Dianthus superbus flavone carbon glycosides, and a method for preparing and using the same

Abstract

The application discloses a flavonoid carbon glycoside compound, a directional separation and preparation method and application thereof. The flavonoid carbon glycoside compound is first extracted and discovered, and is used for preparing a medicine for treating or improving diabetes. The preparation process comprises the following steps: extraction, microporous resin medium pressure chromatography rough separation, reverse phase medium pressure chromatography enrichment, reverse phase preparation column preparation, Fr411 reverse phase preparation liquid chromatography purification, and Fr412 hydrophilic preparation liquid chromatography purification. The flavonoid carbon glycoside compound separated by the application can effectively inhibit alpha-glucosidase activity, has low cost, and has a product purity greater than 95%. The technical means adopted by the application can be used for large-scale production, and is easy to scale. The reverse phase preparation liquid chromatography or the hydrophilic preparation liquid chromatography used in the separation and purification is a rapid isocratic method.

Description

Technical Field

[0001] This invention relates to the field of natural medicinal chemistry, specifically to a class of flavonoid C-glycosides from Dianthus superbus, their directed separation and preparation methods, and their applications. More specifically, it relates to a class of flavonoid C-glycosides containing a novel compound, along with its directed separation and preparation method and its novel application in inhibiting α-glucosidase activity, for use in the preparation of drugs for treating or improving diabetes. Technical Background

[0002] Diabetes is one of the fastest-growing global health emergencies of the 21st century, and among the hundreds of millions of people with diabetes worldwide, 85%–90% have type 2 diabetes (T2DM). T2DM is characterized by hyperglycemia; excessive intake of high-carbohydrate foods causes a rise in postprandial blood glucose, leading to T2DM. However, excessively high postprandial blood glucose not only leads to diabetes and other complications but also increases mortality. Therefore, controlling postprandial blood glucose is a primary way to improve diabetes and can reduce mortality in diabetic patients. Currently, methods for controlling postprandial hyperglycemia mainly involve inhibiting the activity of α-glucosidase and α-amylase, thereby reducing their conversion of sugar into absorbable monosaccharides. Therefore, finding and developing natural active substances with low toxicity that can effectively inhibit α-glucosidase activity will play a positive role in the treatment or improvement of diabetes.

[0003] Dianthus superbus L., a perennial herb belonging to the Caryophyllaceae family and the Dianthus genus, is recorded in the 2015 edition of the Chinese Pharmacopoeia. It possesses diuretic and blood-activating properties, and is used for conditions such as urinary tract infections, hematuria, urinary stones, difficulty urinating, painful urination, and amenorrhea due to blood stasis. Dianthus superbus contains various chemical components including saponins, cyclic peptides, flavonoids, polyphenols, and alkaloids. Modern pharmacological studies have shown that it has various effects, including antibacterial, nephroprotective, anti-early pregnancy, anti-tumor, immunosuppressive, and neuroprotective effects. However, no flavonoid C-glycosides have been isolated, purified, and identified from Dianthus superbus, and there are no literature reports on its inhibitory effects on α-glucosidase activity and blood glucose. Therefore, there is an urgent need to establish a simple method for the large-scale preparation of flavonoid C-glycosides from Dianthus superbus and to determine their α-glucosidase inhibitory activity. Summary of the Invention

[0004] Based on the above problems, the purpose of this invention is to provide a dianthus flavonoid C-glycoside compound, a method for its directional separation and preparation, and its application. Specifically, it provides a dianthus flavonoid C-glycoside compound with α-glucosidase activity, a method for its directional separation and preparation, and its application in preparing a blood glucose inhibitor, as well as its application in preparing a drug for treating or improving diabetes.

[0005] This is the first extraction and discovery of a flavonoid C-glycoside compound from Dianthus superbus, and its structural formula is shown below:

[0006]

[0007] The application of Dianthus flavonoid C-glycosides in the preparation of α-glucosidase inhibitors, the structural formula of which is shown below:

[0008]

[0009] The above-mentioned Dianthus flavonoid C-glycosides are used in the preparation of blood glucose-inhibiting drugs, or in the preparation of drugs for treating or improving diabetes, and similar drugs.

[0010] A method for the directional separation and preparation of flavonoid C-glycosides from Dianthus superbus includes the following steps:

[0011] Step 1, Extraction: Dry the whole Dianthus superbus in the shade, coarsely crush it, and extract it with ethanol at a ratio of 1g:5-100mL. Extract 2-4 times at room temperature, each time for 2-4 hours. Filter and combine the filtrates, which is filtrate A. Mix filtrate A with silica gel at a ratio of 1:5-15 and dry under reduced pressure to obtain the Dianthus superbus extract sample.

[0012] Step 2, coarse separation by medium-pressure chromatography with microporous resin: The sample of Dianthus chinensis extract was separated by medium-pressure chromatography with microporous resin. The sample was detected by a UV detector with a wavelength of 210 nm. Six chromatographic peak fractions were collected and dried under reduced pressure and labeled as Dianthus chinensis components Fr1, Fr2, Fr3, Fr4, Fr5 and Fr6.

