Use of mulberry extract in preparation of medicament for treating and / or preventing diabetic vascular complications

Abstract

Use of a mulberry extract in the preparation of a medicament for treating and / or preventing diabetic vascular complications. The mulberry extract comprises an alkaloid, and the alkaloid comprises 1-deoxynojirimycin. The diabetic vascular complications include diabetic foot or diabetic atherosclerosis. The mulberry extract has a significant ameliorating effect on the diabetic vascular complications.

Description

Use of mulberry extract in preparation of medicine for treating and / or preventing diabetic vascular complications TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and relates to use of mulberry extract in preparation of medicine for treating and / or preventing diabetic vascular complications. BACKGROUND

[0002] The Global Diabetes Map 10th Edition released by the International Diabetes Federation at the end of 2021 reports that diabetes is one of the fastest growing global public health emergencies. Diabetes is a chronic metabolic disease, and high blood sugar does not cause death, but its complications can bring death risk.

[0003] The pathogenesis of diabetic vascular complications is a complex process involving multiple factors, including high blood sugar, glycosylation end products, oxidative stress and inflammation. Long-term high blood sugar can damage vascular endothelial cells, leading to vascular dysfunction and increased permeability. At the same time, high blood sugar leads to the accumulation of glycosylation end products formed by the combination of glucose and protein in the blood vessel wall. In addition, diabetic patients often have increased oxidative stress, leading to increased production of free radicals, which damages vascular endothelial cells and blood vessel walls. In general, the various pathophysiological changes caused by high blood sugar are the main mechanisms of diabetic vascular complications, and vascular damage and abnormal blood circulation both play important roles in this process.

[0004] Endothelial cells are a continuous monolayer of cells covering the inner walls of arteries, veins and other blood vessels. Their total area is 400 square meters. In addition to the mechanical barrier function, vascular endothelial cells can also synthesize and secrete substances that regulate the coagulation-fibrinolysis system, factors that regulate vascular tone, and cytokines, etc., and play an important role in blood vessel formation, wound healing, anti-thrombosis and inflammatory response. Therefore, once the endothelial function is impaired, it will affect various organs and functions. High blood sugar is an important factor affecting vascular endothelial function, and vascular endothelial cell dysfunction is also the initiating factor of diabetic vascular complications, both of which contribute to the development of the disease. Addressing the vascular endothelial damage caused by high blood sugar has become an important way to prevent and treat diabetic vascular complications.

[0005] Experimental studies and clinical applications have confirmed that intensive blood glucose control cannot reduce the incidence of diabetic vascular complications, and the use of hypoglycemic drugs alone cannot achieve satisfactory therapeutic effect, and some complications will still progress. The use of drugs with multiple vascular endothelial protection effects is an effective treatment strategy, which can achieve the effect of treating diabetic vascular complications on the basis of improving vascular endothelial cell function. SUMMARY

[0006] The present invention aims to provide the use of mulberry extract in the preparation of medicaments for treating and / or preventing vascular complications of diabetes.

[0007] In one embodiment of the present invention, a method for treating and / or preventing diabetic vascular complications is provided, comprising administering an effective amount of mulberry extract to a subject in need.

[0008] In one embodiment of the present invention, the use of mulberry extract in the treatment and / or prevention of diabetic vascular complications is provided.

[0009] In one embodiment of the present invention, a mulberry extract is also provided, which is used to treat and / or prevent diabetic vascular complications.

[0010] In this invention, based on the weight percentage of the sum of all components of the mulberry extract as 100%, the mulberry extract contains alkaloids at a weight content of 3% or more (optionally containing alkaloids at a weight content of 3-99%, further optionally containing alkaloids at a weight content of 15-99%, further optionally containing alkaloids at a weight content of 30-99%, further optionally containing alkaloids at a weight content of 40-99%, further optionally containing alkaloids at a weight content of 50-99%, further optionally containing alkaloids at a weight content of 60-99%).

[0011] And / or contains polysaccharides with a weight content not exceeding 70% (optionally containing 0.2-70% polysaccharides, further optionally containing 0.2-50% polysaccharides, further optionally containing 0.2-35% polysaccharides, further optionally containing 0.2-25% polysaccharides, further optionally containing 0.2-23% polysaccharides, further optionally containing 20-25% polysaccharides).

[0012] And / or contains no more than 10% by weight of flavonoids (optionally containing 0.05-5% by weight of flavonoids, further optionally containing 0-2% by weight of flavonoids, further optionally containing 0.05-2% by weight of flavonoids, further optionally containing 0.5-1.5% by weight of flavonoids, further optionally containing 0-1% by weight of flavonoids, further optionally containing 0.05-1% by weight of flavonoids),

[0013] And / or contains amino acids at a weight content not exceeding 50% (optionally containing 0-30% amino acids, further optionally containing 0-25% amino acids, further optionally containing 0-20% amino acids, further optionally containing 0-5% amino acids, further optionally containing 3-25% amino acids, or further optionally containing 5-20% amino acids).

[0014] And / or other components (optionally 0-25% by weight, further optional 0-20% by weight, further optional 0-15% by weight, further optional 0-11% by weight, further optional 2-20% by weight, further optional 4-8% by weight).

[0015] In a preferred embodiment of the present invention, the treatment and / or prevention of diabetic vascular complications manifests as improvement of vascular endothelial cell dysfunction; preferably specifically manifested as improvement of any one or more of the following:

[0016] (1) Promotes the proliferation of vascular endothelial cells;

[0017] (2) Promotes the migration of vascular endothelial cells;

[0018] (3) Promotes angiogenesis;

[0019] (4) Inhibits apoptosis of vascular endothelial cells;

[0020] (5) Promotes NO accumulation in endothelial cells;

[0021] (6) Upregulate the expression of vasoactive factors, preferably including CD31 (platelet-endothelial cell adhesion factor-1), VEGF (vascular endothelial growth factor) (preferably VEGF-A) and / or eNOS (endothelial nitric oxide synthase);

[0022] (7) Relieve vascular endothelial inflammation, preferably by inhibiting the adhesion of vascular endothelial cells to monocytes, and more preferably by inhibiting the secretion of vascular endothelial cell adhesion factors, wherein the adhesion factors include P-selectin and / or E-selectin;

[0023] (8) Increase the number of mitochondria in vascular endothelial cells.

[0024] Furthermore, the treatment and / or prevention of diabetic vascular complications works by improving endothelial cell dysfunction caused by high glucose, high insulin, and / or inflammatory conditions, wherein the high glucose conditions include high concentrations of glucose and / or glycation products (such as advanced glycation end products, AGEs), and the inflammatory conditions include induction and / or stimulation by inflammatory factors (such as TNF-α).

[0025] Preferably, the improvement in vascular endothelial cell function is further manifested in promoting the expression of NRF2 (nuclear factor erythroid lineage 2-related factor 2) and / or HO-1 (heme oxygenase 1).

[0026] In a preferred embodiment of the present invention, the diabetic vascular complications include diabetic foot or diabetic atherosclerosis.

[0027] In a preferred embodiment, the diabetic foot includes the presence or absence of ulcers, ulcers, or symptoms such as infection or gangrene.

[0028] Preferably, the treatment and / or prevention of diabetic foot is manifested in any one or more of the following:

[0029] (1) Promotes wound healing, including promoting wound regeneration, re-epithelialization, and / or collagen production.

[0030] (2) Reduces cell apoptosis in the wound area

[0031] (3) Promotes cell migration and / or proliferation in the wound area.

[0032] (4) Promotes angiogenesis, remodeling and / or repair in the wound area, preferably promotes the expression of CD31, VEGF (preferably VEGF-A) and / or eNOS.

[0033] Preferably, the treatment and / or prevention of diabetic atherosclerosis is manifested in any one or more of the following:

[0034] (1) Reduce the formation of arterial plaques

[0035] (2) Improves arterial blood flow,

[0036] (3) Relieve vascular endothelial inflammation, preferably by reducing the level of vascular endothelial cell adhesion factors, including P-selectin and / or E-selectin.

[0037] In a preferred embodiment of the present invention, taking the total weight percentage of all components of the mulberry extract as 100%, the weight percentage of each component in the mulberry extract is as follows:

[0038] Preferably, with the sum of the weight percentages of all components of the mulberry extract being 100%, the weight percentages of each component in the mulberry extract are as follows:

[0039] Preferably, with the sum of the weight percentages of all components of the mulberry extract being 100%, the weight percentages of each component in the mulberry extract are as follows:

[0040] Preferably, with the sum of the weight percentages of all components of the mulberry extract being 100%, the weight percentages of each component in the mulberry extract are as follows:

[0041] Preferably, with the sum of the weight percentages of all components of the mulberry extract being 100%, the weight percentages of each component in the mulberry extract are as follows:

[0042] Preferably, with the sum of the weight percentages of all components of the mulberry extract being 100%, the weight percentages of each component in the mulberry extract are as follows:

[0043] Preferably, with the sum of the weight percentages of all components of the mulberry extract being 100%, the weight percentages of each component in the mulberry extract are as follows:

[0044] More preferably, based on the sum of the weight percentages of all components of the mulberry extract as 100%, the weight percentages of each component in the mulberry extract are as follows:

[0045] More preferably, based on the sum of the weight percentages of all components of the mulberry extract as 100%, the weight percentages of each component in the mulberry extract are as follows:

[0046] Preferably, the alkaloid comprises 1-deoxynojirimycin (DNJ), N-methyl-1-deoxynojirimycin, fagomine (FAG), 3-epi-fagomine, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), calystegin B2, calystegin C1, 2-oxo-(α-D-) One or more of the following: galactopyranosyl-1-deoxynojirimycin, 6-oxo-(β-D-glucopyranosyl)-1-deoxynojirimycin, and 1,4-dideoxy-1,4-imino-(2-oxo-β-D-glucopyranosyl)-D-arabinitol.

[0047] Preferably, the weight percentage of DNJ is not less than 50% of the alkaloids (preferably 60-99%).

[0048] Preferably, the heavy metal content of the mulberry extract does not exceed 10 ppm.

[0049] The drug is an oral dosage form; optionally, the drug is a tablet, capsule, tablet-containing tablet, powder, oral solution, oral emulsion, pill, granule, syrup or powder.

[0050] In this invention, the mulberry extract can be provided in the form of commercially available mulberry twig alkaloid tablets (National Drug Approval Number Z20200002).

[0051] Optionally, the mulberry extract can be prepared according to the method described in CN 110393738A.

[0052] Optionally, in this invention, the preparation of the mulberry extract includes the following steps:

[0053] 1) Prepare a crude extract of mulberry plants; 2) Separate the crude extract using a cation exchange resin and / or optionally an anion exchange resin to obtain a resin eluent; Optional step 3): Perform alcohol precipitation on the resin eluent from step 2) and collect the supernatant; 4) Concentrate and / or dry the supernatant. Optionally, the resin eluent from step 2) is concentrated and / or dried before alcohol precipitation.

[0054] Preferably, the extraction method further includes: a step of concentrating the crude plant extract before separation in step 2); preferably, the crude plant extract is concentrated to a solids concentration of 1-15%, more preferably 2-10%. The solids refer to the solid substances remaining after water is removed from the solution.

