A dryopteris laeta extract, a preparation method and application thereof

Abstract

The application relates to a Drynaria rhizome extract and a preparation method and application thereof, and the preparation method comprises the following steps: extracting Drynaria rhizome with ethanol with a volume fraction of 70-95% to obtain an ethanol extract; suspending the ethanol extract in water, extracting with petroleum ether, and concentrating to obtain a petroleum ether extract; and after the petroleum ether extract is further purified, concentrating and drying to obtain the product. The preparation method is simple in operation, can be industrialized, can realize effective extraction of effective components from the Drynaria rhizome, has high purity, and has wide application value.

Description

Technical Field

[0001] This application relates to the field of natural product chemistry technology, and more specifically, to an extract of *Dryopteris crassirhizoma*, its preparation method, and its application. Background Technology

[0002] Ferns are among the earliest terrestrial plants with a long history of medicinal use. The genus *Dryopteris* is a large genus of ferns in my country, rich in medicinal resources. It was first recorded in the Chinese classic *Shennong Bencao Jing* and later included in the pharmacopoeias of countries such as the UK, Germany, and Japan. Studies have reported that a homogeneous polysaccharide purified from the rhizome of *Dryopteris gracilis* inhibits key enzymes in lipid metabolism; other studies have found that different acyl-repinephrine monomers in this plant inhibit fatty acid synthase activity to varying degrees. Furthermore, our team previously discovered that repinephrine compounds isolated from *Dryopteris macrocarpa* can inhibit the expression of genes related to fatty acid uptake, thus reducing lipid levels in cells.

[0003] *Dryopteris atrata*, a plant belonging to the genus *Dryopteris* in the family Dryopteridaceae, is found in provinces south of the Yangtze River in my country, extending east to Taiwan, north to Gansu, and southwest to Tibet. It is also distributed in India and Myanmar. Its rhizome is used medicinally; it is cold in nature and bitter in taste. The *Xinhua Materia Medica* records its main functions as treating dysfunctional uterine bleeding and expelling roundworms. Therefore, its primary application in current technology is the treatment of dysfunctional uterine bleeding, and no other applications have been reported.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The primary objective of this application is to provide a method for preparing extracts of *Dryopteris fasciatus*, which is simple to operate, industrially feasible, and can effectively extract active ingredients from *Dryopteris fasciatus* with high purity and wide application value.

[0006] The second objective of this application is to provide an extract of *Dryopteris fasciatus* prepared by the above-described method, which has high purity and broad medicinal value.

[0007] The third objective of this application is to provide the application of the above-mentioned extract of *Dryopteris crassirhizoma*, which has been found through extensive practice to have excellent application in the preparation of drugs with lipid-lowering effects.

[0008] To achieve the above objectives, the present invention provides a method for preparing an extract of *Dryopteris crassirhizoma*, comprising the following steps: The ethanol extract was obtained by extracting the dark-scaled hairy fern with 70% to 95% ethanol by volume. The ethanol extract was then suspended in water and extracted and concentrated with petroleum ether to obtain a petroleum ether extract. The petroleum ether extract was further purified, concentrated, and dried to obtain the final product.

[0009] Preferably, the volume fraction of ethanol is 95%.

[0010] Preferably, the extraction is performed 1 to 6 times.

[0011] More preferably, the extraction is performed 3 to 5 times.

[0012] Preferably, as a further feasible option, the ratio of ethanol to *Dryopteris erythropterus* is (10-20 L): 1 kg.

[0013] Preferably, as a further feasible option, the ratio of the ethanol extract to water is (0.1-1L):1kg, and more preferably the ratio is 0.3L:1kg.

[0014] Preferably, as a further feasible option, the volume ratio of the petroleum ether to the suspension formed by the ethanol extract and water is (3-10):1, more preferably 6:1.

[0015] Preferably, as a further feasible option, the step of further purifying the petroleum ether extract includes: first eluting with a silica gel column, concentrating and drying to obtain a first eluted component, then eluting the first eluted component with a gel column, concentrating and drying to obtain a second eluted component, eluting the second eluted component with an ODS column to obtain a third eluted component, and finally concentrating and drying to obtain the extract of *Dryopteris tarda*.

[0016] Preferably, as a further feasible option, the step of eluting with silica gel column, concentrating and drying to obtain the first eluent component includes: first eluting with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 100:(0-5) at a ratio of 5-15 column volumes; then eluting with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 100:(10-50) at a ratio of 5-15 column volumes, collecting the eluent, concentrating and drying.