[0013] Step 3, reversed-phase medium-pressure chromatography enrichment: The target component Fr4 is mixed with silica gel to obtain a sample. The sample is separated by a reversed-phase medium-pressure chromatography column packed with silica gel matrix material and detected by an ultraviolet detector with a detection wavelength of 210 nm. The fraction of the first chromatographic peak in the preparation chromatogram is collected. The fraction is dried under reduced pressure to obtain component Fr41 containing the target component.

[0014] Step 4, reversed-phase preparation column preparation: The component Fr41 containing the target component is dissolved in a methanol-water solution with a volume fraction of 50-100% to prepare a sample concentration of 20.0-50.0 mg / mL. The solution is filtered through a 0.45 μm microporous membrane to obtain filtrate B. Filtrate B is prepared by reversed-phase preparation column and detected by a UV detector with a detection wavelength of 210 nm. The two chromatographic peak fractions in the reversed-phase preparation chromatogram of filtrate B are collected. The chromatographic peak fractions are dried under reduced pressure to obtain components Fr411 and Fr412 containing the target component.

[0015] Step 5, reversed-phase preparative liquid chromatography purification of Fr411: The component Fr411 containing the target component was dissolved in a 100% methanol solution to prepare a sample concentration of 25.0 mg / mL. The solution was filtered through a 0.45 μm microporous membrane to obtain the filtrate, i.e., filtrate C. Filtrate C was purified by reversed-phase high-performance liquid chromatography and detected by a UV detector with a detection wavelength of 210 nm. The chromatographic peak fraction Fr4111 in the reversed-phase high-performance preparative chromatogram of filtrate C was collected. This chromatographic peak fraction was dried under reduced pressure to obtain 2”-O-rhamnosyllutonarin with a purity greater than 95%.

[0016] Step 6, hydrophilic preparative high-performance liquid chromatography (HPLC) purification of Fr412: The component Fr412 containing the target component was dissolved in a 100% methanol solution to prepare a sample concentration of 25.0 mg / mL. The solution was filtered through a 0.45 μm microporous membrane to obtain filtrate, i.e., filtrate D. Filtrate D was purified by hydrophilic high-performance liquid chromatography (HPLC) and detected by a UV detector with a detection wavelength of 210 nm. The corresponding chromatographic peak fractions Fr4121, Fr4122, and Fr4123 in the hydrophilic preparative chromatogram of filtrate D were collected. The chromatographic peak fractions Fr4121, Fr4122, and Fr4123 were dried under reduced pressure to obtain Luteolin 6-C-glucoside-7-O-glucoside, Luteolin 6-C-(6”-O-β-D-glucoside)-glucoside, and 6”'-O-rhamnosyllutonarin with purities greater than 95%, respectively.

[0017] Furthermore, in steps 1, 2, 3, 4, 5, and 6, the conditions for vacuum drying are: vacuum degree 50–250 mbar and temperature 40–60 °C.

[0018] Furthermore, in step 2, the separation parameters for the crude fraction obtained by medium-pressure chromatography using microporous resin are as follows: column length 460 mm, diameter 49 mm, stationary phase of microporous resin column is HP20SS, mobile phase A is water, B is methanol, C is dichloromethane, chromatographic conditions are 0–20 min, 0% B, 20–240 min, 0%–100% B, 240–300 min, 100% B, 300–420 min, 0–100% C, injection volume is 10–80 g, and flow rate is 40–60 mL / min;

[0019] Furthermore, in step 3, the operating parameters for reversed-phase medium-pressure chromatography enrichment are as follows: column length 500 mm, diameter 50 mm, reversed-phase preparative column stationary phase is 50 μm Spherical C18, mobile phase A is water, mobile phase B is methanol, chromatographic conditions are 0–120 min, 20%–65% B, injection volume is 10–30 g, and flow rate is 40–60 mL / min.

[0020] Furthermore, in step 4, the working parameters for preparing the reversed-phase preparation column are as follows: the column size is 250×20mm, the stationary phase of the reversed-phase preparation column is a 7μm reversed-phase column 7-X10, and the mobile phase is a 6% methanol-water solution.

[0021] Furthermore, in step 5, the working parameters for reversed-phase preparative liquid chromatography purification are: column length 250 mm, diameter 20 mm, stationary phase of reversed-phase preparative column is 5 μm reversed-phase column SunFire C18, and mobile phase is 20% methanol-water solution;

[0022] Furthermore, in step 6, the operating parameters for hydrophilic preparative liquid chromatography purification are as follows: column size is 250×20mm, stationary phase is 5μm zwitterionic ClickXION column, and mobile phase is 90% acetonitrile-5% trifluoroacetic acid aqueous solution.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] (1) This invention has low cost and high product purity. The extraction solvents used, as well as the solvents used in the microporous resin medium-pressure chromatography crude separation, reversed-phase medium-pressure chromatography enrichment, reversed-phase, and hydrophilic chromatography purification, can all be recycled and reused; the chromatographic separation materials used (microporous resin, reversed-phase preparative liquid chromatography, and hydrophilic preparative liquid chromatography separation materials) can all be reused; the recycled solvents and reused separation materials ensure that the average cost of the separation process is relatively low, and the two-step medium-pressure separation (microporous resin medium-pressure chromatography crude separation and reversed-phase medium-pressure chromatography enrichment) and high-pressure liquid chromatography purification can ensure that the purity of the product is greater than 95%.

