Preparation method of wangzaozi polysaccharide and application thereof

Abstract

The present disclosure provides a preparation method of Wangzaozi polysaccharide and its application. The present disclosure provides an extraction and separation process of Wangzaozi polysaccharide, and 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.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a method for preparing polysaccharides from jujube seeds and their application. Background Technology

[0002] Wangzaozi (Isodon amethystoides (Benth) Cy Wu et Hsuan) is a plant belonging to the genus Isodon in the family Lamiaceae. It mainly grows in shady, rocky areas of northern Anhui province. It possesses antibacterial, anti-inflammatory, and antitumor properties. The main chemical components of Wangzaozi include diterpenoids, triterpenoids, and flavonoids. It is commonly used to treat various diseases such as carbuncles, tumors, snake bites, colds, prostatitis, acute infectious hepatitis, and gynecological inflammation.

[0003] However, no methods for isolating polysaccharides from jujube seeds or the physiological effects of jujube seed polysaccharides have been reported. Further development of deep processing methods for jujube seeds is urgently needed, and the medicinal or health-promoting value of jujube seed extracts also requires in-depth exploration.

[0004] Plant polysaccharides are encased within the tough cell walls of plants, making it difficult to extract them completely using conventional methods (mechanical disruption, heat treatment, acid / alkali treatment, enzymatic hydrolysis). Plants often contain numerous impurities coexisting with polysaccharides, such as proteins, lipids, pigments, inorganic salts, monosaccharides / oligosaccharides, nucleic acids, and small molecule metabolites, which are difficult to remove completely, especially proteins or pigments tightly bound to polysaccharides, resulting in low purity of the final product. Furthermore, traditional separation methods (precipitation, dialysis, ion exchange, gel filtration) have limited resolution, small throughput, and are time-consuming. Therefore, it is necessary to efficiently and selectively disrupt these physical barriers to release polysaccharides while avoiding excessive disruption that could lead to polysaccharide degradation or the dissolution of large amounts of impurities. Choosing novel separation and purification methods can yield high-purity plant polysaccharides.

[0005] The prevalence of metabolic diseases such as obesity, hyperlipidemia, and fatty liver is constantly increasing. Hyperlipidemia is defined as an abnormality in lipid metabolism or transport, resulting in elevated levels of one or more lipids in the plasma. Lipids are insoluble or only slightly soluble in water and must bind to proteins to exist in the form of lipoproteins. Therefore, hyperlipidemia is often hyperlipoproteinemia, manifesting as hypercholesterolemia, hypertriglyceridemia, or both. The incidence of hyperlipidemia is a significant public health problem, and its rate of increase is alarming. It significantly increases 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 currently the most common and widely used treatment for hyperlipidemia. However, due to their high cost and side effects, research has begun to develop more effective and safer alternatives or adjunctive lipid-lowering products. Summary of the Invention

[0006] To address at least one of the above problems, this disclosure provides a method for preparing polysaccharides from jujube seeds and their applications.

[0007] According to one aspect of this disclosure, a method for preparing polysaccharides from jujube seeds is provided, comprising the following steps:

[0008] This invention provides a method for preparing polysaccharides from jujube seeds, comprising the following steps:

[0009] 1) Mix the dried powder obtained by crushing the leaves of the Chinese jujube with water, and extract using ultrasonic extraction to obtain an extract;

[0010] 2) The extract was concentrated and mixed with anhydrous ethanol, allowed to stand, and then centrifuged to obtain the precipitate;

[0011] 3) Dissolve the precipitate in water, filter it through a ceramic membrane, and collect the permeate;

[0012] 4) After filtering the permeate with an organic membrane, the liquid is collected and dried to obtain jujube polysaccharide.

[0013] In some embodiments, the fresh jujube leaves are dried before pulverizing in step 1) to obtain dried jujube leaves.

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

[0015] Those skilled in the art will understand that any known pulverizing method can be used for pulverization. Non-limiting examples of pulverization include mechanical impact pulverization, air jet pulverization, ball milling or vibratory milling, high pressure pulverization, cryogenic pulverization, ultrafine pulverization, nano pulverization, etc., all of which are common choices for those skilled in the art.

[0016] In some embodiments, the particle size of the pulverized dry powder in step 1) is 5-100 μm, preferably 5-50 μm, and more preferably 5-10 μm.

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

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

[0019] In some embodiments, the mass-to-volume ratio of the pulverized dry powder to water in step 1) is 1g:(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, 11 1, 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, 15 6, 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 200-1500W, preferably 900W-1500W, and most preferably 900W.

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

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

[0023] In some embodiments, the ultrasonic extraction time in step 1) is 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, or 74 min. 75min, 76min, 77min, 78min, 79min, 80min, 81min, 82min, 83min, 84min, 85min, 86min, 87min, 88min, 89min, 90min, 91min, 92min, 93min, 94min, 95min, 96min, 97min, 98min, 99min, 100min, 101min, 102min, 103min, 104min, 105min, 106min, 107min, 108min, 109min, 110min, 111min, 112min, 113min, 114min, 115min, 116min, 117min, 118min, 119min, or 120min.

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

[0025] In some embodiments, the extraction temperature of the ultrasonic extraction method in step 1) is 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℃, or 60℃.

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

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

[0028] In some embodiments, the ultrasonic extraction method in step 1) has an ultrasonic power of 900W, an ultrasonic time of 45min, and an extraction temperature of 45℃.

[0029] In some embodiments, the concentration in step 2) includes concentrating and dehydrating to a paste-like state.

[0030] Those skilled in the art will understand that any known concentration method can be used for concentration. Non-limiting examples of concentration include evaporation concentration, freeze concentration, membrane separation concentration, adsorption concentration, extraction concentration, electrodialysis concentration, centrifugal concentration, etc. Evaporation concentration includes atmospheric pressure evaporation concentration, vacuum evaporation concentration (hereinafter referred to as vacuum concentration), and thin film evaporation concentration, all of which are common choices for those skilled in the art.

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

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

[0033] In some implementations, the settling time in step 2) is 12 to 48 hours, for example, 24 hours.