[0055] Optionally, the concentrated crude extract can be subjected to alcohol precipitation before the resin separation treatment in step 2). During alcohol precipitation, ethanol is added to the crude extract, stirred until homogeneous, and then allowed to stand for a certain period to allow the insoluble substances to precipitate. Optionally, the volume-to-mass ratio of ethanol added to the plant raw material is 0.2-20 times, preferably 0.4-10 times, based on L / kg. Further, optionally, an alcohol precipitation tank is used for alcohol precipitation. Optionally, the stirring speed during alcohol precipitation is 10-600 rpm, preferably 40-500 rpm, and further preferably 80-400 rpm or 300 rpm.

[0056] Optionally, the Moraceae plants mentioned are: *Morus multicaulis* Perrott., *Morus alba* L., *Morus atropurpurea* Roxb., *Morus mizuho* Hotta, *Morus wittiorum* Hand Mazz., *Morus laevigata* Wall., *Morus nigra* Linn., *Morus cathayana* Hemsi., *Morus serrata* Roxb., *Morus mongolica* Schneid., *Morus bombycis* Koidz., *Morus notabilis* Schneid., *Morus nigriformis* Koidz., *Morus yunnanensis* Koidz., *Morus australis* Poir., and *Morus mongolica(Bur.)Schneid var. diabolica*. Mulberry species including Koidz., large-leaved mulberry, weeping mulberry (Morus alba var. Pendula Dippel), white-veined mulberry, and mulberry varieties bred from the above species, as well as hybrid mulberry species selected from within or between species of the above species.

[0057] Optionally, the mulberry plant is selected from one or more of Guangdong mulberry, Lu mulberry, white mulberry, fine-toothed mulberry, mountain mulberry, or hybrid mulberry. The hybrid mulberry can be selected from Yue Sang 11, Gui Sang You 62, or Sang Te You 2. Various parts of the mulberry plant, such as leaves, roots, branches, bark, buds, stems, and fruits, can be used, and mulberry branches, leaves, or white bark can be selected.

[0058] In this invention, the mulberry extract may be selected as mulberry twig extract, mulberry bark extract, mulberry leaf extract, or a mixture thereof.

[0059] Optionally, the crude extraction of the mulberry plant can be performed using solvents such as alcohol-water, water, alkaline aqueous solution, or acidic aqueous solution. During extraction, it is preferable to pulverize the plant and then add it to the solvent for thermal extraction. Optionally, the extraction can be repeated 1-3 times, with the extraction time preferably being 0.5-3 hours each time, and more preferably 1-3 hours each time.

[0060] In a preferred embodiment, the pulverized plant material can be added to an extraction tank for extraction.

[0061] Preferably, the extract is filtered to remove insoluble matter, yielding a crude plant extract.

[0062] In one embodiment, the mulberry extract is prepared according to the following steps: pulverizing mulberry plants, extracting them by heating and reflux with water and / or alcohol solution or acidic water, the solvent volume being 3-20 times (optionally 4-15 times) of the original medicinal material, repeating the extraction 1-3 times (extraction time can be 0.5-3 hours each time, further optionally 1-3 hours each time), combining the extracts, concentrating, loading onto a cation exchange resin, eluting with 0.2-3N ammonia water, loading the eluent onto anion exchange resin, collecting the non-adsorbed portion, adding ethanol, precipitating to remove impurities, concentrating and / or drying to obtain the extract.

[0063] In one embodiment, the mulberry extract is prepared according to the following steps: pulverizing mulberry plants, extracting them by heating and reflux with water and / or alcohol solution or acidic water, the solvent volume being 3-20 times (preferably 4-15 times) of the original medicinal material, repeating the extraction 1-3 times (the extraction time can be selected as 0.5-3 hours each time, and further selected as 1-3 hours each time), combining the extracts, concentrating, loading onto a cation exchange resin, eluting with 0.2-3N ammonia water, loading the eluent onto anion exchange resin, collecting the non-adsorbed portion, concentrating and / or drying to obtain the extract.

[0064] In one embodiment, the mulberry extract is prepared according to the following steps: pulverizing mulberry plants, extracting them by heating and reflux with water and / or alcohol solution or acidic water, the solvent volume being 3-20 times (optionally 4-15 times, further optionally 4-12 times) of the original medicinal material, repeating the extraction 1-3 times (extraction time optional 0.5-3 hours each time, further optionally 1-3 hours each time), combining the extracts, concentrating, loading onto a cation exchange resin, eluting with 0.2-3N ammonia water, concentrating and / or drying the eluent to obtain the extract.

[0065] Optionally, after packing the cation exchange resin column, activation is performed in the order of acidic solution washing, alkaline solution washing, and then acidic solution washing again. Optionally, alkaline solution washing is performed until the pH of the eluent is 8.0-9.5, preferably 8.5-9.5; optionally, the alkaline solution is selected from ammonia solution, sodium hydroxide solution, potassium hydroxide solution, or sodium carbonate solution; optionally, the concentration of the alkaline solution is 0.5-4 mol / L, preferably 1-2 mol / L. Optionally, acidic solution washing is performed until the pH of the eluent is 3.0-7.0, preferably 4.5-6.5. Optionally, the acidic solution is selected from hydrochloric acid solution, phosphoric acid solution, or disodium hydrogen phosphate-citric acid buffer; optionally, the concentration of the acidic solution is 0.5-4 mol / L, preferably 1.5-2 mol / L. Optionally, after the final acidic solution washing, the cation exchange resin can also be rinsed with 3-5 column volumes of deionized water.

[0066] Preferably, the cation exchange resin is selected from one or more combinations of strongly acidic cation exchange resins, weakly acidic cation exchange resins, and strongly basic quaternary ammonium cation exchange resins. Optionally, the cation exchange resin is one or more combinations of 732 type strongly acidic styrene-based cation exchange resin, 002SC type strongly acidic styrene-based cation exchange resin, 734 type strongly acidic styrene-based cation exchange resin, D001 type macroporous strongly acidic styrene-based cation exchange resin, or D113 type macroporous weakly acidic cation exchange resin and D254 type macroporous strongly basic quaternary ammonium cation exchange resin. Preferably, the cation exchange resin is one or more combinations of 732 type strongly acidic styrene-based cation exchange resin, 734 type strongly acidic styrene-based cation exchange resin, 002SC type strongly acidic styrene-based cation exchange resin, D113 type macroporous weakly acidic cation exchange resin, and D001 type macroporous strongly acidic styrene-based cation exchange resin. Preferably, the cation exchange resin is one or more of the following: 002SC type strong acid styrene-based cation exchange resin, D113 type macroporous weak acid cation exchange resin, and D001 type macroporous strong acid styrene-based cation exchange resin.

[0067] Optionally, the ratio of the amount of cationic resin to the weight of the plant raw materials is 1:1-30 (optionally, 1:1-25, 1:2-20, 1:2-15, 1:2-10, 1:2-7, 1:2-3, 1:4-15).

[0068] After loading the crude plant extract onto the cation exchange resin, the resin is eluted with an eluent. Optionally, the eluent is a salt solution or alkaline solution containing cations, and may be one or more of sodium chloride, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonia, potassium chloride, and sodium hydroxide.

[0069] Optionally, the concentration of cations in the eluent is 0.04-5 mol / L (optionally 0.5-2.5 mol / L, 0.2-3 mol / L, and further optionally 0.5-2.5 mol / L).

[0070] Optionally, the eluent flow rate is 1-15 BV / h (optionally 5-10 BV / h, further optionally 5-6 BV / h).

[0071] Optionally, the eluent used for cation exchange is 0.1-30 times the weight of the plant raw material; alternatively, the eluent is 0.5-10 times the weight of the plant raw material; and further alternatively, 1-10 times.

[0072] The collection starting point can be determined based on the pH of the cation exchange resin effluent. For example, when using an alkaline solution such as ammonia for elution, the eluent can be collected when the pH of the cation exchange column effluent is >7. Alternatively, the collection starting point can be determined based on colorimetric or precipitation reactions. Optionally, collection can be stopped when the volume of the collected liquid reaches 0.1-10 times (more preferably 0.6-10 times, or 0.2-5 times) the weight of the plant raw material. The collected liquid can then be optionally purified using an anion exchange column. To improve the separation efficiency of the cation exchange resin, multiple separations can be performed, for example, 2-5 times.

[0073] During anion exchange column purification, optionally, after packing the anion exchange resin column, activation can be performed in the order of washing with alkaline solution, acidic solution, and alkaline solution again.

[0074] Optionally, the alkaline solution is used to wash until the pH of the washing solution is 8.0-9.5, preferably pH 8.5-9.5;

[0075] Optionally, the alkaline solution is selected from ammonia solution, sodium hydroxide solution, potassium hydroxide solution, or sodium carbonate solution; optionally, the concentration of the alkaline solution is 0.5-4 mol / L, and optionally 1-2 mol / L.

[0076] Optionally, the acidic solution is used to wash until the pH of the washing solution is 3.0-7.0, preferably 3.5-6.5. Optionally, the acidic solution is selected from hydrochloric acid solution, phosphoric acid solution, and disodium hydrogen phosphate-citrate buffer solution. Optionally, the concentration of the acidic solution is 0.5-4 mol / L, preferably 1-2 mol / L.

[0077] Preferably, the anion exchange resin is selected from one or more combinations of strongly basic anion exchange resins, weakly basic anion exchange resins, or weakly acidic anion exchange resins. Optionally, the anion exchange resin is selected from one or more combinations of 711 type strongly basic styrene-based anion exchange resin, 717 type strongly basic styrene-based anion exchange resin, D201 type macroporous strongly basic styrene-based anion exchange resin, D218 type macroporous strongly basic acrylic anion exchange resin, D301-G type macroporous weakly acidic styrene-based anion exchange resin, and D301 type macroporous weakly basic styrene-based anion exchange resin. Preferably, the anion exchange resin is selected from one or more combinations of 711 type strongly basic styrene-based anion exchange resin, 717 type strongly basic styrene-based anion exchange resin, D201 type macroporous strongly basic styrene-based anion exchange resin, and D218 type macroporous strongly basic acrylic anion exchange resin. Preferably, the anion exchange resin is one or a combination of 711 type strong base styrene-based anion exchange resin, D201 type macroporous strong base styrene-based anion exchange resin, and D218 type macroporous strong base acrylic anion exchange resin.

[0078] Optionally, the ratio of the amount of anion exchange resin to the weight of the plant raw materials is 1:1-80 (optionally, 1:1-64, 1:1-32, 1:1-24, 1:5-16, 1:3).

[0079] Collection begins when the liquid flows out of the anion exchange resin. Optionally, collection stops when the volume of the collected liquid reaches 0.05-10 times (optionally 0.1-5 times) the weight of the plant raw material. Optionally, to improve the separation effect of the anion exchange resin, multiple separations can be performed, for example, 2-4 times.

[0080] Optionally, the weight ratio of ethanol to plant raw materials used in the alcohol precipitation treatment is 1:4-600 (optionally 1:20-300, further options include 1:20-200, 1:20-50, 1:40, 1:80, 1:22, 1:200). During the alcohol precipitation treatment, the stirring speed is 10-600 rpm (optionally 40-500 rpm, 80-400 rpm, 300-400 rpm). The alcohol precipitation treatment time is 12-24 hours.

[0081] Furthermore, prior to the alcohol precipitation treatment, the process includes centrifuging or microfiltration to remove impurities from the anion exchange resin effluent, followed by concentration via a reverse ion exchange membrane. The specific gravity of the concentrated liquid can be 1.0-1.3, optionally 1.1-1.25. This specific gravity refers to the mass ratio of the concentrated liquid to water under the same volume conditions.