[0025] (2) The preparation method of the present invention can meet the needs of large-scale production. The raw materials are not demanding, the cost is low, and they are very easy to obtain. Generally cultivated or commercially available Dianthus superbus medicinal materials are sufficient, making it easy to prepare materials in batches. Ethanol extraction at room temperature is easy to operate. The separation uses a microporous resin column for coarse separation. This microporous resin separation material can be packed into a medium-pressure column chromatography system, making it easy to achieve large-scale production. The reversed-phase preparative liquid chromatography or hydrophilic preparative liquid chromatography used in the separation and purification are rapid isocratic methods, which are very suitable for large-scale production.

[0026] (3) The flavonoid C-glycosides from Dianthus superbus described in this invention are pure natural, plant-derived, non-toxic, and non-irritating substances that can significantly inhibit α-glucosidase activity. This invention provides the application of Dianthus superbus flavonoid C-glycosides in the preparation of drugs for treating or improving diabetes, and provides the application in the preparation of drugs that inhibit α-glucosidase. Attached Figure Description

[0027] Figure 1 This is a chromatogram of microporous resin separation of the sample of the present invention;

[0028] Figure 2 This is a reversed-phase medium-pressure chromatogram of the microporous resin separation component Fr4 of the present invention.

[0029] Figure 3 This is a reversed-phase preparative liquid chromatography (RP-LC) separation diagram of the target component Fr41 from Dianthus superbus in this invention.

[0030] Figure 4 This is a reversed-phase preparative liquid chromatography (RP-LC) separation diagram of the target component Fr411 from Dianthus superbus in this invention.

[0031] Figure 5 This is a liquid chromatography separation diagram of the hydrophilic preparation of the target component Fr412 from Dianthus superbus in this invention;

[0032] Figure 6 Chromatograms for purity verification of the flavonoid C-glycosides Fr4111 (2”-O-rhamnosyllutonarin), Fr4121 (Luteolin 6-C-glucoside-7-O-glucoside), Fr4122 (Luteolin 6-C-(6”-O-β-D-glucoside)-glucoside), and Fr4123 (6”'-O-rhamnosyllutonarin) from Dianthus superbus in this invention;

[0033] Figure 7 This is the high-resolution mass spectrum of 2”-O-rhamnosyllutonarin obtained by the present invention;

[0034] Figure 8 2”-O-rhamnosyllutonarin obtained by the present invention 1 HNMR MRI;

[0035] Figure 9 2”-O-rhamnosyllutonarin obtained by the present invention 13 C10 NMR MRI;

[0036] Figure 10The infrared spectrum of 2”-O-rhamnosyllutonarin obtained by the present invention;

[0037] Figure 11 The UV spectrum of 2”-O-rhamnosyllutonarin obtained by the present invention;

[0038] Figure 12 This is a low-resolution mass spectrum of Luteolin 6-C-glucoside-7-O-glucoside obtained in this invention.

[0039] Figure 13 Luteolin 6-C-glucoside-7-O-glucoside isolated in this invention 1 H NMR MRI;

[0040] Figure 14 Luteolin 6-C-glucoside-7-O-glucoside isolated in this invention 13 C10 NMR MRI;

[0041] Figure 15 The infrared spectrum of Luteolin 6-C-glucoside-7-O-glucoside obtained by the present invention is shown below.

[0042] Figure 16 The image shows the ultraviolet spectrum of Luteolin 6-C-glucoside-7-O-glucoside obtained in this invention.

[0043] Figure 17 This is the low-resolution mass spectrum of Luteolin 6-C-(6”-O-β-D-glucoside)-glucoside obtained in this invention;

[0044] Figure 18 Luteolin 6-C-(6”-O-β-D-glucoside)-glucoside isolated in this invention 1 HNMR MRI;

[0045] Figure 19 Luteolin 6-C-(6”-O-β-D-glucoside)-glucoside isolated in this invention 13 CNMR (nuclear magnetic resonance) image;

[0046] Figure 20The infrared spectrum of Luteolin 6-C-(6”-O-β-D-glucoside)-glucoside obtained by the present invention;

[0047] Figure 21 The UV spectrum of Luteolin 6-C-(6”-O-β-D-glucoside)-glucoside obtained in this invention;

[0048] Figure 22 This is the low-resolution mass spectrum of 6”'-O-rhamnosyllutonarin obtained by the present invention;

[0049] Figure 23 The 6”'-O-rhamnosyllutonarin isolated in this invention 1 HNMR MRI;

[0050] Figure 24 The 6”'-O-rhamnosyllutonarin isolated in this invention 13 C10 NMR MRI;

[0051] Figure 25 The infrared spectrum of 6”'-O-rhamnosyllutonarin obtained by the present invention;

[0052] Figure 26 The UV spectrum of 6”'-O-rhamnosyllutonarin obtained by the present invention;

