Use of mulberry extract in preparing drug which treats and / or prevents retinopathy

Abstract

A use of a mulberry extract in preparing a drug which treats and / or prevents retinopathy, the mulberry extract including an alkaloid having an amount by weight of 3% or more, and / or including a polysaccharide having an amount by weight not higher than 70%, and / or including a flavonoid having an amount by weight not higher than 10%, and / or including an amino acid having an amount by weight not higher than 50%, and / or other components. Preparation of the mulberry extract comprises: 1) preparing a crude extract of a moraceae plant; 2) performing separation on the crude extract by means of a cationic resin and / or an optional anionic resin, to obtain a resin effluent; optional step 3): performing alcohol precipitation treatment on the resin effluent of step 2), and collecting a supernatant; 4) performing concentration and / or drying treatment on the supernatant; and optionally, before performing alcohol precipitation treatment on the resin effluent of step 2), performing concentration and / or drying treatment. The mulberry extract has a significant effect on improving retinopathy.

Description

Use of mulberry extract in the preparation of a drug for treating and / or preventing retinopathy TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to use of mulberry extract in the preparation of a drug for treating and / or preventing retinopathy. TECHNICAL BACKGROUND

[0002] The retina is the initial site of vision formation and is the lesion site of various blinding eye diseases. Common retinal diseases include retinal vascular disease, macular disease, retinal detachment, hereditary retinal dystrophy, etc. Part of the retinal diseases can cause irreversible visual function damage. At present, the causes of most diseases are still unclear, and there is no effective treatment method. The development of related drug research is also relatively slow.

[0003] Vascular retinopathy is a group of diseases characterized by retinal hemorrhage, exudation and abnormal proliferation of blood vessels. Among them, retinal pathological angiogenesis (vascular proliferation) and retinal vascular leakage often occur in some common blinding eye diseases, such as wet age-related macular degeneration (wet age-related macular degeneration, wAMD), diabetic retinopathy (Diabetic retinopathy, DR) and retinal vein occlusion (retinal vein occlusion) and so on. Pathological retinal neovascularization (retinal neovascularization, RNV) refers to a pathological condition caused by the imbalance between pro-angiogenic factors such as vascular endothelial cell growth factor (vascular endothelial cell growth factor, VEGF), tumor necrosis factor alpha (tumor necrosis factor alpha, TNFα), interleukin 1-β and various immunoglobulins and anti-angiogenic factors due to retinal ischemia, hypoxia and other factors. RNV can occur in many retinal diseases such as diabetic retinopathy, retinopathy of prematurity and age-related macular degeneration. So far, anti-VEGF drugs have gradually become the first-line drugs for treating RNV, which can achieve the effect of anti-neovascularization by blocking VEGF. However, intravitreal injection of anti-VEGF drugs may cause intraocular inflammation, intraocular hemorrhage, retinal detachment and other complications or other side effects. When the retina provides the necessary nutrients and oxygen, the components in the blood are selectively permeable into the retinal neural tissue through the blood vessels. This selective permeability is mainly achieved by the blood-retina barrier (Blood-Retina Barrier, BRB), which plays an important protective role in the health of the retina. The presence of BRB provides a stable microenvironment for retinal neurons to ensure their normal physiological functions. When the integrity of the retinal blood vessels is damaged, some components in the blood will leak into the retinal tissue, causing a series of inflammatory reactions and damaging the normal function of the retina.

[0004] At present, for retinopathy, clinical treatment urgently needs to explore safer and more effective drugs. SUMMARY

[0005] The present application aims to provide a use of mulberry extract in the preparation of a medicament for treating and / or preventing retinopathy.

[0006] In one embodiment, the present application provides a method for treating and / or preventing retinopathy, comprising administering to a subject in need thereof an effective amount of mulberry extract.

[0007] In one embodiment, the present application provides use of a mulberry extract in treating and / or preventing retinopathy.

[0008] In one embodiment, the present application provides a mulberry extract for treating and / or preventing retinopathy.

[0009] In the present application, the mulberry extract comprises alkaloids in a weight content of 3% or more (optionally comprising alkaloids in a weight content of 3-99%, further optionally comprising alkaloids in a weight content of 15-99%, further optionally comprising alkaloids in a weight content of 30-99%, further optionally comprising alkaloids in a weight content of 40-99%, further optionally comprising alkaloids in a weight content of 50-99%, further optionally comprising alkaloids in a weight content of 60-99%) based on the weight percentage content of the sum of the components of the mulberry extract being 100%,

[0010] and / or polysaccharides in a weight content of not more than 70% (optionally comprising polysaccharides in a weight content of 0.2-70%, further optionally comprising polysaccharides in a weight content of 0.2-50%, further optionally comprising polysaccharides in a weight content of 0.2-35%, further optionally comprising polysaccharides in a weight content of 0.2-25%, further optionally comprising polysaccharides in a weight content of 0.2-23%, further optionally comprising polysaccharides in a weight content of 20-25%),

[0011] and / or flavonoids in a weight content of not more than 10% (optionally comprising flavonoids in a weight content of 0.05-5%, further optionally comprising flavonoids in a weight content of 0-2%, further optionally comprising flavonoids in a weight content of 0.05-2%, further optionally comprising flavonoids in a weight content of 0.5-1.5%, further optionally comprising flavonoids in a weight content of 0-1%, further optionally comprising flavonoids in a weight content of 0.05-1%),

[0012] and / or amino acids in a weight content of not more than 50% (optionally comprising amino acids in a weight content of 0-30%, further optionally comprising amino acids in a weight content of 0-25%, further optionally comprising amino acids in a weight content of 0-20%, further optionally comprising amino acids in a weight content of 0-5%, further optionally comprising amino acids in a weight content of 3-25% or further optionally comprising amino acids in a weight content of 5-20%),

[0013] and / or other components (optionally in a weight content of 0-25%, further optionally in a weight content of 0-20%, further optionally in a weight content of 0-15%, further optionally in a weight content of 0-11%, further optionally in a weight content of 2-20%, further optionally in a weight content of 4-8%).

[0014] In a preferred embodiment of the present application, the treatment and / or prevention of retinopathy comprises improving any one or several of the following (1)-(11):

[0015] (1) inhibiting retinal pathological angiogenesis and / or reducing the central avascular area;

[0016] (2) protecting retinal tissue structure;

[0017] (3) inhibiting retinal vascular leakage;

[0018] (4) reducing the expression level of VEGF in retinal tissue;

[0019] (5) reducing the expression level of EGLN1 and / or EPHA2 in retinal tissue;

[0020] (6) inhibiting retinal vascular endothelial cell tube formation and / or migration;

[0021] (7) improving the permeability and / or integrity of the retinal vascular endothelial cell layer;

[0022] (8) reducing the expression level of PI3K in retinal tissue;

[0023] (9) reducing the expression level of Akt in retinal tissue;

[0024] (10) increasing the expression level of Occludin in retinal tissue;

[0025] (11) reducing the increase in retinal vascular endothelial cell viability caused by high glucose.

[0026] In a preferred embodiment, the retinopathy is induced by oxygen, VEGF and / or high blood glucose.

[0027] In a preferred embodiment, the retinopathy comprises proliferative retinopathy and / or exudative retinopathy.

[0028] In a preferred embodiment, the retinopathy comprises diabetic retinopathy, age-related macular degeneration, retinal vein occlusion, retinal vasculitis or retinopathy of prematurity.

[0029] In an embodiment, the treatment and / or prevention of retinopathy is the treatment and / or prevention of oxygen-induced retinopathy, which comprises improving any one or several of the following (1)-(10):

[0030] (1) inhibiting retinal pathological angiogenesis and / or reducing the central avascular area;

[0031] (2) protecting retinal tissue structure;

[0032] (3) inhibiting retinal vascular leakage;

[0033] (4) reducing the expression level of VEGF in retinal tissue;

[0034] (5) reducing the expression level of EGLN1 and / or EPHA2 in retinal tissue;

[0035] (6) inhibiting retinal vascular endothelial cell tube formation and / or migration;

[0036] (7) improving the permeability and / or integrity of retinal vascular endothelial cell layer;

[0037] (8) reducing the expression level of PI3K in retinal tissue;

[0038] (9) reducing the expression level of Akt in retinal tissue;

[0039] (10) increasing the expression level of Occludin in retinal tissue.

[0040] In one embodiment, the treatment and / or prevention of retinal disorder is the treatment and / or prevention of VEGF-induced retinal disorder, which comprises improving any one or several of (1)-(5) below:

[0041] (1) inhibiting retinal pathological angiogenesis and / or reducing central avascular area;

[0042] (2) protecting retinal tissue structure;

[0043] (3) inhibiting retinal vascular leakage;

[0044] (4) inhibiting retinal vascular endothelial cell tube formation and / or migration;

[0045] (5) improving the permeability and / or integrity of retinal vascular endothelial cell layer.

[0046] In one embodiment, the treatment and / or prevention of retinal disorder is the treatment and / or prevention of hyperglycemia-induced retinal disorder, which comprises improving any one or several of (1)-(11) below:

[0047] (1) inhibiting retinal pathological angiogenesis and / or reducing central avascular area;

[0048] (2) protecting retinal tissue structure;

[0049] (3) inhibiting retinal vascular leakage;

[0050] (4) reducing the expression level of VEGF in the retinal tissue;

[0051] (5) reducing the expression level of EGLN1 and / or EPHA2 in the retinal tissue;

[0052] (6) inhibiting the tube formation and / or migration of retinal vascular endothelial cells;

[0053] (7) improving the permeability and / or integrity of the retinal vascular endothelial cell layer;

[0054] (8) reducing the expression level of PI3K in the retinal tissue;

[0055] (9) reducing the expression level of Akt in the retinal tissue;

[0056] (10) increasing the expression level of Occludin in the retinal tissue;

[0057] (11) reducing the increase of retinal vascular endothelial cell viability caused by high glucose.

[0058] Optionally, the high glucose-induced retinopathy is diabetic retinopathy.

[0059] In one embodiment, the treatment and / or prevention of retinopathy is treatment and / or prevention of age-related macular degeneration, retinal vein occlusion, retinal vasculitis or retinopathy of prematurity, which is manifested as improvement in any one or several of the following (1)-(10):

[0060] (1) inhibiting retinal pathological angiogenesis and / or reducing the central avascular area;

[0061] (2) protecting the retinal tissue structure;

[0062] (3) inhibiting retinal vascular leakage;

[0063] (4) reducing the expression level of VEGF in the retinal tissue;

[0064] (5) reducing the expression level of EGLN1 and / or EPHA2 in the retinal tissue;

[0065] (6) inhibiting the tube formation and / or migration of retinal vascular endothelial cells;

[0066] (7) improving the permeability and / or integrity of the retinal vascular endothelial cell layer;

[0067] (8) reducing the expression level of PI3K in the retinal tissue;

[0068] (9) reducing the expression level of Akt in retinal tissue;

[0069] (10) increasing the expression level of Occludin in retinal tissue.

[0070] In a preferred embodiment of the present application, the weight content of each component in the mulberry extract is, based on 100% of the weight percentage content of the sum of the components of the mulberry extract:

[0071] Preferably, the weight content of each component in the mulberry extract is, based on 100% of the weight percentage content of the sum of the components of the mulberry extract:

[0072] Preferably, the weight content of each component in the mulberry extract is, based on 100% of the weight percentage content of the sum of the components of the mulberry extract:

[0073] Preferably, the weight content of each component in the mulberry extract is, based on 100% of the weight percentage content of the sum of the components of the mulberry extract:

[0074] Preferably, the weight content of each component in the mulberry extract is, based on 100% of the weight percentage content of the sum of the components of the mulberry extract:

[0075] Preferably, the weight content of each component in the mulberry extract is, based on 100% of the weight percentage content of the sum of the components of the mulberry extract:

[0076] Preferably, the weight content of each component in the mulberry extract is, based on 100% of the weight percentage content of the sum of the components of the mulberry extract:

[0077] Preferably, the weight content of each component in the mulberry extract is, based on 100% of the weight percentage content of the sum of the components of the mulberry extract:

[0078] More preferably, the weight content of each component in the mulberry extract is, based on 100% of the weight percentage content of the sum of the components of the mulberry extract:

[0079] More preferably, the weight content of each component in the mulberry extract is, based on 100% of the weight percentage content of the sum of the components of the mulberry extract:

[0080] Preferably, 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-(a-D-galactopyranosyl)-1-deoxynojirimycin, 6-O-(b-D-glucopyranosyl)-1-deoxynojirimycin, 1,4-dideoxy-1,4-imino-(2-O-b-D-glucopyranosyl)-D-arabinitol.

