Application of lysimachia christinae hance plant extract in preparation of medicine for preventing and treating diabetic retinopathy

Abstract

The invention relates to the field of biological medicine, in particular to application of lysimachia christinae extract to preparation of medicine for preventing and treating diabetic retinopathy. According to the invention, zebra fish is taken as a model animal, and the lysimachia christinae extract is subjected to safety evaluation; evaluating the protective effect of the lysimachia christinae extract on the retinal vascular injury by taking the diameter of the retinal vascular of the zebra fish as an index; the inhibition activity of the lysimachia christinae series plant extract on AR is investigated; the method comprises the following steps: acquiring mass spectrum data of the lysimachia christinae extract by adopting an HPLC-Q-TOF / MS technology, and characterizing the mass spectrum data; the identification compound of the lysimachia christinae series plant extract is researched; the anti-DR related activity of the lysimachia christinae extract and the content of the labeled compound are regressed, so that the active ingredients of the lysimachia christinae extract are determined. Results show that the lysimachia christinae extract is high in biological safety, has a protective effect on retinal blood vessels and can inhibit AR activity, and 21 active ingredients are defined.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to application of a radix lutea plant extract in preparing a medicine for preventing and treating diabetic retinopathy. Background Art

[0002] Diabetic retinopathy (DR) is an ocular microvascular complication caused by long-term hyperglycemia. It is the most serious of the common retinal vascular diseases and can lead to irreversible retinal dysfunction or even blindness, greatly reducing the quality of life of patients. With the increase in the number of diabetic patients, DR has become a major public health challenge. Therefore, the prevention and treatment of DR, especially the prevention and treatment strategies for aldose reductase (AR), are of great significance to protect the health of patients. At present, there are few reports on the research on the improvement of high sugar-induced retinal vascular damage in zebrafish by using plants of the Passiflora family.

[0003] Rhizoma Dianthus has obvious effects of expelling stones, promoting bile secretion, and inhibiting bacteria. Studies have shown that Rhizoma Dianthus can effectively inhibit acute and chronic inflammatory reactions such as increased vascular permeability, ear inflammation, and joint swelling. It is often used as the main medicine for diseases such as damp-heat jaundice, hepatobiliary stones, and urinary tract infections. Rhizoma Dianthus Guangxi has the effects of clearing heat and dampness, expelling stones and relieving stranguria; Rhizoma Dianthus Badong and Rhizoma Dianthus Zhejiang also have the effects of clearing heat and detoxifying, promoting diuresis and relieving stranguria, and reducing swelling and dispersing blood stasis. They are widely used in folk medicine to treat hot stranguria, sand stranguria, icteric hepatitis and other diseases. Fresh grass is crushed and used externally to treat snake bites, furuncles, and boils. The above-mentioned more common Rhizoma Dianthus plants have similar pharmacological activities. Phytochemical studies have shown that they all contain flavonoids, phenolic acids, terpenes, polysaccharides, and volatile oils. In the current literature reports, the research on different Rhizoma Dianthus plants is mainly focused on garden applications, and the research on the Rhizoma Dianthus plant extracts for the prevention and treatment of diabetic retinopathy is still very rare. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a plant extract of the Rhizoma Cibotii family in the preparation of a drug for preventing and treating diabetic retinopathy, so as to solve the problems existing in the above-mentioned prior art. The plant extract of the Rhizoma Cibotii family provided by the present invention can effectively prevent and treat diabetic retinopathy.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides an application of a flavonoid plant extract in preparing a medicine for preventing and treating diabetic retinopathy. The flavonoid plants include flavonoids from Guangxi, flavonoids from Zhejiang, flavonoids from Dian Gland and flavonoids from Badong.

[0007] Preferably, the method for preparing the extract of the Rhizoma Corydalis family plants comprises the steps of sequentially pulverizing, alcohol extraction and freeze-drying the Rhizoma Corydalis family plants.

[0008] Preferably, the alcohol extraction includes a first alcohol extraction, a second alcohol extraction and a third alcohol extraction;

[0009] The first alcohol extraction comprises the steps of mixing the powder of the radix lutea plant obtained after crushing with a methanol solution, performing extraction, and obtaining a first alcohol extract and a first alcohol extraction residue;

[0010] The second alcohol extraction comprises the steps of mixing the first alcohol extraction residue with a methanol solution and performing extraction to obtain a second alcohol extract and a second residue;

[0011] The third alcohol extraction comprises the steps of mixing the second alcohol extraction residue and a methanol solution, performing extraction, and obtaining a third alcohol extract and a third residue.

[0012] Preferably, when performing the first alcohol extraction, the solid-liquid ratio is 1 g:5 mL;

[0013] And / or, when performing the first alcohol extraction, the volume percentage of methanol in the methanol solution is 50%.

[0014] Preferably, during the second alcohol extraction and the third alcohol extraction, the solid-liquid ratio is 1 g:1 mL;

[0015] And / or, during the second alcohol extraction and the third alcohol extraction, the volume percentage of methanol in the methanol solution is 50%.

[0016] The invention provides a medicine for preventing and treating diabetic retinopathy. The medicine comprises extracts of plants of the Rhizoma Cibotii family. The Rhizoma Cibotii family plants include Rhizoma Cibotii Guangxiensis, Rhizoma Cibotii Zhejiangensis, Rhizoma Cibotii dactyli and Rhizoma Cibotii Badongensis.

[0017] Preferably, the method for preparing the extract of the Rhizoma Corydalis family plants comprises the steps of sequentially pulverizing, alcohol extraction and freeze-drying the Rhizoma Corydalis family plants.

[0018] Preferably, the alcohol extraction includes a first alcohol extraction, a second alcohol extraction and a third alcohol extraction;

[0019] The first alcohol extraction comprises the steps of mixing the powder of the radix lutea plant obtained after crushing with a methanol solution, performing extraction, and obtaining a first alcohol extract and a first alcohol extraction residue;

[0020] The second alcohol extraction comprises the steps of mixing the first alcohol extraction residue with a methanol solution and performing extraction to obtain a second alcohol extract and a second residue;

[0021] The third alcohol extraction comprises the steps of mixing the second alcohol extraction residue and a methanol solution, performing extraction, and obtaining a third alcohol extract and a third residue.

[0022] Preferably, when performing the first alcohol extraction, the solid-liquid ratio is 1 g:5 mL;

[0023] And / or, when performing the first alcohol extraction, the volume percentage of methanol in the methanol solution is 70%.

[0024] Preferably, when performing the second alcohol extraction and the third alcohol extraction, the material-liquid ratio is 1g:1mL;

[0025] And / or, during the second alcohol extraction and the third alcohol extraction, the volume percentage of methanol in the methanol solution is 50%.

[0026] As an additional embodiment, the present invention provides L-phenylalanine, isoleucine, aurantiamide acetate, L-proline, quercitrin, myricetin, quercetin, vicenin-III, III), kaempferol-3-O-rutinoside, quercetin 3-O-glucuronide, geniposide, hyperoside, chlorogenic acid, catechin, quinic acid, ixoside, quercetin-3-O-rutinoside, myricitrin, coumaroyl quinicacid and rosmarinic acid in the preparation of a drug for preventing and treating diabetic retinopathy.