[0082] The concentration process of the present invention may include thermal concentration, nanofiltration membrane concentration, reverse ion osmosis membrane concentration, or combinations thereof. Preferably, centrifugation, ultrafiltration membrane filtration, or microfiltration membrane filtration are performed to remove impurities before reverse ion osmosis membrane and nanofiltration membrane concentration.

[0083] Optionally, the drug may further include a pharmaceutically acceptable carrier. The carrier is an inactive ingredient that is non-toxic to humans and conforms to the route of administration or method of delivery. The carrier may be a solid or liquid excipient. Solid excipients include, for example, microcrystalline cellulose, mannitol, lactose, pregelatinized starch, low-substituted hydroxypropyl cellulose, crospovidone, sodium carboxymethyl starch, aspartame, calcium hydrogen phosphate, sodium lactate, poloxamer, sodium lauryl sulfate, sodium carboxymethyl cellulose, gelatin, xanthan gum, povidone, starch, magnesium stearate, sodium carboxymethyl starch, and talc; liquid excipients include, for example, water, ethanol, syrup, and glycerin.

[0084] The beneficial effects of this invention are:

[0085] The mulberry extract of the present invention can promote the proliferation and migration of vascular endothelial cells, inhibit apoptosis, promote angiogenesis, and protect vascular endothelial cells from AGEs-induced oxidative stress damage.

[0086] The mulberry extract of this invention regulates endothelial cell function by activating the NRF2 / HO-1 / eNOS signaling pathway, including promoting the expression of NRF2 and HO-1, increasing the NO content of endothelial cells, and promoting the expression of vasoactive factors, including promoting the expression of CD31, VEGF-A, or eNOS.

[0087] The mulberry extract of this invention can inhibit the adhesion of HUVECs to monocytes induced by high concentrations of glucose and TNF-α, thereby alleviating the local amplification of inflammatory responses.

[0088] The mulberry extract of the present invention can improve endothelial cell dysfunction induced by high insulin, increase the number of endothelial cell mitochondria, improve mitochondrial damage, and improve vascular endothelial cell function.

[0089] Animal experiments have shown that the mulberry extract of this invention can promote wound healing in diabetic foot ulcers, including promoting wound regeneration and re-epithelialization, and promoting collagen production; reducing cell apoptosis in the wound area, promoting cell proliferation, promoting angiogenesis or vascular remodeling and / or repair, and promoting the expression of VEGF-A, CD31, and / or eNOS. The mulberry extract of this invention can alleviate diabetic atherosclerosis: reducing arterial plaque formation, improving arterial blood flow, and lowering the levels of vascular endothelial adhesion factors, including P-selectin and / or E-selectin. Attached Figure Description

[0090] Figure 1 shows the effect of different concentrations of SZ-A on the viability of HUVECs in Experiment 1. Statistical analysis was performed using GraphPad Prism 8. Data are expressed as mean ± SD. **P<0.01.

[0091] Figure 2 shows the effect of SZ-A on AGEs-induced functional impairment of HUVECs in Experimental Example 1.

[0092] a and b are microscopic images of tube formation determination and quantitative analysis of tube segment length, respectively (n=3).

[0093] c) Microscopic images of cell scratches observed at 0, 8, 16, and 24 h; d, e, and f) Graphs showing quantitative analysis of wound closure rates at 8, 16, and 24 h, respectively (n=5).

[0094] g and h are microscopic images of cell migration in the Transwell assay and quantitative analysis images, respectively (n=4).

[0095] i and j are the results of flow cytometry and the quantitative analysis of apoptosis, respectively (n=3).

[0096] k and l are TUNEL staining map and quantitative analysis map, respectively (n=4), *p<0.05, **p<0.01, ***p<0.001.

[0097] Figure 3 shows the effect of SZ-A on the NRF2 signaling pathway in HUVECs treated by AGEs in Experiment Example 1.

[0098] Where a, b, and c are Western blotting and quantitative statistical graphs of the relative levels of NRF2 and HO-1 proteins in HUVECs after SZ-A intervention and AGEs treatment, respectively (n=4); d and e are immunofluorescence graphs and fluorescence intensity statistical analysis graphs of NRF2 in HUVECs after SZ-A intervention and AGEs treatment, respectively (n=4). *p<0.05, **p<0.01.

[0099] Figure 4 shows the effect of SZ-A on vasoactivity-related factors in HUVECs treated with AGEs in Experiment 1.

[0100] Wherein, a, b, c, and d are Western blotting and quantitative statistical graphs of the relative protein levels of CD31, VEGF-A, and eNOS in HUVECs after SZ-A intervention and AGEs treatment, respectively (n=4); e and f are immunofluorescence and quantitative statistical graphs of NO levels in HUVECs determined by DAF-FM DA method, respectively (n=4); g, h, i, and j are immunofluorescence and statistical analysis graphs of CD31 and VEGF-A, respectively (n=3); k, l, m, n, o, and p are Western blotting and statistical results of relative protein levels of NRF2, HO-1, CD31, VEGF-A, and eNOS after 24h of intervention with or without ML385, respectively (n=4). *p<0.05, **p<0.01, ***p<0.001, and ns indicate no significant difference.

[0101] Figure 5 shows the effect of SZ-A on the adhesion of HUVECs to Thrp-1 induced by high glucose and TNF-α in Experiment Example 2;

[0102] Where a is a microscopic image of HUVECs with green fluorescent labeling Thp-1, b is a quantitative statistical graph of fluorescence intensity within the same area (n=4), ** indicates P<0.01 compared with the model, and *** indicates P<0.001 compared with the model.

[0103] Figure 6 shows the effect of SZ-A on apoptosis of HUVECs induced by high glucose and TNF-α in Experiment Example 2;

[0104] Where a is a flow cytometry analysis graph, b is a late apoptosis rate statistical graph (n=4), and * indicates P<0.05 compared with the model.

[0105] Figure 7 shows the effect of SZ-A on scratch healing of HUVECs induced by high glucose and TNF-α in Experiment 2;

[0106] Where a is a visual image of cell scratches under a microscope, and b is a statistical graph of the healing rate of each group of scratches (n=4). * indicates P<0.05 compared with the model, ** indicates P<0.01 compared with the model, and *** indicates P<0.001 compared with the model.

[0107] Figure 8 shows the effect of SZ-A on hyperinsulinemia-induced mitochondrial damage in HUVECs in Experiment Example 3.

[0108] Where a is a mitochondrial fluorescence microscopy image of HUVECs in each group, b is a statistical graph of fluorescence intensity in each group, and * indicates P<0.05 compared with the model.

[0109] Figure 9 shows the effect of SZ-A on wound healing in diabetic mice in Experiment 4;

[0110] In the figure, a and b are TUNEL staining images and quantitative fluorescence intensity maps of pancreatic tissue of mice on day 7 after STZ administration (n=4, ***P<0.001 compared with C57). c and d are representative images and schematic diagrams of the wound healing process of mice in each group on days 0, 3, 5, 7 and 9 after surgery. e is a quantitative assessment map of wound healing rate (n=10). f, g and h are H&E staining images and quantitative evaluations of the width of neoepithelial space and the degree of reepithelialization represented by horizontal black lines (n=3). i and j are Masson trichrome staining images and quantitative maps of collagen deposition volume (n=3). Statistical analysis was performed using GraphPad Prism 8. Data are expressed as mean ± SD. **P<0.01, ***P<0.001, ns indicates no significant difference.

[0111] Figure 10 shows the effect of SZ-A on vascular function during wound healing in diabetic mice in Experiment 4.

[0112] In the figure, a and b are representative immunofluorescence images and quantitative analysis images of cell apoptosis in skin wounds on day 9 after SZ-A intervention (n=3), c and d are representative immunofluorescence images and quantitative analysis images of cell proliferation in skin wounds on day 9 after SZ-A intervention (n=3), e and f are immunofluorescence staining images and quantitative analysis images of CD31 labeled with Alexa Fluor 488 and α-SMA labeled with Alexa Fluor 555, respectively, DAPI-stained cell nuclei (n=3), g, h and i, j are representative immunofluorescence images and quantitative analysis images of VEGF-A and eNOS in skin wounds on day 9 after SZ-A intervention (n=3). Statistical analysis was performed using GraphPad Prism 8. Data are expressed as mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ns indicates no statistical significance.

[0113] Figure 11 shows the effect of SZ-A on a single high-dose STZ injection model of diabetes AS in Experiment 5.

[0114] Where a is the weight change curve; b is the postprandial random blood glucose bar graph; c is the fasting blood glucose bar graph; d is the gross Oil Red O staining map of the arteries after treatment; e is the gross Oil Red O staining area statistical bar graph of the arteries; f is the Oil Red O staining map of the arterial root after treatment; g is the serum E-selectin statistical graph; h is the serum P-selectin statistical graph; * indicates P < 0.05 compared with the model; ** indicates P < 0.01 compared with the model; *** indicates P < 0.001 compared with the model.

[0115] Figure 12 shows the effect of SZ-A on a diabetic AS model induced by five low-dose STZ injections in Experiment 5; where a is the survival curve; b is the weight change curve; c is the gross Oil Red O staining map of the arteries after treatment; d is the statistical bar chart of the gross Oil Red O staining area of ​​the arteries; e is the random blood glucose bar chart; f is the glycated hemoglobin bar chart; g is the peak blood flow bar chart of the ascending aorta; h is the peak blood flow bar chart of the descending aorta; i is the statistical chart of E-selectin in serum; j is the statistical chart of P-selectin in serum; * indicates P < 0.05 compared with the model; ** indicates P < 0.01 compared with the model; *** indicates P < 0.001 compared with the model. Detailed Implementation

[0116] The present invention will be further described in detail below through examples. Through these exemplary descriptions, the features and advantages of the present invention will become clearer and more apparent.

[0117] In this context, the technical term "exemplary" means "used as an example, embodiment, or illustration." Any embodiment illustrated herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0118] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0119] Preparation of mulberry extract

[0120] Example 1 of the preparation of mulberry extract

[0121] Take 1000 kg of fresh mulberry branches (Fine-toothed Mulberry, Yue Sang No. 11), crush them, add 4000 L of water, and extract by reflux for 2 hours. Combine the extracts, filter to remove insoluble matter, and obtain crude extract. Concentrate the crude extract by heat until the solids content reaches 4%, and keep it at 50℃ as the loading solution for cation exchange resin column.

[0122] A 150 kg column of D113 macroporous weakly acidic styrene-propylene cation exchange resin was packed. The column was washed with 2 mol / L hydrochloric acid until the eluent pH reached 4.5; then with 1 mol / L sodium hydroxide solution until the eluent pH reached 8.5; and finally with 2 mol / L hydrochloric acid until the eluent pH reached 4.5. Activation was then achieved by rinsing with 5 column volumes of deionized water. The concentrated extract was loaded onto the column, followed by elution with 1000 L of 2.5 mol / L ammonia solution at a rate of 6 BV / h. The eluent was collected when the pH of the cation exchange column eluent was greater than 7. Collection was stopped when 900 L of eluent was collected, and the collected eluent was directly purified using an anion exchange column.