[0081] Preferably, the weight percentage of DNJ is not less than 50% (preferably 60-99%) of the alkaloid.

[0082] Preferably, the heavy metal content of the mulberry extract is not more than 10 ppm.

[0083] The medicine is in the form of oral administration; alternatively, the medicine is in the form of tablets, capsules, lozenges, powders, oral solutions, oral emulsions, pills, granules, syrups or powders.

[0084] In the present application, the mulberry extract can be provided in the form of commercially available mulberry twig total alkaloid tablets (Guo Yao Zhun Zi Z20200002).

[0085] Alternatively, the mulberry extract can be prepared according to the method described in CN 110393738A.

[0086] Alternatively, in the present application, the preparation of the mulberry extract comprises the following steps:

[0087] 1) preparing a crude extract of Moraceae plant; 2) separating the crude extract by cation resin and / or optional anion resin to obtain resin effluent, optional step 3) alcohol precipitation treatment to the resin effluent of step 2) and collecting supernatant; 4) concentrating and / or drying treatment to the supernatant. Optionally, before alcohol precipitation treatment to the resin effluent of step 2), concentration and / or drying treatment is performed.

[0088] Preferably, the extraction method further comprises a step of concentrating the crude extract of plant before the separation of step 2); preferably, the crude extract of plant is concentrated to a mass concentration of 1-15%, preferably 2-10% of solid in solution. The solid refers to the solid substance left after removal of water in solution.

[0089] Optionally, alcohol precipitation treatment can also be performed to the concentrated crude extract before resin separation treatment of step 2). In alcohol precipitation treatment, ethanol is added to the crude extract, stirred and mixed, agitation is stopped and left for a certain period of time to allow the insoluble substance to precipitate. Optionally, the volume mass ratio of added ethanol to plant raw material is 0.2-20 times, optionally 0.4-10 times, calculated in L / kg. Further optionally, alcohol precipitation tank is used for alcohol precipitation treatment. Optionally, the stirring speed in alcohol precipitation treatment is 10-600 rpm, optionally 40-500 rpm, further optionally 80-400 rpm or 300 rpm.

[0090] 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. Mulberry varieties bred from the above species, including var. diabolica 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 varieties selected from within or between species of the above species;

[0091] 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.

[0092] In this invention, the mulberry extract may be selected as mulberry twig extract, mulberry bark extract, mulberry leaf extract, or a mixture thereof.

[0093] 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.

[0094] In a preferred embodiment, the pulverized plant material can be added to an extraction tank for extraction.

[0095] Preferably, the extract is filtered to remove insoluble matter, yielding a crude plant extract.

[0096] 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.

[0097] 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, concentrating and / or drying to obtain the extract.

[0098] 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.

[0099] 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.

[0100] Preferably, the cationic resin is selected from the group consisting of one or more of strong acid cation exchange resin, weak acid cation exchange resin and strong base quaternary ammonium type cation resin. Alternatively, the cationic resin is one or more of 732 type strong acid styrene cation exchange resin, 002SC type strong acid styrene cation resin, 734 type strong acid styrene cation exchange resin, D001 type macroporous strong acid styrene cation exchange resin or D113 type macroporous weak acid cation resin and D254 type macroporous strong base quaternary ammonium type cation exchange resin. Preferably, the cationic resin is one or more of 732 type strong acid styrene cation exchange resin, 734 type strong acid styrene cation exchange resin, 002SC type strong acid styrene cation resin, D113 type macroporous weak acid cation resin and D001 type macroporous strong acid styrene cation exchange resin. Preferably, the cationic resin is one or more of 002SC type strong acid styrene cation resin, D113 type macroporous weak acid cation resin and D001 type macroporous strong acid styrene cation exchange resin.

[0101] Alternatively, the amount of cationic resin used is 1:1-30 (alternatively, 1:1-25, 1:2-20, 1:2-15, 1:2-10, 1:2-7, 1:2-3, 1:4-15) by weight of the plant raw material.

[0102] After loading the plant crude extract onto the cationic resin, the loaded cationic resin is eluted with an eluent. Alternatively, the eluent is a salt solution or an alkaline solution containing cations, which are alternatively one or more of sodium chloride, ammonium chloride, ammonium sulfate, ammonium nitrate, aqueous ammonia, potassium chloride and sodium hydroxide.

[0103] Alternatively, the concentration of cations in the eluent is 0.04-5 mol / L (alternatively, 0.5-2.5 mol / L, 0.2-3 mol / L, further alternatively, 0.5-2.5 mol / L).

[0104] Alternatively, the flow rate of the eluent is 1-15 BV / h (alternatively, 5-10 BV / h, further alternatively, 5-6 BV / h).

[0105] Alternatively, the weight of the eluent used for the separation of the cationic resin is 0.1-30 times the weight of the plant raw material, which is alternatively 0.5-10 times the weight of the plant raw material, further alternatively, 1-10 times.

[0106] The starting point of collection can be determined according to the pH of the cation resin effluent, for example, when an alkaline solution such as ammonia is used for elution, the effluent is collected when the pH of the cation column effluent is greater than 7, or the starting point of collection of the effluent is determined according to a color reaction or precipitation reaction; alternatively, when the volume of the collected solution reaches 0.1-10 times (further alternatively, 0.6-10 times, or 0.2-5 times) the weight of the plant raw material, the collection is stopped, and the collected solution is optionally purified by passing through an anion column. In order to improve the separation effect of the cation resin, the cation resin can also be used for multiple separations, for example, 2-5 times.

[0107] When purified by passing through an anion column, the anion resin column can be activated in the order of alkaline solution washing, acidic solution washing, and alkaline solution washing.

[0108] Alternatively, the alkaline solution washing is performed until the pH of the eluate is 8.0-9.5, which can be 8.5-9.5.

[0109] Alternatively, the alkaline solution is selected from an ammonia solution, a sodium hydroxide solution, a potassium hydroxide solution, or a sodium carbonate solution; and the concentration of the alkaline solution is 0.5-4 mol / L, which can be 1-2 mol / L.

[0110] Alternatively, the acidic solution washing is performed until the pH of the eluate is 3.0-7.0, which can be 3.5-6.5. Alternatively, the acidic solution is selected from a hydrochloric acid solution, a phosphoric acid solution, a sodium hydrogen phosphate-citric acid buffer solution; and the concentration of the acidic solution is 0.5-4 mol / L, which can be 1-2 mol / L.

[0111] Preferably, the anion resin is one or a combination of strong basic anion exchange resin, weak basic anion exchange resin, or weak acid anion exchange resin. Alternatively, the anion resin is one or a combination of 711 type strong basic styrene anion resin, 717 type strong basic styrene anion exchange resin, D201 type macroporous strong basic styrene anion exchange resin, D218 type macroporous strong basic acrylic anion exchange resin, D301-G type macroporous weak acid styrene anion exchange resin, and D301 type macroporous weak basic styrene anion exchange resin. Preferably, the anion resin is one or a combination of 711 type strong basic styrene anion resin, 717 type strong basic styrene anion exchange resin, D201 type macroporous strong basic styrene anion exchange resin, and D218 type macroporous strong basic acrylic anion exchange resin. Preferably, the anion resin is one or a combination of 711 type strong basic styrene anion resin, D201 type macroporous strong basic styrene anion exchange resin, and D218 type macroporous strong basic acrylic anion exchange resin.

[0112] Optionally, the amount of the anion resin is 1:1-80 (optionally, 1:1-64, 1:1-32, 1:1-24, 1:5-16, 1:3) relative to the weight of the plant raw material.

[0113] The collection is started when the liquid flows out of the anion resin. Optionally, the collection is stopped when the volume of the collected liquid reaches 0.05-10 times (optionally, 0.1-5 times) of the weight of the plant raw material. Optionally, to improve the separation effect of the anion resin, the anion resin can be used for multiple times of separation, for example, 2-4 times of separation.

[0114] Optionally, the amount of the ethanol used in the alcohol precipitation treatment is 1:4-600 (optionally, 1:20-300, further optionally, 1:20-200, 1:20-50, 1:40, 1:80, 1:22, 1:200) relative to the weight of the plant raw material. In the alcohol precipitation treatment, the stirring speed is 10-600 rpm (optionally, 40-500 rpm, 80-400 rpm, 300-400 rpm). The time for the alcohol precipitation treatment is 12-24 h.

[0115] Further, before the alcohol precipitation treatment, the method further comprises the steps of centrifugal impurity removal or microfiltration membrane filtration impurity removal of the anion resin effluent, and reverse ion permeation membrane concentration. The specific gravity of the concentrated liquid is 1.0-1.3, optionally, 1.1-1.25. The specific gravity refers to the mass ratio of the concentrated liquid to water under the same volume condition.

[0116] The concentration treatment method of the present application can comprise heat concentration, nanofiltration membrane, reverse ion permeation membrane concentration, and combinations thereof. Preferably, centrifugal, ultrafiltration membrane filtration or microfiltration membrane filtration impurity removal is performed before reverse ion permeation membrane and nanofiltration membrane concentration.

[0117] Optionally, the medicament further comprises a pharmaceutically acceptable carrier. The carrier is a non-active ingredient that is non-toxic to the human body in accordance with the medication route or administration method. The carrier can be a solid or liquid auxiliary material. The solid auxiliary material, for example, includes microcrystalline cellulose, mannitol, lactose, pregelatinized starch, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, 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; the liquid auxiliary material, for example, includes water, ethanol, sugar syrup, and glycerol.

[0118] Beneficial effects: The mulberry extract of the present application can inhibit retinal pathological angiogenesis and / or reduce the central avascular area; protect the retinal tissue structure; inhibit retinal vascular leakage; reduce the expression level of VEGF in the retinal tissue; reduce the expression level of EGLN1 and / or EPHA2 in the retinal tissue; inhibit the tube formation and / or migration of retinal vascular endothelial cells; improve the permeability and integrity of the retinal vascular endothelial cell layer, etc., and can effectively treat and / or prevent retinal lesions. BRIEF DESCRIPTION OF DRAWINGS

[0119] Figure 1 is a whole retinal flat mount image (10x) of P17 mice in Test Example 1 (Control group and OIR group) (the circled part in the figure is a new blood vessel cluster, and the area indicated by the arrow is the central avascular area);

[0120] Figure 2 is the ratio of new blood vessels and avascular area to the whole retinal area of P17 mice in Test Example 1 (n=6) (note: *** indicates P<0.001 compared with the Control group);

[0121] Figure 3 is a whole retinal flat mount image (10x) of mice in each administration group in Test Example 1 after administration;

[0122] Figure 4 is an HE staining image of mice in each administration group in Test Example 1 after administration;

[0123] Figure 5 is a VEGF immunohistochemical image of mice in each group in Test Example 1 after administration;

[0124] Figure 6 is an immunofluorescence photograph of the expression of EGLN1 protein in the retinal tissue of mice in each group in Test Example 1;

[0125] Figure 7 is a bar chart of the average immunofluorescence intensity of the photographs of each group in Figure 6 ((bar=50μm) n=6, `x±s.*P<0.05 vs Control group; # P<0.05, ## P<0.01 vs OIR group; ΔΔ P<0.01 vs Bevacizumab group; ns:P>0.05 vs Bevacizumab group);

[0126] Figure 8 is a result graph of the expression of EphA2, PI3K and Akt in the retinal tissue of mice in each group in Test Example 1 (n=3, x`±s.*P<0.05, **P<0.01, ***P<0.001 vs Control group, # P<0.05, ## P<0.01, ### P<0.001, ####P<0.0001 vs OIR group; ΔΔ P<0.01, ΔΔΔΔ P<0.0001, ns: P>0.05 vs Bevacizumab group);

[0127] Figure 9 is an immunofluorescence photograph of Occludin protein expression in the retinal tissue of mice in each group in Test Example 1;

[0128] Figure 10 is a bar graph of the average immunofluorescence intensity of the photographs in each group in Figure 9 (bar = 50 μm, n = 7, x ± s. *P<0.05 vs Control group; ns: P>0.05 vs Bevacizumab group);

[0129] Figure 11 is a graph of the retinal permeability of model mice in each group in Test Example 2, to which VEGF was injected into the vitreous body at different doses;

[0130] Figure 12 is an HE staining photograph of mice in each group in Test Example 2 after administration of the drug;

[0131] Figure 13 is an HE staining photograph of KKAy mice in each group in Test Example 3;

[0132] Figure 14 is a graph of the effect of mulberry extract on the viability of HRMEC cells induced by high glucose in Test Example 4 *** P<0.001, **** P<0.0001 indicates comparison with the Control group; # P<0.05, ## P<0.01, ### P<0.001 indicates comparison with the HG group);

[0133] Figure 15 is a photograph under a microscope of HRMECs in each group in the tube formation test in Test Example 4;