[0027] As an additional scheme, the present invention provides a drug for preventing and treating diabetic retinopathy, which includes any one or more of L-phenylalanine, isoleucine, citrus acetate, L-proline, quercetin, myricetin, quercetin, vesinenin-III, kaempferol-3-O-rutinoside, quercetin 3-O-glucuronide, geniposide, hyperoside, chlorogenic acid, catechin, quinic acid, quassin, quercetin-3-O-rutinoside, myricetin, coumaroylquinic acid and rosmarinic acid.

[0028] As an additional scheme, the present invention provides the use of any one or more of L-phenylalanine, isoleucine, citrulline acetate, L-proline, quercetin, myricetin, quercetin, vesellarin-III, kaempferol-3-O-rutinoside, quercetin 3-O-glucuronide and geniposide in the preparation of a drug for preventing and treating retinal vascular damage.

[0029] As an additional scheme, the present invention provides a drug for preventing and treating retinal vascular damage, which includes any one or more of L-phenylalanine, isoleucine, citrus acetate, L-proline, quercetin, myricetin, quercetin, vesin-III, kaempferol-3-O-rutinoside, quercetin 3-O-glucuronide and geniposide.

[0030] As an additional embodiment, the present invention provides the use of any one or more of hyperoside, chlorogenic acid, quercetin, quercetin, myricetin, catechin, quinic acid, quassin, quercetin-3-O-rutinoside, myricetin, coumaroylquinic acid, rosmarinic acid, quercetin 3-O-glucuronide and geniposide in the preparation of aldose reductase inhibitors.

[0031] As an additional embodiment, the present invention provides an aldose reductase inhibitor, which includes any one or more of hyperoside, chlorogenic acid, quercetin, quercetin, myricetin, catechin, quinic acid, quassin, quercetin-3-O-rutinoside, myricetin, coumaroylquinic acid, rosmarinic acid, quercetin 3-O-glucuronide and geniposide.

[0032] The present invention discloses the following technical effects:

[0033] 1. Biosafety: Extracts from Rhizoma Dioscoreae plants had no significant effect on the growth and development of zebrafish embryos at concentrations below 10 μg / mL, indicating that these extracts have good biosafety.

[0034] 2. Retinal vascular protection: All extracts from the Rhizoma Cibotii family showed protective effects on high-sugar-induced retinal vascular damage in zebrafish. Specifically, Rhizoma Cibotii plant extracts were able to reduce the increase in retinal vascular diameter induced by high sugar in zebrafish, and Rhizoma Cibotii plant extracts at a concentration of 10 μg / mL could significantly reduce the increase in vascular diameter to less than 88%.

[0035] 3. Aldose reductase (AR) inhibitory activity: Extracts from Rhizoma Dioscoreae plants showed inhibitory activity against AR, with an IC 50 The values ​​ranged from 8.35 μg / mL to 13.17 μg / mL, indicating that these plant extracts have potential anti-diabetic retinopathy activity.

[0036] 4. Identification of active ingredients: 206 compounds were identified and 36 marker compounds were screened through UPLC-Q-TOF / MS technology and molecular network analysis. Further activity association analysis identified 21 active ingredients that were closely related to the anti-DR activity of Rhizoma Dioscoreae plant extracts.

[0037] 5. The present invention provides a new strategy for the prevention and treatment of DR, especially for AR, which is of great significance for protecting the health of diabetic patients. At the same time, it provides a scientific basis for the in-depth development and utilization of Rhizoma Dioscoreae plants, which may drive the development of related medicines and health care products industries, and generate social and economic benefits.

[0038] In summary, the present invention proves the intervention effect of Glehnia littoralis plants on DR, clarifies its active ingredients, provides new ideas for the prevention and treatment of DR, and also provides a basis for the in-depth development and utilization of Glehnia littoralis plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 The effects of extracts from Rhizoma Cibotii plants on the survival rate of zebrafish embryos; (A) shows the effects of different concentrations of LAH on the survival rate of zebrafish embryos; (B) shows the effects of different concentrations of LCC on the survival rate of zebrafish embryos; (C) shows the effects of different concentrations of LCH on the survival rate of zebrafish embryos; (D) shows the effects of different concentrations of LHM on the survival rate of zebrafish embryos; (E) shows the effects of different concentrations of LPH on the survival rate of zebrafish embryos; ※ indicates a significant difference compared with the control group (p<0.05); LAH: Rhizoma Cibotii Guangxiensis; LCC: Rhizoma Cibotii Zhejiangensis; LHM: Rhizoma Cibotii punctata; LPH: Rhizoma Cibotii Badongensis; LCH: Herba Lysimachiae;

[0041] Figure 2The effects of extracts from the family of Rhizoma Cibotii on the hatching rate of zebrafish embryos; (A) shows the effects of different concentrations of LAH on the hatching rate of zebrafish embryos; (B) shows the effects of different concentrations of LCC on the hatching rate of zebrafish embryos; (C) shows the effects of different concentrations of LCH on the hatching rate of zebrafish embryos; (D) shows the effects of different concentrations of LHM on the hatching rate of zebrafish embryos; (E) shows the effects of different concentrations of LPH on the hatching rate of zebrafish embryos; ※ indicates a significant difference compared with the control group (p<0.05); LAH: Guangxi Rhizoma Cibotii; LCC: Zhejiang Rhizoma Cibotii; LHM: Rhizoma Cibotii; LPH: Rhizoma Cibotii; LCH: Herba Lysimachiae;

[0042] Figure 3 The effects of extracts from Rhizoma Cibotii plants and 130 mM glucose on the survival of zebrafish embryos on the 6th day; (A) shows the effects of different concentrations of LAH and 130 mM glucose on the survival of zebrafish embryos on the 6th day; (B) shows the effects of different concentrations of LCC and 130 mM glucose on the survival of zebrafish embryos on the 6th day; (C) shows the effects of different concentrations of LCH and 130 mM glucose on the survival of zebrafish embryos on the 6th day; (D) shows the effects of different concentrations of LHM and 130 mM glucose on the survival of zebrafish embryos on the 6th day; (E) shows the effects of different concentrations of LPH and 130 mM glucose on the survival of zebrafish embryos on the 6th day; ※ indicates a significant difference compared with the control group (p<0.05); LAH: Rhizoma Cibotii Guangxiensis; LCC: Rhizoma Cibotii Zhejiangensis; LHM: Rhizoma Cibotii punctata; LPH: Rhizoma Cibotii Badongensis; LCH: Herba Lysimachiae;