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

[0035] In some embodiments, the centrifugation time in step 2) is 5 to 30 minutes, preferably 10 to 20 minutes, and more preferably 15 minutes.

[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 centrifugal speed in step 2) is 10,000 to 20,000 rpm.

[0038] In some embodiments, the centrifugal 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), and 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 to 1 μm, preferably 0.2 to 0.5 μm, and 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 mL / min to 200 mL / min, preferably 150 to 200 mL / min, and most preferably 150 mL / min.

[0044] 3) The membrane flow rate 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,152mL / min, 153mL / min, 154mL / min, 155mL / min, 156mL / min, 157mL / min, 158mL / min, 159mL / min, 160mL / min, 161mL / min, 162mL / min, 163mL / min, 16 4mL / min, 165mL / min, 166mL / min, 167mL / min, 168mL / min, 169mL / min, 170mL / min, 171mL / min, 172mL / min, 173mL / min, 174mL / min, 175mL / min, 176mL / min, 177mL / min, 178mL / min, 179mL / min, 180mL / min, 181mL / min, 182mL / min, 183mL / min, 184mL / min, 185mL / min, 186mL / min, 187mL / min, 188mL / m in, 189mL / min, 190mL / min, 191mL / min, 192mL / min, 193mL / min, 194mL / min, 195mL / min, 196mL / min, 197mL / min, 198mL / min, 199mL / min or 200mL / min. ,

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

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

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

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

[0049] In some embodiments, the retention capacity of the organic membrane in step 4) is 5,000 to 50,000 Daltons, preferably 10,000 to 30,000 Daltons, and most preferably 10,000 Daltons.

[0050] In some embodiments, the retention capacity of the organic membrane in step 4) is 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 10500, 11000, 11500, 12000, 12500, 13000, 13500, 14000, 14500, or 1500. 0, 15500, 16000, 16500, 17000, 17500, 18000, 18500, 19000, 19500, 20000, 20500, 21000, 21500, 22000, 22500, 23000, 23500, 24000, 24500, 25000, 25500, 26000, 26500, 27 000, 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 flow rate of the organic membrane in step 4) is 50 mL / min to 250 mL / min, preferably 100 to 150 mL / min, and most preferably 100 mL / min.

[0052] In some embodiments, the flow rate of the organic membrane in step 4) is 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 to 50000 Daltons and a flow rate of 50 mL / min to 250 mL / min.

[0054] In some embodiments, the organic membrane in step 4) has a retention capacity of 10,000 to 30,000 Daltons and a flow rate of 100 mL / min to 250 mL / min.

[0055] In some embodiments, the organic membrane in step 4) has a retention capacity of 10,000 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.

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

[0058] In some embodiments, the cold trap temperature for 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 level is 0.1 to 1 MPa, more preferably 0.2 MPa.

[0060] In some implementations, the vacuum level 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 implementations, the microwave power is 800–1200W, preferably 1000W.

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

[0067] In some implementations, the method includes the following steps:

[0068] 1) The dry powder with a particle size of 5-100μm obtained by crushing the leaves of the Chinese jujube was mixed with water at a ratio of 1g:(5-200)mL and extracted by ultrasonic extraction to obtain an extract. The ultrasonic power was 200W-1500W, the ultrasonic time was 30min-120min, and the extraction temperature was 30-60℃.

[0069] 2) Concentrate and dehydrate the extract to a paste-like consistency, mix it with anhydrous ethanol at a volume ratio of 1:(3-10), let it stand for 12-48 hours, and then centrifuge for 5-30 minutes at a speed of 10,000-20,000 rpm to obtain the precipitate.

[0070] 3) Dissolve the precipitate in water at a mass ratio of precipitate to water of 1:(1-20), filter it through a ceramic membrane with a pore size of 0.1-1 μm at a flow rate of 50 mL / min-200 mL / min, and collect the permeate;

[0071] 4) The permeate is filtered through an organic membrane with a retention capacity of 5000 to 50000 Daltons at a flow rate of 50 mL / min to 250 mL / min. The liquid is collected and dried to obtain jujube polysaccharide.

[0072] In some implementations, the method includes the following steps:

[0073] 1) The dried powder with a particle size of 5-50 μm obtained by crushing the leaves of the Chinese jujube was mixed with water at a ratio of 1g:(10-200)mL and extracted by ultrasonic extraction to obtain an extract. The ultrasonic power was 900W-1500W, the ultrasonic time was 45min-105min, and the extraction temperature was 45-60℃.

[0074] 2) Concentrate and dehydrate the extract to a paste-like state, mix it with anhydrous ethanol at a volume ratio of 1:(3-5), let it stand for 12-48 hours, and then centrifuge for 10-20 minutes at a speed of 10000-20000 rpm to obtain the precipitate.

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

[0076] 4) The permeate is filtered through an organic membrane with a retention capacity of 10,000 to 30,000 Daltons at a flow rate of 100 mL / min to 150 mL / min. The liquid is collected and dried to obtain jujube polysaccharide.

[0077] In some implementations, the method includes the following steps:

[0078] 1) The dry powder with a particle size of 10μm obtained by crushing the leaves of the Chinese jujube was mixed with water at a ratio of 1g:150mL and extracted by ultrasonic extraction to obtain the extract. The ultrasonic power was 900W, the ultrasonic time was 45min, and the extraction temperature was 45℃.

[0079] 2) Concentrate and dehydrate the extract to a paste-like consistency, mix it with anhydrous ethanol at a volume ratio of 1:5, let it stand for 24 hours, and then centrifuge for 15 minutes at a speed of 10,000 to 20,000 rpm to obtain the precipitate.

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

[0081] 4) The permeate was filtered through an organic membrane with a retention capacity of 10,000 Daltons at a flow rate of 100 mL / min. The liquid was collected and dried to obtain jujube polysaccharide.

[0082] According to another aspect of this disclosure, a polysaccharide from jujube seeds prepared according to the method described herein is provided.

[0083] According to another aspect of this disclosure, a medicament is provided comprising the jujube polysaccharide.

