dry powder after crushing is 5-100 μm, preferably 5-50 μm, and more preferably 5-10 μm; and / or, the mass-volume ratio of the dry powder after crushing to water is 1 g: (5-200) mL, preferably 1 g: (10-200) mL, more preferably 1 g: (125-200) mL, and most preferably 1 g: 150 mL; and / or, the ultrasonic power of the ultrasonic extraction is 200-1500 W, preferably 900-1500 W, and most preferably 900 W; and / or, the ultrasonic time of the ultrasonic extraction is 30 min-120 min, preferably 45 min-105 min, and most preferably 45 min; and / or, the extraction temperature of the ultrasonic extraction is 30-60 ℃, preferably 45-60 ℃, and most preferably 45 ℃. 3.The method according to claim 1, wherein, in the step 2), the volume ratio of the extract to anhydrous ethanol is 1: (3-10), preferably 1: (3-5) ; and / or, the standing time is 12-48 h, preferably 24 h; and / or, the centrifugation time is 5-30 min, preferably 10-20 min, and more preferably 15 min; and / or, the centrifugation speed is 10000-20000 rpm. 4.The method according to claim 1, wherein, in the step 3), the mass ratio of the precipitate to water is 1: (1-20), preferably 1: (1-10), and more preferably 1: (1-5) ; and / or, the pore size of the ceramic membrane is 0.1-1 μm, preferably 0.2-0.5 μm, and most preferably 0.2 μm; and / or, the flow rate of the ceramic membrane is 50-200 mL / min, preferably 150-200 mL / min, and most preferably 150 mL / min. 5.The method according to claim 1, wherein, in the step 4), the cut-off capacity of the organic membrane is 5000-50000 Da, preferably 10000-30000 Da, and most preferably 10000 Da; and / or, the flow rate of the organic membrane is 50-250 mL / min, preferably 100-150 mL / min, and most preferably 100 mL / min. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ 6. The preparation method of claim 1 or 2, wherein the drying comprises one or more of the following: freeze-drying, hot air drying, spray drying, steam drying, vacuum drying, microwave drying or vacuum microwave drying. Preferably, the freeze-drying has a cold trap temperature of -60 to -50°C, more preferably -55°C, and a vacuum degree of 0.1 to 1 Mpa, more preferably 0.2 Mpa. Preferably, the hot air drying has a temperature of 40 to 60°C, more preferably 50°C. Preferably, the vacuum microwave drying has a temperature of 40 to 50°C, more preferably 45°C, and a microwave power of 800 to 1200 W, more preferably 1000 W.

7. A polysaccharide of Fructus Psidii prepared by the method of any one of claims 1 to 6.

8. Use of the preparation method of any one of claims 1 to 6 or the polysaccharide of Fructus Psidii of claim 7 in the preparation of a medicament for preventing, treating, ameliorating, controlling or alleviating hyperlipidemia, dyslipidemia, obesity, and / or improving lipid metabolism.

9. The use of claim 8, wherein the use comprises one or more of the following: (1) weight loss or weight control; (2) prevention, treatment, amelioration, control or alleviation of hyperlipidemia; (3) improvement of lipid metabolism; (4) control or reduction of triglyceride, total cholesterol and low-density lipoprotein cholesterol levels; (5) increase of high-density lipoprotein cholesterol levels; (6) control or reduction of body fat, local fat or fat mass ratio; (7) prevention, treatment, amelioration, control or alleviation of liver function impairment; (8) prevention, treatment, amelioration, control or alleviation of atherosclerosis.

10. Use of the preparation method of any one of claims 1 to 6 or the polysaccharide of Fructus Psidii of claim 7 in the preparation of a health product for helping to maintain a healthy level of blood lipids.

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