[0017] More preferably, the extract of *Dryopteris tarda* is loaded onto a silica gel column and eluted first with 10 column volumes of a mixed solvent consisting of petroleum ether and ethyl acetate in a volume ratio of 100:5; then eluted again with 10 column volumes of a mixed solvent consisting of petroleum ether and ethyl acetate in a volume ratio of 100:20, and the eluted fraction is collected, concentrated, and dried to obtain the effective fraction of *Dryopteris tarda* obtained by silica gel column chromatography. Through continuous purification using silica gel column chromatography, the lipid-lowering effect of the purified substance is further significantly enhanced.

[0018] Preferably, as a further feasible option, the step of eluting the first eluent component by gel column elution, concentration and drying to obtain the second eluent component includes: eluting the first eluent component with a mixed solvent of chloroform and methanol in a volume ratio of (1~3):1, collecting the eluent of 3 to 10 column volumes, and concentrating and drying it.

[0019] More preferably, the effective fraction of *Dryopteris tarda* is loaded onto a gel column for silica gel column chromatography, eluted with a mixed solvent of chloroform and methanol in a volume ratio of 1:1, and the eluent of the 5th to 7th column volumes is collected, concentrated and dried.

[0020] The preferred choice for the above-mentioned gel column is cross-linked dextran gel LH-20.

[0021] Preferably, as a further feasible option, the step of eluting the second eluent component to obtain the third eluent component by using an ODS column includes: first eluting with a mixed solvent of methanol and water in a volume ratio of (80:40) to (75:25) of 10 to 30 column volumes; and then eluting with a mixed solvent of methanol and water in a volume ratio of (90:20) to (90:10) of 10 to 30 column volumes.

[0022] More preferably, the effective fraction of *Dryopteris tarda* gel column chromatography is loaded onto an ODS column, and eluted first with a mixed solvent system of methanol and water in a volume ratio of 70:30 (20 column volumes); then eluted again with a mixed solvent system of methanol and water in a volume ratio of 85:15 (20 column volumes), and the eluted fraction is collected, concentrated, and dried to obtain the effective fraction of *Dryopteris tarda* ODS column chromatography.

[0023] Preferably, as a further feasible option, the volume ratio of the petroleum ether to the suspension formed by the ethanol extract and water is (3-10):1.

[0024] The preparation method of this invention involves extraction followed by purification through a series of chromatographic methods. The resulting extract of *Dryopteris crassirhizoma* exhibits the effects of reducing intracellular lipid levels in macrophages and inhibiting aortic plaque formation in APOE- / - atherosclerotic model mice. Therefore, its use as an active ingredient in the preparation of drugs with lipid-lowering effects has significant application value. The present invention also provides an extract of *Dryopteris crassirhizoma* prepared by the above preparation method, which has strong application potential in the preparation of lipid-lowering drugs.

[0025] As is well known, atherosclerosis (AS), as the main pathological basis for disability and death in cardiovascular disease (CVD), has become a major global health threat. It is essentially a chronic, progressive vascular disease driven by lipid infiltration, inflammatory immune responses, and fibrosis. In its complex pathogenesis, abnormal lipid metabolism is considered to play a key pathogenic role in the pathological process of AS. Therefore, statins, which exert lipid-lowering effects by inhibiting 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), are considered the first-line drugs for treating AS. First-generation statins, such as lovastatin, are considered the "prototype" of this class of drugs, initially isolated from the fermentation products of Aspergillus terreus and Monascus purpureus. Therefore, finding new anti-AS drugs based on natural products remains a potentially promising exploratory strategy. The extract of *Dryopteris tarda* prepared by the present invention fills the aforementioned technological gap.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention is the first to discover that the ethanol extract of *Tetracentron sinense* has a certain effect in reducing cellular lipid levels. Further research revealed that the *Tetracentron sinense* extract, the effective fraction obtained by silica gel column chromatography, the effective fraction obtained by gel column chromatography, and the effective fraction obtained by ODS column chromatography prepared according to the method of this invention exhibit progressively and significantly improved effects in reducing cellular lipid levels; in particular, the *Tetracentron sinense* extract obtained by further elution via ODS column chromatography has an excellent effect in reducing cellular lipid levels. Since the ethanol extract, extract, effective fraction obtained by silica gel column chromatography, effective fraction obtained by gel column chromatography, and the extract obtained by the method of this invention all have the effect of reducing cellular lipid levels, they have significant application value as active ingredients in the preparation of drugs with lipid-lowering effects.