[0084] According to another aspect of this disclosure, the preparation method or the use of the jujube polysaccharide in the preparation of medicaments for the prevention, treatment, improvement, control or relief of hyperlipidemia, dyslipidemia, obesity and / or improvement of lipid metabolism is provided.

[0085] According to another aspect of this disclosure, the preparation method or the use of the jujube polysaccharide in the prevention, treatment, improvement, control or relief of hyperlipidemia, dyslipidemia, obesity and / or improvement of lipid metabolism is provided.

[0086] In some implementations, the application includes one or more of the following:

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

[0088] (2) To prevent, treat, improve, control or alleviate hyperlipidemia;

[0089] (3) Improves lipid metabolism;

[0090] (4) Control or reduce triglyceride, total cholesterol and low-density lipoprotein cholesterol levels;

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

[0092] (6) Control or reduce body fat, local fat or fat-to-body ratio;

[0093] (7) To prevent, treat, improve, control or reduce liver function damage;

[0094] (8) To prevent, treat, improve, control or reduce atherosclerosis.

[0095] In some embodiments, the liver dysfunction includes at least one of the following diseases: fatty liver, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver fibrosis, cirrhosis, liver cancer, or hepatocyte damage.

[0096] In some embodiments, the causes of liver function impairment include, but are not limited to, at least one of a high-fat diet, a high-cholesterol diet, high blood lipids, or high cholesterol.

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

[0098] According to another aspect of this disclosure, the preparation method or the application of the jujube polysaccharide in the preparation of health products that help maintain healthy blood lipid levels is provided.

[0099] According to another aspect of this disclosure, the preparation method or the application of the jujube polysaccharide in maintaining healthy blood lipid levels is provided.

[0100] Beneficial effects:

[0101] There is currently limited research on polysaccharides from jujube seeds. This paper discloses an extraction and separation process for jujube seed polysaccharides to obtain high-content polysaccharides. These polysaccharides have functions such as lowering blood lipids, reducing body fat, local fat or lipid body ratio, preventing, treating, improving or reducing liver damage. Attached Figure Description

[0102] Figure 1 The Fourier transform infrared spectrum of the polysaccharide from jujube seed in Example 4 is shown.

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

[0104] Figure 3 The image shows a tissue section of the mouse liver after the administration of the drug via gavage in Example 6. Detailed Implementation

[0105] definition

[0106] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0107] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references.

[0108] The term "about" as used herein is as understood by one of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If one of ordinary skill in the art is unfamiliar with the use of this term in the context in which it is used, "about" will mean a particular value plus or minus 10%.

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

[0110] As used in this disclosure, the term "polysaccharide" refers to a polymeric carbohydrate molecule consisting of long chains of monosaccharide units linked together by glycosidic bonds and which, upon hydrolysis, yield component monosaccharides or oligosaccharides. This includes glycans or oligosaccharides, as well as the glycosidic portion of glycocomplexes. Oligosaccharides are glycopolymers containing a small number (typically 3-10) of glycosidic components (monosaccharides). Polysaccharides are typically composed of monosaccharides linked by O- or N-glycosidic bonds to amino acid side chains or lipid moieties in suitable proteins. The glycocomplexes include, but are not limited to, glycoproteins, glycolipids, proteoglycans, and glycophosphosphingolipids, or other known or unknown glycocomplexes.

[0111] The term "liver coefficient" used in this disclosure refers to the ratio of an animal's liver weight to its body weight. It is a commonly used indicator in pharmacology. The larger the liver coefficient, the more severe the edema, hyperplasia and hypertrophy of the liver in the body.

[0112] As used in this disclosure, the term "fat body ratio" is the ratio of fat weight to body weight. A higher ratio indicates a higher fat content and a more obese body. In some embodiments, the fat body ratio can be calculated using the ratio of peritesticular fat content to body weight.

[0113] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. The actual scope of protection of this invention is set forth in the claims. In the following description, descriptions of well-known structures and techniques are omitted to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications. Unless otherwise specified, the equipment, instruments, reagents, and / or kits used in the following embodiments are commercially available or obtained through conventional methods known to those skilled in the art.

[0114] Example

[0115] Methods for determining polysaccharide content:

[0116] Preparation of standard stock solution: Accurately weigh glucose standard (molecular weight 180, purity greater than 95%, Shanghai Yuanye Biotechnology Co., Ltd.) dried to constant weight at 105℃, add it to a 100mL volumetric flask, add an appropriate amount of water to dissolve, dilute to the mark, and shake well for later use.

[0117] Preparation of the standard curve: Accurately measure 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL, 3.0 mL, 3.5 mL, and 4.0 mL of the above standard stock solution into 50 mL volumetric flasks, add water to the mark, and shake well to obtain the standard solutions. Accurately measure 2.0 mL of each standard solution into a test tube, add 1 mL of 5% phenol solution to each, mix well, and quickly add 7.0 mL of concentrated sulfuric acid. Shake well. Incubate in a 40℃ water bath for 30 min, then place in an ice-water bath for 5 min. Use the first sample as a blank. Measure the absorbance at a wavelength of 490 nm. Construct a standard curve with absorbance on the ordinate and concentration (mg / mL) on the abscissa.

[0118] Preparation of sample solution: Accurately weigh 0.1g of the sample to be tested and place it in a 50mL heart-shaped bottle. Add approximately 30mL of water, reflux for 2 hours, filter, and dilute to 50mL. Shake well and take 1mL of the solution into a 10mL volumetric flask. Add water to the mark, mix well, and use as the test solution.

[0119] Determination of sample solution: Accurately measure 2.0 mL of the prepared test solution into test tubes. Then, under the "Preparation of Standard Curve" section, start from "Add 1 mL of 5% phenol solution". Measure the absorbance at a wavelength of 490 nm. Detect the polysaccharide concentration (mg / mL) using the external standard method and calculate the polysaccharide content.