[0053] Figure 27 The structural diagrams of the flavonoid C-glycosides Fr4111(2”-O-rhamnosyllutonarin), Fr4121(Luteolin 6-C-glucoside-7-O-glucoside), Fr4122(Luteolin 6-C-(6”-O-β-D-glucoside)-glucoside), and Fr4123(6”'-O-rhamnosyllutonarin) isolated in this invention are shown. Detailed Implementation

[0054] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0055] Example 1

[0056] A method for the directional separation and preparation of flavonoid C-glycosides from Dianthus superbus, comprising the following steps:

[0057] Step 1, Extraction: Take 4.0 kg of the whole Dianthus superbus herb after it has been air-dried, crush it, add 10 times its weight of ethanol, extract 4 times at room temperature, 2 hours each time, filter, and combine the filtrates, which is filtrate A. Mix filtrate A with silica gel at a ratio of 1:15 and dry under reduced pressure to obtain 827 g of Dianthus superbus ethanol extract sample. The conditions for reduced pressure drying are a vacuum degree of 50 mbar and a temperature of 40℃.

[0058] Step 2, coarse separation by medium-pressure chromatography using microporous resin: The sample of Dianthus chinensis ethanol extract was separated by a medium-pressure chromatographic column packed with microporous resin material. The fractions were detected by a UV detector with a wavelength of 210 nm, and six main chromatographic peaks were collected. After drying under reduced pressure, these fractions were labeled as Dianthus chinensis components Fr1: 74g, Fr2: 78g, Fr3: 66g, Fr4: 155g, Fr5: 26g, and Fr6: 15g (attached). Figure 1 (as shown);

[0059] The conditions for vacuum drying were a vacuum degree of 50 mbar and a temperature of 40℃. The operating parameters for crude fractionation by medium-pressure chromatography using microporous resin were as follows: column length 460 mm, diameter 49 mm, stationary phase HP20SS, mobile phase A: water, B: methanol, C: dichloromethane, chromatographic conditions: 0–20 min, 0% B, 20–240 min, 0%–100% B, 240–300 min, 100% B, 300–420 min, 0–100% C, injection volume 80 g, and flow rate 60 mL / min.

[0060] Step 3, Reversed-Phase Medium-Pressure Chromatography Enrichment: The target component Fr4 is mixed with silica gel to obtain a sample. This sample is separated by a reversed-phase medium-pressure chromatographic column packed with silica gel matrix material. Detection is performed using a UV detector with a detection wavelength of 210 nm. The fraction Fr41 (attached) of the first major chromatographic peak in the preparative chromatogram is collected. Figure 2 (As shown), the fraction was dried under reduced pressure to obtain component Fr41: 20.5g containing the target component;

[0061] The conditions for vacuum drying were a vacuum degree of 50 mbar and a temperature of 40 ℃. The separation parameters for reversed-phase medium-pressure chromatography enrichment were: column length of 500 mm and diameter of 50 mm, stationary phase of 50 μm Spherical C18 for reversed-phase preparative column, mobile phase A of water and mobile phase B of methanol, chromatographic conditions of 0–120 min, 20%–65% B, injection volume of 30 g, and flow rate of 60 mL / min.

[0062] Step 4, Reversed-Phase Preparative Column Preparation: Component Fr41 containing the target ingredient was dissolved in a 50% (v / v) methanol-water solution to prepare a sample concentration of 50.0 mg / mL. The solution was filtered through a 0.45 μm microporous membrane to obtain filtrate B. Filtrate B was then processed using a reversed-phase preparative column and detected with a 210 nm UV detector. The two main chromatographic peaks in the reversed-phase preparative chromatogram of filtrate B were collected. These peaks were dried under reduced pressure to obtain components Fr411 and Fr412 containing the target ingredient (see attached image). Figure 3 (as shown);

[0063] The conditions for vacuum drying were a vacuum degree of 50 mbar and a temperature of 40 °C. The working parameters for the preparation of the reversed-phase preparative column were: column size of 250 × 20 mm, stationary phase of 7 μm reversed-phase column 7-X10, mobile phase of 6% methanol-water solution, injection volume of 4 mL, and flow rate of 19 mL / min.

[0064] Step 5, Reversed-Phase Preparative Liquid Chromatography Purification of Fr411: The component Fr411 containing the target ingredient was dissolved in 100% methanol solution to prepare a sample concentration of 25.0 mg / mL. The solution was filtered through a 0.45 μm microporous membrane to obtain filtrate C. Filtrate C was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and detected by a 210 nm UV detector. The chromatographic peak fraction Fr4111 from the RP-HPLC chromatogram of filtrate C was collected. Figure 4 (As shown), the chromatographic peak fraction was dried under reduced pressure to obtain 3.1 g of 2”-O-rhamnosyllutonarin with a purity greater than 95%;

[0065] The conditions for vacuum drying were a vacuum degree of 50 mbar and a temperature of 40 °C. The working parameters for the reversed-phase preparative liquid chromatography purification of Fr411 were: column length of 250 mm and diameter of 20 mm, stationary phase of 5 μm reversed-phase column SunFire C18, mobile phase of 20% methanol-water solution, injection volume of 4 mL, and flow rate of 19 mL / min.