[0134] Figure 16 is a graph of the effect of SZ-A on the tube formation length of HRMECs induced by high glucose in Test Example 4 **** P<0.0001 indicates comparison with the Control group; # P<0.05, ## P<0.01, ### P<0.001, #### P<0.0001 indicates comparison with the HG group);

[0135] Figure 17 is a comparison graph of the effect of different SZ-A on the angiogenic ability of HRMECs in Test Example 4 (*P<0.05 vs SZ-A-1 group);

[0136] Figure 18 is a microscope image of the effect of SZ-A on high glucose-induced HRMEC cell migration in Example 4;

[0137] Figure 19 is a graph of the effect of SZ-A on high glucose-induced HRMEC cell migration in Example 4; ** P<0.01, *** P<0.001 vs Control group; # P<0.05, ## P<0.01, ### P<0.001 vs HG group;

[0138] Figure 20 is a graph of the ability of different SZ-A to inhibit high glucose-induced HRMEC cell migration rate in Example 4 (*P<0.05 vs SZ-A-1 group);

[0139] Figure 21 is a graph of the effect of SZ-A on the barrier function of HRMECs in vitro in the TEER experiment of Example 4; *** P<0.001 vs Control group; # P<0.05, ## P<0.01 vs HG group;

[0140] Figure 22 is a graph of the ability of different mulberry extracts to improve the blood-retinal barrier in the TEER experiment of Example 4 (**P<0.01 vs SZ-A-1 group);

[0141] Figure 23 is a graph of the effect of SZ-A on high glucose-induced HRMECs cell FITC-dextran permeability in Example 4; *** P<0.001 vs Control group; ## P<0.01 vs HG group;

[0142] Figure 24 is a graph of the ability of different mulberry extracts to inhibit FITC-dextran leakage rate. DETAILED DESCRIPTION

[0143] The present application is further described in detail by the following examples. The particulars shown herein are by way of example and for purposes of illustrative discussion of the present application only and are not intended to limit the scope of the application to the practice only of the particulars described herein.

[0144] The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations.

[0145] Moreover, the technical features involved in the different implementations of the present application described hereinafter can be combined with each other when they do not conflict with each other.

[0146] Preparation of mulberry extract

[0147] Preparation example 1 of mulberry extract

[0148] Take fresh mulberry branches (Yueshang No. 11) 1000 kg, crush, add 4000 L water, extract by heating reflux method for 2 h, combine the extract, filter to remove insoluble matter, and obtain crude extract. Heat the crude extract to concentrate to a solid content of 4%, and keep at 50℃ as the sample liquid for cation resin column.

[0149] Use D113 type macroporous weak acid phenylpropylene cation resin 150 kg to load the column, use 2 mol / L hydrochloric acid solution to wash until the pH of the washing liquid is 4.5; use 1 mol / L sodium hydroxide solution to wash until the pH of the washing liquid is 8.5; use 2 mol / L hydrochloric acid solution to wash until the pH of the washing liquid is 4.5; and then use 5 times the column volume of deionized water to rinse, and complete the activation. Load the concentrated extract, and then use 1000 L 2.5 mol / L ammonia water to elute, with an elution speed of 6 BV / h, collect the eluate when the pH of the cation column effluent is >7, and stop collecting when the collected liquid reaches 900 L. The collected liquid is directly purified by an anion column.

[0150] Use D218 type macroporous strong base acrylic anion resin 62.5 kg to load the column, use 1.5 mol / L sodium hydroxide solution to wash until the pH of the washing liquid is 9.0; use 1.5 mol / L hydrochloric acid solution to wash until the pH of the washing liquid is 3.5; use 1.5 mol / L sodium hydroxide solution to wash until the pH of the washing liquid is 9.0; and complete the activation. Load the collected cation resin eluate to the anion resin, and collect the effluent until the effluent reaches 920 L.

[0151] Centrifuge the collected liquid to remove impurities, and then concentrate by reverse ion permeation membrane. The specific gravity of the concentrated liquid is 1.25, which is transferred to an alcohol precipitation tank, and 25 L of anhydrous ethanol is added under stirring at 500 rpm. Stop stirring after the addition of ethanol is completed, alcohol precipitation for 24 h, take the supernatant, and concentrate under reduced pressure to obtain mulberry branch extract infusion (i.e. mulberry extract SZ-A).

[0152] In the mulberry branch extract infusion, the mass percentage of alkaloids is 52%, the mass percentage of polysaccharides is 22%, the mass percentage of flavonoids is 0.8%, and the mass percentage of amino acids is 20%. Among the alkaloids, the content of 1-DNJ is 60%, the content of FAG is 17%, and the content of DAB is 15%.

[0153] Preparation example 2 of mulberry extract

[0154] Take fresh mulberry branches (Mulberry Tiyou No. 2) 10 kg, after crushing, add 150 L water, add 2 times, each time extract 3 h by decoction method, combine the extract, filter out the insoluble. The extract is hot concentrated to the mass percentage of solid content of 8%, it is transferred to the alcohol precipitation tank, the stirring paddle 300 rpm is added 2367.9 g of anhydrous ethanol (3 L). After the ethanol is added, stop stirring, alcohol precipitation 24 h, take the supernatant as the sample liquid of cation resin column. Use 002SC type strong acid styrene cation resin 5 kg to load column, activate the cation resin according to the method of preparation example 1. Sample the concentrated alcohol precipitation treated extract, then use 100 L 5 mol / L potassium chloride to elute, the elution speed is 5 BV / h, use 20% silicotungstic acid to detect the effluent, when white precipitate is generated, start to collect, when the collected liquid reaches 25 L, stop collecting, the collected liquid is directly purified by anion column.

[0155] Use 711 type strong basic styrene anion resin 10 kg to load column, activate the anion resin according to the method of preparation example 1. Sample the collected cation resin eluent to the anion resin, collect the effluent until the effluent reaches 15 L to end. Resample the collected liquid to the cation resin, and separate twice again by using the cation resin and the anion resin according to the above method.

[0156] Centrifuge the collected liquid obtained after three column separations to remove impurities, and then concentrate by reverse ion permeation membrane. The specific gravity of the concentrated liquid is 1.25, which is transferred to the alcohol precipitation tank, and 125 g of anhydrous ethanol is added under the stirring paddle of 1000 rpm. After the ethanol is added, stop stirring, alcohol precipitation 24 h, take the supernatant, and concentrate under reduced pressure to obtain the extract infusion. In addition, fresh mulberry bark and mulberry leaves (Mulberry Tiyou No. 2) are extracted, and the extraction method and parameters are the same as the above method.

[0157] The mass percentage of alkaloids in the obtained mulberry branch extract is 98%, the mass percentage of polysaccharides is 0.2%, the mass percentage of flavonoids is 0.05%, and the mass percentage of amino acids is 0%. Among the alkaloids, the content of 1-DNJ is 99%, FAG is 0.5%, and DAB is 0.4%.

[0158] The mass percentage of alkaloids in the obtained mulberry bark extract is 95%, the mass percentage of polysaccharides is 2%, the mass percentage of flavonoids is 0.1%, and the mass percentage of amino acids is 1%. Among the alkaloids, the content of 1-DNJ is 96%, FAG is 1.5%, and DAB is 1.4%.

[0159] The obtained mulberry leaf extract contains 90% alkaloids, 4% polysaccharides, 0.1% flavones and 3% amino acids by mass percentage. In the alkaloids, the content of 1-DNJ is 91%, the content of FAG is 3.1%, and the content of DAB is 2.8%.

[0160] Preparation Example 3 of mulberry extract

[0161] Take 1000 kg of fresh mulberry branches (Guangdong mulberry) and crush them. Then add 11500 L of water and reflux extract for 2 hours. Combine the extract and filter to remove insoluble substances to obtain a crude extract. The crude extract is first centrifuged to remove impurities, and then concentrated to a solid content of 1% by mass using a reverse ion permeable membrane, which is used as the sample solution for the cation resin column.

[0162] Use 300 kg of D001 macroporous strong acid styrene cation resin to fill the column, and activate the cation resin according to the method of Preparation Example 1. Load the concentrated crude extract, and use 5000 L of 0.04 mol / L ammonium nitrate to elute at a speed of 5 BV / h. Use 20% tungsten silicide to detect the effluent, and start collecting when white precipitate is formed. Stop collecting when the collected solution reaches 1000 L.

[0163] Concentrate the collected solution obtained after cation column separation using a nanofiltration membrane, and concentrate under reduced pressure to obtain the extract infusion.

[0164] The obtained mulberry branch extract contains 15% alkaloids, 20% polysaccharides, 7% flavones and 45% amino acids by mass percentage. In the alkaloids, the content of 1-DNJ is 55%, the content of FAG is 23%, and the content of DAB is 10%.

[0165] Preparation Example 4 of mulberry extract

[0166] Take 333 kg of dried mulberry branches (Yue mulberry No. 11) and crush them. Then add 4000 L of water and reflux extract twice, each time for 1 hour. Combine the extract and filter, and concentrate the extract to 1 kg of crude drug per L.

[0167] Use D113 type macroporous weak acid phenylpropyl cation resin 150 kg column, using 2 mol / L hydrochloric acid solution to wash until the effluent pH is 4.5; using 1 mol / L sodium hydroxide solution to wash until the effluent pH is 8.5; 2 mol / L hydrochloric acid solution to wash until the effluent pH is 4.5; and then using 5 times the column volume of deionized water to rinse, complete the activation. Sample the concentrated extract, then use 1000L 2.5 mol / L ammonia water to elute, the elution rate is 6BV / h, collect the eluate when the cation column effluent pH>7, when the collected liquid reaches 900L, stop collecting, and directly purify the collected liquid through the anion column.

[0168] Use D218 type macroporous strong base acrylic anion resin 125 kg column, using 1.5 mol / L sodium hydroxide solution to wash until the effluent pH is 9.0; using 1.5 mol / L hydrochloric acid solution to wash until the effluent pH is 3.5; 1.5 mol / L sodium hydroxide solution to wash until the effluent pH is 9.0; complete the activation. Sample the collected cation resin eluate to the anion resin, collect the effluent with pH greater than 8 until the effluent reaches 870L.

[0169] The collected liquid obtained after separation through the anion column is filtered through a microfiltration membrane to remove impurities, and then concentrated using a reverse ion permeation membrane. The specific gravity of the concentrated liquid is 1.1. The concentrated liquid is transferred to an alcohol precipitation tank, and 15 kg of anhydrous ethanol is added under stirring at 400 rpm. After the addition of ethanol is completed, the stirring is stopped, and the alcohol precipitation is carried out for 24 hours. The supernatant is collected and concentrated under reduced pressure to obtain a mulberry branch extract. The sample content: the mass percentage content of alkaloids is 80%, the mass percentage content of polysaccharides is 5%, the mass percentage content of flavonoids is 0.1%, and the mass percentage content of amino acids is 4%. Among the alkaloids, the content of 1-DNJ is 75%, the content of FAG is 12%, and the content of DAB is 10%.

[0170] Preparation example 5 of mulberry extract

[0171] Take 400 kg of dry mulberry branches (Yuesang No. 11), crush them, and then extract them twice using the heating reflux method, with 4000L of water each time, refluxing for 1 hour each time. Combine the extract and filter it. Concentrate the extract to 1 kg of crude drug per liter.

[0172] Use D218 type macroporous strong base acrylic anion resin 62.5 kg column, using 1.5 mol / L sodium hydroxide solution to wash until the effluent pH is 9.0; using 1.5 mol / L hydrochloric acid solution to wash until the effluent pH is 3.5; 1.5 mol / L sodium hydroxide solution to wash until the effluent pH is 9.0; complete the activation. Sample the collected extract concentrate to the anion resin, and collect the effluent.

[0173] The collected solution after anion column separation was filtered by microfiltration membrane to remove impurities, and then concentrated by reverse ion permeation membrane. The mulberry branch extract extract was obtained by further concentration under reduced pressure and drying. The sample content was: the mass percentage of alkaloids was 3%, the mass percentage of polysaccharides was 70%, the mass percentage of flavonoids was 10%, and the mass percentage of amino acids was 10%. In the alkaloids, the content of 1-DNJ was 68%, the content of FAG was 17%, and the content of DAB was 8%.

[0174] Preparation example 6 of mulberry extract

[0175] Take 1500 kg of fresh mulberry branches (Yuesang No. 11), crush them, add 6000 L of water, and extract them by heating reflux for 2 hours. Combine the extract and filter to remove insoluble substances to obtain a crude extract. Heat the crude extract to concentrate it to a solid content of 4%, and keep it at 50°C as the sample solution for the cation resin column.