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

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

[0122] Example 1: Extraction method of polysaccharides from jujube seeds

[0123] Method: (1) Fresh jujube leaves were dried to constant weight to obtain dried jujube leaves;

[0124] (2) The dried leaves of the jujube were pulverized into dry powder with a particle size of 5μm using an ultra-micro pulverizer;

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

[0126] (4) The extract was concentrated under reduced pressure (50-90℃, vacuum pressure 0.07MPa) and then dehydrated to a paste-like state to obtain a concentrated solution;

[0127] (5) Add three times the volume of anhydrous ethanol to the concentrate for precipitation. After standing for 24 hours, centrifuge for 10 minutes at a speed of 10,000 rpm and collect precipitate A to obtain crude polysaccharide from jujube.

[0128] (6) The crude polysaccharide of jujube was dissolved in pure water at a ratio of 1:1 by weight, and then impurities were removed by a ceramic membrane (Hefei Shijie Membrane Equipment). The ceramic membrane had a pore size of 0.1 μm and a flow rate of 50 mL / min. The permeate was collected.

[0129] (7) The permeate is filtered by an organic membrane with a pore size of 10,000 Daltons and a flow rate of 50 mL / min.

[0130] (8) The permeate was freeze-dried at a cold trap temperature of -55°C and a vacuum degree of 0.2 MPa. After drying, a high content of polysaccharide was obtained.

[0131] (9) Weigh the mass of the high-content polysaccharide obtained in step (8), calculate the ratio of its mass to the mass of the fresh jujube leaves in step (1) as the yield, the yield is 25%, determine the polysaccharide concentration, and calculate the polysaccharide content as 78%.

[0132] Example 2: Extraction method of polysaccharides from jujube seeds

[0133] Method: (1) Fresh jujube leaves were dried to constant weight to obtain dried jujube leaves;

[0134] (2) The dried leaves of the jujube were pulverized into dry powder with a particle size of 10 μm using an ultra-micro pulverizer;

[0135] (3) Add water to the dry powder at a liquid-to-solid ratio of 30mL:1g, and then extract it by ultrasonic extraction. The power of ultrasonic extraction is 400W, the temperature of ultrasonic extraction is 40℃, and the time of ultrasonic extraction is 90min to obtain the extract.

[0136] (4) The extract was concentrated under reduced pressure (50-90℃, vacuum pressure 0.07MPa) and then dehydrated to a paste-like state to obtain a concentrated solution;

[0137] (5) Add 4 times its volume of anhydrous ethanol to the concentrate for precipitation, let it stand for 24 hours, then centrifuge for 20 minutes at a speed of 20,000 rpm, and collect precipitate A to obtain crude polysaccharide from jujube.

[0138] (6) The crude polysaccharide of jujube was dissolved in twice its weight of pure water, and then impurities were removed by a ceramic membrane with a pore size of 0.2 μm and a flow rate of 200 mL / min. The permeate was collected.

[0139] (7) The permeate was filtered through an organic membrane with a pore size of 50,000 Daltons and a flow rate of 100 mL / min.

[0140] (8) The permeate is dried with hot air at a temperature of 50°C. After drying, a high content of polysaccharide is obtained.

[0141] (9) The yield obtained by the method described in Example 1 was 23%, and the polysaccharide content was 82%.

[0142] Example 3: Extraction method of polysaccharides from jujube seeds

[0143] Method: (1) Fresh jujube leaves were dried to constant weight to obtain dried jujube leaves;

[0144] (2) The dried leaves of the jujube were pulverized into dry powder with a particle size of 100μm using an ultra-micro pulverizer;

[0145] (3) Add water to the dry powder at a liquid-to-solid ratio of 200mL:1g, and then extract it by ultrasonic extraction. The power of ultrasonic extraction is 1000W, the temperature of ultrasonic extraction is 50℃, and the time of ultrasonic extraction is 120min to obtain the extract.

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

[0147] (5) Add 4 times its volume of anhydrous ethanol to the concentrate for precipitation, let it stand for 24 hours, then centrifuge for 15 minutes at a speed of 20,000 rpm, and collect precipitate A to obtain crude polysaccharide from jujube.

[0148] (6) The crude polysaccharide of jujube was dissolved in pure water at a ratio of 1:1, and then impurities were removed by a ceramic membrane with a pore size of 0.5 μm and a flow rate of 100 mL / min. The permeate was collected.

[0149] (7) The permeate was filtered through an organic membrane with a pore size of 20,000 Daltons and a flow rate of 150 mL / min.

[0150] (8) The permeate was vacuum microwave dried at a temperature of 45°C and a microwave power of 1000W. After drying, a high content of polysaccharide was obtained.

[0151] (9) The yield obtained by the method described in Example 1 was 22%, and the polysaccharide content was 78%.

[0152] Comparative Example 1

[0153] A polysaccharide from jujube seeds prepared by a method similar to that in Example 1, the difference being that ultrasonic extraction in step 1 was not performed, but a hot reflux extraction method was used, with a reflux extraction temperature of 80°C and a material-to-liquid ratio of 1:20, resulting in a yield of 19% and a polysaccharide content of 46%.

[0154] Comparative Example 2

[0155] A polysaccharide from jujube seeds prepared by a method similar to that in Example 1 was obtained, except that the ceramic membrane impurity removal step 1 was not performed, resulting in a yield of 22% and a polysaccharide content of 38%.

[0156] Comparative Example 3

[0157] A polysaccharide from jujube seeds prepared by a method similar to that in Example 1, the difference being that the organic membrane purification in step 1 was not performed, resulting in a yield of 23% and a polysaccharide content of 45%.

[0158] Comparative Example 4

[0159] Method: (1) Fresh jujube leaves were dried to constant weight to obtain dried jujube leaves;

[0160] (2) The dried leaves of the jujube were pulverized into dry powder with a particle size of 10 μm using an ultra-micro pulverizer;

[0161] (3) Add water to the dry powder at a liquid-to-solid ratio of 10 mL: 1 g, and then extract it using ultrasonic extraction. The ultrasonic extraction power is 900 W, the ultrasonic extraction temperature is 45 °C, and the ultrasonic extraction time is 45 min to obtain the extract.

[0162] (4) Extract the liquid and then remove impurities using a ceramic membrane with a pore size of 0.2 μm and a flow rate of 1500 mL / min. Collect the permeate.