[0027] Other features and advantages of this application will be described in detail in the following detailed description section. Attached Figure Description

[0028] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This illustrates the effects of different extracts from Experiment 1 of this invention on the levels of TC and TG in macrophages of each group. Figure 2 This is a schematic diagram of the results of Oil Red O staining of the aorta in different groups of APOE- / - mice in Experiment Example 2 of the present invention. Detailed Implementation

[0030] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are only some embodiments of the present invention, not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0031] Example 1: Preparation of extract from *Dryopteris tarda* (1) The dark-scaled hairy fern was extracted by immersion in 95% ethanol for 3 days. After the extraction, the extract was concentrated and dried to obtain the dark-scaled hairy fern ethanol extract. The ratio of ethanol to dark-scaled hairy fern was 10 L: 1 Kg. (2) The ethanol extract of *Dryopteris fasciatus* obtained in step (1) is suspended in water, with a water-to-extract ratio of 0.3 L:1 kg. Extraction is performed with petroleum ether at a volume 5 times that of the suspension, with a volume ratio of petroleum ether to the suspension formed by the ethanol extract and water of 6:1. The petroleum ether layer extract is collected and concentrated. The extraction and recovery steps are repeated 4 times, and the extracts are combined to obtain *Dryopteris fasciatus* extract. (3) The extract of *Dryopteris fasciatus* obtained in step (2) was loaded onto a silica gel column (the silica gel column was filled with 300 mesh silica gel). First, it was eluted with a mixed solvent system of petroleum ether and ethyl acetate with a volume ratio of 100:5 (10 column volumes) to remove impurities. Then, it was eluted with a mixed solvent system of petroleum ether and ethyl acetate with a volume ratio of 100:20 (10 column volumes) and the eluted fraction was collected. After concentration and drying, the effective fraction of *Dryopteris fasciatus* obtained by silica gel column chromatography was obtained. (4) The effective fraction of *Dryopteris tarda* obtained in step (3) was loaded onto a gel column (cross-linked dextran gel LH-20) and eluted with a mixed solvent system of chloroform and methanol in a volume ratio of 1:1. The eluent was collected in the 5th to 7th column volumes, concentrated and dried to obtain the *Dryopteris tarda* extract.

[0032] (5) The effective fraction of *Dryopteris tarda* obtained in step (4) is loaded onto an ODS column and eluted first with a mixed solvent system of methanol and water with a volume ratio of 70:30 and 20 column volumes; then eluted again with a mixed solvent system of methanol and water with a volume ratio of 85:15 and 20 column volumes, and the eluted fraction is collected, concentrated and dried to obtain the extract of *Dryopteris tarda*.

[0033] Example 2 (1) The dark-scaled hairy fern was extracted by maceration with 70% ethanol for 3 days. After the extraction, the extract was concentrated and dried to obtain the dark-scaled hairy fern ethanol extract. The ratio of ethanol to dark-scaled hairy fern was 20L:1Kg. (2) The ethanol extract of *Dryopteris fasciatus* obtained in step (1) is suspended in water at a ratio of 1 L to 1 kg. Extraction is performed with petroleum ether at a volume 5 times that of the suspension, with a volume ratio of petroleum ether to the suspension formed by the ethanol extract and water of 10:1. The petroleum ether layer extract is collected and concentrated. The extraction and recovery steps are repeated 3 times, and the extracts are combined to obtain *Dryopteris fasciatus* extract. (3) The extract of *Dryopteris fasciatus* obtained in step (2) was loaded onto a silica gel column (the silica gel column was filled with 300 mesh silica gel). First, it was eluted with a mixed solvent system of petroleum ether and ethyl acetate with a volume ratio of 100:2 (5 column volumes) to remove impurities. Then, it was eluted with a mixed solvent system of petroleum ether and ethyl acetate with a volume ratio of 100:10 (15 column volumes) and the eluted fraction was collected. After concentration and drying, the effective fraction of *Dryopteris fasciatus* obtained by silica gel column chromatography was obtained. (4) The effective fraction of *Dryopteris fasciatus* obtained in step (3) was loaded onto a gel column (cross-linked dextran gel LH-20) and eluted with a mixed solvent system of chloroform and methanol in a volume ratio of 2:1. The eluent was collected in the 5th to 7th column volumes, concentrated and dried to obtain the *Dryopteris fasciatus* extract.