[0176] Use D113 type macroporous weak acid phenylpropylene cation resin 100 kg to load the column, use 2 mol / L hydrochloric acid solution to wash until the pH of the eluate is 4.5; use 1 mol / L sodium hydroxide solution to wash until the pH of the eluate is 8.5; use 2 mol / L hydrochloric acid solution to wash until the pH of the eluate is 4.5; then use 5 times the column volume of deionized water to rinse, and complete the activation. Load the concentrated extract, then use 1000 L of 2.5 mol / L ammonia water to elute, with an elution rate of 6 BV / h. When the pH of the cation column effluent is greater than 7, collect the eluate. When the collected solution reaches 900 L, stop collecting, and directly purify the collected solution by anion column.

[0177] Use D218 type macroporous strong basic acrylic anion resin 62.5 kg to load the column, use 1.5 mol / L sodium hydroxide solution to wash until the pH of the eluate is 9.0; use 1.5 mol / L hydrochloric acid solution to wash until the pH of the eluate is 3.5; use 1.5 mol / L sodium hydroxide solution to wash until the pH of the eluate is 9.0; complete the activation. Load the collected cation resin eluate onto the anion resin, and collect the effluent until the effluent reaches 870 L. Concentrate the effluent under reduced pressure to obtain mulberry branch extract extract, wherein the mass percentage of alkaloids is 30%, the mass percentage of polysaccharides is 35%, the mass percentage of flavonoids is 2%, and the mass percentage of amino acids is 25%. In the alkaloids, the content of 1-DNJ is 62%, the content of FAG is 20%, and the content of DAB is 13%.

[0178] Preparation example 7 of mulberry extract

[0179] Take fresh mulberry branches (Yuesang No. 11) 1000 kg, after crushing, add 4000 L water, extract by heating reflux method for 2 h, combine the extract, filter to remove insoluble matter, and obtain the crude extract. Heat the crude extract to concentrate to a solid content of 4%, and keep at 50℃ as the sample solution for cation resin column.

[0180] Use D113 type macroporous weak acid phenylpropylene cation resin 100 kg to load the column, use 2 mol / L hydrochloric acid solution to wash until the pH of the effluent is 4.5; use 1 mol / L sodium hydroxide solution to wash until the pH of the effluent is 8.5; use 2 mol / L hydrochloric acid solution to wash until the pH of the effluent is 4.5; and then use 5 times the column volume of deionized water to rinse, and complete the activation. Load the concentrated extract, and then use 1000 L 2.5 mol / L ammonia water to elute, with an elution rate of 6 BV / h, and collect the eluate when the pH of the cation column effluent is greater than 7; when the collected solution reaches 900 L, stop collecting, and directly purify the collected solution through an anion column.

[0181] Use D218 type macroporous strong base acrylic anion resin 62.5 kg to load the column, use 1.5 mol / L sodium hydroxide solution to wash until the pH of the effluent is 9.0; use 1.5 mol / L hydrochloric acid solution to wash until the pH of the effluent is 3.5; use 1.5 mol / L sodium hydroxide solution to wash until the pH of the effluent is 9.0; and complete the activation. Load the collected cation resin eluate to the anion resin, and collect the effluent until the effluent reaches 870 L. Reduce the pressure to concentrate the effluent to obtain mulberry branch extract extract, wherein the mass percentage of alkaloids is 40%, the mass percentage of polysaccharides is 25%, the mass percentage of flavonoids is 0.5%, and the mass percentage of amino acids is 25%. Among the alkaloids, the content of 1-DNJ is 57%, the content of FAG is 24%, and the content of DAB is 16%.

[0182] Preparation Example 8 of mulberry extract

[0183] Take dry mulberry branches (Yuesang No. 11) 333 kg, crush, and then add 4000 L water. Extract by heating reflux method for 1 h twice, combine the extract, filter, and concentrate the extract to 1 kg crude drug amount / L.

[0184] The D113 type macroporous weak acid phenylpropyl cation resin 150 kg was used to load column, 2 mol / L hydrochloric acid solution was used to wash until the pH of the eluate was 4.5; 1 mol / L sodium hydroxide solution was used to wash until the pH of the eluate was 8.5; 2 mol / L hydrochloric acid solution was used to wash until the pH of the eluate was 4.5; and then 5 times column volume of deionized water was used to rinse, and the activation was completed. The concentrated extract was loaded, and then 1000 L 2.5 mol / L ammonia water was used to elute, the elution rate was 6 BV / h, the pH of the cation column effluent was detected, and when the pH was greater than 7, the eluate was collected; when the collected liquid reached 900 L, the collection was stopped, and the collected liquid was directly purified through the anion column.

[0185] The D218 type macroporous strong base acrylic anion resin 62.5 kg was used to load column, 1.5 mol / L sodium hydroxide solution was used to wash until the pH of the eluate was 9.0; 1.5 mol / L hydrochloric acid solution was used to wash until the pH of the eluate was 3.5; 1.5 mol / L sodium hydroxide solution was used to wash until the pH of the eluate was 9.0; and the activation was completed. The collected cation resin eluate was loaded into the anion resin, and the effluent with pH greater than 8 was collected until the effluent reached 870 L.

[0186] The collected liquid obtained after separation through the anion column was subjected to impurity removal through microfiltration membrane filtration and then concentrated through reverse ion permeation membrane, the specific gravity of the concentrated liquid was 1.1, the concentrated liquid was transferred to an alcohol precipitation tank, 15 kg of anhydrous ethanol was added under the stirring of a stirring paddle at 400 rpm. After the addition of ethanol was completed, the stirring was stopped, alcohol precipitation was carried out for 24 h, the supernatant was taken, and the mulberry branch extract extract was obtained by vacuum concentration. The sample content: the mass percentage content of alkaloids was 63%, the mass percentage content of polysaccharides was 23%, the mass percentage content of flavones was 1%, and the mass percentage content of amino acids was 5%.

[0187] In the alkaloids, the content of 1-DNJ was 61.9%, the content of FAG was 16.6%, and the content of DAB was 11.1%.

[0188] Preparation example 9 of mulberry extract

[0189] 1000 kg of fresh mulberry branches (Yuesang No. 11) were taken, crushed, 4000 L of water was added, and the extraction was carried out by heating reflux method for 2 h, the extract was combined, and the insoluble substances were removed by filtration to obtain a crude extract. The crude extract was concentrated at 50 ℃ until the solid content reached 4%, and the concentrated extract was used as the loading liquid of the cation resin column.

[0190] The D113 type macroporous weak acid phenylpropyl cation resin 120 kg was used to load the column, 2 mol / L hydrochloric acid solution was used to wash until the pH of the eluate was 4.5; 1 mol / L sodium hydroxide solution was used to wash until the pH of the eluate was 8.5; 2 mol / L hydrochloric acid solution was used to wash until the pH of the eluate was 4.5; and then 5 times the column volume of deionized water was used to rinse, and the activation was completed. The concentrated extract was loaded, and then 1000 L of 2.5 mol / L ammonia water was used to elute, the elution rate was 6 BV / h, and the eluate was collected when the pH of the cation column effluent was greater than 7; when the collected liquid reached 900 L, the collection was stopped, and the collected liquid was directly purified through the anion column.

[0191] The D218 type macroporous strong base acrylic anion resin 45 kg was used to load the column, 1.5 mol / L sodium hydroxide solution was used to wash until the pH of the eluate was 9.0; 1.5 mol / L hydrochloric acid solution was used to wash until the pH of the eluate was 3.5; 1.5 mol / L sodium hydroxide solution was used to wash until the pH of the eluate was 9.0; and the activation was completed. The collected cation resin eluate was loaded into the anion resin, and the effluent was collected until the effluent reached 870 L.

[0192] The collected liquid after separation through the anion column was filtered through a microfiltration membrane to remove impurities, and then concentrated through a reverse ion permeation membrane. The specific gravity of the concentrated liquid was 1.1, which was transferred to an alcohol precipitation tank, 15 kg of anhydrous ethanol was added under the stirring of a paddle at 300 rpm. After the addition of ethanol was completed, the stirring was stopped, and the alcohol precipitation was carried out for 24 h. The supernatant was taken and concentrated under reduced pressure to obtain the extract extract. The sample content: the mass percentage content of alkaloids was 70%, the mass percentage content of polysaccharides was 20%, the mass percentage content of flavones was 0.6%, and the mass percentage content of amino acids was 5%.

[0193] In the alkaloids, the content of 1-DNJ was 70%, the content of FAG was 13%, and the content of DAB was 10%.

[0194] Preparation example 10 of mulberry extract

[0195] 80 kg of fresh mulberry branches (Gui Sang You 62) were taken, crushed, and then extracted with 1000 L of water by heating reflux for 2 h. The extract was combined, filtered to remove insoluble substances, and the crude extract was obtained. The crude extract was concentrated at 50°C until the solid content reached 4%, and then used as the loading liquid for the cation resin column.

[0196] The D113 type macroporous weak acid cationic phenylpropyl resin 50 kg was used to column, 2 mol / L hydrochloric acid solution was used to wash until the pH of the eluate was 4.5; 1 mol / L sodium hydroxide solution was used to wash until the pH of the eluate was 8.5; 2 mol / L hydrochloric acid solution was used to wash until the pH of the eluate was 4.5; and then 5 times column volume of deionized water was used to rinse, and the activation was completed. The concentrated extract was loaded, and then 600 L 2.5 mol / L ammonia water was used to elute, the elution rate was 6 BV / h, the eluate was collected when the pH of the cation column effluent was greater than 7, and the collection was stopped when the collection reached 9800 L, and the collection was directly purified through the anion column.

[0197] The D218 type macroporous strong basic acrylic anion resin 65 kg was used to column, 1.5 mol / L sodium hydroxide solution was used to wash until the pH of the eluate was 9.0; 1.5 mol / L hydrochloric acid solution was used to wash until the pH of the eluate was 3.5; 1.5 mol / L sodium hydroxide solution was used to wash until the pH of the eluate was 9.0; and the activation was completed. The collected cation resin eluate was loaded into the anion resin, and the effluent was collected until the effluent reached 750 L. The collection was reloaded into the cation resin, and the cation resin and the anion resin were separated once again according to the above method.

[0198] The collected solution after two times of separation was filtered through a microfiltration membrane to remove impurities, and then concentrated through a reverse ion permeation membrane. The specific gravity of the concentrated liquid was 1.1, which was transferred to an alcohol precipitation tank, 400 g of anhydrous ethanol was added under the stirring of a paddle at 350 rpm. After the addition of ethanol was completed, the stirring was stopped, and the alcohol precipitation was carried out for 24 h. The supernatant was taken, and the extract was concentrated under reduced pressure to obtain an extract infusion. The sample content: the mass percentage content of alkaloids was 90%, the mass percentage content of polysaccharides was 3%, the mass percentage content of flavones was 0.5%, and the mass percentage content of amino acids was 2%.

[0199] In the alkaloids, the content of 1-DNJ was 94%, the content of FAG was 2%, and the content of DAB was 1%.

[0200] Pharmacological test of mulberry extract

[0201] In the following test examples, the mulberry extract is represented as SZ-A.

[0202] Test Example 1

[0203] 1 Materials and Methods

[0204] 1.1 Experimental Animals

[0205] SPF grade 6-day-old C57BL / 6J nest mice, male and female, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. and co-caged with lactating mother mice. The experimental animal license number is SCXK (Jing) 2019-0010. The mice were bred in the animal experiment center of Beijing University of Chinese Medicine, with a temperature of 22-25°C, a humidity of 50%-70%, natural light, and free water and food. The age of the newborn pups was recorded, with the day of birth recorded as P0. This experiment was reviewed by the Experimental Animal Ethics Committee of Beijing University of Chinese Medicine.

[0206] 1.2 Drugs and main reagents

[0207] Drug: mulberry extract SZ-A prepared in Example 8.

[0208] 2 Experimental methods

[0209] 2.1 Establishment of oxygen-induced retinal neovascularization model

[0210] (1) C56BL / 6J nest mice 6 days after birth were purchased, male and female, and the purchased newborn pups were co-caged with the mother mice to mix and feed, so that their odors were integrated. The newborn pups were fed in a normal environment until the 7th day of age (P7). All pups were randomly divided into a normal air group and a high oxygen group.

[0211] (2) On the 7th day of age (P7), the high oxygen group pups were placed in an intelligent oxygen control chamber with the lactating mother mice, and the oxygen concentration of the chamber was adjusted to 75%±2. The normal air group pups and lactating mother mice were continued to be fed in a normal environment, and the environmental temperature in the animal room was maintained at 22-25°C, the humidity was 50%-70%, and the natural light was provided. The mother mice were provided with sufficient breeding feed and drinking water every day.