[0043] Figure 4 The effect of the extract of the plant family of Rhizoma Cibotii on the retinal blood vessels of zebrafish induced by 130 mM glucose; (A) is a staining image, three points were selected in each image to measure the diameter of blood vessels, and the results were expressed as mean ± standard deviation (n = 40 per group); data with different letters are significantly different from each other (p < 0.05), optic disc is represented by yellow *, and the scale bar is = 20 μm; (B) is a statistical graph; Glu: 130 mM glucose; LAH: Guangxi Rhizoma Cibotii; LCC: Zhejiang Rhizoma Cibotii; LHM: Dot-gland Rhizoma Cibotii; LPH: Badong Rhizoma Cibotii; LCH: Lysimachia chinensis;

[0044] Figure 5 The total ion chromatogram (TIC) of the extracts of the Rhizoma Cibotii family in positive (A) and negative (B) modes; LAH: Rhizoma Cibotii from Guangxi; LCC: Rhizoma Cibotii from Zhejiang; LHM: Rhizoma Cibotii from Dian Gland; LPH: Rhizoma Cibotii from Badong; LCH: Herba Lysimachiae;

[0045] Figure 6 Visualization of the molecular network of Rhizoma Dioscoreae plants in positive ion mode;

[0046] Figure 7 Visualization of the molecular network of Rhizoma Dioscoreae plants in negative ion mode;

[0047] Figure 8 Principal component analysis (PCA) of five L. philadelphica plant extracts in positive (A) and negative (B) ion modes; LAH: L. philadelphica from Guangxi; LCC: L. philadelphica from Zhejiang; LCH: Lysimachia chinensis; LHM: L. philadelphica from schizonepeta; LPH: L. philadelphica from Padang; QC: quality control;

[0048] Fig. 9 OPLS-DA scatter plots ((A) and (C)) and S-plot scores ((B) and (D)) of the extract of L. guangxiensis and the extract of Herba Lysimachiae in positive and negative ion modes; LCH: Herba Lysimachiae; LAH: L. guangxiensis;

[0049] Fig.10 OPLS-DA scatter plots ((A) and (C)) and S-plot scores ((B) and (D)) of the extract of Lysimachia zeylanicum and Lysimachia chinensis in positive and negative ion modes; LCH: Lysimachia zeylanicum; LCC: Lysimachia zeylanicum;

[0050] Fig.11 OPLS-DA scatter plots ((A) and (C)) and S-plot scores ((B) and (D)) of Lysimachia chinensis extract and L. philadelphica extract in positive and negative ion modes; LCH: Lysimachia chinensis; LHM: L. philadelphica;

[0051] Fig.12 OPLS-DA scatter plots ((A) and (C)) and S-plot scores ((B) and (D)) of Lysimachia chinensis extract and Leptospermum officinale extract in positive and negative ion modes; LCH: Lysimachia chinensis; LPH: Leptospermum officinale;

[0052] Fig.13 An overview of the relationship between active ingredients, sources and activities. DETAILED DESCRIPTION

[0053] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0054] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0055] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0056] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0057] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0058] Example 1

[0059] 1. Preparation of Extracts from Plants of the Radix Glehniae Family

[0060] The dried Herba Lysimachiae, Herba Lysimachiae Guangxiae, Herba Lysimachiae Zhejiangae, Herba Lysimachiae Roots, and Herba Lysimachiae Badongae were crushed and passed through an 80-mesh sieve to prepare them in a dry powder state. Subsequently, the powder of Herba Lysimachiae plants was fully mixed and stirred with 50% (v / v) methanol at a ratio of 1g:5mL, and the extraction was assisted by an ultrasonic cleaner at 150W power at room temperature for 20min. Subsequently, the extract was filtered and the filtrate and residue were collected, and the residue was fully mixed and stirred with an equal amount of 50% methanol in the first extraction, and the extraction was repeated twice according to the above steps. The filtrates of the three extractions were collected and mixed evenly, and the extracts were concentrated at 37°C by a rotary evaporator. Finally, the concentrated extract was transferred to a plate for freeze drying at a freeze drying temperature of -50°C for 24h, and the extracts of Herba Lysimachiae plants (herba Lysimachiae extract, Herba Lysimachiae Guangxiae, Herba Lysimachiae Zhejiangae, Herba Lysimachiae Roots, and Herba Lysimachiae Badongae) were collected, labeled, and sealed and stored at -20°C for future use.

[0061] 2. Preparation of culture medium

[0062] (2.1), 130mM glucose culture medium

[0063] Glucose powder was dissolved in embryo culture medium at a concentration of 130 mM, mixed evenly with a magnetic stirrer, and used as a reserve solution.

[0064] (2.2) Culture medium of extracts from Rhizoma Corydalis plants

[0065] In order to evaluate the biosafety of extracts from the Glechoma series plants, appropriate amounts of crude extracts from each Glechoma series plant were weighed and fully dissolved in embryo culture medium to prepare an extract solution of the Glechoma series plants with a concentration of 500 μg / mL. Subsequently, appropriate amounts of embryo culture medium were added to dilute the extracts to prepare extract solutions of 100 μg / mL, 50 μg / mL, 25 μg / mL, 10 μg / mL, 5 μg / mL and 1 μg / mL of Glechoma series plants, respectively, for use.

[0066] To evaluate the effects of extracts from Glechoma longituba on retinal vascular diameters of zebrafish embryos exposed to 130 mM glucose, appropriate amounts of extracts from Glechoma longituba were weighed and fully dissolved in 130 mM glucose culture medium to prepare a 500 μg / mL solution of Glechoma longituba extract and 130 mM glucose. Appropriate amounts of 130 mM glucose culture medium were then added to dilute the extracts to prepare 100 μg / mL, 50 μg / mL, 25 μg / mL, 10 μg / mL, 5 μg / mL and 1 μg / mL solutions of Glechoma longituba extract and 130 mM glucose, respectively, for later use.

[0067] 3. Zebrafish Culture

[0068] (3.1) Zebrafish culture conditions

[0069] Zebrafish were kept in a fish tank controlled at about 26.0°C, with light provided according to a light:dark cycle of 14h:10h, and were fed brine shrimp eggs twice, at 11:45-12:15 noon and 6:45-7:15 pm.

[0070] (3.2) Hatching of brine shrimp eggs

[0071] Weigh about 13g of sea salt and place it in a large beaker, add 1L of first-grade water, and after it is fully dissolved, weigh 5g of unhatched shrimp eggs and put them into an air pump to allow the brine shrimp to obtain the oxygen required for hatching. After 30-36 hours, remove the air pump and let it stand, waiting for the upper shrimp shells and the lower shrimp eggs to be completely separated, slowly pour out the brown floating objects, and the orange precipitate at the bottom is the hatched shrimp eggs, and then put an appropriate amount of shrimp eggs in the fish tank according to the number of zebrafish.

[0072] (3.3) Zebrafish breeding

[0073] Wild-type zebrafish embryos and TG-type fluorescent transgenic zebrafish embryos were obtained by mating of parent fish. The experiment of the protective effect of different concentrations of lutea plant extracts on high-sugar-induced zebrafish retinal blood vessels used TG-type fluorescent transgenic zebrafish embryos, and the remaining experiments used wild-type zebrafish embryos. The night before zebrafish spawning, 2 male and female wild-type / transgenic zebrafish were randomly selected and placed in the spawning tank as needed and separated by partitions. After illumination the next day, the partitions were removed to stimulate spawning. After waiting for 2-4 hours to finish spawning, impurities in the water were removed, zebrafish embryos were collected, placed in culture medium, and cultivated into adult fish in a constant temperature incubator at 27.0°C or used for subsequent experiments.