[0034] (5) The effective fraction of *Dryopteris tarda* obtained in step (4) is loaded onto an ODS column and eluted first with a mixed solvent system of methanol and water with a volume ratio of 40:75 and 10 column volumes; then eluted again with a mixed solvent system of methanol and water with a volume ratio of 90:10 and 30 column volumes and the eluted fraction is collected, concentrated and dried to obtain the extract of *Dryopteris tarda*.

[0035] Example 3 (1) The dark-scaled hairy fern was extracted by immersion in 80% ethanol for 3 days. After the extraction, the extract was concentrated and dried to obtain the dark-scaled hairy fern ethanol extract. The ratio of ethanol to dark-scaled hairy fern was 15L:1Kg. (2) The ethanol extract of *Dryopteris fasciatus* obtained in step (1) is suspended in water, with a water-to-extract ratio of 0.1 L:1 kg. Extraction is performed with petroleum ether at a volume 5 times that of the suspension, with a volume ratio of petroleum ether to the suspension formed by the ethanol extract and water of 3:1. The petroleum ether layer extract is collected and concentrated. The extraction and recovery steps are repeated 5 times, and the extracts are combined to obtain *Dryopteris fasciatus* extract. (3) The extract of *Dryopteris fasciatus* obtained in step (2) was loaded onto a silica gel column (the silica gel column was filled with 300 mesh silica gel). First, it was eluted with 15 column volumes of petroleum ether solvent to remove impurities. Then, it was eluted with 5 column volumes of a mixed solvent system consisting of petroleum ether and ethyl acetate in a volume ratio of 100:50 and the eluted fraction was collected. After concentration and drying, the effective fraction of *Dryopteris fasciatus* obtained by silica gel column chromatography was obtained. (4) The effective fraction of *Dryopteris tarda* obtained in step (3) was loaded onto a gel column (cross-linked dextran gel LH-20) and eluted with a mixed solvent system of chloroform and methanol in a volume ratio of 3:1. The eluent was collected in the 5th to 7th column volumes, concentrated and dried to obtain the *Dryopteris tarda* extract.

[0036] (5) The effective fraction of *Dryopteris tarda* obtained in step (4) is loaded onto an ODS column. First, it is eluted with a mixed solvent system of methanol and water with a volume ratio of 80:25 and a column volume of 30 times. Then, it is eluted with a mixed solvent system of methanol and water with a volume ratio of 20:90 and a column volume of 10 times. The eluted fraction is collected, concentrated and dried to obtain the *Dryopteris tarda* extract.

[0037] Experiment Example 1: Activity Test for Reducing Intracellular Lipid Levels The following samples were tested: the extract of *Dryopteris fasciatus* prepared according to the method of Example 1 (experimental group 1), the ethanol extract of *Dryopteris fasciatus* prepared according to step (1) of Example 1 (experimental group 2), the extract of *Dryopteris fasciatus* prepared according to step (2) of Example 1 (experimental group 3), the effective fraction of *Dryopteris fasciatus* prepared according to step (3) of Example 1 (experimental group 4), and the effective fraction of *Dryopteris fasciatus* prepared according to step (4) of Example 1 (experimental group 5).

[0038] Experimental Methods: RAW264.7 cells in logarithmic growth phase were seeded in 12-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. The experiment consisted of a control group, a model group (ox-LDL), and an extract group. Different extracts from each group were added to the extract groups, while the other groups received an equal volume of culture medium. After 24 h of incubation, the original culture medium was discarded, and except for the control group, each well was added to culture medium containing 50 μg / mL ox-LDL for another 24 h. Cells from each group were collected and homogenized by sonication under ice bath conditions. The total cholesterol (TC) and triglyceride (TG) levels in the cell homogenates were measured using a commercially available kit (enzymatic / colorimetric method) following the manufacturer's instructions.

[0039] like Figure 1 As shown, by Figure 1 Data on intracellular TC and TG levels after treatment with different groups of extracts showed that the ethanol extract of *Dryopteris glomeratus* obtained by ethanol extraction exhibited a certain effect in reducing intracellular lipid levels. The lipid-reducing effect of the extract obtained by extracting the ethanol extract of *Dryopteris glomeratus* with petroleum ether was significantly enhanced. Furthermore, the lipid-reducing effect of the effective fraction of *Dryopteris glomeratus* obtained by eluting the extract under the silica gel column elution conditions described in this method was further significantly enhanced. In particular, the extract of *Dryopteris glomeratus* obtained by eluting the effective fraction of *Dryopteris glomeratus* by gel column elution under the ODS column elution conditions described in this invention exhibited excellent lipid-reducing effects, which were further significantly enhanced.