[0212] (3) The chamber door was opened every 12 hours to replace the breeding feed and drinking water, and the normal air group and high oxygen group mother mice were exchanged to prevent the mother mice from having decreased lactation ability or death due to oxygen poisoning. The oxygen volume fraction of the oxygen chamber was monitored at regular intervals. The pups were fed in a high oxygen environment for 5 days, during which the activity and growth of the normal air group and high oxygen group pups were observed every day, and the mother mice and pups were observed for abnormal death, and relevant experimental records were made.

[0213] (4) On the 12th day of age (P12), the high oxygen group pups were taken out of the oxygen chamber and continued to be fed with the normal air group pups until the 17th day of age (P17). The body weight of the pups was measured daily, and the activity and growth of the pups were closely observed, and relevant experimental records were made.

[0214] 2.2 Animal grouping and drug administration

[0215] Experimental grouping: The experiment was divided into normal air group (Control), on the 12th day of the mouse age (P12), the high oxygen group of the mouse taken out of the oxygen cabin was randomly divided into model group (OIR), SZ-A low-dose treatment group (SZ-A-L), SZ-A medium-dose treatment group (SZ-A-M), SZ-A high-dose treatment group (SZ-A-H), and positive drug (Bevacizumab) treatment group according to the body weight. There were 6-7 in each group.

[0216] Dose and mode of administration: On the 13th day of the mouse age (P13), according to the results of the pre-experiment, the SZ-A low, medium and high dose treatment groups were administered by gavage at a dose of 6.25 mg / kg, 12.5 mg / kg and 25 mg / kg (calculated according to the total base) dissolved in normal saline, with a volume of 0.1 ml / 10g. The administration was stopped on the 17th day of the mouse age (P17) and lasted for 5 consecutive days. The positive drug (Bevacizumab) treatment group was administered by intraperitoneal injection at a dose of 25 mg / kg dissolved in normal saline, with a volume of 0.1 ml / 10g, and the administration was given for 1 day on the 13th day of the mouse age (P13).

[0217] 2.3 Isagglutinin GS-IB4 retinal staining smear observation

[0218] (1) Enucleation and fixation: On the 17th day of the mouse age (P17), all groups of young mice were rapidly killed by intraperitoneal injection of sodium pentobarbital, and the eyeballs were removed and placed in 4% paraformaldehyde for 24h at room temperature. When the eyeballs of the young mice were removed, the operation should be careful and gentle to prevent damage to the integrity of the retina.

[0219] (2) Dissection of the retina: After the eyeball was fixed for 24h, it was washed with PBS solution for 3 times, 5min each time. After washing, the anterior segment was removed by ophthalmic microsurgical scissors, the lens was removed, the iris tissue was carefully separated and removed by ophthalmic forceps, and the complete retinal cup was peeled off. The remaining debris in the retinal cup was carefully removed with ophthalmic forceps, and the peeled retina was placed in a 96-well plate containing 4% paraformaldehyde at 4°C overnight.

[0220] (3) GS-IB4 staining: The fixed retinal tissue was washed with PBS for 3 times, 5min each time. The agglutinin staining solution (3% goat serum + 0.2% Triton X-100 + agglutinin (1:200)) was prepared, and the washed retinal tissue was placed in a 96-well plate containing the agglutinin mixed staining solution and incubated at 4°C on a shaking table for 3 days in the dark.

[0221] (4) Retinal flat mount: The retinas were washed with PBS solution for 5 times, 5 min each time. The stained retinas were placed on a glass slide, and PBS solution was added to make the retinas into a bowl shape. The retinas were radially cut with an ophthalmic scissors about 1 mm away from the optic disc to the optic nerve, so that the retinas were evenly divided into 4 pieces. The edge iris tissue was removed, and the retinas were flat-mounted on a glass slide. The excess liquid was absorbed, and finally PBS solution was added again. The cover glass was carefully covered to prevent air bubbles, and the surrounding was sealed with nail polish. The retinal flat mount was stored in a 4°C refrigerator in the dark.

[0222] (5) Laser confocal microscope photographing: The retinal flat mount was photographed using a laser confocal microscope. The retinal flat mount was scanned under a laser confocal microscope using a 10x objective lens.

[0223] (6) Quantitative analysis: The area ratio of retinal pathological neovascular clusters and central non-perfusion area in each group was calculated using ImageJ software, and the data was statistically analyzed.

[0224] 2.4 Paraffin section of mouse eyeball

[0225] Preparation of paraffin section: On the 17th day of the mouse, the pups were quickly killed by intraperitoneal injection of sodium pentobarbital, and the eyeballs were carefully removed and placed in 4% paraformaldehyde for room temperature fixation for 48 h. After fixation, gradient ethanol dehydration, paraffin embedding, and sectioning at 4-5 pm thickness were performed.

[0226] 2.5 HE staining

[0227] After the paraffin section was baked in an oven, it was hydrated with xylene, anhydrous ethanol, and gradient ethanol, and then washed with tap water. The cell nucleus was stained with hematoxylin, and the differentiation was performed with hydrochloric acid ethanol. Ammonia was used for blue returning. Then the cytoplasm was stained with eosin staining solution, and further anhydrous ethanol dehydration, xylene transparency, and neutral gum sealing were performed. The observation was performed under an optical microscope.

[0228] 2.6 PCR

[0229] The RNA in the sample was extracted according to the Trizol kit instructions. The primer sequence was designed according to the gene sequence on the NCBI website. The cDNA was reverse transcribed according to the reverse transcription kit instructions, and the qPCR was performed with the cDNA as the template. The reaction system was 20 pL: 0.4 pL of upper and lower primers, 1 pL of cDNA, 10 pL of Taq SYBR Green qPCR Premix, and 8.2 pL of DEPC water. The reaction parameters were set as follows: 95°C pre-denaturation for 30 s, 95°C for 10 s, 60°C for 10 s, 72°C for 30 s, 40 cycles. Beta-actin was used as an internal reference gene, and the data was processed according to 2 -△△Ct Method for processing data.

[0230] Primer design sequence

[0231] 2.7 Immunohistochemistry

[0232] The retinal tissue sections were baked, dewaxed, washed with PBS, and then subjected to antigen repair using 1x antigen repair solution. Endogenous peroxidase blocking agent was added and placed in a 37°C oven for 1 h. After completion, blocking solution was added and placed in a 37°C oven for 30 min. After completion of the blocking, the tissue sections were added with PBS-diluted primary antibody (VEGF antibody was diluted 1:100) and incubated overnight in a 4°C refrigerator. The next day, after washing with PBS, biotin-labeled secondary antibody working solution was added and placed in a 37°C oven for 30 min. After completion, PBS was added for washing, streptomycin working solution was added and placed in a 37°C oven for 10 min, DAB was used for color development, pure water was used for termination of staining, and hematoxylin was used for re-staining. After dehydration and transparency treatment, neutral resin was used for mounting. Optical microscope was used for photographing, 4-6 fields of view were randomly selected for each section, deep brown staining on the tissue was used as positive expression, and ImageJ software was used for data analysis.

[0233] 2.8 Immunofluorescence detection

[0234] After the mouse eyeball specimens were fixed with 4% paraformaldehyde, gradient ethanol dehydration, paraffin embedding, and sectioning at a thickness of 5 μm, the paraffin sections were baked, then subjected to xylene, anhydrous ethanol, and gradient ethanol hydration, and tap water washing. 2 mg / ml glycine solution was used for incubation and soaking for 10 min, and then 1x antigen repair solution was used for antigen repair. Sheep serum (sheep serum working solution + 0.3% Triton X-100) was used for blocking, and after incubation for 30 min, EGLN1 or Occludin antibody was added at a dilution of 1:600 for primary antibody, and incubated overnight at 4°C. The next day, after washing with PBS, the corresponding secondary antibody (1:400) was added and incubated for 1 h at room temperature in the dark. After completion of the secondary antibody, DAPI mounting agent was added, and careful mounting was performed to avoid the generation of air bubbles. Laser confocal microscope was used for scanning and photographing at 400x magnification, 4-6 photos were randomly taken for each section, ImageJ software was used for analyzing the average fluorescence intensity of the photos of each group, and data statistical analysis was performed.

[0235] 2.9 Western blotting experiment

[0236] Take the mouse retina sample by grinding to prepare the sample, extract the protein with RIPA lysis buffer containing protease inhibitor and phosphatase inhibitor, determine the protein concentration by BCA method and quantify, heat denaturation, prepare 10% SDS-PAGE precast gel, electrophoresis (150V-45min), transfer membrane (300mA-90min), room temperature rocking bed blocking 2h, incubate EphA2 (1:2000), PI3K (1:2000), Akt (1:2000) and GAPDH (1:10000) primary antibody, β-actin (1:10000), the next day use horseradish peroxidase (HRP) labeled rabbit secondary antibody (1:5000) and mouse secondary antibody (1:5000), incubate at room temperature for 2h, then wash the membrane and develop, use ImageJ software to analyze the gray value of the band, and the gray value ratio of the target protein band to the internal reference GAPDH or internal reference actin band in each group is used as the relative expression amount of the protein and statistical analysis.

[0237] 3Results

[0238] 3.1 Successful construction of oxygen-induced retinopathy model (OIR)

[0239] Oxygen-induced retinopathy model (OIR) is a classic model for studying pathological retinal angiogenesis. After establishing the OIR model according to the experimental steps, the retinal blood vessels were stained with green fluorescent IB4 antibody to determine whether the retinal neovascularization model was successfully constructed. Observation of P17 mouse retinal smears showed that the retinal blood vessels in the Control group were evenly distributed from the optic disc, the blood vessels were clear, and there was no abnormal neovascularization and avascular area. The retinal blood vessels in the OIR group were distributed in disorder, and there were expansion, tortuosity and other phenomena, and a large number of neovascular clusters (as shown in the circled area in Figure 1) were produced, and a large area of avascular area appeared in the central area of the retina (as shown by the arrow in Figure 1). The high-intensity fluorescent points appearing at the junction of the neovascular area and the avascular area were determined as pathological neovascular clusters. After statistical analysis, it was found that the proportion of retinal neovascular clusters and avascular area in the OIR group was higher than that in the normal air group, and the difference was statistically significant (P<0.001) (as shown in Figure 2). The experimental results showed that the oxygen-induced retinal neovascularization model was successfully constructed.

[0240] 3.2 Effect of SZ-A intervention on the general growth and body weight of OIR mice

[0241] 3.2.1 General growth of mice

[0242] No abnormal death occurred in each group of mice before and after modeling. When not modeling, the mice grew well, and the activity response and hair were basically consistent. After the modeling ended, the hair of the mice in the Control group was thick, bright and smooth, the activity response was agile, and the food intake was good. The hair of the mice in the OIR group was sparse, the response was slower, the spirit was not good, and the food intake was less than normal. After SZ-A intervention, the above conditions improved.

[0243] 3.2.2 Changes in body weight of mice

[0244] To evaluate whether SZ-A intervention affects the body weight of mice, the body weight changes of mice at P13, P14, P15, P16 and P17 after SZ-A drug intervention were measured. The experimental results are shown in Table 1. The results show that after the modeling ended, the average body weight of mice in the OIR group and each drug treatment group was lower than that in the Control group. From P14, the average body weight of mice in the OIR group and each drug treatment group gradually approached that in the Control group as the mice grew older. At P17, the average body weight of mice in the OIR group was still significantly lower than that in the Control group, and the difference was statistically significant (P<0.05). * Compared with the OIR group, the average body weight of mice in the SZ-A-L group, the SZ-A-M group and the SZ-A-H group was significantly increased, and the difference was statistically significant (P<0.01 or P<0.001). Compared with the Bevacizumab group, the average body weight of mice in the different dose groups of SZ-A was significantly increased, and the difference was statistically significant (P<0.0001).

[0245] Table 1 Body weight growth of mice in each group after SZ-A intervention (g) n=6 Note: Compared with the Control group, *** P<0.001, ** P<0.01, * P<0.05; compared with the OIR group, ## P<0.01, # P<0.05; compared with the Bevacizumab group, ΔΔΔΔ P<0.0001.