[0066] Step 6, hydrophilic preparative high-performance liquid chromatography (HPLC) purification of Fr412: The component Fr412 containing the target ingredient was dissolved in 100% methanol solution to prepare a sample concentration of 25.0 mg / mL. The solution was filtered through a 0.45 μm microporous membrane to obtain filtrate D. Filtrate D was purified by hydrophilic HPLC and detected by a UV detector at a wavelength of 210 nm. The corresponding peak fractions Fr4121, Fr4122, and Fr4123 (see attached image) from the hydrophilic preparative HPLC chromatogram of filtrate D were collected. Figure 5As shown), the chromatographic peak fractions Fr4121, Fr4122, and Fr4123 were dried under reduced pressure to obtain Luteolin 6-C-glucoside-7-O-glucoside 765.4 mg, Luteolin 6-C-(6”-O-β-D-glucoside)-glucoside 8.9 mg, and 6”'-O-rhamnosyllutonarin 204.7 mg, respectively, with purities greater than 95%.

[0067] The conditions for vacuum drying were a vacuum degree of 50 mbar and a temperature of 40 °C. The working parameters for the hydrophilic preparative liquid chromatography purification of Fr412 were as follows: column size of 250 × 20 mm, stationary phase of 5 μm zwitterionic ClickXION column, mobile phase of 90% acetonitrile-5% trifluoroacetic acid aqueous solution, injection volume of 4 mL, and flow rate of 19 mL / min.

[0068] Among them, Fr4111, Fr4121, Fr4122, and Fr4123 were obtained as 2”-O-rhamnosyllutonarin, Luteolin 6-C-glucoside-7-O-glucoside, Luteolin 6-C-(6”-O-β-D-glucoside)-glucoside, and 6”'-O-rhamnosyllutonarin samples with purities greater than 95%, respectively. Purity verification chromatograms are attached. Figure 6 (as shown);

[0069] The structural characterization and structures of the flavonoid C-glycosides 2”-O-rhamnosyllutonarin, Luteolin 6-C-glucoside-7-O-glucoside, Luteolin 6-C-(6”-O-β-D-glucoside)-glucoside and 6”'-O-rhamnosyllutonarin obtained from Dianthus superbus are attached. Figures 7-27 As shown.

[0070] Example 2

[0071] A method for the directional separation and preparation of flavonoid C-glycosides from Dianthus superbus includes the following steps:

[0072] Step 1, Extraction: Take 10 kg of the whole Dianthus superbus herb after it has been air-dried, crush it, add 10 times its weight of ethanol, extract it 3 times at room temperature, 3 hours each time, filter, and combine the filtrates, which is filtrate A. Mix filtrate A with silica gel at a ratio of 1:8 and dry it under reduced pressure to obtain 2.66 kg of Dianthus superbus ethanol extract sample. The conditions for reduced pressure drying are a vacuum degree of 250 mbar and a temperature of 60℃.

[0073] Step 2, coarse separation by medium-pressure chromatography with microporous resin: The sample of Dianthus chinensis ethanol extract is mixed and separated by a medium-pressure chromatographic column packed with microporous resin material. The sample is detected by an ultraviolet detector with a detection wavelength of 210 nm, and six main chromatographic peak fractions are collected. After being dried under reduced pressure, they are labeled as Dianthus chinensis components Fr1: 184 g, Fr2: 193 g, Fr3: 161 g, Fr4: 392 g, Fr5: 7.1 g and Fr6: 4.0 g.

[0074] The conditions for vacuum drying were a vacuum degree of 250 mbar and a temperature of 60℃. The operating parameters for crude fractionation by medium-pressure chromatography using microporous resin were as follows: column length 460 mm, diameter 49 mm, stationary phase HP20SS, mobile phase A: water, B: methanol, C: dichloromethane, chromatographic conditions: 0–20 min, 0% B, 20–240 min, 0%–100% B, 240–300 min, 100% B, 300–420 min, 0–100% C, injection volume 40 g, and flow rate 50 mL / min.

[0075] Step 3, reversed-phase medium-pressure chromatography enrichment: The target component Fr4 is mixed with silica gel to obtain a sample. The sample is separated by a reversed-phase medium-pressure chromatography column packed with silica gel matrix material and detected by an ultraviolet detector with a detection wavelength of 210 nm. The first major chromatographic peak fraction Fr41 in the preparation chromatogram is collected. The fraction is dried under reduced pressure to obtain component Fr41 containing the target component: 50.6 g.

[0076] The conditions for vacuum drying were a vacuum degree of 250 mbar and a temperature of 60 ℃. The separation parameters for reversed-phase medium-pressure chromatography enrichment were: column length of 500 mm and diameter of 50 mm, reversed-phase preparative column stationary phase of 50 μm Spherical C18, mobile phase A of water and mobile phase B of methanol, chromatographic conditions of 0–120 min, 20%–65% B, injection volume of 15 g, and flow rate of 45 mL / min.