[0040] Experimental Example 2: Test of Anti-atherosclerotic Effect Experimental methods: Six-week-old male C57BL / 6 wild-type males were purchased (as a control, fed with standard feed) and ApoE. – / – Mice were fed a high-fat diet with ApoE. – / – Mice were fed until 12 weeks of age, and then ApoE mice were randomly sacrificed. – / – In mice, the formation of aortic plaques was observed to determine if the model was successful; other ApoE... – / –Mice were randomly divided into 3 groups: (1) ApoE - / - + Extract group (DA): The extract obtained in Example 1 was dissolved in 1% DMSO and phosphate buffer solution and administered intraperitoneally at a dose of 50 mg / kg / d; (2) ApoE - / - Group + Atorvastatin Calcium (AC): Daily intraperitoneal injection of atorvastatin calcium (Lipitor, 20 mg / kg); (3) ApoE - / - Group (Model): Mice were administered an equal volume of solvent via intraperitoneal injection daily. After 12 weeks of continuous drug administration, mice were sacrificed, and the aortic root was harvested for Oil Red O staining. Figure 2 The results of Oil Red O staining of the aorta in different groups of AS model mice showed that the extract obtained in Example 1 had a very good effect in preventing the formation of aortic plaques in mice.

[0041] When the extracts of *Dryopteris crassirhizoma* obtained in other Examples 2-3 were used to conduct the experiments in Examples 1-2, the same experimental results were achieved, proving that the extracts of *Dryopteris crassirhizoma* of the present invention have significant application value in the preparation of lipid-lowering drugs.

[0042] It should be noted that, where there is no conflict, the features in the embodiments of this application can be combined with each other.

[0043] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing an extract of *Dryopteris crassirhizoma*, characterized in that, Includes the following steps: The ethanol extract was obtained by extracting the dark-scaled hairy fern with 70% to 95% ethanol by volume. The ethanol extract was then suspended in water and extracted and concentrated with petroleum ether to obtain a petroleum ether extract. The petroleum ether extract was further purified, concentrated, and dried to obtain the final product.

2. The method for preparing the extract of *Dryopteris crassirhizoma* according to claim 1, characterized in that, The ratio of ethanol to *Dryopteris erythropterus* was (10–20 L): 1 kg.

3. The method for preparing the extract of *Dryopteris crassirhizoma* according to claim 1, characterized in that, The ratio of the ethanol extract to water is (0.1-1L):1 kg.

4. The method for preparing the extract of *Dryopteris crassirhizoma* according to claim 1, characterized in that, The steps for further purifying the petroleum ether extract include: first eluting with a silica gel column, concentrating and drying to obtain a first eluted component; then eluting the first eluted component with a gel column, concentrating and drying to obtain a second eluted component; eluting the second eluted component with an ODS column to obtain a third eluted component; and finally concentrating and drying to obtain the extract of *Dryopteris tarda*.

5. The method for preparing the extract of *Dryopteris crassirhizoma* according to claim 4, characterized in that, The steps of eluting with silica gel column, concentrating and drying to obtain the first eluent component include: first eluting with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 100:(0-5) at a ratio of 5-15 column volumes; then eluting with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 100:(10-50) at a ratio of 5-15 column volumes, collecting the eluent, concentrating and drying.

6. The method for preparing the extract of *Dryopteris crassirhizoma* according to claim 4, characterized in that, The step of eluting the first eluent using a gel column, concentrating and drying to obtain the second eluent includes: eluting the first eluent with a mixed solvent of chloroform and methanol in a volume ratio of (1~3):1, collecting the eluent in volumes 3 to 10, and concentrating and drying it.

7. The method for preparing the extract of *Dryopteris crassirhizoma* according to claim 4, characterized in that, The steps for eluting the second eluent component to obtain the third eluent component using an ODS column include: first eluting with a mixed solvent of methanol and water in a volume ratio of (80:40) to (75:25) of 10 to 30 column volumes; and then eluting with a mixed solvent of methanol and water in a volume ratio of (90:20) to (90:10) of 10 to 30 column volumes.

8. The method for preparing the extract of *Dryopteris crassirhizoma* according to claim 1, characterized in that, The volume ratio of the suspension formed by the petroleum ether and the ethanol extract with water is (3-10):

1.

9. The *Dryopteris tarda* extract prepared by the method according to any one of claims 1-8.

10. The use of the *Dryopteris fasciatus* extract prepared by the method of any one of claims 1-8 and the *Dryopteris fasciatus* extract of claim 9 in the preparation of lipid-lowering drugs.

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