[0246] 3.3 SZ-A inhibits pathological retinal neovascularization in OIR mice

[0247] To further investigate the effect of SZ-A on retinal neovascularization in OIR mice, different doses of SZ-A were administered to OIR mice, and IB4 antibody was used to label retinal blood vessels to observe the effect of SZ-A on retinal neovascularization. The whole-mount retinal flat-mount showed that the blood vessels in the Control group were evenly distributed, with normal and natural direction, and the blood vessels in each layer of the retina were clear, and no neovascular tufts or avascular areas were observed; the blood vessels in the OIR group were sparse and disordered, and a large number of neovascular tufts and central avascular areas appeared; after drug intervention, the retinal neovascular tufts and avascular areas were reduced compared with the OIR group, as shown in Figure 3. The relative area of neovascular tufts and avascular areas was analyzed by Photoshop software, and the experimental results are shown in Table 2. Compared with the Control group, the relative area of neovascular tufts in the OIR group was significantly increased (P<0.001); compared with the OIR group, the relative area of neovascular tufts in the SZ-A-L, SZ-A-M, SZ-A-H, and Bevacizumab groups was significantly reduced (P<0.01 or P<0.001). Compared with the Control group, the relative area of avascular perfusion area in the OIR group was significantly increased (P<0.001); compared with the OIR group, the relative area of avascular perfusion area in the SZ-A-L and SZ-A-M groups was significantly reduced (P<0.05 or P<0.01). The above experimental results suggest that SZ-A can effectively inhibit the generation of retinal pathological neovascularization and reduce the central avascular area.

[0248] Table 2 Relative area of neovascular tufts and avascular perfusion area in each group (%) ) Note: compared with the Control group, *** P<0.001, ** P<0.01, * P<0.05; compared with the OIR group, ### P<0.001, ## P<0.01, # P<0.05; compared with the Bevacizumab group, ΔΔΔ P<0.001, ΔΔ P<0.01

[0249] 3.4 SZ-A intervention can reduce the number of vascular endothelial cell nuclei breaking through the internal limiting membrane in OIR mice

[0250] The results of retinal HE staining showed that the internal limiting membrane of the Control group was smooth and complete, and the cells were arranged in order, and no obvious new blood vessel nuclei breaking through the internal limiting membrane into the vitreous body was observed. A large number of pathological new blood vessel endothelial cell nuclei breaking through the internal limiting membrane were observed in the OIR group, and the cells under the internal limiting membrane of the retina appeared abnormal proliferation and disordered arrangement. Compared with the OIR group, the internal limiting membrane of the SZ-A-L group, the SZ-A-M group, the SZ-A-H group and the bevacizumab group was relatively smooth, and the number of new blood vessel endothelial cell nuclei breaking through the internal limiting membrane was reduced (Figure 4). Counting analysis and statistics showed that compared with the Control group, the number of new blood vessel endothelial cell nuclei breaking through the internal limiting membrane in the OIR group was significantly increased (P<0.01); compared with the OIR group, the number of new blood vessel endothelial cell nuclei breaking through the internal limiting membrane in the SZ-A-L group, the SZ-A-M group, the SZ-A-H group and the bevacizumab group was significantly reduced, and the difference was statistically significant (P<0.05 or P<0.01), as shown in Table 3.

[0251] Table 3: Number of new blood vessel endothelial cell nuclei breaking through the internal limiting membrane in each group n=6) Note: compared with the Control group, *** P<0.001, ** P<0.01, * P<0.05; compared with the OIR group, ### P<0.001, ## P<0.01, # P<0.05.

[0252] 3.5 SZ-A inhibits the expression of VEGF in the retinal tissue of OIR mice

[0253] VEGF is a highly specific pro-vascular endothelial cell growth factor, which has important biological functions such as increasing vascular permeability, accelerating extracellular matrix degeneration, promoting vascular endothelial cell migration, proliferation and angiogenesis.

[0254] The results of qPCR experiment showed that compared with the Control group, the expression of VEGF mRNA in the retinal tissue of the OIR group was significantly increased (P<0.001) in the OIR mouse model; compared with the OIR group, the expression of VEGF mRNA in the retinal tissue of the SZ-A medium and high dose groups and the bevacizumab group was significantly decreased, and the difference was statistically significant (P<0.05 or P<0.01), as shown in Table 4.

[0255] The immunohistochemical experiment results show that, as shown in FIG. 5 and Table 5, compared with the Control group, the expression level of VEGF protein in the retinal tissue of the OIR group is significantly increased (P<0.05); compared with the OIR group, the expression levels of VEGF protein in the retinal tissue of the SZ-A low, medium and high dose groups and the bevacizumab group are all decreased (P<0.001). The experiment at the gene level and the protein level both confirms that, in the OIR mouse model, SZ-A reduces the expression of VEGF.

[0256] Table 4: Relative expression amount of VEGF mRNA in the retinal tissue of each group Note: compared with the Control group, *** P<0.001, ** P<0.01, * P<0.05; compared with the OIR group, ## P<0.01, # P<0.05; compared with the bevacizumab group, Δ P<0.05.

[0257] Table 5: Expression amount of VEGF protein in the retinal tissue of each group Note: compared with the Control group, * P<0.05; compared with the OIR group, ### P<0.001.

[0258] 3.6 SZ-A regulates the expression of EGLN1 and EPHA2 in the retinal tissue of OIR mice

[0259] EGLN1 (egl-9 family hypoxia inducible factor 1, also known as PHD2) is an oxygen-sensitive factor and plays an important role in regulating angiogenesis. EPHA2 is a kind of receptor tyrosine kinase and is widely expressed in various human tissues or cell lines, and can regulate cell proliferation, apoptosis, migration and angiogenesis.

[0260] The qPCR experiment results show that, compared with the Control group, the expression levels of EGLN1 and EPHA2 mRNA in the OIR group are both significantly increased, and the difference is statistically significant (P<0.01); compared with the OIR group, the expression levels of EGLN1 mRNA in the SZ-A medium and high dose groups and the bevacizumab group are all significantly decreased (P<0.05 or P<0.01 or P<0.001); the expression levels of EPHA2 mRNA in the SZ-A low and high dose groups and the bevacizumab group are all significantly decreased (P<0.05), as shown in Table 6.

[0261] Table 6 Relative expression of EGLN1, EPHA2 mRNA in retinal tissue of each group Note: compared with the Control group, ** P < 0.01; compared with the OIR group, ### P < 0.001, ## P < 0.01, # P < 0.05.

[0262] 3.7 Effect of SZ-A on expression of EGLN1 protein in retinas of OIR mice

[0263] The results of immunofluorescence experiments are shown in Figures 6 and 7. Compared with the OIR group, the average fluorescence intensity of EGLN1 was reduced in the SZ-A medium and high dose groups, and the difference was statistically significant (P < 0.05 or P < 0.01). However, after treatment with bevacizumab, the average fluorescence intensity of EGLN1 was reduced, but the difference was not statistically significant.

[0264] 3.8 Effect of SZ-A on expression of EphA2, PI3K, and Akt proteins in retinas of OIR mice

[0265] The results of Western blot experiments are shown in Figure 8. Compared with the OIR group, the relative expression of EphA2, PI3K, and Akt proteins was significantly reduced in the SZ-A medium and high dose groups, and the difference was statistically significant (P < 0.05, P < 0.01, P < 0.001, P < 0.0001).

[0266] 3.9 Effect of SZ-A on expression of Occludin protein in retinal tissue of OIR mice

[0267] The results of immunofluorescence experiments are shown in Figures 9 and 10. In the Control group, the Occludin fluorescence staining was bright and strong in the inner plexiform layer and retinal pigment epithelial layer. In the OIR group, the Occludin fluorescence staining was relatively dim, and the average fluorescence intensity was significantly reduced, and the expression of Occludin was decreased (P < 0.05). After treatment with SZ-A and bevacizumab, the fluorescence staining brightness was enhanced, and the average fluorescence intensity of Occludin protein was increased, and the expression was increased, but the difference was not statistically significant (P > 0.05) compared with the OIR group.

[0268] Test Example 2

[0269] Effect of SZ-A intervention on VEGF-induced retinal vascular leakage model

[0270] 1 Materials and Methods

[0271] 1.1 Experimental animals

[0272] 8-week-old SPF male C57BL / 6J mice weighing 22 ± 2 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (license number: SYXK (jing) 2023-0011) and raised in the animal experimental center of Beijing University of Chinese Medicine at a temperature of 22-25°C, a humidity of 50%-70%, natural light, and free water and food. After all the experimental animals were adaptively fed for 7 days, the subsequent experimental operation was carried out. This experiment was reviewed by the experimental animal ethics committee of Beijing University of Chinese Medicine.

[0273] 1.2 Construction of mouse retinal vascular leakage model

[0274] (1) VEGF 165 Protein injection dose: VEGF 165 Protein was injected into the vitreous body at a dose of 200 ng / eye and a volume of 2 μL / eye.

[0275] (2) Vitreous body injection: The mice were anesthetized by intraperitoneal injection of pentobarbital solution, the eyes of the mice were disinfected by applying iodophor around the eyes, sufficient mydriasis was achieved by dropping appropriate amount of compound tropicamide eye drops, and the mouse eye surface was locally anesthetized by dropping obidoxime hydrochloride eye drops. Adjust the posture of the mouse head, use a Hamilton microsyringe (5 μL) and a needle with a size of 33 for vitreous body injection, protrude the eyeball, expose the sclera, carefully clamp the sclera with microforceps to fix the eyeball, and vertically insert the needle at the temporal side 1-2 mm posterior to the corneoscleral junction. After the needle breaks through, the needle is tilted to avoid the lens, and then slowly injected. After injection, the needle is left for about 30 seconds to prevent liquid from flowing out, and then the needle is slowly withdrawn. Gently put the eye back into the eye socket and apply carbomer gel.

[0276] 1.3 Experimental animal grouping and drug administration

[0277] Experimental grouping: After the experimental animals were adaptively fed for 7 days, the body weight of the mice was measured and recorded, and then randomly divided into normal group (Control), model group, SZ-A low-dose treatment group (SZ-A-L), SZ-A medium-dose treatment group (SZ-A-M), SZ-A high-dose treatment group (SZ-A-H), and positive drug (Bevacizumab) treatment group (Bevacizumab) according to the body weight. Each group has 7-8 mice.

[0278] Drug: Morus extract SZ-A of Preparation Example 8.

[0279] Dose and mode of administration: According to the results of the pre-experiment, the SZ-A low, medium and high dose treatment groups were administered at a dose of 6.25 mg / kg, 12.5 mg / kg, 25 mg / kg (based on total alkaloids), dissolved in physiological saline, and administered by gavage, with a volume of 0.1 ml / 10g, for 7 consecutive days. The positive drug (bevacizumab) treatment group was administered at a dose of 25 μg / eye, with a volume of 1 μL / eye, and was administered by intravitreal injection on the first day of the experiment.

[0280] 1.4 Retinal permeability analysis

[0281] (1) Evans blue injection: 24h after VEGF intravitreal injection modeling, the mice were weighed and the body weight was recorded. Evans blue powder was dissolved in physiological saline, and was injected into the tail vein at a dose of 45 mg / kg and a concentration of 20 mg / ml. The injection was circulated in the body for 2h.

[0282] (2) Tail vein injection: The mice were placed in a special fixator, the mouse tail was soaked in warm water at an appropriate temperature to fill the blood vessels and soften the cuticle, making it easier to insert the needle. Then, a tail vein injection instrument was used to find a thicker blood vessel, and the 1 / 3-1 / 2 segment from the tail tip was used as the needle insertion point. The needle tip was inclined upward, and the needle was inserted at an angle of 30° with the mouse tail. When the needle tip inclined surface just completely entered the cuticle, the angle was adjusted to be parallel to the mouse tail, and a small amount of drug solution was slowly injected. The injection should be smooth and unobstructed, and the liquid should be seen pushing the venous blood forward. After the injection was completed, the mouse immediately turned blue, and the most obvious parts were the lips, nails, and ears, indicating that the injection was successful.

[0283] (3) Sample collection: After 2h of Evans blue circulation in the body, the mice were anesthetized, and blood was taken from the inner canthus. Each mouse took about 500 μL of blood and was stored in a 1.5 ml centrifuge tube containing heparin sodium. After the mouse was fixed, the abdomen was opened, and the needle was inserted from the apex of the heart. Sodium citrate buffer (PH = 3.5) preheated at 37°C was used for heart perfusion, with a perfusion volume of 60 ml. The perfusion was uniform and continued until the mouse's liver turned white. After perfusion, the eyeball was removed, the retina was peeled off, and the excess tissue was removed and stored in a 1.5 ml centrifuge tube in the dark (the centrifuge tube for storing the retina needs to be weighed in advance and recorded).

[0284] (4) Sample processing: After the sampling is completed, all the retinal tissues are placed in a 37°C oven and dried for 12 h. After drying, the dry weight of the retinal tissues is weighed and recorded. Then, 120 μL of formamide is added to the dried retinal tissues, which are incubated in a 70°C oven for 18 h. The collected blood samples are centrifuged at 4°C and 14,000 rpm for 20 min, and the supernatant is collected and diluted 100 times with formamide. Then, 100 μL of the diluted sample is added to each well of a 96-well plate, and the plate is protected from light to avoid bubble formation. After the incubation of the retinal samples, the samples are centrifuged for 2 min, and the supernatant is collected and centrifuged at 4°C and 14,000 rpm for 90 min. After centrifugation, the supernatant is collected and added to a 96-well plate, with 45 μL of the supernatant added to each well.