[0074] (3.4) Experimental Animal Grouping

[0075] ① In this example, when evaluating the safety of the extract of the Rhizoma Cibotii plant, zebrafish embryos of 0-96 hpf were used as the research objects, and the group incubated with methylene blue embryo culture medium was the normal group, with 40 embryos in each group. The grouping of the normal group and the group of extracts of the Rhizoma Cibotii plant of different concentrations is shown in Table 1.

[0076] Table 1 Grouping of biosafety evaluation of extracts from Rhizoma Dioscoreae plants

[0077]

[0078]

[0079] ② In this example, when investigating the effects of the extracts of Rhizoma Cibotii plants on the survival of zebrafish at 6 dpf induced by high sugar and on the diameter of retinal blood vessels, 2.5 dpf zebrafish embryos were used as the research objects, with 40 embryos in each group, and the grouping is shown in Table 2.

[0080] Table 2 Effects of extracts from Rhizoma Cyperi plants on six-day survival of zebrafish / retinal blood vessel diameter Grouping

[0081] Grouping Culture solution Con / Normal Methylene blue embryo culture medium incubation Glu 130 mM glucose Glu+1μg / mLLAH 130mM glucose + 1μg / mL Guangxi Herba Lysimachiae extract intervention Glu+5μg / mLLAH 130mM glucose + 5μg / mL Guangxi Herba Lysimachiae extract intervention Glu+10μg / mLLAH 130mM glucose + 10μg / mL Guangxi Herba Lysimachiae extract intervention Glu+1μg / mL LCC 130mM glucose + 1μg / mL Zhejiang radix scutellariae extract intervention Glu+5μg / mL LCC 130mM glucose + 5μg / mL Zhejiang radix scutellariae extract intervention Glu+10μg / mL LCC 130mM glucose + 10μg / mL Zhejiang radix scutellariae extract intervention solution Glu+1μg / mL LHM 130mM glucose + 1μg / mL Psoralea corylifolia extract intervention Glu+5μg / mL LHM 130mM glucose + 5μg / mL Psoralea corylifolia extract intervention Glu+10μg / mL LHM 130mM glucose + 10μg / mL Psoralea corylifolia extract intervention Glu+1μg / mL LPH 130mM glucose + 1μg / mL Radix Glehniae extract intervention Glu+5μg / mL LPH 130mM glucose + 5μg / mL Radix Glehniae extract intervention solution Glu+10μg / mL LPH 130mM glucose + 10μg / mL Radix Glehniae extract intervention Glu+1μg / mL LCH 130mM glucose + 1μg / mL Lysimachia sibiricum extract intervention Glu+5μg / mL LCH 130mM glucose + 5μg / mL Lysimachia sibiricum extract intervention Glu+10μg / mL LCH 130 mM glucose + 10 μg / mL Lysimachia sibiricum extract intervention

[0082] (3.5) Zebrafish embryo culture

[0083] A 24-well plate was used as a culture vessel, and 10 randomly selected zebrafish embryos fertilized for 4 hours were placed in each well. After the residual embryo culture solution was absorbed, the 24-well plate was grouped according to Table 1 and Table 2 and the corresponding culture solution was added. The eggs were incubated in an embryo incubator at 27.5°C with a light-dark cycle of 14:10 h. Dead eggs were removed in time and fresh culture solution was replaced once a day.

[0084] (3.6) Collection of experimental animal samples

[0085] ① In this example, when evaluating the biosafety of the extracts from the Rhizoma Cibotii family, the survival and incubation conditions of zebrafish embryos were observed and recorded under a stereomicroscope for 4 consecutive days.

[0086] ② In this example, when exploring the effects of different concentrations of Rhizoma Cibotii plant extracts on the six-day survival of zebrafish induced by high sugar, the number of surviving and dead zebrafish was observed and recorded at 6 dpf.

[0087] ③ In this example, when exploring the effects of different concentrations of extracts from Rhizoma Cibotii plants on the diameter of zebrafish retinal vessels induced by high glucose, embryos were collected at 6 dpf.

[0088] 4. Survival and hatching rates of zebrafish embryos

[0089] In order to explore the biosafety of extracts from Rhizoma Corydalis plants, the effects of different concentrations of extracts (1, 5, 10, 25, 50 and 100 μg / mL) on the survival rate and hatching rate of zebrafish embryos were investigated. The experiment was carried out according to the following steps.

[0090] (4.1) Obtain zebrafish samples

[0091] The zebrafish embryos were grouped according to Table 1, and the zebrafish were cultured according to the method in step “(3.5) Zebrafish embryo culture”.

[0092] (4.2) Calculation of survival rate and hatching rate

[0093] The survival rate and hatching rate were calculated according to the formula, wherein the survival rate was calculated as follows: survival rate (%) = number of surviving embryos / total number of embryos (40) × 100; the hatching rate was calculated as follows: hatching rate (%) = number of hatched embryos / total number of embryos (40) × 100.

[0094] The survival rate and hatching rate of zebrafish embryos in each group were calculated to evaluate the biosafety of R. passerineii plants.

[0095] 5. Zebrafish survival on day 6

[0096] The zebrafish embryos were grouped according to Table 2, with 4-well zebrafish embryos set as one group. The zebrafish were cultured according to the method in step “(3.5) Zebrafish embryo culture” for a total of 40 embryos. The number of surviving and dead zebrafish was recorded at 6 dpf.

[0097] 6. Measurement of Zebrafish Retinal Blood Vessel Diameter

[0098] (6.1) Obtain zebrafish samples

[0099] The zebrafish embryos were grouped according to Table 2, and the zebrafish were cultured according to the method in step “(3.5) Zebrafish embryo culture”.

[0100] (6.2) Fixation and hydrolysis of zebrafish embryos

[0101] Zebrafish at 6 dpf were anesthetized with tricaine and placed in a 5 mL centrifuge tube. The tricaine was then aspirated and 4% paraformaldehyde was added to fix overnight at 4°C. The fixed larvae were gently washed with double distilled water three times to remove the fixative and placed at 37°C for 40 min with 0.25% trypsin EDTA solution. They were fixed in 4% paraformaldehyde at 4°C overnight. The fixed larvae were washed with double distilled water three times and placed in a water bath with 0.25% trypsin EDTA solution at 37°C for 40 min. The retinal vessels of the larvae were then dissected under a stereo microscope and transferred to a slide containing 4% methylcellulose gel for fixation. Phenotypic evaluation images were collected using a Zeiss inverted fluorescence microscope (Axio Vert.A1). Image J software was used to quantify the vessel diameter and the number of fluorescent nuclei.