[0163] (5) The extract is concentrated under reduced pressure (50-90℃, vacuum pressure 0.07MPa) and then dehydrated to a paste state to obtain a concentrated solution; the volume ratio of the concentrated solution to the extract is 1:6.

[0164] (6) Add three times the volume of anhydrous ethanol to the concentrate to precipitate it. After standing for 24 hours, centrifuge for 15 minutes at a speed of 20,000 rpm and collect the precipitate.

[0165] (7) Dissolve the precipitate with pure water of 1 times its mass, and filter it through an organic membrane of 10,000 Daltons at a flow rate of 100 mL / min to obtain the filtrate.

[0166] (8) The filtrate was concentrated and then freeze-dried at a cold trap temperature of -55℃ and a vacuum degree of 0.2Mpa to obtain jujube polysaccharide.

[0167] (9) The yield was 25%, and the polysaccharide content was 43%.

[0168] Example 4: Infrared Spectroscopy Determination of Polysaccharides

[0169] The polysaccharide from jujube seeds prepared in Example 2 was thoroughly mixed with KBr using an agate mortar and pressed into thin sheets. Fourier transform infrared spectroscopy was used to measure the polysaccharide, with a scanning range of 4000–4000 cm⁻¹. -1 The infrared spectrum obtained is as follows: Figure 1 As shown in the figure. It can be seen that at a wavenumber of 3297.16 cm⁻¹... -1 A strong hydroxyl stretching vibration peak is observed at 2928.39 cm⁻¹. -1 The peak at 1641.7 cm⁻¹ represents the CH stretching vibration. -1 The obvious absorption peak at 1400.9 cm⁻¹ may be due to the stretching vibration of the amide bond (I>C=O) or the bending of the peptide-amine bond (CN). Additionally, the peak at 1400.9 cm⁻¹... -1 The absorption peak at [location] is due to the extension of the >C=O group in the carboxyl group. The presence of characteristic hydroxyl groups preliminarily indicates that the extracted sample is a polysaccharide. Furthermore, in the fingerprint region of the spectrum, the main absorption peak is at 1034.3 cm⁻¹. -1 This is the CO absorption peak of the hydroxyl group of an alcohol, at 1231.6 cm⁻¹. -1 The weaker peak is the absorption peak of the COC group. Infrared spectroscopy shows that the extracted jujube polysaccharide sample contains most of the characteristic absorption peaks related to polysaccharides, and can be identified as a polysaccharide.

[0170] Example 5: Single-factor comparison experiment of process parameters in the extraction method of polysaccharides from jujube leaves:

[0171] 1. Selection of feed-to-liquid ratio:

[0172] Several portions of jujube seeds were weighed and pulverized into a dry powder with a particle size of 10 μm. Different volumes of water were added to each powder, with material-to-liquid ratios of 1g:5mL, 1g:25mL, 1g:50mL, 1g:75mL, 1g:100mL, 1g:125mL, 1g:150mL, 1g:175mL, 1g:200mL, 1g:225mL, and 1g:250mL, respectively. Each powder was then extracted using ultrasound at a power of 900W for 60 minutes at a temperature of 50℃. The extracts were then concentrated and dissolved in anhydrous ethanol at a ratio of 1... The mixture was prepared in a 5:1 ratio, allowed to stand for 24 hours, and then centrifuged for 15 minutes at 10,000 rpm to obtain a precipitate. The precipitate was dissolved in pure water at a ratio of 1:1 by weight and then filtered through a ceramic membrane (0.2 μm pore size, 100 mL / min). The permeate was collected. The permeate was then filtered through an organic membrane (20,000 Dalton pore size, 150 mL / min). The liquid was collected, freeze-dried, and the polysaccharide was obtained. The yield was calculated and the polysaccharide content was determined according to the method described in Example 1. The specific material-liquid ratio test results are shown in Table 1.

[0173] Table 1. Results of Single-Factor Comparison Experiment on Feed-to-Liquid Ratio

[0174]

[0175] The results are shown in Table 1. When the material-to-liquid ratio is between 1g:5mL and 1g:200mL, the polysaccharide content reaches over 65% and the yield is over 17%. When the material-to-liquid ratio is between 1g:125mL and 1g:200mL, the polysaccharide content reaches over 72% and the yield is over 21%. When the material-to-liquid ratio is 1g:150mL, the polysaccharide content is the highest at 75%, and the yield is the highest at 25%.

[0176] 2. Extraction power selection:

[0177] Several portions of jujube seeds were weighed and pulverized into a dry powder with a particle size of 10 μm. Different volumes of water were added to each sample, with a solid-liquid ratio of 1:150. Ultrasonic extraction was performed at power levels of 200W, 500W, 700W, 900W, 1200W, and 1500W for 45 minutes at 45℃ to obtain the extract. The extract was concentrated and mixed with anhydrous ethanol at a ratio of 1:5. After standing for 24 hours, the mixture was centrifuged for 15 minutes at 10,000 rpm. The precipitate was obtained; the precipitate was dissolved in pure water at a mass equal to its weight and then filtered through an inorganic membrane with a pore size of 0.2 μm and a flow rate of 150 mL / min. The permeate was collected. The permeate was then filtered through an organic membrane with a pore size of 10,000 Daltons and a flow rate of 100 mL / min. The liquid was collected and freeze-dried to obtain jujube polysaccharide. The yield was calculated and the polysaccharide content was determined according to the method described in Example 1. The specific material-liquid ratio test results are shown in Table 2.

[0178] Table 2. Results of Single-Factor Comparison Experiment on Extraction Power

[0179]

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

[0181] 3. Ultrasound timing selection:

[0182] Several portions of jujube seeds were weighed and pulverized into a dry powder with a particle size of 10 μm. Different volumes of water were added to each sample, with a solid-liquid ratio of 1:150. Ultrasonic extraction was performed at a power of 900 W for 15 min, 30 min, 45 min, 60 min, 75 min, 90 min, 105 min, and 120 min, at a temperature of 45℃ to obtain the extract. The extract was concentrated and mixed with anhydrous ethanol at a ratio of 1:5. After standing for 24 h, the mixture was centrifuged for 15 min. The flow rate was 0000 rpm to obtain a precipitate. The precipitate was dissolved in pure water at a volume equal to its weight and then filtered through an inorganic membrane with a pore size of 0.2 μm and a flow rate of 150 mL / min. The permeate was collected. The permeate was then filtered through an organic membrane with a pore size of 10000 Daltons and a flow rate of 100 mL / min. The liquid was collected and freeze-dried to obtain jujube polysaccharide. The yield was calculated and the polysaccharide content was determined according to the method described in Example 1. The specific material-liquid ratio test results are shown in Table 3.