[0285] (5) Preparation of a standard curve: Standard solutions of Evans blue with concentrations of 5,000, 25,000, 10,000, 5,000, 1,000, 500, 250, 125 and 0 ng / ml are prepared, and the absorbance at 620 nm and 740 nm is detected by adding the solutions to a 96-well plate. The difference between the OD values at the two wavelengths is calculated. Two replicate wells are prepared for each sample.

[0286] The penetration rate of Evans blue dye in the retinal sample is calculated according to the following formula: Penetration rate [μl / (g·h)] = [retinal Evans blue content (μg) / retinal dry weight (g)] / [Evans blue blood concentration (μg / μl) x circulation time (h)].

[0287] 1.5 Paraffin section of a mouse eyeball

[0288] Preparation of a paraffin section: VEGF 165 After 24 h of intravitreal injection, the mice in each group are quickly sacrificed by intraperitoneal injection of sodium pentobarbital, and the eyeballs are carefully removed and fixed in 4% paraformaldehyde at room temperature for 48 h. The remaining experimental procedures are the same as those described in 2.4 of Test Example 1.

[0289] 1.6 HE staining

[0290] The experimental procedures are the same as those described in 2.5 of Test Example 1.

[0291] 2 Results

[0292] 2.1 Successful construction of a VEGF-induced mouse retinal vascular leakage model

[0293] C57 mice were injected with VEGF in the vitreous body to induce retinal vascular leakage. After consulting the literature, two injection doses of 100 ng / eye and 200 ng / eye were set to find the best modeling dose condition. 24 hours after the end of vitreous body injection, Evans blue tail vein injection was performed, the injection dose of Evans blue was 45 mg / kg, the injection concentration was 20 mg / ml, and the dye circulated in the mouse body for 2 hours after the end of injection. The mice were sacrificed, the retina was peeled off and the penetration rate of Evans blue dye in the retinal specimen was calculated to evaluate whether the model was successfully constructed. Both 100 ng / eye and 200 ng / eye injection doses can increase retinal vascular leakage in mice, as shown in Figure 11.

[0294] The following experiment selected VEGF vitreous body injection as the model group of mice with 200 ng / eye for research.

[0295] 2.2 SZ-A intervention can reduce VEGF-induced retinal vascular leakage in mice

[0296] After 7 days of SZ-A administration, Evans blue dye was injected into the tail vein to analyze the Evans blue penetration rate in retinal tissue and study the effect of SZ-A on retinal vascular leakage. The experimental results are shown in Table 7. Compared with the Control group, the Evans blue concentration in the model group was significantly increased (P<0.001); compared with the model group, the Evans blue concentration in the SZ-A-L (6.25 mg / kg), SZ-A-M (12.5 mg / kg), SZ-A-H (25 mg / kg), and Bevacizumab groups was significantly reduced (P<0.05), with the most obvious effect in the SZ-A-H group. The experimental results suggest that the SZ-A treatment group can effectively protect the retinal blood vessels and inhibit vascular leakage.

[0297] Table 7 Retinal vascular leakage in each group after vitreous body injection of VEGF-A165 Note: Compared with the Control group, *** P<0.001; compared with the OIR group, # P<0.05.

[0298] 2.3 Effect of SZ-A intervention on VEGF-induced retinal morphology in mice

[0299] HE staining is shown in Figure 12. Observation found that the Control group retinal layers showed clear and complete structure, with orderly, uniform and dense cell arrangement. The retinal cells in the model group were relatively disordered, and the layers were not clearly distributed. After treatment with SZ-A and Bevacizumab, the retinal tissue structure improved.

[0300] Pharmacodynamic experiment of mulberry extract on retinopathy of diabetic animals

[0301] 3.1 KKAy mouse model

[0302] Spontaneous type 2 diabetic KKAy mice, 15 weeks old, female, purchased from Beijing Huafukang Technology Co., Ltd., Animal License No.: SCXK(Jing)2019-0008. The animals were raised in SPF animal room, room temperature 20-26℃, air humidity 40%-60%, 12h cycle light, animals free to eat and drink. The experimental operation process was in accordance with the requirements of the Standard Operation Procedure of the Institute of Pharmaceutical Research of the Chinese Academy of Medical Sciences and the Animal Management and Animal Welfare Committee.

[0303] After the KKAy mice were settled, they were adaptively fed in the animal room for one week, and then fed with high-fat feed. After two weeks of high-fat feed, the mice were randomly divided into three groups according to fasting blood glucose (FBG), postprandial blood glucose (PBG), oral glucose tolerance test (OGTT) 30min blood glucose increase percentage (Glycemia increase at 30min in OGTT), blood triglyceride (TG), blood total cholesterol (TC) and body weight (BW). That is, the model control group (Control), the SZ-A 100mg / kg group and the SZ-A 200mg / kg group (calculated according to the weight of alkaloids, mulberry extract obtained from Preparation Example 8). Each group has 8 mice. Each administration group was given the corresponding drug by gavage, and the Control group was given the same volume of distilled water, once a day, for 10 consecutive weeks.

[0304] The eyeballs of the mice were removed and fixed in 4% paraformaldehyde at room temperature for 48h, and then HE staining was performed, according to the method described in 2.5 of Test Example 1.

[0305] The results are shown in Figure 13. Observation of HE staining showed that the cells in the retinal layers of the Model group were sparse and disordered, the cells were not closely connected, and there were vacuolar degeneration. After SZ-A drug intervention, the above phenomena were improved.

[0306] 3.2 STZ-induced diabetic mouse model

[0307] 8-week-old SPF C57BL / 6J and ApoE- / -mice were adaptively fed for one week. C57BL / 6J was the control group, fed with normal standard feed; ApoE- / -mice were the modeling group, fed with high cholesterol feed (40kcal% fat, 1.25% cholesterol), after 6 weeks, STZ was injected once at a dose of 100mg / kg, blood glucose was measured at 3 days after injection, ≥11 was modeling success, used for experiment. After modeling, the mice were randomly divided into groups according to body weight, blood glucose, and total cholesterol level, and the experimental groups were as follows, and the drug treatment was as follows, for a total of 6 weeks (sang extract SZ-A was obtained from Preparation Example 8, the dose of SZ-A administration group was as shown in Table 8, based on the weight of alkaloids).

[0308] Table 8

[0309] The eyeballs of the mice were extracted and fixed in 4% paraformaldehyde at room temperature for 48h, and HE staining was performed, according to the method described in 2.5 of Test Example 1. Results: SZ-A drug intervention can improve the retinal tissue structure to a certain extent.

[0310] Test Example 4

[0311] Cell experiment

[0312] Human retinal vascular endothelial cells (hRECs) were cultured in a special medium for human retinal vascular endothelial cells after conventional resuscitation, and the culture conditions were 37°C constant temperature, 5% volume fraction of carbon dioxide in a humidified incubator. The culture medium was replaced every two days, and when the cells covered more than 80% of the bottle bottom, they could be passaged, frozen or used for subsequent experiments.

[0313] A. The experimental groups were Control group (5.5mmol / L glucose), HG group (25mmol / L glucose), Mannitol group (5.5mmol / L glucose + 19.5mmol / L mannitol), HG+SZ-A-1 group (SZ-A-1 was prepared from Preparation Example 6), HG+SZ-A-2 group (SZ-A-2 was prepared from Preparation Example 8), HG+SZ-A-3 group (SZ-A-3 was prepared from Preparation Example 10), and SZ-A-1, SZ-A-2 and SZ-A-3 were administered at low (50μg / mL), medium (100μg / mL), and high (200μg / mL) doses (based on the weight of the extract).

[0314] 1) CCK8 method for detecting the effect of mulberry extract on the cell viability of high glucose-induced retinal vascular endothelial cells (HRMEC):

[0315] HRMEC cells were seeded in 96-well plates at a density of 5000 cells per well. After the cells adhered, the old culture medium was discarded and replaced with ECM medium containing 1% FBS to starve the cells for 8 h. The cells were divided into the following groups: Control group (5.5 mmol / L glucose), HG group (25 mmol / L glucose), Mannitol group (5.5 mmol / L glucose + 19.5 mmol / L mannitol), HG+SZ-A-1 group, HG+SZ-A-2 group, HG+SZ-A-3 group, and the drug concentrations of SZ-A-1, SZ-A-2, and SZ-A-3 were 50 μg / mL, 100 μg / mL, and 200 μg / mL (based on mulberry extract), respectively. After 24 h of high glucose and drug intervention, 10 μL of CCK-8 solution was added to each well, and the cells were incubated in the cell incubator for 2 h in the dark. The absorbance value was measured at 450 nm using a microplate reader. The effect of the drug on cell viability was calculated according to the formula: cell viability (%) = (treatment group - blank group) / (control group - blank group) x 100%.

[0316] Figure 14 shows that compared with the Control group, the HRMEC cell viability of the HG group increased significantly; compared with the HG group, the HRMEC cell viability after intervention with 50 μg / mL, 100 μg / mL, and 200 μg / mL doses of SZ-A-1, SZ-A-2, and SZ-A-3 decreased significantly. This suggests that mulberry extract can significantly reduce high glucose-induced HRMEC cell viability (cell proliferation ability).

[0317] 2) Vessel proliferation experiment:

[0318] 2.1) Tube formation experiment:

[0319] 2.1.1) HRMECs of 3-7 passages were used, and after 8 h of starvation with serum-free medium, the cells were divided into groups and administered Control group (5.5 mmol / L glucose), HG group (25 mmol / L glucose), Mannitol group (5.5 mmol / L glucose + 19.5 mmol / L mannitol), HG+SZ-A-1, 2, 3 groups (50 μg / mL, 100 μg / mL, 200 μg / mL), and then replaced with serum-free medium for culture and drug administration for 24 h.

[0320] 2.1.2) Matrigel was taken out from -80°C, thawed overnight at 4°C, and 20 uL of matrigel was added to each well of a 24-well plate. Before seeding the cells, the plate was placed in the incubator to solidify.

[0321] 2.1.3) The cells treated with drugs were seeded into a 24-well plate at a density of 1 x 10 5Cells were cultured in an incubator for 4-6 hours, observed under a microscope, and photographed. ImageJ software was used to analyze the length of the tube formation in each group of images, and the length of the tube formation per pore was used to represent the cell's angiogenesis capacity.

[0322] Figures 15 and 16 show that, compared with the Control group, the channel length in the HG group was significantly increased (P < 0.0001); compared with the HG group, the channel formation length was significantly reduced after intervention with medium and high doses of drug SZ-A-1 (P < 0.01, P < 0.05); the channel formation length was significantly reduced after intervention with low, medium, and high doses of drug SZ-A-2 (P < 0.05, P < 0.001, P < 0.0001); and the channel formation length was significantly reduced after intervention with low, medium, and high doses of drug SZ-A-3 (P < 0.001, P < 0.0001).

[0323] Figure 17 shows that SZ-A-2 and SZ-A-3 are better than SZ-A-1 in promoting angiogenesis in HRMECs, with SZ-A-3 showing the best ability to promote angiogenesis in HRMECs.

[0324] 2.2) Effect of mulberry extract (SZ-A) on high glucose-induced HRMEC cell migration as detected by cell scratch assay:

[0325] Streaking cell culture plates: First, use a marker pen to draw evenly spaced horizontal lines on the back of the 6-well plate with a ruler, approximately one line every 1 cm. Draw 3 lines per well.

[0326] Cell seeding: Seed 2 × 10⁶ cells per well in a 6-well plate. 5 The cells were cultured overnight for 24 hours to achieve a 100% fusion rate.

[0327] Cell starvation: Replace with serum-free culture medium and starve for 8 hours.

[0328] Cell streaking: On the second day, use a 200μL sterile pipette tip to streak lines on the cell layer perpendicular to the lines on the back of the well plate.

[0329] Cell washing: After scratching, the cells were washed three times with sterile PBS to remove the cells detached during scratching. The cells were then divided into groups and administered the following drugs: Control group (5.5 mmol / L glucose), HG group (25 mmol / L glucose), Mannitol group (5.5 mmol / L glucose + 19.5 mmol / L mannitol), and HG+SZ-A-1, 2, and 3 groups (50 μg / mL (low-dose group L), 100 μg / mL (medium-dose group M), and 200 μg / mL (high-dose group H)). The cells were then cultured in serum-free medium for 24 hours after drug administration.