[0102] (6.3) Observation and quantification of zebrafish retinal blood vessels

[0103] The zebrafish was placed on a flat dish, the eyeball was separated from the body, and the vitreous was then peeled off using a dissecting needle and transferred to a glass slide with 4% methylcellulose gel for fixation. An inverted fluorescence microscope was used to collect images of the zebrafish retina, and the retinal blood vessel diameter was quantified using Image J.

[0104] 7. Extraction of Aldose Reductase from Rat Eyeballs

[0105] SD rats were killed by cervical dislocation and the eyeballs were removed. The iris wrapped around the lens was removed using sterilized scissors and forceps. 0.5 mL of buffer solution (1.272 g of potassium dihydrogen phosphate and 0.66 g of potassium dihydrogen phosphate dissolved in 100 mL of water) was added to each lens, and the eyes were continuously ground in a mortar placed on an ice box until no obvious tissue state was observed. The eyes were centrifuged at 11,000 g and 4°C in a low-temperature centrifuge for 20 min, and the supernatant was collected.

[0106] 8. Aldose reductase inhibition assay

[0107] In this example, DL-glyceraldehyde was used as a substrate to explore the AR inhibitory activity of the extract of the Rhizoma Corydalis family. The experimental steps are as follows: 192 μL of buffer solution (2.04 g of potassium dihydrogen phosphate and 0.38 g of sodium hydroxide dissolved in 300 mL of water), 15 μL of aldose reductase, 30 μL of NADPH (0.4 mM), 3 μL of sample (Rhizoma Corydalis family plant extract), 30 μL of ammonium sulfate solution (528 mg / mL), 30 μL of DL-glyceraldehyde (9 mg / mL), and the total volume is 300 μL. After mixing and vortexing with a pipette, the change in absorbance of NADPH within 3 minutes was immediately measured by a UV spectrophotometer at 340 nm. The sample without AR and the control group were blank control groups, the sample without AR was the control group, and quercetin was the positive control. The aldose reductase inhibition rate (%) was calculated according to the following formula.

[0108] Aldose reductase inhibition rate (%) = [1-(ΔA1-ΔA2) / (ΔA2-ΔA0)]*100%; wherein ΔA0 represents the absorbance change value of the blank control group within 3 minutes, ΔA1 represents the absorbance change value of the sample group within 3 minutes, and ΔA2 represents the absorbance change value of the control group within 3 minutes.

[0109] 9. LC-MS analysis

[0110] LC-MS (Thermo, Vanquish, QExactive HF) was used for liquid chromatography-mass spectrometry analysis, and the liquid phase analysis column was C 18 Chromatographic column (Zorbax Eclipse C 18 (1.8μm*2.1mm*100mm)).

[0111] (9.1) Mobile phase gradient and liquid phase parameter settings

[0112] The gradient elution program (2 μL) was: 0-2 min, 5% B; 2-6 min, 5-30% B; 6-7 min, 30% B; 7-12 min, 30-78% B; 12-14 min, 78% B; 14-17 min, 78-95% B; 17-20 min, 95% B, the mobile phase was 0.1% formic acid aqueous solution (A) and acetonitrile (B), the flow rate was 0.3 mL / min; column temperature: 40°C; injection volume: 2 μL.

[0113] (9.2) Setting of mass spectrometry parameters

[0114] The mass spectrometry data of the extracts of Radix Glehniae plants were collected in the positive and negative ion modes, respectively. The optimized mass spectrometer parameters were: heater temperature: 325°C; purge gas flow rate: 1arb; electrospray voltage 3.5KV; auxiliary gas flow rate: 15arb; capillary temperature 330°C; sheath gas flow rate, 45arb; S-Lens RF Level: 55%; scanning mode: full scan (FullScan, m / z100-1500) and data-dependent secondary mass spectrometry scan (dd-MS2, TopN=5); resolution: 120000 (primary mass spectrometry) and 60000 (secondary mass spectrometry); collision mode: high energy collision dissociation (HCD).

[0115] (9.3) Preparation of test solution

[0116] Weigh 100 mg of each mixed extract of Radix Glehniae plants into a 2 mL centrifuge tube, add 1 mL of 70% methanol and 3 mm steel beads, and use a fully automatic sample rapid grinder (JXFSTPRP-48, 70 Hz) to shake and crush for 3 min. After cooling, perform low-temperature ultrasound for 10 min, and centrifuge at 12000 rpm for 10 min at a low temperature of 4°C. Take the supernatant and pass it through a 0.22 μm PTFE filter for detection.

[0117] 10. Construction of molecular network and identification of metabolic components

[0118] The mass spectrometry data of the extracts of the Rhizoma Glehniae family were converted into mzML format by MSConvert, and feature quantification processing such as retention time correction, peak identification, peak extraction, peak integration, and peak alignment was performed by MZmine2, and the mzML file, MGF file, and feature quantification table were uploaded. Subsequently, a feature-based molecular network (FBMN) was created on the GNPS platform (https: / / gnps.ucsd.edu) and the metabolites in the extracts of the Rhizoma Glehniae family were identified based on the GNPS database. The parameters of FBMN were set according to the cosine score of 0.7, the minimum number of matching fragment ions of 6, topK10, and the mass deviation of the parent ion and the daughter ion of 0.02Da. Subsequently, MS2LDA and DEREPLICATOR+ processing were performed on the GNPS platform, and the parameters were set as follows: precursor ion mass tolerance of 0.005Da, fragment ion mass tolerance of 0.01Da, maximum charge of 2, minimum score of 9.0, and fragmentation mode of probe. The results of FBMN, MS2LDA and DEREPLICATOR+ constructed in positive and negative ion modes of the extracts of the Rhizoma Dioscoreae family were then combined with MonNetEnhancer to improve the structural annotation of the metabolic components and perfect the molecular network. Finally, the repeated information in the integrated molecular network was processed and visualized using Cytoscape3.4.0 software.

[0119] 11. Analysis of Differential Metabolism

[0120] The mass spectrometry data were subjected to peak extraction, denoising, deconvolution, and peak alignment to obtain a three-dimensional data matrix containing retention time, mass-to-charge ratio, and mass spectrum peak area. The obtained three-dimensional data matrix was imported into SIMCA-P13.0 software for multivariate statistical analysis, including PCA and OPLS-DA, and S-plot was used to identify potential marker compounds that contributed more to the difference.

[0121] 12. Activity association analysis

[0122] The active ingredients in the extracts of the Rhizoma Dioscoreae family were identified by partial least squares analysis (PLS) using SIMCA-P 13.0 software. In this example, the mass spectrometry peak areas of the marker compounds in the anion and cation modes of Rhizoma Dioscoreae Guangxi, Rhizoma Dioscoreae Zhejiang, Rhizoma Dioscoreae Dotae, and Rhizoma Dioscoreae Badong were used as X variables, and the intervention effects of the extracts of the Rhizoma Dioscoreae family on the retinal vascular damage induced by high sugar in zebrafish and the IC values ​​of the extracts on aldose reductase were respectively used. 50 The value was used as the Y variable and imported into SIMCA-P 13.0 software to establish a PLS model between the chemical components of the extracts of Rhizoma Dioscoreae and their anti-DR activity, and the results were presented using Biplot. The contribution of the identified compounds of the extracts of Rhizoma Dioscoreae to the anti-DR activity was predicted by the positive and negative regression coefficients, and the active ingredients were identified.