[0123] Activation was completed by packing a column with 62.5 kg of D218 macroporous strong basic acrylic anion exchange resin. The column was washed with 1.5 mol / L sodium hydroxide solution until the eluent pH reached 9.0; then with 1.5 mol / L hydrochloric acid solution until the eluent pH reached 3.5; and finally with 1.5 mol / L sodium hydroxide solution until the eluent pH reached 9.0. The collected cation exchange resin eluent was then loaded onto the anion exchange resin, and the eluent was collected until a total of 920 L was reached.

[0124] The collected liquid was centrifuged to remove impurities and then concentrated using a reverse ion osmosis membrane. The specific gravity of the concentrated liquid was 1.25. It was then transferred to an alcohol precipitation tank, and 25 L of anhydrous ethanol was added under stirring at 500 rpm. After the ethanol was added, stirring was stopped, and alcohol precipitation was carried out for 24 h. The supernatant was collected and concentrated under reduced pressure to obtain mulberry twig extract (i.e., mulberry extract SZ-A).

[0125] The mulberry twig extract contains 52% alkaloids, 22% polysaccharides, 0.8% flavonoids, and 20% amino acids by mass. Among the alkaloids, 1-DNJ accounts for 60%, FAG for 17%, and DAB for 15%.

[0126] Example 2 of the preparation of mulberry extract

[0127] Take 10 kg of fresh mulberry branches (Sangtuo No. 2), crush them, add 150 L of water in two portions, and extract by decoction for 3 hours each time. Combine the extracts and filter to remove insoluble matter. Concentrate the extract by heat until the solid content reaches 8%, transfer it to an alcohol precipitation tank, and add 2367.9 g of anhydrous ethanol (3 L) at 300 rpm. After the ethanol is added, stop stirring and precipitate for 24 hours. Take the supernatant as the loading solution for the cation exchange resin column. Pack 5 kg of 002SC type strong acid styrene-based cation exchange resin into the column and activate the cation exchange resin according to the method of Preparation Example 1. Load the concentrated alcohol-precipitated extract onto the column, and then elute with 100 L of 5 mol / L potassium chloride at a rate of 5 BV / h. Monitor the eluent with 20% silicotungstic acid. Start collecting when a white precipitate forms and stop collecting when 25 L of the collected solution is reached. Purify the collected solution directly through an anion exchange column.

[0128] A 10 kg column of 711 type strong base styrene-based anion exchange resin was packed, and the anion exchange resin was activated according to the method in Preparation Example 1. The collected cation exchange resin eluent was loaded onto the anion exchange resin, and the eluent was collected until 15 L was reached. The collected eluent was then loaded back onto the cation exchange resin, and the separation was repeated twice more using cation exchange resin and anion exchange resin in sequence, following the method described above.

[0129] The collected liquid obtained after three column separations was centrifuged to remove impurities and then concentrated using a reverse ion permeation membrane. The specific gravity of the concentrated liquid was 1.25. It was then transferred to an alcohol precipitation tank, and 125g of anhydrous ethanol was added with a stirrer at 1000rpm. After the ethanol was added, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain the extract. Fresh mulberry bark and leaves (Sangtuo No. 2) were also used for extraction, using the same extraction methods and parameters as described above.

[0130] The obtained mulberry twig extract contained 98% alkaloids, 0.2% polysaccharides, 0.05% flavonoids, and 0% amino acids. Among the alkaloids, 1-DNJ accounted for 99%, FAG for 0.5%, and DAB for 0.4%.

[0131] The obtained mulberry bark extract contained 95% alkaloids, 2% polysaccharides, 0.1% flavonoids, and 1% amino acids. Among the alkaloids, 1-DNJ accounted for 96%, FAG for 1.5%, and DAB for 1.4%.

[0132] The obtained mulberry leaf extract contained 90% alkaloids, 4% polysaccharides, 0.1% flavonoids, and 3% amino acids by mass. Among the alkaloids, 1-DNJ accounted for 91%, FAG for 3.1%, and DAB for 2.8%.

[0133] Example 3 of the preparation of mulberry extract

[0134] Take 1000 kg of fresh mulberry branches (Guangdong mulberry), crush them, add 11500 L of water, heat and reflux for 2 h, combine the extracts, filter to remove insoluble matter, and obtain crude extract. The crude extract is first centrifuged to remove impurities, and then concentrated with a reverse ion permeation membrane until the solids content reaches 1%, which is then used as the loading solution for the cation exchange resin column.

[0135] A column was packed with 300 kg of D001 type macroporous strong acid styrene-based cation exchange resin, and the resin was activated according to the method in Preparation Example 1. The concentrated crude extract was loaded onto the column and eluted with 5000 L of 0.04 mol / L ammonium nitrate at a rate of 5 BV / h. The eluent was monitored with 20% silicotungstic acid; collection began when a white precipitate formed and stopped when 1000 L of eluent was collected.

[0136] The collected solution obtained after cation exchange was concentrated by nanofiltration membrane and then concentrated under reduced pressure to obtain an extract paste.

[0137] The obtained mulberry twig extract contained 15% alkaloids, 20% polysaccharides, 7% flavonoids, and 45% amino acids by mass. Among the alkaloids, 1-DNJ accounted for 55%, FAG for 23%, and DAB for 10%.

[0138] Example 4 of the preparation of mulberry extract

[0139] Take 333 kg of dried mulberry branches (Yue Sang No. 11), crush them, add 4000 L of water, and extract them twice by heating and reflux, refluxing for 1 hour each time. Combine the extracts, filter them, and concentrate the extract to 1 kg of raw medicinal material per L.

[0140] A 150 kg column of D113 macroporous weakly acidic styrene-propylene cation exchange resin was packed. The column was washed with 2 mol / L hydrochloric acid until the eluent pH reached 4.5; then with 1 mol / L sodium hydroxide solution until the eluent pH reached 8.5; and finally with 2 mol / L hydrochloric acid until the eluent pH reached 4.5. Activation was then achieved by rinsing with 5 column volumes of deionized water. The concentrated extract was loaded onto the column, followed by elution with 1000 L of 2.5 mol / L ammonia solution at a rate of 6 BV / h. The eluent was collected when the pH of the cation exchange column eluent was greater than 7. Collection was stopped when 900 L of eluent was collected, and the collected eluent was directly purified using an anion exchange column.

[0141] Activation was completed by packing a 125 kg column with D218 macroporous strong-basic acrylic anion exchange resin. The column was washed with 1.5 mol / L sodium hydroxide solution until the eluent pH reached 9.0; then with 1.5 mol / L hydrochloric acid solution until the eluent pH reached 3.5; and finally with 1.5 mol / L sodium hydroxide solution until the eluent pH reached 9.0. The collected cation exchange resin eluent was then loaded onto the anion exchange resin, and the eluent with a pH greater than 8 was collected until 870 L of eluent was reached.

[0142] The collected solution obtained after anion exchange column separation was filtered through a microfiltration membrane to remove impurities and then concentrated using a reverse ion osmosis membrane. The specific gravity of the concentrated liquid was 1.1. It was then transferred to an alcohol precipitation tank, and 15 kg of anhydrous ethanol was added at 400 rpm with a stirring paddle. After the ethanol addition was complete, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain mulberry twig extract. Sample content: 80% alkaloids, 5% polysaccharides, 0.1% flavonoids, and 4% amino acids. Among the alkaloids, 1-DNJ accounted for 75%, FAG for 12%, and DAB for 10%.

[0143] Example 5 of the preparation of mulberry extract

[0144] Take 400 kg of dried mulberry branches (Yue Sang No. 11), crush them, add 4000 L of water, and extract them twice by heating and reflux, refluxing for 1 hour each time. Combine the extracts, filter them, and concentrate the extract to 1 kg of raw medicinal material per L.

[0145] Activation was completed by packing a 62.5 kg column with D218 macroporous strong-basic acrylic anion exchange resin. The column was washed with 1.5 mol / L sodium hydroxide solution until the eluent pH reached 9.0; then with 1.5 mol / L hydrochloric acid solution until the eluent pH reached 3.5; and finally with 1.5 mol / L sodium hydroxide solution until the eluent pH reached 9.0. The collected extract concentrate was loaded onto the anion exchange resin, and 40 L of eluent was collected.

[0146] The collected solution obtained after anion exchange column separation was filtered through a microfiltration membrane to remove impurities, then concentrated using a reverse ion exchange membrane, and further concentrated under reduced pressure and dried to obtain mulberry twig extract. Sample content: alkaloids 3% by mass, polysaccharides 70% by mass, flavonoids 10% by mass, and amino acids 10% by mass. Among the alkaloids, 1-DNJ accounted for 68%, FAG 17%, and DAB 8%.

[0147] Example 6: Preparation of Mulberry Extract

[0148] Take 1500 kg of fresh mulberry branches (Fine-toothed Mulberry, Yue Sang No. 11), crush them, add 6000 L of water, and extract by reflux for 2 hours. Combine the extracts, filter to remove insoluble matter, and obtain crude extract. Concentrate the crude extract by heat until the solids content reaches 4%, and keep it at 50℃ as the loading solution for cation exchange resin column.

[0149] A 100kg column of D113 macroporous weakly acidic styrene-propylene cation exchange resin was packed. The column was washed with 2mol / L hydrochloric acid until the eluent pH reached 4.5; then with 1mol / L sodium hydroxide solution until the eluent pH reached 8.5; and finally with 2mol / L hydrochloric acid solution until the eluent pH reached 4.5. Activation was then achieved by rinsing with 5 column volumes of deionized water. The concentrated extract was loaded onto the column, followed by elution with 1000L of 2.5mol / L ammonia solution at a rate of 6 BV / h. The eluent was collected when the pH of the cation exchange column eluent was greater than 7. Collection was stopped when 900L of eluent was collected, and the collected eluent was directly purified using an anion exchange column.

[0150] A 62.5 kg column of D218 macroporous strong-basic acrylic anion exchange resin was packed. The column was washed with 1.5 mol / L sodium hydroxide solution until the eluent pH reached 9.0; then with 1.5 mol / L hydrochloric acid solution until the eluent pH reached 3.5; and finally with 1.5 mol / L sodium hydroxide solution until the eluent pH reached 9.0, thus completing activation. The collected cation exchange resin eluent was loaded onto the anion exchange resin, and the eluent was collected until 870 L was reached. The eluent was concentrated under reduced pressure to obtain a mulberry twig extract, containing 30% alkaloids, 35% polysaccharides, 2% flavonoids, and 25% amino acids. Among the alkaloids, 1-DNJ accounted for 62%, FAG for 20%, and DAB for 13%.

[0151] Example 7: Preparation of Mulberry Extract

[0152] Take 1000 kg of fresh mulberry branches (Fine-toothed Mulberry, Yue Sang No. 11), crush them, add 4000 L of water, and extract by reflux for 2 hours. Combine the extracts, filter to remove insoluble matter, and obtain crude extract. Concentrate the crude extract by heat until the solids content reaches 4%, and keep it at 50℃ as the loading solution for cation exchange resin column.

[0153] A 100kg column of D113 macroporous weakly acidic styrene-propylene cation exchange resin was packed. The column was washed with 2mol / L hydrochloric acid until the eluent pH reached 4.5; then with 1mol / L sodium hydroxide solution until the eluent pH reached 8.5; and finally with 2mol / L hydrochloric acid solution until the eluent pH reached 4.5. Activation was then achieved by rinsing with 5 column volumes of deionized water. The concentrated extract was loaded onto the column, followed by elution with 1000L of 2.5mol / L ammonia solution at a rate of 6 BV / h. The eluent was collected when the pH of the cation exchange column eluent was greater than 7. Collection was stopped when 900L of eluent was collected, and the collected eluent was directly purified using an anion exchange column.