[0330] Cell culture, observation: Incubate in the cell incubator with 5% CO2 for 24h. Take out the cells at the time point (0h, 24h), observe under microscope and measure the width of the scratch.

[0331] Result analysis: Use Image J software to calculate the migration rate of each group of cells. The calculation formula is as follows: cell migration rate = (0h scratch area-24h scratch area) / 0h scratch area x 100%.

[0332] Figures 18 and 19 show that the migration ability of HRMEC cells in the HG group is significantly increased compared with the Control group, and the migration ability of HRMEC cells after intervention of SZ-A-1, SZ-A-2 and SZ-A-3 is significantly reduced.

[0333] The results show that SZ-A can inhibit the migration of HRMEC cells induced by high glucose.

[0334] Figure 20 shows that the ability of SZ-A-2 and SZ-A-3 to inhibit the migration rate of HRMEC cells induced by high glucose is better than that of SZ-A-1.

[0335] 3) Vascular leakage experiment:

[0336] 3.1) Transendothelial electrical resistance (TEER) detection

[0337] HRMECs cells were seeded in the upper chamber of 12-well plate Transwell chamber to establish a blood-retinal inner barrier model (500 μL and 1.5 mL of endothelial cell special culture medium were added to the upper and lower chambers, respectively), and the endothelial cell Transwell hole was used as a blank control group. After culture to form a complete blood-retinal barrier (the resistance value tends to be stable), after 8h of starvation, the groups were divided and administered Control group (5.5 mmol / L glucose), HG group (25 mmol / L glucose), Mannitol group (5.5 mmol / L glucose + 19.5 mmol / L mannitol), HG+SZ-A1, 2, 3 group (50 μg / mL (low dose group L), 100 μg / mL (medium dose group M), 200 μg / mL (high dose group H)), and the culture medium was replaced with serum-free medium for 24h of culture and administration. The electrodes of the transendothelial cell resistance detector were wiped with 75% alcohol, passed through a PBS, and finally immersed in the culture medium and taken out to dry. Then the hole plate was taken out of the incubator, and after 30min of standing, the transendothelial cell resistance detector was used to measure each hole, and each hole was measured repeatedly three times. The calculation formula of TEER value is as follows: TEER (Ω·cm 2 ) = (average value of TEER per hole - value of cell-free blank hole) x cell growth area (1.12 cm 2 ).

[0338] Figure 21 shows that the TEER value of HG group was significantly decreased compared with Control group (P<0.001); compared with HG group, the TEER value of SZ-A-1, SZ-A-2 and SZ-A-3 groups had a trend of increase, and the TEER value of high dose groups of SZ-A-2 and SZ-A-3 was significantly increased (P<0.05, P<0.01) (Figure 22).

[0339] This indicates that SZ-A can improve the blood-retinal barrier to some extent and inhibit vascular leakage.

[0340] 3.2) Fluorescein isothiocyanate-dextran (FITC-dextran) leakage test

[0341] After the cells were treated for 24 hours according to the foregoing method, the cells were taken out from the cell incubator; the original culture medium in the upper chamber of the Transwell was discarded, 200 μL of 1 mg / mL fluorescein isothiocyanate-dextran labeled culture medium was added to the upper chamber of the Transwell, and 1 mL of normal DMEM culture medium was added to the lower chamber; then it was placed in the cell incubator for continued incubation for 4 hours; after taking out, 100 μL of culture medium in four different areas of the lower chamber of the Transwell was sucked into a 96-well plate; at the same time, the fluorescein isothiocyanate-dextran labeled culture medium was diluted to prepare a standard (the standard concentrations were 1000, 500, 250, 125, 62.5, 31.25, 0 μg / mL, respectively). The fluorescence intensity of fluorescein isothiocyanate-dextran was detected by an enzyme-labeled instrument (excitation light 490 nm, emission light 520 nm), and the standard curve was drawn according to the fluorescence intensity and the concentration of fluorescein isothiocyanate-dextran to calculate the concentration of fluorescein isothiocyanate-dextran in the lower chamber of the Transwell.

[0342] Figure 23 shows that the FITC-dextran permeability of HG group was significantly increased compared with Control group (P<0.001); compared with HG group, the FITC-dextran permeability of SZ-A-1, SZ-A-2 and SZ-A-3 groups had a trend of decrease, and the FITC-dextran permeability of high dose group of SZ-A-3 was significantly decreased (P<0.01), which indicates that the intervention of SZ-A can reduce the FITC-dextran permeability of HRMEC cells induced by high glucose, and the effects of SZ-A-2 and SZ-A-3 on reducing the FITC-dextran permeability are better than that of SZ-A-1, and the effect of SZ-A-3 is the best (Figure 24).

[0343] The above experiments show that SZ-A can maintain the integrity of the blood-retinal barrier in vitro to some extent.

[0344] The results show that SZ-A can improve the permeability and integrity of the vascular endothelial cell layer, and inhibit vascular leakage.

[0345] B. hRECs cells were seeded in a six-well plate at a density of 1.2 x 10 5 The cells of the 3rd to 5th generations were treated with normal glucose (NG, 5.5 mmol / L), normal glucose plus mannitol (M, 24.4 mmol / L), and different concentrations of glucose (15-30 mmol / L) for 2 h, 4 h, 6 h, 12 h, 24 h, and 48 h, and the SZ-A extract of Morus alba L. prepared in Preparation Examples 1-10 was added during the induction of the cells.

[0346] 1) Determination of the effective glucose-induced DR concentration and modeling time.

[0347] 2) Effect of SZ-A on angiogenesis and vascular permeability of hRECs cells

[0348] The pathological neovascularization of the induced hRECs cells is caused by the elevated levels of growth factors such as VEGF and its related pathways (VEGF, VEGFR2, Src, VEcadherin signaling pathway), insulin-like growth factor I (IGFI), and placental growth factor (PlGF). VEGF can reduce the expression of adhesion junction proteins (such as VE-Cadherin and β-catenin) and tight junction proteins (such as occludin, ZO-1, and occludin). The expression of related proteins such as VEGF, IGFI, PlGF, VE-Cadherin, β-catenin, occludin, ZO-1, and occludin was detected by WB experiments.

[0349] According to the detection results of the related proteins, compared with the control group, SZ-A can significantly reduce the expression of growth factors, inhibit pathological regeneration of blood vessels, and reduce cell permeability.

[0350] 3) Effect of the Morus alba L. extract on metabolic abnormalities of hRECs cells

[0351] The hRECs cells induced for different times were collected, and the ELISA kit was used to detect the elevated levels of reactive oxygen species (ROS) and advanced glycation end products (AGEs) in the cells after induction. According to the ELISA detection results, SZ-A can significantly reduce the oxidative damage to the retina caused by the elevated level of ROS.

[0352] 4) Effect of the Morus alba L. extract on apoptosis of hRECs cells

[0353] hRECs cells were collected, washed with PBS, resuspended with 195 μL buffer, and then added with 5 μL propidium iodide and 5 μL Annexin-FITC. After mixing, the mixture was reacted at room temperature for 10 min in the dark. The apoptosis rate of each group was detected within 1 h. The experiment was repeated 3 times.

[0354] WB experiment was used to detect the expression of apoptosis-related proteins. After high-glucose culture, the expression of apoptosis-related proteins BAX and cleaved-caspase-3 was significantly up-regulated, and the expression of anti-apoptosis-related protein Bcl-2 was significantly down-regulated.

[0355] The results showed that mulberry extract could significantly inhibit cell apoptosis.

[0356] 5) Effect of mulberry extract on potential neuroprotective factors

[0357] hRECs cells were collected, and Takara reverse transcription kit was used to convert RNA into cDNA. The reverse transcription product was diluted 10 times, and the target genes RUNX, OSX, COL1A1 and OCN were detected by Real-time PCR. Each group was independently tested in 3 replicates, and the reaction conditions were as follows: 95°C pre-denaturation for 2 min; 95°C for 15 s, 60°C for 1 min (40 cycles), and then a melting curve was established after reaction.

[0358] The expression levels of related endogenous neuroprotective agents, including insulin-like growth factor 1, pigment epithelium-derived factor (PEDF), somatostatin (SST), pituitary adenylate-cyclase-activated polypeptide (PACAP), glucagon-like peptide-1 (GLP-1), and brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were detected.

[0359] The results showed that mulberry extract could promote the expression of neurotrophic protective factors.

[0360] 6) Anti-inflammatory effect of mulberry extract

[0361] hRECs cells were collected, and Takara reverse transcription kit was used to convert RNA into cDNA. The reverse transcription product was diluted 10 times, and the target genes RUNX, OSX, COL1A1 and OCN were detected by Real-time PCR. Each group was independently tested in 3 replicates, and the reaction conditions were as follows: 95°C pre-denaturation for 2 min; 95°C for 15 s, 60°C for 1 min (40 cycles), and then a melting curve was established after reaction.

[0362] The related pro-inflammatory factors (IL-18, IL-1β, TNF-α and IL-6), monocyte chemotactic protein 1 (MCP-1) and cyclooxygenase-2 are detected.

[0363] The expression of NLRP3 inflammatory signaling pathway related proteins (NLRP3, ASC, Caspase1), NF-κB signaling pathway related proteins (IκB, p-IκB, p65, p-p65), protein kinase C pathway and protein kinase (MAP kinase) are detected by WB experiment.

[0364] The detection results show that the mulberry extract has significant anti-inflammatory effect.

Claims

1. Use of a mulberry extract for the manufacture of a medicament for the treatment and / or prevention of retinopathy, characterized in that, Based on the total weight percentage 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%). 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). 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), 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). 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).

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 retinopathy, characterized in that, The treatment and / or prevention of retinopathy includes improvement of any one or more of the following (1)-(11): (1) Inhibit pathological angiogenesis in the retina and / or reduce the central avascular zone; (2) Protects the structure of retinal tissue; (3) Inhibits retinal vascular leakage; (4) Reduce the expression level of VEGF in retinal tissue; (5) Reduce the expression levels of EGLN1 and / or EPHA2 in retinal tissue; (6) Inhibits tubule formation and / or migration of retinal vascular endothelial cells; (7) Improves the permeability and / or integrity of the retinal vascular endothelial cell layer; (8) Reduces the expression level of PI3K in retinal tissue; (9) Reduce the expression level of Akt in retinal tissue; (10) Increases the expression level of Occludin in retinal tissue; (11) Reduce the increased activity of retinal vascular endothelial cells caused by high sugar.

3. Use of the extract of Morus alba according to claim 1 for the preparation of a medicament for the treatment and / or prevention of retinopathy, characterized in that, The retinal diseases include proliferative retinopathy and / or exudative retinopathy.

4. The use of the mulberry extract according to claim 1 in the preparation of a medicament for treating and / or preventing retinopathy, characterized in that, The retinal diseases mentioned include diabetic retinopathy, age-related macular degeneration, retinal vein occlusion, retinal vasculitis, or retinopathy of prematurity.

5. The use of the mulberry extract according to any one of claims 1-4 in the preparation of a medicament for treating and / or preventing retinopathy, characterized in that, Based on 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: 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: 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: 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: 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: 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: 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: 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: 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:

6. The use of the mulberry extract according to any one of claims 1-5 in the preparation of a medicament for treating and / or preventing retinopathy, characterized in that, The alkaloids include 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, and 2-oxo-(α-D-galactoside) One or more of the following: 2-O-(α-D-galactopyranosyl)-1-deoxynojirimycin, 6-O-(β-D-glucopyranosyl)-1-deoxynojirimycin, and 1,4-dideoxy-1,4-imino-(2-O-β-D-glucopyranosyl)-D-arabinitol; Optionally, the weight percentage of DNJ is not less than 50% of the total alkaloids (optionally 60-99%).

7. The use of the mulberry extract according to any one of claims 1-6 in the preparation of a medicament for the treatment and / or prevention of retinopathy, characterized in that, The preparation of the mulberry extract includes the following steps: 1) Prepare a crude extract of mulberry plants; 2) Separate the crude extract with a cation exchange resin and / or optionally an anion exchange resin to obtain a resin eluent; optionally, step 3) subject the resin eluent of step 2) to alcohol precipitation and collect the supernatant; 4) Concentrate and / or dry the supernatant; optionally, concentrate and / or dry the resin eluent of step 2) before alcohol precipitation.

8. The use of the mulberry extract according to any one of claims 1-7 in the preparation of a medicament for treating and / or preventing retinopathy, characterized in that, The mulberry extract is effective in humans or mammals.

9. The use of the mulberry extract according to any one of claims 1-8 in the preparation of a medicament for treating and / or treating retinopathy, characterized in that, The drug is an oral dosage form; optionally, the drug is a tablet, capsule, oral solution, oral emulsion, pill, granule, syrup or powder.

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