[0123] 13. Statistical Analysis

[0124] In this embodiment, the experimental results are expressed as mean ± standard deviation (Mean ± SD), GraphPad Prism 9.0 and Origin 2021 are used for graphing, SPSS 24.0 is used for statistical analysis, and t-test or one-way analysis of variance (ANOVA) is used for significance test as needed. The difference is considered to be statistically significant when p < 0.05.

[0125] 14. Results

[0126] (14.1) Effects of extracts from Rhizoma Cyperi plants on the survival rate and hatching rate of zebrafish embryos

[0127] When zebrafish embryos were exposed to different concentrations of extracts from the Rhizoma Dioscoreae family, their survival rates varied between 0 and 96 hpf. Figure 1 As shown. In the normal group (Normal), the survival rate of zebrafish embryos showed a downward trend over time and reached the lowest at 96hpf. When zebrafish embryos were exposed to the extract solution, the trend of their survival rate over time was consistent with that of the normal group. However, it is worth noting that the survival rate of zebrafish embryos exposed to the extract solution of the Rhizoma Corydalis at 96hpf decreased with the increase of the extract concentration, indicating that the toxic effect increased with the increase of the extract concentration. Among them, the Rhizoma Corydalis plant extracts at concentrations of 1, 5 and 10 μg / mL had no significant effect on the survival rate of zebrafish embryos, while the Rhizoma Corydalis plant extracts at concentrations of 25, 50 and 100 μg / mL significantly affected the survival rate of zebrafish embryos.

[0128] When zebrafish embryos were exposed to different concentrations of the extracts of the Rhizoma Cyperi plant, the hatching rate varied between 0 and 96 hpf. Figure 2 As shown. In the normal group, the hatching rate of zebrafish embryos showed an upward trend over time and reached the highest at 96hpf. When zebrafish embryos were exposed to the extract solution, the trend of their hatching rate over time was consistent with that of the normal group. However, it is worth noting that the hatching rate of zebrafish embryos exposed to the extract solution of the Rhizoma Corydalis at 96hpf decreased with the increase of the extract concentration, indicating that the toxic effect increased with the increase of the extract concentration. Among them, the Rhizoma Corydalis plant extracts at concentrations of 1, 5 and 10 μg / mL had no significant effect on the hatching rate of zebrafish embryos, while the Rhizoma Corydalis plant extracts at concentrations of 25, 50 and 100 μg / mL significantly affected the hatching rate of zebrafish embryos.

[0129] (14.2) Effects of co-exposure of different concentrations of Rhizoma Dioscoreae plant extracts and 130 mM glucose on the survival of zebrafish embryos on day 6

[0130] In order to determine the appropriate intervention concentration of the Rhizoma Dioscoreae plant extract, this example investigated the effects of co-exposure of different concentrations of Rhizoma Dioscoreae plant extract and 130 mM glucose on the survival of zebrafish embryos on day 6. This example counted the number of surviving and dead zebrafish embryos at 6 dpf. The results are as follows: Figure 3 As shown. The number of surviving zebrafish embryos in the normal group (Normal) at 6dpf was 38-39, while the number of surviving zebrafish embryos exposed to 130mM glucose solution was 37-38, which was not significantly different from the normal group. When zebrafish embryos were exposed to 130mM glucose and extract solution, their survival was closely related to the concentration of the extract. When the extract concentration was 1, 5 and 10μg / mL, the number of surviving zebrafish embryos was not significantly different from that of the normal group; when the extract concentration was 25, 50, and 100μg / mL, the number of surviving zebrafish embryos was significantly lower than that of the normal group. Therefore, in subsequent experiments, 1, 5, and 10μg / mL were selected as the intervention concentrations of the extract of the Rhizoma Dioscoreae plant to explore its intervention effect on high sugar-induced retinal vascular damage in zebrafish.

[0131] (14.3) Protective effects of different concentrations of Rhizoma Cibotii plant extracts on high glucose-induced retinal angiogenesis in zebrafish

[0132] Using an inverted fluorescence microscope to capture images of zebrafish retinal blood vessels, Figure 4 In (A), the "*" indicates the location of the optic disc, and the scale bar is 20 μm. When zebrafish embryos were exposed to 130 mM glucose, their retinal blood vessels became significantly thicker, while after intervention with the five kinds of lutea plant extracts, the diameter of the retinal blood vessels became thinner ( Figure 4 (A) in the figure.

[0133] Image J software was used to quantify the diameter of zebrafish retinal blood vessels. Figure 4As shown in (B), the diameter of retinal blood vessels in zebrafish embryos in the 130 mM glucose group (100%) was significantly thickened compared with the normal group (Normal, 74.60%) (p<0.05); under the intervention of 1, 5, and 10 μg / mL Guangxi Herba Lysimachiae extract, the diameter of retinal blood vessels decreased to 91.58%, 89.17%, and 88.00%, respectively; under the intervention of 1, 5, and 10 μg / mL Zhejiang Herba Lysimachiae extract, the diameter of retinal blood vessels decreased to 91.14%, 89.57%, and 89.60%, respectively. and 88.55%; under the intervention of 1, 5, 10μg / mL Lysimachia chinensis extract, the retinal vessel diameter decreased to 78.67%, 75.14% and 74.62%, respectively; under the intervention of 1, 5, and 10μg / mL Passerella punctata extract, the retinal vessel diameter decreased to 88.56%, 84.43% and 82.13%, respectively; under the intervention of 1, 5, 10μg / mL Passerella padi extract, the retinal vessel diameter decreased to 86.34%, 85.81% and 83.42%, respectively.

[0134] By comparing the improvement effects of five extracts from the Glechoma longituba plant family on high sugar-induced thickening of retinal vascular diameter in zebrafish, it was found that the intervention effect of the Lysimachia chinensis extract was significantly lower than that of the other four Glechoma longituba plant extracts; the intervention effect of the Glechoma punctata extract at higher concentrations (5 and 10 μg / mL) was better than that of the Glechoma paduan extract, and the intervention effect at low concentrations (1 μg / mL) was weaker than that of the Glechoma paduan extract, but there was no significant difference (p<0.05).

[0135] (14.4) Using quercetin as a positive control, the inhibitory effects of five Rhizoma Cibotii plant extracts at different concentrations on AR activity were investigated.

[0136] Table 3 Aldose reductase inhibitory activity of extracts from Rhizoma Corydalis plants

[0137]

[0138]

[0139] Note: IC 50 represents the sample concentration required to reduce the initial substrate concentration by 50%; LAH: Guangxi Lycopodiella; LCC: Zhejiang Lycopodiella; LHM: Lycopodiella punctata; LPH: Lycopodiella cuneata; LCH: Lysimachia chinensis; the results are expressed as the mean standard deviation (n=3), and the values ​​in the columns marked with different letters are significantly different (p<0.05); quercetin was used as a positive control for aldose reductase inhibition.