[0154] Activation was completed by packing a 62.5 kg column with D218 macroporous strong-basic acrylic anion exchange resin. The column was washed with 1.5 mol / L sodium hydroxide solution until the eluent pH reached 9.0; then with 1.5 mol / L hydrochloric acid solution until the eluent pH reached 3.5; and finally with 1.5 mol / L sodium hydroxide solution until the eluent pH reached 9.0. The collected cation exchange resin eluent was loaded onto the anion exchange resin, and the eluent was collected until 870 L was reached. The eluent was concentrated under reduced pressure to obtain a mulberry twig extract, containing 40% alkaloids, 25% polysaccharides, 0.5% flavonoids, and 25% amino acids. Among the alkaloids, 1-DNJ accounted for 57%, FAG for 24%, and DAB for 16%.

[0155] Example 8: Preparation of Mulberry Extract

[0156] Take 333 kg of dried mulberry branches (Yue Sang No. 11), crush them, add 4000 L of water, and extract them twice by heating and reflux, refluxing for 1 hour each time. Combine the extracts, filter them, and concentrate the extract to 1 kg of raw medicinal material per L.

[0157] A 150 kg column of D113 macroporous weakly acidic styrene-propylene cation exchange resin was packed. The column was washed with 2 mol / L hydrochloric acid until the eluent pH reached 4.5; then with 1 mol / L sodium hydroxide solution until the eluent pH reached 8.5; and finally with 2 mol / L hydrochloric acid until the eluent pH reached 4.5. Activation was then achieved by rinsing with 5 column volumes of deionized water. The concentrated extract was loaded onto the column, followed by elution with 1000 L of 2.5 mol / L ammonia solution at a rate of 6 BV / h. The eluent was collected when the pH of the cation exchange column eluent was greater than 7. Collection was stopped when 900 L of eluent was collected, and the collected eluent was directly purified using an anion exchange column.

[0158] Activation was completed by packing a column with 62.5 kg of D218 macroporous strong-basic acrylic anion exchange resin. The column was washed with 1.5 mol / L sodium hydroxide solution until the eluent pH reached 9.0; then with 1.5 mol / L hydrochloric acid solution until the eluent pH reached 3.5; and finally with 1.5 mol / L sodium hydroxide solution until the eluent pH reached 9.0. The collected cation exchange resin eluent was then loaded onto the anion exchange resin, and the eluent with pH greater than 8 was collected until 870 L of eluent was reached.

[0159] The collected solution obtained after anion exchange column separation was filtered through a microfiltration membrane to remove impurities and then concentrated using a reverse ion osmosis membrane. The specific gravity of the concentrated liquid was 1.1. It was then transferred to an alcohol precipitation tank, and 15 kg of anhydrous ethanol was added at 400 rpm with a stirring paddle. After the ethanol addition was complete, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain mulberry twig extract. Sample content: alkaloids 63% by mass, polysaccharides 23% by mass, flavonoids 1% by mass, and amino acids 5% by mass.

[0160] The alkaloids contained 61.9% 1-DNJ, 16.6% FAG, and 11.1% DAB.

[0161] Example 9: Preparation of mulberry extract

[0162] Take 1000 kg of fresh mulberry branches (Yue Sang 11), crush them, add 4000 L of water, and extract by reflux for 2 hours. Combine the extracts, filter to remove insoluble matter, and obtain crude extract. Concentrate the crude extract by heat until the solid content reaches 4%, and keep it at 50℃ as the loading solution for cation exchange resin column.

[0163] A 120 kg column of D113 macroporous weakly acidic styrene-propylene cation exchange resin was packed. The column was washed with 2 mol / L hydrochloric acid until the eluent pH reached 4.5; then with 1 mol / L sodium hydroxide solution until the eluent pH reached 8.5; finally, with 2 mol / L hydrochloric acid solution until the eluent pH reached 4.5. Activation was then achieved by rinsing with 5 column volumes of deionized water. The concentrated extract was loaded onto the column and then eluted with 1000 L of 2.5 mol / L ammonia solution at a rate of 6 BV / h. The eluent was collected when the pH of the cation exchange column eluent was greater than 7. Collection was stopped when 900 L of eluent was collected, and the collected eluent was directly purified using an anion exchange column.

[0164] Activation was completed by packing a 45kg column with D218 macroporous strong-basic acrylic anion exchange resin. The column was washed with 1.5mol / L sodium hydroxide solution until the eluent pH reached 9.0; then with 1.5mol / L hydrochloric acid solution until the eluent pH reached 3.5; and finally with 1.5mol / L sodium hydroxide solution until the eluent pH reached 9.0. The collected cation exchange resin eluent was loaded onto the anion exchange resin, and the eluent was collected until a flow rate of 870L was reached.

[0165] The collected solution obtained after anion exchange column separation was filtered through a microfiltration membrane to remove impurities and then concentrated using a reverse ion exchange membrane. The specific gravity of the concentrated liquid was 1.1. It was then transferred to an alcohol precipitation tank, and 15 kg of anhydrous ethanol was added at 300 rpm with a stirring paddle. After the ethanol addition was complete, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain the extract. Sample content: 70% alkaloids, 20% polysaccharides, 0.6% flavonoids, and 5% amino acids.

[0166] The alkaloids contain 70% 1-DNJ, 13% FAG, and 10% DAB.

[0167] Example 10 of the preparation of mulberry extract

[0168] Take 80 kg of fresh mulberry branches (Gui Sang You 62), crush them, add 1000 L of water, and extract by reflux for 2 hours. Combine the extracts, filter to remove insoluble matter, and obtain crude extract. Concentrate the crude extract by heat until the solid content reaches 4%, and keep it at 50℃ as the loading solution for cation exchange resin column.

[0169] A 50 kg column of D113 macroporous weakly acidic styrene-propylene cation exchange resin was packed. The column was washed with 2 mol / L hydrochloric acid until the eluent pH reached 4.5; then with 1 mol / L sodium hydroxide solution until the eluent pH reached 8.5; finally, with 2 mol / L hydrochloric acid solution until the eluent pH reached 4.5. Activation was then achieved by rinsing with 5 column volumes of deionized water. The concentrated extract was loaded onto the column and then eluted with 600 L of 2.5 mol / L ammonia solution at a rate of 6 BV / h. The eluent was collected when the pH of the cation exchange column eluent was greater than 7. Collection was stopped when 800 L of eluent was collected, and the collected eluent was directly purified using an anion exchange column.

[0170] A 65 kg column of D218 macroporous strong-basic acrylic anion exchange resin was packed. The column was washed with 1.5 mol / L sodium hydroxide solution until the eluent pH reached 9.0; then with 1.5 mol / L hydrochloric acid solution until the eluent pH reached 3.5; and finally with 1.5 mol / L sodium hydroxide solution until the eluent pH reached 9.0. Activation was then complete. The collected cation exchange resin eluent was loaded onto the anion exchange resin, and the eluent was collected until 750 L was reached. The collected eluent was then reloaded onto the cation exchange resin, and the process was repeated once more using both cation and anion exchange resins.

[0171] The collected liquid, after two separations, was filtered through a microfiltration membrane to remove impurities and then concentrated using a reverse ion osmosis membrane. The specific gravity of the concentrated liquid was 1.1. It was then transferred to an alcohol precipitation tank, and 400 g of anhydrous ethanol was added with a stirrer at 350 rpm. After the ethanol was added, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain the extract. Sample content: alkaloids 90% by mass, polysaccharides 3% by mass, flavonoids 0.5% by mass, and amino acids 2% by mass.

[0172] The alkaloids contain 94% 1-DNJ, 2% FAG, and 1% DAB.

[0173] Pharmacological test of mulberry extract

[0174] In the following experimental examples, "SZ-A" represents mulberry extract.

[0175] Cell experiments

[0176] Experimental Example 1: Effects of mulberry extract on AGEs (advanced glycation end products)-induced functional impairment of HUVECs

[0177] Cell culture and SZ-A intervention

[0178] Human umbilical vein endothelial cells (HUVECs) were obtained from Procell (Wuhan, China). HUVECs were incubated in a CO2 incubator at 37°C and 5% humidity in 11.1 mM glucose 1640 medium (supplemented with 1% antibiotics (penicillin / streptomycin) and 10% fetal bovine serum). Subsequent experiments involved treating HUVECs with SZ-A and incubating them for 24 h with 2 μg / mL AGEs (bs-1158P, bioss) to simulate the diabetic microenvironment. The following experiments used HUVECs from passages 3 to 6.

[0179] 1. Cell viability assay

[0180] HUVECs were cultured for 24 h in a medium containing different concentrations of SZ-A (mulberry extract from Preparation Example 1, with concentrations of 0.1, 0.25, 0.5, 0.75, 1, 1.5, and 2 mg / mL of alkaloids, respectively). Cell viability was then measured using a Cell Counting Kit-8 (CCK-8) and read at 450 nm using a Synergy H1 microplate reader. The results are shown in Figure 1.

[0181] The results showed that SZ-A promoted the proliferation of HUVECs cells.

[0182] 2. Tube formation determination

[0183] HUVECs were pretreated with 1 mg / mL SZ-A (mulberry extract from Preparation Example 1, calculated as alkaloids) for 3 h. They were then cultured for 24 h with or without 2 μg / mL AGEs. HUVECs without SZ-A intervention were designated the Ctrl group, those with SZ-A intervention were designated the SZ-A group, HUVECs without AGEs treatment were designated the Vehicle group, and HUVECs with AGEs treatment were designated the AGEs group. For example, the SZ-A group within the AGEs treatment group consisted of HUVECs pretreated with 1 mg / mL SZ-A for 3 h, followed by culture with 2 μg / mL AGEs for 24 h.

[0184] Tube formation assays were performed using a matrix gel (356234, Corning) to evaluate the angiogenic potential of HUVECs. 70 μL of liquid gel was uniformly added to each well of a 48-well plate placed on ice, and then incubated at 37 °C for 30 min to allow the gel to solidify. Subsequently, drug-treated HUVECs were implanted into the solidified gel, and tube formation was monitored and recorded under a microscope over 6 h. The results are shown in Figures 2a and 2b.

[0185] The results showed that, without SZ-A intervention (Ctrl), the AGEs group had a significantly shorter tube segment length compared to the Vehicle group. After SZ-A intervention, both the Vehicle and AGEs groups showed significantly longer tube segments.

[0186] 3. Cell migration

[0187] Cell scratch assay: HUVECs were seeded into 6-well plates and pretreated with 1 mg / mL SZ-A (mulberry extract from Preparation Example 1, calculated as alkaloids) for 3 h, followed by intervention with or without AGEs for 24 h. After intervention, cells were scratched with the tip of a 20 μL pipette. The migration process of HUVECs was observed and recorded within 24 h using a Lionheart FX automated microscope (Biotek, USA), and the degree of wound closure was indicated by a black horizontal line. The results are shown in Figure 2c. Quantitative analysis of wound closure rates at 8 h, 16 h, and 24 h is shown in Figures 2d, e, and f.