[0077] Step 4, reversed-phase preparation column preparation: The component Fr41 containing the target component is dissolved in an 80% methanol-water solution to prepare a sample concentration of 40.0 mg / mL. The solution is filtered through a 0.45 μm microporous membrane to obtain filtrate B. Filtrate B is prepared by reversed-phase preparation column and detected by a UV detector with a detection wavelength of 210 nm. The two main chromatographic peak fractions in the reversed-phase preparation chromatogram of filtrate B are collected. The chromatographic peak fractions are dried under reduced pressure to obtain components Fr411 and Fr412 containing the target component.

[0078] The conditions for vacuum drying were a vacuum degree of 250 mbar and a temperature of 60 ℃; the working parameters for the preparation of the reversed-phase preparative column were: column size of 250 × 20 mm, stationary phase of 7 μm reversed-phase column 7-X10, mobile phase of 6% methanol-water solution, injection volume of 4 mL, and flow rate of 19 mL / min.

[0079] Step 5, reversed-phase preparative liquid chromatography purification of Fr411: The component Fr411 containing the target component was dissolved in a 100% methanol solution to prepare a sample concentration of 25.0 mg / mL. The solution was filtered through a 0.45 μm microporous membrane to obtain the filtrate, i.e., filtrate C. Filtrate C was purified by reversed-phase high-performance liquid chromatography and detected by a UV detector with a detection wavelength of 210 nm. The chromatographic peak fraction Fr4111 in the reversed-phase high-performance preparative chromatogram of filtrate C was collected. This chromatographic peak fraction was dried under reduced pressure to obtain 7.6 g of 2”-O-rhamnosyllutonarin with a purity greater than 95%.

[0080] The conditions for vacuum drying were a vacuum degree of 250 mbar and a temperature of 60 °C. The working parameters for the reversed-phase preparative liquid chromatography purification of Fr411 were: column length of 250 mm and diameter of 20 mm, stationary phase of 5 μm reversed-phase column SunFire C18, mobile phase of 20% methanol-water solution, injection volume of 4 mL, and flow rate of 19 mL / min.

[0081] Step 6, hydrophilic preparative high-performance liquid chromatography (HPLC) purification of Fr412: The component Fr412 containing the target ingredient was dissolved in 100% methanol solution to prepare a sample concentration of 25.0 mg / mL. The solution was filtered through a 0.45 μm microporous membrane to obtain filtrate D. Filtrate D was purified by hydrophilic HPLC and detected by a UV detector at a wavelength of 210 nm. The corresponding peak fractions Fr4121, Fr4122, and Fr4123 in the hydrophilic preparative chromatogram of filtrate D were collected. These peak fractions were dried under reduced pressure to obtain 1.9 g of Luteolin 6-C-glucoside-7-O-glucoside and 1.9 g of Luteolin 6-C-(6”-O-β-D-glucoside)-glucoside with a purity greater than 95%, respectively. 22.2 mg and 6”'-O-rhamnosyllutonarin 507.5 mg;

[0082] The conditions for vacuum drying were a vacuum degree of 250 mbar and a temperature of 60 °C. The working parameters for the hydrophilic preparative liquid chromatography purification of Fr412 were as follows: column size of 250 × 20 mm, stationary phase of 5 μm zwitterionic ClickXION column, mobile phase of 90% acetonitrile-5% trifluoroacetic acid aqueous solution, injection volume of 4 mL, and flow rate of 19 mL / min.

[0083] Example 3

[0084] A method for the directional separation and preparation of flavonoid C-glycosides from Dianthus superbus includes the following steps:

[0085] Step 1, Extraction: Take 1 kg of the whole Dianthus superbus herb after it has been air-dried, crush it, add 10 times its weight of ethanol, extract twice at room temperature for 4 hours each time, filter, and combine the filtrates, which is filtrate A. Mix filtrate A with silica gel at a ratio of 1:5 and dry under reduced pressure to obtain 338 g of Dianthus superbus ethanol extract sample. The conditions for reduced pressure drying are a vacuum degree of 150 mbar and a temperature of 50℃.

[0086] Step 2, coarse separation by medium-pressure chromatography using microporous resin: The sample of Dianthus chinensis ethanol extract is mixed and separated by a medium-pressure chromatographic column packed with microporous resin material. The sample is detected by an ultraviolet detector with a detection wavelength of 210 nm, and six main chromatographic peak fractions are collected. After being dried under reduced pressure, they are labeled as Dianthus chinensis components Fr1: 18.6 g, Fr2: 19.6 g, Fr3: 16.4 g, Fr4: 38.7 g, Fr5: 6.5 g, and Fr6: 3.7 g.

[0087] The conditions for vacuum drying were a vacuum degree of 150 mbar and a temperature of 50 °C. The operating parameters for crude fractionation by medium-pressure chromatography using microporous resin were as follows: column length 460 mm, diameter 49 mm, stationary phase HP20SS, mobile phase A: water, B: methanol, C: dichloromethane, chromatographic conditions: 0–20 min, 0% B, 20–240 min, 0%–100% B, 240–300 min, 100% B, 300–420 min, 0–100% C, injection volume 10 g, and flow rate 40 mL / min.

[0088] Step 3, reversed-phase medium-pressure chromatography enrichment: The target component Fr4 is mixed with silica gel to obtain a sample. The sample is separated by a reversed-phase medium-pressure chromatography column packed with silica gel matrix material and detected by an ultraviolet detector with a detection wavelength of 210 nm. The first major chromatographic peak fraction Fr41 in the preparation chromatogram is collected. The fraction is dried under reduced pressure to obtain component Fr41 containing the target component: 5.0 g.