[0140] The results are shown in Table 3. The inhibition rates of 5, 10 and 50 μg / mL Guangxi Herba Lysimachiae extract on AR were 27.86%, 42.57% and 83.58%, respectively; the inhibition rates of 5, 10 and 50 μg / mL Zhejiang Herba Lysimachiae extract on AR were 27.77%, 36.79% and 84.70%, respectively; the inhibition rates of 5, 10 and 50 μg / mL Lysimachia chinensis extract on AR were 12.59%, 26.80% and 6. 1.56%; 5, 10 and 50 μg / mL of the extract of Glehnia littoralis had an inhibitory rate of 31.96%, 61.07% and 86.04% respectively; 5, 10 and 50 μg / mL of the extract of Glehnia littoralis had an inhibitory rate of 25.81%, 42.68% and 77.20% respectively; 5, 10 and 50 μg / mL of quercetin had an inhibitory rate of 45.14%, 77.52% and 94.53% respectively. When the final concentration of the five Glehnia littoralis plant extracts was 50 μg / mL, the inhibitory rate of AR exceeded 50%, which was comparable to quercetin (IC 50 The value was 5.28±0.09μg / mL), compared with the extract of Psoralea corylifolia (IC 50 The value was 8.35±0.40μg / mL), Guangxi Herba Lysimachiae extract (IC 50 The value was 12.01±0.87μg / mL), Zhejiang radix scutellariae extract (IC 50 The value was 13.10±0.17μg / mL), and the extract of Radix Glehniae Padangensis (IC 50 value was 13.17±0.97μg / mL) and Lysimachia chinensis extract (IC 50 The value was 31.15±1.11μg / mL) and all showed AR inhibitory activity, and showed a dose-dependent relationship in which the inhibitory effect increased with the increase of extract concentration. Among the five extracts of Rhizoma Corydalis, the AR inhibitory activity of Rhizoma Corydalis extract was significantly better than that of other Rhizoma Corydalis extracts (p<0.05), the AR inhibitory activity of Lysimachia chinensis extract was significantly weaker than that of other Rhizoma Corydalis extracts (p<0.05), and there was no significant difference in the AR inhibitory activity of Rhizoma Corydalis Guangxi, Rhizoma Corydalis Zhejiang, and Rhizoma Corydalis Badong.

[0141] (14.5) Analysis of extracts from Rhizoma Cyperi plants in positive and negative ion modes

[0142] The total ion current chromatogram is a graph obtained by summing up the intensities of all ions in the mass spectrum at each time point and plotting them continuously, reflecting the overall information of the sample. By comparing and observing the total ion chromatograms of the extracts of the Rhizoma Corydalis family, we can intuitively see that there are differences in the metabolic components of the extracts of the Rhizoma Corydalis family, and the degree of difference between different extracts is also different ( Figure 5). In positive ion mode, the molecular network map of the extract of Rhizoma Cyperi is as follows Figure 6 As shown in the figure, it is composed of 21 components, including flavonoids, triterpenes, alkaloids, coumarins, phenolic acids, phenylpropanoids, small peptides, lignans, monoterpenes, sugars, organic acids and their derivatives, organic oxygen compounds, phenols, fatty acids, etc. Figure 7 As shown in Table 4, the metabolites of the extracts of Rhizoma Corydalis in negative ion mode were divided into 16 categories, including flavonoids, triterpenes, alkaloids, coumarins, lignins, fatty acids, etc. In positive and negative ion modes, a total of 206 compounds were identified in the metabolites of the extracts of Rhizoma Corydalis through GNPS database matching. The detailed information such as the name, molecular formula, relative molecular mass, and source of the compounds are shown in Table 4.

[0143] Table 4 206 compounds identified from five Rhizoma Cyperi plants extracts based on GNPS platform

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156] Principal Component Analysis:

[0157] By analyzing the sources of the metabolites in the extracts of the five Rhizoma Corydalis plants, it was found that the types of metabolites in the extracts were relatively small, and most of the identified compounds were widely present in the five Rhizoma Corydalis plants. However, the differences in the metabolic components between plants of similar genera are closely related to activity. Therefore, it is of great significance to further clarify the marker compounds in the extracts of the five Rhizoma Corydalis plants through multivariate statistical analysis methods to explore the components that exert anti-DR activity. The results of the principal component analysis are shown in the figure. Figure 8 As shown. In both positive and negative ion modes, the quality control (QC) and five Rhizoma Passerinae plant extract samples clustered with each other, and the QC was almost located at the center of the PCA graph, indicating that the sample analysis was reproducible and that the distances between different extract samples were caused by differences in metabolic components, and had nothing to do with the influence of the instrument system and the environment. The five Rhizoma Passerinae plant extract samples were relatively discrete from each other, again indicating that there were differences in the metabolic components of the five Rhizoma Passerinae plant extracts. From the analysis of the degree of dispersion, it can be seen that the metabolic components of the extracts of Lysimachia chinensis, Rhizoma Passerinae Badong, and Rhizoma Passerinae Guangxi were quite different from those of other samples, while the extracts of Rhizoma Passerinae indicum and Rhizoma Passerinae Zhejiang were less different from each other.

[0158] OPLS-DA statistical model analysis:

[0159] OPLS-DA and S-plot were used to perform differential discriminant analysis on the metabolic components of the five Rhizoma Corydalis plants. The specific comparative discriminant models and parameters are shown in Table 5. Each model has a high ability to explain variation (R 2 The OPLS-DA model, parameters and number of selected marker compounds are shown in Table 2.

[0160] Table 5 OPLS-DA model, parameters and number of selected marker compounds

[0161]

[0162] Note: LAH: Guangxi Lysimachia extract; LCC: Zhejiang Lysimachia extract; LCH: Lysimachia chinensis extract; LHM: Lysimachia punctata extract; LPH: Lysimachia patula extract; Comp.: components, same as the following table.

[0163] The OPLS-DA analysis results of Guangxi Herba Lysimachiae extract and Lysimachia chinensis extract in positive and negative ion modes are shown in Figure 2. Fig. 9As shown in the figure, in the S-plot mode, the X-axis represents the contribution of variation and the Y-axis represents the correlation; each point represents a metabolite, and the closer the point is to the two ends of the "S", the greater the difference in contribution. Therefore, the points distributed in the upper right corner (1) and the lower left corner (-1) are often the compounds of focus, and they are the candidate marker compounds in the discriminant mode and are marked in red. Among them, the components identified by GNPS were screened as the marker compounds of the extracts of the Rhizoma Corydalis plants. The marker compounds identified in the extract of Rhizoma Corydalis in Guangxi are Hyperoside, Coumarin, Quinic acid, Chlorogenic acid, Myricitrin and Coumaroylquinic acid; the marker compounds identified in the extract of Lysimachia sibiricum are Aurantiamide acetate, Aurantiamide, Betaine, Pyroglutamic acid, alpha-Linolenicacid, Kaempferol-3-O-rutinoside and L-Phenylalanine.