[0188] The results showed that, without SZ-A treatment, the wound closure rate of HUVECs was significantly lower at 16 h and 24 h after AGEs treatment, and the cell migration ability was reduced compared with the Vehicle group. After SZ-A intervention, the wound closure rate of both the Vehicle group and the AGEs group was significantly increased (Figure 2c, d, e, f), and the effect of SZ-A on the cell migration of HUVECs treated with AGEs was more obvious.

[0189] Transwell assay: HUVECs were pretreated with 1 mg / mL SZ-A (mulberry extract from Preparation Example 1, calculated as alkaloids) for 3 h, followed by intervention with or without AGEs for 24 h. After intervention, cells were cultured in 4 × 10⁶ cells per 100 μL serum-free medium. 5 / mL was inoculated into the upper chamber of a Transwell plate (3422, Corning), and 10% fetal bovine serum culture medium was added to the lower chamber. After incubation at 37°C for 24 h, the Transwell plate was removed, fixed with 4% paraformaldehyde for 15 min, and then stained with Coomassie brilliant blue for 15 min. The chamber was cleaned with PBS, wiped clean with cotton swabs, and air-dried. Then, it was observed, photographed, and counted using an inverted microscope. The results are shown in Figure 2g and h.

[0190] The results showed that after SZ-A intervention, the number of cells migrating increased in both the Vehicle and AGEs groups.

[0191] 4. Apoptosis assay

[0192] Flow cytometry: HUVECs were pretreated with 1 mg / mL SZ-A (mulberry extract from Preparation Example 1, calculated as alkaloids) for 3 h, followed by intervention with or without AGEs for 24 h. All procedures were performed according to the instructions provided with the apoptosis assay kit (FXP023, 4Abio). Apoptosis levels in each group were assessed using the TUNEL apoptosis assay kit (KGA702, KeyGen Biotech).

[0193] The results showed that after SZ-A intervention, apoptotic cells in HUVECs (AGEs group) under simulated hyperglycemic pathological conditions were significantly reduced, and SZ-A exhibited a significant anti-apoptotic effect (Fig. 2i, j). TUNEL staining results showed that apoptosis in the AGEs group was inhibited after SZ-A intervention (Fig. 2k, l).

[0194] 5. Effects of SZ-A on vasoactivity-related factors in HUVECs treated with AGEs

[0195] Protein isolation and Western blotting analysis: Proteins were extracted from HUVECs cells using RIPA lysis buffer with added protease (sc-364162, Santa Cruz Biotechnology) and phosphatase inhibitor (B15001, Selleck). Total protein content was quantified using a BCA protein assay kit (ZJ102, Epizyme). Proteins were separated by polyacrylamide gel electrophoresis, then transferred to a PVDF membrane (Millipore, Billeria), blocked in TBST with 5% milk for 1 hour, and incubated overnight at 4°C with primary antibodies against NRF2 (AF0639, Affinity Bioscience), HO-1 (T55113, Abmart), eNOS (345227, Zen Bio), CD31 (R10021, Zen Bio), VEGF-A (WL00009b, WanLeiBio), and β-Tubulin (10094-1-AP, Proteintech). After washing three times with TBST buffer, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (31430, Thermo Fisher Scientific). The antigen was then visualized using a chemiluminescence kit (P2300, NCM Biotech). Immunoreactive bands were visualized using a ChemiDoc Touch (Bio-Rad, USA) chemiluminescence imaging system and analyzed using ImageJ.

[0196] Endogenous nitric oxide (NO) assay: As a key regulator of NO synthesis, the importance of eNOS in vascular function regulation is widely recognized. The intracellular NO accumulation in HUVECs was further detected using the DAF-FM DA kit (S0019S, Beyotime). HUVECs were pretreated with 1 mg / mL SZ-A (mulberry extract from Preparation Example 1, calculated as alkaloids) for 3 h, followed by exposure to AGEs or blank control conditions for 24 h. After PBS recovery and washing, DAF-FM DA was added to a final concentration of 2.5 μM. The samples were then incubated at 37 °C in the dark for 20 min, followed by washing three times with PBS. Visualization was performed using fluorescence microscopy, and quantitative analysis was performed using ImageJ.

[0197] The levels of NRF2, CD31 and VEGF-A were detected by immunofluorescence assay.

[0198] Furthermore, HUVECs cells were pretreated with 1 mg / mL SZ-A (mulberry extract from Preparation Example 1, calculated as alkaloids) for 3 hours, followed by intervention with ML385 or without ML385 (2 μg / mL) for 24 hours. The expression levels of NRF2, HO-1, CD31, VEGF-A, and eNOS in HUVECs were detected by Western blot. ML385 is an NRF2 inhibitor.

[0199] Experimental results:

[0200] Figure 3 shows that under simulated hyperglycemic pathological conditions (HUVECs treated with AGEs), SZ-A promoted NRF2 expression compared to the blank control group (Ctrl group) (Figure 3a, b), consistent with the results of immunofluorescence quantitative analysis (Figure 3d, e); and SZ-A upregulated the expression level of downstream effector molecule HO-1 (heme oxygenase 1) (Figure 3a, c). This indicates that SZ-A can regulate the NRF2 / HO-1 signaling pathway.

[0201] Figure 4 shows that SZ-A significantly enhanced the levels of CD31 (also known as platelet-endothelial cell adhesion factor-1) and VEGF-A (vascular endothelial growth factor-A), and significantly upregulated the expression of eNOS (endothelial nitric oxide synthase) (Figure 4a, b, c, d). After SZ-A intervention, the NO content in the Vehicle group and the AGEs group was significantly increased (Figure 4e, f), and the expression levels of CD31 and VEGF-A were significantly enhanced (Figure 4g, h, i, j). In simulated hyperglycemic pathological conditions (HUVECs treated with AGEs), in untreated HUVECs (Vehicle group), administration of SZ-A significantly promoted the expression of NRF2, HO-1, CD31, VEGF-A, and eNOS; in HUVECs treated with ML385 (ML385 group), the promoting effect of SZ-A on the expression of NRF2, HO-1, CD31, VEGF-A, and eNOS was inhibited (Figure 4k, l, m, n, o, p).

[0202] These results indicate that SZ-A-mediated endothelial cell protection is closely related to the NRF2 / HO-1 / eNOS signaling pathway and improves endothelial cell function by promoting the expression of vasoactive-related factors (such as eNOS, CD31, and VEGF-A).

[0203] In conclusion, SZ-A can effectively inhibit endothelial cell dysfunction caused by AGEs.

[0204] Experimental Example 2: Effects of mulberry extract on HUVECs damage induced by high concentrations of glucose and TNF-α

[0205] Inflammation and high glucose levels can induce the secretion of endothelial cell adhesion factors, leading to the adhesion of monocytes and macrophages and amplifying the local endothelial cell inflammatory response. This study investigated the effects of mulberry extract on HUVECs damage by constructing an in vitro model induced by high glucose concentrations (referred to as high glucose) and tumor necrosis factor-α (TNF-α).

[0206] 1. Cell adhesion

[0207] Experimental cells: Human umbilical vein endothelial cells (HUVEC) and human monocytic leukemia cells (THP-1) (HUVEC was provided by the Union Cell Bank of the Chinese Academy of Medical Sciences; THP-1 was purchased from Wuhan Pronosei Life Science Technology Co., Ltd.)

[0208] Take the inoculation at 10mm 2HUVEC cells in culture dishes were digested at 0.25%, collected, centrifuged at 300×g for 5 min, the supernatant was discarded, and the cells were resuspended in complete culture medium (DMEM-H (cellmax CGM113.05) + 10% FBS (Sino Biological FBS FBS01)) and counted. The resuspended HUVEC cells were then diluted to 10⁻⁶. 5 HUVEC cells were seeded at a concentration of [number] cells / mL in 12-well plates; pretreated with medium containing 50 mmol glucose and 20 ng / mL TNF-α, and then treated with 500 μg / mL mulberry extract SZ-A (prepared from Preparation Examples 3, 6, 8, and 10, calculated as alkaloids) for 24 h. After the cells reached confluence, the following pretreatment Thrp-1 was added.

[0209] Thp-1 pretreatment staining:

[0210] (1) Centrifuge THP-1 cells at 300×g for 5 min, and resuspend the cell pellet in serum-free RPMI-1640 medium at a density of 5×10⁻⁶ cells / mL. 6 cells / mL;

[0211] (2) Add CFSE fluorescent probe (Beyotime C1031) to Thp-1 cells under light-protected conditions to make the final concentration of CFSE 5 μM (dissolved in DMSO to prepare a stock solution with a concentration of 5 mM).

[0212] (3) Place the THP-1 cells containing the probe into a CO2 cell culture incubator and incubate in the dark for 15 min;

[0213] (4) Add an equal volume of RPMI-1640 medium containing 20% ​​fetal bovine serum to the Thp-1 cells containing the probe to terminate the reaction for 10 min.

[0214] (5) Centrifuge CFSE-labeled Thrp-1 cells at 300×g for 5 min, resuspend in PBS, centrifuge again, and discard the supernatant;

[0215] (6) Resuspend CFSE-labeled THP-1 cells in RPMI-1640 medium containing 10% fetal bovine serum to a cell density of 1×10⁻⁶ cells / year. 6 cells / mL;

[0216] (7) Add the above-labeled Thp-1 to HUVECs cells (1 mL of endothelial cell culture medium plus 20 μL of labeled Thp-1 cells) and culture them together in the dark for 6 h.

[0217] The unbound Thp-1 cells were washed away with PBS, and then the adhesion of HUVEC cells to Thp-1 cells was observed under a fluorescence microscope after adding an appropriate amount of PBS. The number of green fluorescent cells was used for evaluation, and the results are shown in Figure 5.

[0218] The results showed that HUVECs (model group) treated with high concentrations of glucose and TNF-α had the highest number of adhesions to Thp-1. After treatment with SZ-A, all treatment groups could inhibit the adhesion of HUVECs to Thp-1 to some extent, indicating that mulberry extract can alleviate local inflammation of vascular endothelium by inhibiting the adhesion between monocytes and endothelial cells.

[0219] 2. Apoptosis

[0220] Take the inoculation at 10mm 2 HUVEC cells in culture dishes were digested with 0.25% nitric acid, collected, centrifuged at 300×g for 5 min, the supernatant was discarded, and the cells were resuspended in complete culture medium and counted. The resuspended HUVEC cells were then diluted to 10⁻¹⁰. 5 Cells were seeded at a concentration of [number] cells / mL in 6-well plates and cultured for 24 h. Then, medium containing 50 mmol glucose and 20 ng / mL TNF-α was added, followed by intervention with 500 μg / mL mulberry extract (prepared from Preparation Examples 6, 8, and 10, calculated as alkaloids) for 24 h. Flow cytometry was then used to detect the apoptosis rate. Annexin V fluorescent probe labeling was used, with FITC and PI double-positive cells as apoptotic cells. One-way ANOVA was used to compare differences between groups. The flow cytometry results of HUVEC cell apoptosis are shown in Figure 6, and the late apoptosis rate is shown in Table 1.

[0221] Table 1. Results of the effect of mulberry extract on the late apoptosis rate of HUVECs induced by high glucose and TNF-α (n=4)

[0222] The results showed that the mulberry extract of the present invention can inhibit HUVECs cell apoptosis to a certain extent.