[0183] Table 3. Results of Single-Factor Comparison Experiment on Ultrasonic Extraction Time

[0184]

[0185] The results are shown in Table 3. When the ultrasonic time is between 45 min and 105 min, the polysaccharide content reaches more than 63% and the yield is more than 20%. When the ultrasonic time is 45 min, the polysaccharide content is the highest, at 73%, and the yield is 22%.

[0186] 4. Ultrasonic temperature selection:

[0187] Several portions of jujube seeds were weighed and pulverized into dry powder with a particle size of 10 μm. Different volumes of water were added to each sample, with a material-to-liquid ratio of 1:150. Extraction was performed using ultrasound at a power of 900 W for 45 min at temperatures of 30℃, 45℃, 60℃, 75℃, and 90℃ to obtain extracts. The extracts were concentrated and mixed with anhydrous ethanol at a ratio of 1:5. After standing for 24 h, the mixture was centrifuged for 15 min at 10,000 rpm to obtain a precipitate. The precipitate was dissolved in an equal volume of pure water and filtered through an inorganic membrane with a pore size of 0.2 μm and a flow rate of 150 mL / min. The permeate was collected. The permeate was then filtered through an organic membrane with a pore size of 10,000 Daltons and a flow rate of 100 mL / min. The liquid was collected and freeze-dried to obtain jujube polysaccharide. The yield was calculated and the polysaccharide content was determined according to the method described in Example 1. The specific material-to-liquid ratio results are shown in Table 4.

[0188] Table 4. Results of Single-Factor Comparison Experiment on Ultrasonic Extraction Temperature

[0189]

[0190] The results are shown in Table 4. When the ultrasonic temperature is between 45℃ and 60℃, the polysaccharide content reaches more than 50% and the yield is more than 23%. When the ultrasonic temperature is 45℃, the polysaccharide content is the highest, at 65%, and the yield is 23%.

[0191] 5. Selection of ceramic membrane pore size:

[0192] Several portions of jujube seeds were weighed and pulverized into dry powder with a particle size of 10 μm. Different volumes of water were added to each sample, with a material-to-liquid ratio of 1:150. Extraction was performed using ultrasound at a power of 900 W for 45 min at a temperature of 45 °C. The extract was concentrated and mixed with anhydrous ethanol at a ratio of 1:5. After standing for 24 h, the mixture was centrifuged for 15 min at 10,000 rpm to obtain a precipitate. The precipitate was dissolved in an equal volume of pure water and filtered through inorganic membranes with pore sizes of 0.1 μm, 0.2 μm, 0.5 μm, and 1 μm, at a flow rate of 100 mL / min. The permeate was collected. The permeate was then filtered through an organic membrane with a pore size of 10,000 Daltons at a flow rate of 100 mL / min. The liquid was collected and freeze-dried to obtain jujube polysaccharide. The yield was calculated and the polysaccharide content was determined. The specific material-to-liquid ratio results are shown in Table 5.

[0193] Table 5. Results of Single-Factor Comparison Experiment on Pore Size of Ceramic Membranes

[0194]

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

[0196] 6. Ceramic membrane flow rate selection:

[0197] Several portions of jujube seeds were weighed and pulverized into a dry powder with a particle size of 10 μm. Different volumes of water were added to each sample, with a material-to-liquid ratio of 1:150. Ultrasonic extraction was performed separately at a power of 900 W for 45 min at a temperature of 45℃ to obtain the extract. The extract was concentrated and mixed with anhydrous ethanol at a ratio of 1:5. After standing for 24 h, the mixture was centrifuged for 15 min at 10,000 rpm to obtain the precipitate. The precipitate was dissolved in an equal volume of pure water and then filtered through an inorganic membrane. The pore size of the inorganic membrane was... The permeate was collected at flow rates of 50 mL / min, 100 mL / min, 150 mL / min, 200 mL / min, 250 mL / min, and 300 mL / min. The permeate was then filtered through an organic membrane with a pore size of 10,000 Daltons at a flow rate of 100 mL / min. The liquid was collected and freeze-dried to obtain jujube polysaccharide. The yield was calculated and the polysaccharide content was determined according to the method described in Example 1. The specific material-liquid ratio test results are shown in Table 6.

[0198] Table 6. Results of Single-Factor Comparison Experiment on Ceramic Membrane Flow Rate

[0199]

[0200] The results are shown in Table 6. When the ceramic membrane flow rate is between 150 mL / min and 200 mL / min, the polysaccharide content reaches 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, at 78%, and the yield is 28%.

[0201] 7. Selection of organic membrane pore size:

[0202] Several portions of jujube seeds were weighed and pulverized into a dry powder with a particle size of 10 μm. Different volumes of water were added to each sample, with a material-to-liquid ratio of 1:150. Each sample was then extracted using ultrasound at a power of 900 W for 45 min at a temperature of 45℃. The extracts were concentrated and mixed with anhydrous ethanol at a ratio of 1:5. After standing for 24 h, the mixture was centrifuged for 15 min at 10,000 rpm to obtain a precipitate. The precipitate was dissolved in an equal volume of pure water and then filtered through an inorganic membrane. The inorganic membrane had a pore size of 0.2 μm and a flow rate of 150 mL / min. The permeate was collected. The permeate was then filtered through an organic membrane with pore sizes of 5000 Daltons, 10000 Daltons, 30000 Daltons, 50000 Daltons, and 100000 Daltons and a flow rate of 100 mL / min. The liquid was collected and freeze-dried to obtain jujube polysaccharide. The yield was calculated and the polysaccharide content was determined according to the method described in Example 1. The specific material-liquid ratio test results are shown in Table 7.