[0089] The conditions for vacuum drying were a vacuum degree of 150 mbar and a temperature of 50 °C. The separation parameters for reversed-phase medium-pressure chromatography enrichment were: column length of 500 mm and diameter of 50 mm, reversed-phase preparative column stationary phase of 50 μm Spherical C18, mobile phase A of water and mobile phase B of methanol, chromatographic conditions of 0–120 min, 20%–65% B, injection volume of 10 g, and flow rate of 40 mL / min.

[0090] Step 4, reversed-phase preparation column preparation: The component Fr41 containing the target component is dissolved in a 100% methanol solution to prepare a sample concentration of 20.0 mg / mL. The solution is filtered through a 0.45 μm microporous membrane to obtain filtrate B. Filtrate B is prepared by reversed-phase preparation column and detected by a UV detector with a detection wavelength of 210 nm. The two main chromatographic peak fractions in the reversed-phase preparation chromatogram of filtrate B are collected. The chromatographic peak fractions are dried under reduced pressure to obtain components Fr411 and Fr412 containing the target component.

[0091] The conditions for vacuum drying were a vacuum degree of 150 mbar and a temperature of 50 °C. The working parameters for the preparation of the reversed-phase preparative column were: column size of 250 × 20 mm, stationary phase of 7 μm reversed-phase column 7-X10, mobile phase of 6% methanol-water solution, injection volume of 4 mL, and flow rate of 19 mL / min.

[0092] Step 5, reversed-phase preparative liquid chromatography purification of Fr411: The component Fr411 containing the target component was dissolved in a 100% methanol solution to prepare a sample concentration of 25.0 mg / mL. The solution was filtered through a 0.45 μm microporous membrane to obtain the filtrate, i.e., filtrate C. Filtrate C was purified by reversed-phase high-performance liquid chromatography and detected by a UV detector with a detection wavelength of 210 nm. The chromatographic peak fraction Fr4111 in the reversed-phase high-performance preparative chromatogram of filtrate C was collected. This chromatographic peak fraction was dried under reduced pressure to obtain 751.8 mg of 2”-O-rhamnosyllutonarin with a purity greater than 95%.

[0093] The conditions for vacuum drying were a vacuum degree of 150 mbar and a temperature of 50 °C. The working parameters for the reversed-phase preparative liquid chromatography purification of Fr411 were: column length of 250 mm and diameter of 20 mm, stationary phase of 5 μm reversed-phase column SunFire C18, mobile phase of 20% methanol-water solution, injection volume of 4 mL, and flow rate of 19 mL / min.

[0094] Step 6, hydrophilic preparative high-performance liquid chromatography (HPLC) purification of Fr412: The component Fr412 containing the target ingredient was dissolved in 100% methanol solution to prepare a sample concentration of 25.0 mg / mL. The solution was filtered through a 0.45 μm microporous membrane to obtain filtrate D. Filtrate D was purified by hydrophilic HPLC and detected by a UV detector at a wavelength of 210 nm. The corresponding peak fractions Fr4121, Fr4122, and Fr4123 in the hydrophilic preparative chromatogram of filtrate D were collected. These peak fractions were dried under reduced pressure to obtain 186.7 mg of Luteolin 6-C-glucoside-7-O-glucoside and 186.7 mg of Luteolin 6-C-(6”-O-β-D-glucoside)-glucoside with a purity greater than 95%, respectively. 2.2 mg and 6”'-O-rhamnosyllutonarin 49.9 mg;

[0095] The conditions for vacuum drying were a vacuum degree of 150 mbar and a temperature of 50 °C. The working parameters for the hydrophilic preparative liquid chromatography purification of Fr412 were as follows: column size of 250 × 20 mm, stationary phase of 5 μm zwitterionic ClickXION column, mobile phase of 90% acetonitrile-5% trifluoroacetic acid aqueous solution, injection volume of 4 mL, and flow rate of 19 mL / min.

[0096] Pharmacological tests: Screening for α-glucosidase inhibitory activity of Dianthus flavonoid C-glycosides

[0097] Using 4-Nitrophenyl-β-D-glucopyranoside (PNPG) as the substrate, the experiment was divided into a blank group (without dianthus flavonoid C-glycoside and α-glucosidase solution), a control group (without dianthus flavonoid C-glycoside), a dianthus flavonoid C-glycoside assay group, and a dianthus blank group (without α-glucosidase solution), with 3 replicates in each group.

[0098] Add 50 μL PBS, 50 μL 0.25 U / mL α-glucosidase solution, and 50 μL sample solution (Dianthus superbus monomeric compound sample solutions with concentrations of 1, 10, 50, 100, 500, and 1000 μM) to a 96-well plate, and acarbose concentrations of (1, 5, 20, 50, 100, and 500 μM). Mix at 37°C with shaking for 10 min. Add 50 μL 1 mmol / L PNPG solution, mix at 37°C with shaking for 20 min, and then add 50 μL 0.2 mol / L sodium carbonate solution to terminate the reaction. Measure the absorbance at 405 nm using a microplate reader. Calculate the inhibition rate of α-glucosidase activity according to the formula. Repeat the experiment three times. Calculate the half-maximal inhibitory concentration (IC50) of α-glucosidase activity using SPSS (results are shown in Table 1).