[0223] 3. Cell scratch assay

[0224] Take the inoculation at 10mm 2 HUVEC cells in culture dishes were digested with 0.25% nitric acid, collected, centrifuged at 300×g for 5 min, the supernatant was discarded, and the cells were resuspended in complete culture medium and counted. The resuspended HUVEC cells were then diluted to 10⁻¹⁰. 5 The cells were seeded at a concentration of 100 cells / mL in 12-well plates. After the cells had grown to confluence, a vertical line was drawn at the bottom of the well plate using a 200 μL pipette tip.

[0225] Cells were cultured in a medium containing 50 mmol glucose and 20 ng / mL TNF-α, and then treated with 500 μg / mL mulberry extract (prepared from Preparation Examples 3, 6, 8, and 10, calculated as alkaloids). Cells were washed three times with PBS for 5 min each time, and cell migration was recorded at 0 h, 12 h, and 24 h under an inverted microscope.

[0226] The scratch healing rate of HUVECs over 24 hours was calculated, i.e., the migration rate, as given by the formula: (L0 – Lt) / L0 × 100%. L0 represents the scratch distance at 0 hours, and Lt represents the scratch distance at the set detection time. Phase contrast processing was performed on the images using the built-in microscopy software Gene5. The distance was measured at three randomly selected locations for each scratch using the fluorescence microscope's length measurement function, and the average value was calculated as the scratch distance for that field of view. Data were compared between groups using one-way ANOVA, and the results are shown in Figure 7. The ability of mulberry extract to promote endothelial cell migration and proliferation was evaluated. Specific scratch healing rates are shown in Table 2 below.

[0227] Table 2. Results of scratch healing rate of mulberry extract on high glucose and TNF-α-induced HUVEC damage (n=4)

[0228] The results showed that the mulberry extract of the present invention can significantly increase the migration rate of HUVEC cells to a certain extent and promote the healing of scratches.

[0229] Experimental Example 3: Effects of Mulberry Extract on Hyperinsulin-Induced HUVEC Cell Damage

[0230] Cell culture: Human umbilical vein endothelial cells (HUVECs) were cultured in DMEM medium (10% FBS + 1% antibiotics).

[0231] Cell passage: Digest with 0.25% trypsin, 10 5 Seeds were planted at a density of 1 / mL in 12-well plates and incubated in a cell culture incubator at 37°C and 5% CO2. The culture medium was changed every 2-3 days. Once the cells occupied about 80% of the field of view, the cells were passaged again.

[0232] Model construction: Passaged HUVEC cells were cultured overnight and then randomly divided into six groups: a normal control group, a model group, and a mulberry extract SZ-A group. The normal control group was given DMEM medium (10% FBS + 1% penicillin antibody), the model group (10% FBS + 1% penicillin antibody + 40U insulin), and the SZ-A group (mulberry extract prepared in Example 8, administered at concentrations of 100, 200, 300, and 400 μg / mL based on alkaloids) was given SZ-A (10% FBS + 1% penicillin antibody + 40U insulin + corresponding concentration of SZ-A). After 24 h of culture, mitochondria-specific fluorescent staining was performed using Mito-Tracker Green.

[0233] When cells reach a certain density in cell culture plates or dishes, remove the cell culture medium, add prepared Mito-Tracker Green (mitochondrial green fluorescent probe) working solution, incubate at 37°C for 15-30 minutes, then remove the Mito-Tracker Green working solution and add fresh cell culture medium pre-warmed at 37°C. Observation under a fluorescence microscope reveals bright, strong fluorescent staining of mitochondria. The results are shown in Figure 8.

[0234] The results showed that, compared with the model group, the SZ-A group significantly increased the number of mitochondria in endothelial cells and improved mitochondrial damage, suggesting that mulberry extract can significantly improve endothelial cell function.

[0235] animal testing

[0236] Experiment 4: Effects of mulberry extract on wound healing in diabetic mice

[0237] Construction of a diabetic mouse model:

[0238] Male C57BL / 6J mice aged 6-8 weeks (purchased from Saiyi Biotechnology Co., Ltd. (Suzhou, China)) were housed in a rodent facility under 12-hour light and dark conditions, with free access to food and water. Animal research was approved by the Research Animal Ethics Committee of Chongqing University (COU-IACUC-RE-202308-005). After being fed a high-fat HFD diet (60% fat, D12492, Research Diets) for 4 weeks, mice were intraperitoneally injected with 35 mg / kg streptozotocin (STZ; S0130; Sigma-Aldrich), dissolved in citrate buffer (pH 4.2-4.5), once daily for 5 consecutive days. Blood glucose levels were continuously measured via tail vein sampling. Mice with a random blood glucose level ≥16.7 mmol / L three or more times after one week were classified as diabetic mice and used for the following experiments. On day 7, pancreatic tissues from mice given and not given STZ were collected. The apoptosis level in each group was assessed using the TUNEL cell apoptosis detection kit (KGA702, KeyGen Biotech), and the tissue damage was observed.

[0239] Grouping and administration of experimental animals:

[0240] Diabetic mice were divided into three groups: a normal control (NC) group, an SZ-A-200 group, and an SZ-A-400 group (SZ-A was the mulberry extract from Preparation Example 1, administered at doses of 200 mg / kg and 400 mg / kg, respectively, based on alkaloids). The mice were administered the drug for 15 consecutive days via a mixed-feed administration method (the drug was mixed into a high-fat HDF diet), followed by 6 mm diameter wound modeling in each group.

[0241] Construction of a diabetic mouse skin wound model:

[0242] The model was established as follows: Animals were anesthetized with isoflurane (RDW), and full-thickness wounds were induced bilaterally on the back using sterile 6mm cylindrical perforations. Observation and recording were performed for 9 days, with continuous drug administration during this period. Digital images were acquired on postoperative days 0, 3, 5, 7, and 9. ImageJ software was used to standardize and calibrate wound area measurements and wound closure rates.

[0243] Mice were sacrificed on day 9, and wound tissue was collected through an 8mm skin biopsy well to analyze the expression of mRNA and protein during wound healing.

[0244] Immunofluorescence and morphological staining

[0245] Fixed in 4% paraformaldehyde at 4°C for 16-24 hours, embedded in paraffin, and sectioned to 5 μm. Paraffin sections were dewaxed, antigens were extracted with sodium citrate, washed with PBS, and incubated in blocking buffer. Subsequently, sections were incubated overnight at 4°C with primary antibodies against eNOS (345227, Zen Bio), CD31 (R10021, Zen Bio), α-SMA (55135-I-AP, Proteintech), and VEGF-A (WL00009b, WanLeiBio). Immunofluorescence staining was performed using Alexa Fluor 488 and Alexa Fluor 555 conjugated with secondary antibodies (Thermo Fisher). Fluorescence was then maintained during tissue imaging using DAPI staining solution (C1005, Beyotime) and an anti-fluorescence quenching mounting agent (ab104135, Abcam). Collagen was quantified by analyzing Masson's trichrome staining of skin areas using ImageJ software, based on a Masson's trichrome staining kit (G1346 Solarbio). Cells cultured on coverslips were fixed by treating with 4% paraformaldehyde PBS for 10 min. Subsequently, cells were washed in PBS and infiltrated with PBG-Triton (containing PBS, 0.4% fish skin gelatin, 0.5% bovine serum albumin, and 0.5% Triton X-100) for 1 h. Cells were then incubated overnight with primary antibody at 4°C. After washing with PBS, secondary antibody was applied and incubated at room temperature for 1 h. Coverslips were mounted on slides using ThermoFisher Scientific's Extend Gold Antifade Mountant with DAPI. APEXVIEW was used for analysis. TM Imaging was performed using an APX100 All-in-One microscope (Olympus, Japan). Fluorescence images were quantitatively analyzed using ImageJ software.

[0246] Experimental results:

[0247] 1. SZ-A can promote wound healing in diabetic mice.

[0248] Immunofluorescence images and analysis showed that, compared with normal mice, significant apoptosis occurred in the pancreatic tissue of mice 7 days after STZ administration, indicating pancreatic tissue damage (Fig. 9a, b).

[0249] To reduce wound contraction in rodent models and mimic the taut skin healing process in humans, silicone pads were applied to the wounds to exert an anti-contraction effect, and wound healing was comprehensively evaluated. Figures 9c and 9d show the changes in wound healing on days 0, 3, 5, 7, and 9 after oral administration of different concentrations of SZ-A to diabetic mice. Wound healing rates showed that, compared to the NC group, SZ-A more effectively promoted the healing of diabetic wounds with increasing dosage (Figure 9e). Quantitative analysis of wound length and horizontal black lines in HE-stained images indicated that the SZ-A group was significantly superior to the NC group in terms of wound regeneration and re-epithelialization (Figures 9f, g, h). Masson trichrome staining assays of collagen deposition showed that, compared to the NC group, SZ-A had a stimulating effect on collagen production (Figures 9i and j).

[0250] The above results indicate that SZ-A can promote the healing of diabetic wounds.

[0251] 2. SZ-A can improve vascular function during the wound healing process in diabetic patients.

[0252] Figures 10a and 10b show that, compared with the NC group, on day 9 after SZ-A intervention, the SZ-A-200 and SZ-A-400 groups significantly reduced cell apoptosis in the diabetic wound area. Figures 10c and 10d indicate that SZ-A intervention significantly upregulated the expression level of Ki67 (a nuclear protein closely related to cell proliferation), suggesting that SZ-A promotes cell proliferation in the diabetic wound area.

[0253] Furthermore, CD31 and α-SMA (α-smooth muscle actin) were used as indicators of endothelial cells and smooth muscle cells, respectively, to assess the degree of angiogenesis. Figures 10e and 10f show that SZ-A intervention promoted the angiogenesis process and exhibited a dose-gradient dependence, indicating its potential in promoting angiogenesis and tissue repair.

[0254] Furthermore, the expression levels of VEGF-A and eNOS during diabetic wound healing were evaluated after SZ-A intervention. Figure 10g, h, i, j show that SZ-A intervention significantly upregulated the expression levels of VEGF-A and eNOS, exhibiting a dose-gradient dependence.

[0255] The above experimental results indicate that SZ-A intervention can enhance vascular remodeling and repair.

[0256] Experimental Example 5: Effects of Mulberry Extract on Diabetic Atherosclerosis (AS)

[0257] 1. Animal Model and Grouping

[0258] 1.1 Establishment and grouping of a diabetic atherosclerosis animal model (single high-dose STZ)

[0259] 8-week-old SPF grade C57 BL / 6J and ApoE - / - Mice were acclimatized for one week. C57BL / 6J served as the control group, fed a standard diet; ApoE - / - Mice were used as the model group and fed HFHC (high cholesterol diet, 40% fat, 1.25% cholesterol) for 5 weeks. Then, they were injected intraperitoneally with 100 mg / kg STZ (streptozotocin). Blood glucose was measured on the 3rd day after injection. A blood glucose value ≥11 mmol / L was considered a successful model and used for subsequent experiments.

[0260] After successful modeling, participants were randomly grouped according to their weight and blood glucose levels. The experimental groups and drug administration are shown in Table 3. The drugs were administered as follows for 6 consecutive weeks.

[0261] Table 3. Experimental grouping and administration of diabetic AS animal model (single high-dose STZ)

[0262] 1.2 Establishment and grouping of a diabetic atherosclerosis animal model (five low-dose STZ treatments)

[0263] Eight-week-old SPF-grade ApoE- / - mice were acclimatized for one week. Mice were then divided into a control group and a model group based on body weight and blood glucose levels. The control group was fed a standard diet; the model group was fed HFHC (high-cholesterol diet, 40% fat, 1.25% cholesterol). After six weeks, STZ was injected intraperitoneally once daily at a dose of 50 mg / kg for five consecutive days. Blood glucose levels were measured on days 3, 6, and 9 post-injection. A blood glucose level ≥16.7 mmol / L for three consecutive days was considered a successful model for subsequent experiments.