[0203] Table 7. Results of Single-Factor Comparison Experiment on Pore Size of Organic Membranes

[0204]

[0205]

[0206] The results are shown in Table 7. When the pore size of the organic membrane is between 5,000 Daltons and 50,000 Daltons, the polysaccharide content reaches more than 63% and the yield is more than 25%. When the pore size of the organic membrane is between 10,000 Daltons and 30,000 Daltons, the polysaccharide content reaches more than 76% and the yield is more than 27%. When the pore size of the organic membrane is 10,000 Daltons, the polysaccharide content is the highest at 78% and the yield is the highest at 28%.

[0207] 8. Organic membrane flow rate selection:

[0208] Several portions of jujube seeds were weighed and pulverized into a dry powder with a particle size of 10 μm. Different volumes of water were added to each sample, with a material-to-liquid ratio of 1:150. Each sample was then extracted using ultrasound at a power of 900 W for 45 min at a temperature of 45℃. The extracts were concentrated and mixed with anhydrous ethanol at a ratio of 1:5. After standing for 24 h, the mixture was centrifuged for 15 min at 10,000 rpm to obtain a precipitate. The precipitate was then dissolved in an equal volume of pure water. The permeate was filtered through an inorganic membrane with a pore size of 0.2 μm and a flow rate of 150 mL / min. The permeate was then collected. The permeate was filtered through an organic membrane with a pore size of 10,000 Daltons and flow rates of 50, 100, 150, 200, 250, and 300 mL / min. The liquid was collected, freeze-dried, and the resulting polysaccharide was obtained from jujube. The yield was calculated and the polysaccharide content was determined according to the method described in Example 1. The results of the single-factor comparison experiment of the flow rate of the organic membrane are shown in Table 8.

[0209] Table 8. Results of Single-Factor Comparison Experiment on Organic Membrane Flow Rate

[0210]

[0211] The results are shown in Table 8. When the organic membrane flow rate is between 50 mL / min and 250 mL / min, the polysaccharide content reaches over 70% and the yield is over 23%. When the organic membrane flow rate is between 100 mL / min and 150 mL / min, the polysaccharide content reaches over 78% and the yield is over 27%. When the organic membrane flow rate is 100 mL / min, the polysaccharide content is the highest at 82%, and the yield is the highest at 31%.

[0212] Based on the single-factor comparative experiments 1-8 above, the optimal extraction method for *Ziziphus jujuba* leaf polysaccharides was obtained:

[0213] (1) After drying fresh jujube leaves to constant weight, dried jujube leaves are obtained;

[0214] (2) The dried leaves of the jujube were pulverized into dry powder with a particle size of 10 μm using an ultra-micro pulverizer;

[0215] (3) Add water to the dry powder at a liquid-to-solid ratio of 150mL:1g, and then extract it by ultrasonic extraction. The power of ultrasonic extraction is 900W, the temperature of ultrasonic extraction is 45℃, and the time of ultrasonic extraction is 45min to obtain the extract.

[0216] (4) The extract was concentrated under reduced pressure (50-90℃, vacuum pressure 0.07MPa) and then dehydrated to a paste-like state to obtain a concentrated solution;

[0217] (5) Add 5 times its volume of anhydrous ethanol to the concentrate for precipitation, let it stand for 24 hours, then centrifuge for 15 minutes at a speed of 10,000 rpm, and collect precipitate A to obtain crude polysaccharide from jujube.

[0218] (6) The crude polysaccharide of jujube was dissolved in pure water at a ratio of 1:1 by weight, and then impurities were removed by a ceramic membrane with a pore size of 0.2 μm and a flow rate of 150 mL / min. The permeate was collected.

[0219] (7) The permeate was filtered through an organic membrane with a pore size of 10,000 Daltons and a flow rate of 100 mL / min.

[0220] (8) The permeate is freeze-dried to obtain a high content of polysaccharides.

[0221] Example 6: Jujube polysaccharide has the effects of lowering blood lipids and inhibiting and improving obesity.

[0222] Experimental methods

[0223] Lipid assay: After fasting for 12 hours with free access to water, rats were anesthetized by anhydrous ether inhalation. Approximately 1.2 mL of blood was collected from the rats using a capillary glass tube and transferred to a sterile centrifuge tube. The tube was left to stand for 1 hour until the blood coagulated. The rats were then centrifuged at 10,000 rpm for 15 minutes to separate the serum. The levels of triglycerides (TG), total cholesterol (TCH / T-CHO), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured according to the instructions of the Nanjing Jiancheng Institute of Biology.

[0224] Lee's Index: After the last gavage administration, mice in each treatment group were fasted overnight, their body weight (g) was measured, and their body length (distance from the tip of the nose to the outer edge of the anus, cm) was measured with calipers. Lee's Index (an index used to assess obesity) was calculated. Where: Lee's Index = (body weight (g) / 1 / 3 × 10) / body length (cm).

[0225] Liver coefficient and fat body ratio: Rapidly dissect rats and peel off approximately 1 cm of abdominal skin tissue. 2 Peritesticular fat and liver were separated from rats, quickly rinsed with physiological saline, and dried with sterile coarse filter paper. The liver and fat masses were weighed, and the liver coefficient and fat-body ratio were calculated. Wherein: Liver coefficient = (liver mass / rat body mass) × 100%; Fat-body ratio (%) = (peritesticular fat mass / rat body mass) × 100%.

[0226] Liver and adipose tissue were fixed by soaking in 10% paraformaldehyde, and then fixed according to pathological tissue sampling, embedded, sectioned in paraffin, and frozen. The sections were then washed sequentially with environmentally friendly dewaxing solution I, environmentally friendly dewaxing solution II, anhydrous ethanol, and purified water.