[0164] The OPLS-DA analysis results of Zhejiang radix scutellariae extract and Lysimachia chinensis extract in positive and negative ion modes are shown in Figure 2. Fig.10 As shown in the data, the identified compounds in the extract of Zhejiang Polygonum multiflorum are Rutin, Hyperoside, Ixoside and Quercetin-3-O-rutinoside; the identified compounds in the extract of Lysimachia sibiricum are Aurantiamideacetate, Aurantiamide, Pyroglutamic acid, alpha-Linolenic acid, L-Phenylalanine and Pantothenic acid.

[0165] The OPLS-DA analysis results of Lysimachia chinensis extract and Lysimachia sphaerocephala extract in positive and negative ion modes are shown in Figure 2. Fig.11 As shown, the marker compounds identified in the extract of Lysimachia scabra are Aurantiamide acetate, Aurantiamide, alpha-Linolenic acid, Betaine, Pyroglutamic acid and Pantothenic acid; the marker compounds identified in the extract of Lysimachia scabra are Quercetin, Hyperoside, Chlorogenic acid, Quercitrin, Myricetin, Quinic acid and Myricitrin.

[0166] The OPLS-DA analysis results of Lysimachia chinensis extract and Rhizoma Cyperi padongensis extract in positive and negative ion modes are shown in Figure 2. Fig.12 As shown in the data, the marker compounds identified in the extract of Lysimachia scabra were alpha-Linolenic acid, Aurantiamide and Aurantiamide acetate; the marker compounds identified in the extract of Radix Glechomae Dahuricae were Rutin, Chlorogenic Acid, Quercetin 3-O-glucuronide, Proline, Hyperoside, Quercetin, Vitexin, Catechin, Quercetin-3-O-rutinoside and Quinic acid.

[0167] The OPLS-DA model was used to explore the identifier compounds that contributed significantly to the differences in the extracts of the five Rhizoma Dioscoreae plants, including 24 in the positive ion mode and 12 in the negative ion mode. The detailed information of the compounds is shown in Table 6.

[0168] Table 6 Identifier compounds of five Rhizoma Cyperi plants extracts

[0169]

[0170]

[0171] (14.6) Correlation analysis between marker compounds and activities in extracts of Rhizoma Corydalis plants

[0172] By analyzing the correlation between the marker compounds in the extracts of the Rhizoma Cuscutae and their activities, it was found that 11 compounds, including L-Phenylalanine, Isoleucine, Aurantiamide acetate, L-Proline, Quercitrin, Myricetin, Quercetin, Vicenin III, Kaempferol-3-O-rutinoside, Quercetin 3-O-glucuronide and Geniposide, were the active contributing components of the extracts of the Rhizoma Cuscutae in intervening in high glucose-induced retinal vascular damage in zebrafish; Hyperoside, Chlorogenic A total of 15 compounds, including chlorogenic acid, quercetin, quercitrin, myricetin, catechin, quinicacid, ixoside, quercetin-3-O-rutinoside, myricitrin, coumaroylquinic acid, rosmarinic acid, quercetin 3-O-glucuronide and geniposide, are active contributors to the inhibition of aldose reductase in extracts of the Rhizoma Dioscoreae family. The relationship between the 20 indicative compounds with anti-DR activity in the extracts of the Rhizoma Dioscoreae family and the sources and activities of the extracts is shown in Figure 2. Fig.13 shown.

[0173] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a plant extract of the family Rhizoma Cyperi in the preparation of a drug for preventing and treating diabetic retinopathy, characterized in that: The plants of the Rhizoma Passiflorae series include Rhizoma Passiflorae Guangxi, Rhizoma Passiflorae Zhejiang, Rhizoma Passiflorae Dot-Gland and Rhizoma Passiflorae Badong.

2. The use according to claim 1, characterized in that: The preparation method of the Rhizoma Corydalis plant extract comprises the steps of sequentially crushing, alcohol extraction and freeze-drying the Rhizoma Corydalis plants.

3. The use according to claim 2, characterized in that: The alcohol extraction includes a first alcohol extraction, a second alcohol extraction and a third alcohol extraction; The first alcohol extraction comprises the steps of mixing the powder of the radix lutea plant obtained after crushing with a methanol solution, performing extraction, and obtaining a first alcohol extract and a first alcohol extraction residue; The second alcohol extraction comprises the steps of mixing the first alcohol extraction residue and a methanol solution, performing extraction, and obtaining a second alcohol extract and a second residue; The third alcohol extraction comprises the steps of mixing the second alcohol extraction residue and a methanol solution, performing extraction, and obtaining a third alcohol extract and a third residue.

4. The use according to claim 3, characterized in that: When performing the first alcohol extraction, the solid-liquid ratio is 1 g:5 mL; And / or, when performing the first alcohol extraction, the volume percentage of methanol in the methanol solution is 50%.

5. The use according to claim 3, characterized in that: When performing the second alcohol extraction and the third alcohol extraction, the material-liquid ratio is 1g:1mL; And / or, during the second alcohol extraction and the third alcohol extraction, the volume percentage of methanol in the methanol solution is 50%.

6. A drug for preventing and treating diabetic retinopathy, characterized in that: The medicine includes extracts of plants from the Rhizoma Dioscoreae family; the Rhizoma Dioscoreae family plants include Rhizoma Dioscoreae Guangxi, Rhizoma Dioscoreae Zhejiang, Rhizoma Dioscoreae Indica and Rhizoma Dioscoreae Badong.

7. The drug according to claim 6, characterized in that The preparation method of the Rhizoma Corydalis plant extract comprises the steps of sequentially crushing, alcohol extraction and freeze-drying the Rhizoma Corydalis plants.

8. The drug according to claim 7, characterized in that The alcohol extraction includes a first alcohol extraction, a second alcohol extraction and a third alcohol extraction; The first alcohol extraction comprises the steps of mixing the powder of the radix lutea plant obtained after crushing with a methanol solution, performing extraction, and obtaining a first alcohol extract and a first alcohol extraction residue; The second alcohol extraction comprises the steps of mixing the first alcohol extraction residue and a methanol solution, performing extraction, and obtaining a second alcohol extract and a second residue; The third alcohol extraction comprises the steps of mixing the second alcohol extraction residue and a methanol solution, performing extraction, and obtaining a third alcohol extract and a third residue.

9. The drug according to claim 8, characterized in that When performing the first alcohol extraction, the solid-liquid ratio is 1 g:5 mL; And / or, when performing the first alcohol extraction, the volume percentage of methanol in the methanol solution is 70%.

10. The drug according to claim 8, characterized in that When performing the second alcohol extraction and the third alcohol extraction, the material-liquid ratio is 1g:1mL; And / or, during the second alcohol extraction and the third alcohol extraction, the volume percentage of methanol in the methanol solution is 50%.

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