[0264] After successful modeling, participants were randomly grouped according to their weight and blood glucose levels. The experimental groups and drug administration are shown in Table 4. The drugs were administered as follows for 12 consecutive weeks.

[0265] Table 4. Experimental grouping and administration of diabetic AS animal model (five low-dose STZ treatments)

[0266] 2 Experimental methods and results

[0267] 2.1 SZ-A effectively alleviates disease progression in a single high-dose STZ injection model of diabetes mellitus.

[0268] Three weeks after treatment, blood was collected from the tail tip, and random morning blood glucose levels were measured and recorded using a glucometer. After treatment, mice were fasted overnight, euthanized, and blood was collected from the orbital vein. Fasting blood glucose was measured using a glucometer, and serum was separated from the remaining whole blood. Serum levels of P-selectin and E-selectin were detected using ELISA, and concentration analysis was performed based on a standard curve. The mice were perfused with pre-cooled physiological saline until the outflow was clear. Under a stereomicroscope, the aorta was freed and longitudinally cut. The aorta was fixed with 4% paraformaldehyde, stained with Oil Red O working solution, and incubated. The tissue was washed twice with 70% ethanol. The tissue was immersed in PBS and photographed. The percentage of plaque area was calculated. The experimental results are shown in Figure 11.

[0269] The results showed that, compared with the control group, the body weight of the STZ-injected model mice decreased (Fig. 11a). SZ-A treatment significantly reduced fasting blood glucose (Fig. 11c) and random blood glucose (Fig. 11b), with the high-dose SZ-A group showing comparable effects to the positive control SGLT2i group. Furthermore, the arterial plaque area was significantly reduced in all SZ-A dose groups (Fig. 11d, e, f), with the high-dose SZ-A group showing better results than the SGLT2i group. In addition, SZ-A significantly reduced the levels of E-selectin and P-selectin (Fig. 11g and h).

[0270] 2.2 SZ-A effectively alleviated the disease progression in a diabetic AS model induced by five low-dose STZ injections.

[0271] Five weeks after treatment, blood was collected from the tail tip, and random morning blood glucose levels were measured and recorded using a glucometer. After treatment, mice were fasted overnight and anesthetized, and arterial blood flow was measured using ultrasound. Subsequently, mice were euthanized, and blood was collected from the orbital vein to measure glycated hemoglobin (HBA1c). The remaining serum was separated, and serum P-selectin and E-selectin levels were detected using ELISA, with concentration analysis based on a standard curve. The mice were perfused with pre-cooled physiological saline until the outflow was clear. Under a stereomicroscope, the aorta was freed and longitudinally cut. The aorta was fixed with 4% paraformaldehyde, stained with Oil Red O working solution, and incubated. The tissue was washed twice with 70% ethanol. The tissue was immersed in PBS and photographed. The percentage of plaque area was calculated. The experimental results are shown in Figure 12.

[0272] The results showed that after STZ injection, the model animals died (Fig. 12a) and their body weight decreased (Fig. 12b). Compared with the model group, each dose group of SZ-A significantly reduced random blood glucose (Fig. 12e) and glycated hemoglobin (Fig. 12f), while reducing the formation of arterial plaques (Fig. 12c, d). Compared with the model group, the peak descending aortic blood flow in the SZ-A100 group showed an increasing trend (Fig. 12h, P = 0.052), and the mean blood flow in the ascending and descending aorta increased by 21.79% and 15.73%, respectively, indicating that SZ-A has the effect of improving arterial blood flow. Furthermore, the SZ-A100 group significantly reduced the levels of P-selectin and E-selectin (Fig. 12i, j).

Claims

1. Use of a mulberry extract for the manufacture of a medicament for the treatment and / or prevention of diabetic vascular complications, characterized in that, The mulberry extract contains more than 3% alkaloids by weight (optionally contains 3-99% alkaloids by weight, further optionally contains 15-99% alkaloids by weight, further optionally contains 30-99% alkaloids by weight, further optionally contains 40-99% alkaloids by weight, further optionally contains 50-99% alkaloids by weight, further optionally contains 60-99% alkaloids by weight) based on the total weight percentage of the mulberry extract components; and / or contains not more than 70% polysaccharides by weight (optionally contains 0.2-70% polysaccharides by weight, further optionally contains 0.2-50% polysaccharides by weight, further optionally contains 0.2-35% polysaccharides by weight, further optionally contains 0.2-25% polysaccharides by weight, further optionally contains 0.2-23% polysaccharides by weight, further optionally contains 20-25% polysaccharides by weight), and / or contains not more than 10% flavonoids by weight (optionally contains 0.05-5% flavonoids by weight, further optionally contains 0-2% flavonoids by weight, further optionally contains 0.05-2% flavonoids by weight, further optionally contains 0.5-1.5% flavonoids by weight, further optionally contains 0-1% flavonoids by weight, further optionally contains 0.05-1% flavonoids by weight), and / or contains not more than 50% amino acids by weight (optionally contains 0-30% amino acids by weight, further optionally contains 0-25% amino acids by weight, further optionally contains 0-20% amino acids by weight, further optionally contains 0-5% amino acids by weight, further optionally contains 3-25% amino acids by weight, or further optionally contains 5-20% amino acids by weight), and / or other components (optionally contains 0-25% by weight, further optionally contains 0-20% by weight, further optionally contains 0-15% by weight, further optionally contains 0-11% by weight, further optionally contains 2-20% by weight, further optionally contains 4-8% by weight).

2. Use of the extract of Morus alba according to claim 1 for the preparation of a medicament for the treatment and / or prevention of diabetic vascular complications, characterized in that, The treatment and / or prevention of diabetic vascular complications is manifested by improving the functional impairment of vascular endothelial cells; preferably specifically manifested by improving any one or several of the following: (1) promoting vascular endothelial cell proliferation; (2) promoting vascular endothelial cell migration; (3) promoting angiogenesis; (4) inhibiting vascular endothelial cell apoptosis; (5) promoting NO accumulation in endothelial cells; (6) up-regulating the expression of vasoactive-related factors, preferably the vasoactive-related factors include CD31 (platelet-endothelial cell adhesion factor-1), VEGF (vascular endothelial growth factor) (preferably VEGF-A), and / or eNOS (endothelial nitric oxide synthase); (7) alleviating inflammation of vascular endothelium, preferably by inhibiting adhesion of vascular endothelial cells to monocytes, preferably inhibiting secretion of vascular endothelial cell adhesion factors including P-selectin and / or E-selectin; (8) increasing the number of mitochondria of vascular endothelial cells.

3. Use of the extract of Morus alba according to claim 2 for the preparation of a medicament for the treatment and / or prevention of diabetic vascular complications, characterized in that, The improvement of the impairment of vascular endothelial cell function is also manifested by promoting expression of NRF2 (nuclear factor erythroid 2-related factor 2) and / or HO-1 (heme oxygenase 1).

4. Use of the extract of Morus alba according to claim 1 for the preparation of a medicament for the treatment and / or prevention of diabetic vascular complications, characterized in that, The diabetic vascular complications include diabetic foot or diabetic atherosclerosis.

5. Use of the extract of Morus alba according to claim 4 for the preparation of a medicament for the treatment and / or prevention of diabetic vascular complications, characterized in that, The treatment and / or prevention of diabetic foot is manifested by any one or more of the following: (1) promoting wound healing, including promoting wound regeneration, re-epithelialization and / or collagen production, (2) reducing apoptosis of cells in the wound area, (3) promoting cell migration and / or proliferation in the wound area, (4) promoting angiogenesis, remodeling and / or repair in the wound area, preferably promoting expression of CD31, VEGF (preferably VEGF-A) and / or eNOS.

6. Use of the extract of Morus alba according to claim 4 for the preparation of a medicament for the treatment and / or prevention of diabetic vascular complications, characterized in that, The treatment and / or prevention of diabetic atherosclerosis is manifested by any one or more of the following: (1) reducing the formation of arterial plaques, (2) improving arterial blood flow, (3) alleviating inflammation of vascular endothelium, preferably reducing the level of vascular endothelial cell adhesion factors including P-selectin and / or E-selectin.

7. Use of the extract of Morus alba according to any one of claims 1 to 6 for the preparation of a medicament for the treatment and / or prevention of diabetic vascular complications, characterized in that, The weight content of each component in the mulberry extract is calculated based on the total weight content of all components in the mulberry extract as 100%. Preferably, the weight content of each component in the mulberry extract is, based on 100% of the weight content of the sum of each component of the mulberry extract: Preferably, the weight content of each component in the mulberry extract is, based on 100% of the weight content of the sum of each component of the mulberry extract: Preferably, the weight content of each component in the mulberry extract is, based on 100% of the weight content of the sum of each component of the mulberry extract: Preferably, the weight content of each component in the mulberry extract is, based on 100% of the weight content of the sum of each component of the mulberry extract: Preferably, the weight content of each component in the mulberry extract is, based on 100% of the weight content of the sum of each component of the mulberry extract: Preferably, the weight content of each component in the mulberry extract is, based on 100% of the weight content of the sum of each component of the mulberry extract: More preferably, the weight content of each component in the mulberry extract is, based on the weight content of the sum of each component of the mulberry extract being 100%: More preferably, the weight content of each component in the mulberry extract is, based on the total weight content of the components of the mulberry extract being 100%:

8. Use of the extract of Morus alba according to any one of claims 1 to 7 for the preparation of a medicament for the treatment and / or prevention of diabetic vascular complications, characterized in that, The alkaloid comprises one or more of 1-deoxynojirimycin (DNJ), N-methly-1-deoxynojirimycin, fagomine (FAG), 3-epi-fagomine, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), calystegin B2, calystegin C1, 2-O-(α-D-galactopyranosyl)-1-deoxynojirimycin, 6-O-(β-D-glucopyranosyl)-1-deoxynojirimycin, 1,4-dideoxy-1,4-imino-(2-O-β-D-glucopyranosyl)-D-arabinitol; Optionally, the weight percentage of DNJ is not less than 50% (optionally 60-99%) of the total alkaloid.

9. Use of the extract of Morus alba according to any one of claims 1 to 8 for the preparation of a medicament for the treatment and / or prevention of diabetic vascular complications, characterized in that, The preparation of the mulberry extract comprises the following steps: 1) preparing a crude extract of Moraceae plants; 2) separating the crude extract through a cation resin and / or an optional anion resin to obtain a resin effluent, an optional step 3) : performing alcohol precipitation treatment on the resin effluent of step 2) to collect supernatant; 4) performing concentration and / or drying treatment on the supernatant; optionally, performing concentration and / or drying treatment before performing alcohol precipitation treatment on the resin effluent of step 2).

10. Use of the extract of Morus alba according to any one of claims 1 to 9 for the preparation of a medicament for the treatment and / or prevention of diabetic vascular complications, characterized in that, The mulberry extract acts on humans or mammals; Optionally, the medicine is in the form of oral administration; optionally, the medicine is in the form of tablets, capsules, oral solutions, oral emulsions, pills, granules, syrups or powders.

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