[0227] Frozen sections were removed from the -20°C freezer and brought to room temperature. After fixation with tissue fixative for 15 minutes, they were rinsed with running water. Sections were stained with hematoxylin for 5 minutes, rinsed with tap water, differentiated with differentiation solution, rinsed with tap water, and then bluing with a blue solution, followed by rinsing with running water. Sections were then dehydrated sequentially with 85% and 95% graded alcohol solutions for 5 minutes each, and stained with eosin for 5 minutes. After immersion in anhydrous ethanol, sections were mounted with neutral resin and observed under a light microscope for pathological changes in the liver tissue.

[0228] Experimental setup and detection indicators

[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 invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for preparing polysaccharide from jujube seeds, comprising the following steps: 1) Mix the dried powder obtained by crushing the leaves of the Chinese jujube with water, and extract using ultrasonic extraction to obtain an extract; 2) The extract was concentrated and mixed with anhydrous ethanol, allowed to stand, and then centrifuged to obtain the precipitate; 3) Dissolve the precipitate in water, filter it through a ceramic membrane, and collect the permeate; 4) After filtering the permeate with an organic membrane, the liquid is collected, dried, and then the jujube polysaccharide is obtained. In step 1), the mass-to-volume ratio of the pulverized dry powder to water is 1g:(5-200)mL; The ultrasonic power of the ultrasonic extraction method in step 1) is 200~1500W; The ultrasonic extraction time in step 1) is 45 min to 105 min; The extraction temperature of the ultrasonic extraction method in step 1) is 42~47 ℃; The pore size of the ceramic membrane in step 3) is 0.1~1μm; The flow rate of the ceramic membrane in step 3) is 150 mL / min to 200 mL / min; The retention capacity of the organic membrane in step 4) is 5000~50000 Daltons; The flow rate of the organic membrane in step 4) is 50 mL / min to 250 mL / min.

2. The preparation method according to claim 1, wherein in step 1), the fresh jujube leaves are dried before pulverization to obtain dried jujube leaves; and / or, The particle size of the pulverized dry powder in step 1) is 5–100 μm; and / or, In step 1), the mass-to-volume ratio of the pulverized dry powder to water is 1g:(100-200)mL; and / or, The ultrasonic power of the ultrasonic extraction method in step 1) is 900~1500W; and / or, The ultrasonic extraction time in step 1) is 45 min; and / or, The extraction temperature of the ultrasonic extraction method in step 1) is 45 ℃.

3. According to the preparation method of claim 2, the particle size of the pulverized dry powder in step 1) is 5~50 μm; and / or, In step 1), the mass-to-volume ratio of the pulverized dry powder to water is 1g:(125-200)mL; and / or, The ultrasonic power of the ultrasonic extraction method in step 1) is 900W.

4. According to the preparation method of claim 3, the particle size of the pulverized dry powder in step 1) is 5~10 μm; and / or, In step 1), the mass-to-volume ratio of the pulverized dry powder to water is 1g:150mL.

5. The preparation method according to claim 1, wherein the volume ratio of the extract to anhydrous ethanol in step 2) is 1:(3-10); and / or, The settling time in step 2) is 12~48h; and / or, The centrifugation time in step 2) is 5-30 min; and / or, The centrifugal speed in step 2) is 10,000 to 20,000 rpm.

6. The preparation method according to claim 5, wherein the volume ratio of the extract to anhydrous ethanol in step 2) is 1:(3-5); and / or, The settling time in step 2) is 24 hours; and / or, The centrifugation time in step 2) is 10-20 min.

7. The preparation method according to claim 6, wherein the centrifugation time in step 2) is 15 min.

8. The preparation method according to claim 1, wherein the mass ratio of the precipitate to water in step 3) is 1:(1-20); and / or, The pore size of the ceramic membrane in step 3) is 0.2~0.5μm; and / or, The flow rate of the ceramic membrane in step 3) is 150 mL / min.

9. The preparation method according to claim 8, wherein the mass ratio of the precipitate to water in step 3) is 1:(1-10); and / or, The pore size of the ceramic membrane in step 3) is 0.2 μm.

10. The preparation method according to claim 9, wherein the mass ratio of the precipitate to water in step 3) is 1:(1-5).

11. The preparation method according to claim 1, wherein the organic membrane in step 4) has a retention capacity of 10,000 to 30,000 Daltons; and / or, The flow rate of the organic membrane in step 4) is 100~150mL / min.

12. The preparation method according to claim 11, wherein the organic membrane in step 4) has a retention capacity of 10,000 Daltons; and / or, The flow rate of the organic membrane in step 4) is 100 mL / min.

13. The preparation method according to any one of claims 1 to 12, wherein the drying includes one or more of the following: freeze drying, hot air drying, spray drying, steam drying, vacuum drying, microwave drying, or vacuum microwave drying.

14. The preparation method according to claim 13, wherein the cold trap temperature for freeze drying is -60 to -50 °C, and the vacuum degree is 0.1 to 1 MPa; and / or, The temperature of the hot air drying is 40~60℃; and / or, The vacuum microwave drying temperature is 40~50 ℃; the microwave power is 800~1200W.

15. The preparation method according to claim 14, wherein the freeze-drying cold trap temperature is -55 °C and the vacuum degree is 0.2 MPa; and / or, The hot air drying temperature is 50 °C; and / or, The vacuum microwave drying temperature is 45 ℃; the microwave power is 1000W.

16. Jujube polysaccharide prepared according to any one of claims 1 to 15.

17. The use of the polysaccharide from jujube seed according to claim 16 in the preparation of a medicament for the prevention or treatment of dyslipidemia, obesity, and / or improvement of lipid metabolism.

18. The use of the polysaccharide from jujube seed according to claim 16 in the preparation of a medicament for improving dyslipidemia, obesity, and / or improving lipid metabolism.

19. The use of the polysaccharide from jujube seed according to claim 16 in the preparation of a medicament for controlling dyslipidemia, obesity, and / or improving lipid metabolism.

20. The use of the polysaccharide from jujube seed according to claim 16 in the preparation of a medicament for alleviating dyslipidemia, obesity, and / or improving lipid metabolism.

21. The application according to any one of claims 17-20, characterized in that, The dyslipidemia mentioned above includes hyperlipidemia.

22. The use of the jujube polysaccharide of claim 16 in the preparation of health products that help maintain healthy blood lipid levels.

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