[0099]

[0100] A c : Control group, A b : Blank group, A q Dianthus flavonoid C-glycoside group, A qb : Dianthus chinensis blank group

[0101] Table 1. Screening results of α-glucosidase inhibitory activity of Dianthus flavonoid C-glycosides.

[0102]

[0103]

[0104] Experimental results show that Fr4111 (2”-O-rhamnosyllutonarin), Fr4121 (Luteolin 6-C-glucoside-7-O-glucoside), Fr4122 (Luteolin 6-C-(6”-O-β-D-glucoside)-glucoside), and Fr4123 (6”'-O-rhamnosyllutonarin) exhibit significant α-glucosidase activity, with IC50 values ​​all below 1 mM. Therefore, the flavonoid C-glycosides of the present invention can be used to prepare α-glucosidase inhibitors. Furthermore, since the main method for controlling postprandial hyperglycemia is by inhibiting the activity of α-glucosidase and α-amylase, thereby reducing their conversion of sugar into absorbable monosaccharides, the flavonoid C-glycosides of the present invention can be used to prepare drugs for treating or improving diabetes and play a positive role in the treatment of diabetes.

[0105] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for the directional separation and preparation of flavonoid C-glycosides from Dianthus superbus, characterized in that... Includes the following steps: Step 1, Extraction: Dry the whole Dianthus superbus in the shade, coarsely crush it, and extract it with ethanol at a ratio of 1 g: 5~100 mL. Extract 2~4 times at room temperature, 2~4 hours each time. Filter and combine the filtrates, which is filtrate A. Mix filtrate A with silica gel at a ratio of 1:5~15 and dry under reduced pressure to obtain the Dianthus superbus extract sample. Step 2, coarse separation by medium-pressure chromatography with microporous resin: The sample of Dianthus chinensis extract was separated by medium-pressure chromatography with microporous resin. The sample was detected by a UV detector with a wavelength of 210 nm. Six chromatographic peaks were collected and dried under reduced pressure and labeled as Dianthus chinensis components Fr1, Fr2, Fr3, Fr4, Fr5 and Fr6. Step 3, reversed-phase medium-pressure chromatography enrichment: The target component Fr4 is mixed with silica gel to obtain a sample. The sample is separated by a reversed-phase medium-pressure chromatography column packed with silica gel matrix material and detected by an ultraviolet detector with a detection wavelength of 210 nm. The fraction of the first chromatographic peak in the preparation chromatogram is collected. The fraction is dried under reduced pressure to obtain component Fr41 containing the target component. Step 4, reversed-phase preparation column preparation: The component Fr41 containing the target component is dissolved in a methanol-water solution with a volume fraction of 50-100% to prepare a sample concentration of 20.0-50.0 mg / mL. The solution is filtered through a 0.45 μm microporous membrane to obtain filtrate B. Filtrate B is prepared by reversed-phase preparation column and detected by a UV detector with a detection wavelength of 210 nm. The two chromatographic peak fractions in the reversed-phase preparation chromatogram of filtrate B are collected. The chromatographic peak fractions are dried under reduced pressure to obtain components Fr411 and Fr412 containing the target component. Step 5, reversed-phase preparative liquid chromatography purification of Fr411: The component Fr411 containing the target component was dissolved in a 100% methanol solution to prepare a sample concentration of 25.0 mg / mL. The solution was filtered through a 0.45 μm microporous membrane to obtain the filtrate, i.e., filtrate C. Filtrate C was purified by reversed-phase high-performance liquid chromatography and detected by a UV detector with a detection wavelength of 210 nm. The chromatographic peak fraction Fr4111 in the reversed-phase high-performance preparative chromatogram of filtrate C was collected. This chromatographic peak fraction was dried under reduced pressure to obtain 2''-O-rhamnosyllutonarin with a purity greater than 95%. Step 6, hydrophilic preparative high-performance liquid chromatography (HPLC) purification of Fr412: The component Fr412 containing the target component was dissolved in a 100% methanol solution to prepare a sample concentration of 25.0 mg / mL. The solution was filtered through a 0.45 μm microporous membrane to obtain filtrate, i.e., filtrate D. Filtrate D was purified by hydrophilic high-performance liquid chromatography (HPLC) and detected by a UV detector with a detection wavelength of 210 nm. The corresponding chromatographic peak fractions Fr4121, Fr4122, and Fr4123 in the hydrophilic preparative chromatogram of filtrate D were collected. The chromatographic peak fractions Fr4121, Fr4122, and Fr4123 were dried under reduced pressure to obtain Luteolin 6-C-glucoside-7-O-glucoside, Luteolin 6-C-(6''-O-β-D-glucoside)-glucoside, and 6'''-O-rhamnosyllutonarin with purities greater than 95%, respectively. The structural formulas of the following compounds are shown below: 2''-O-rhamnosyllutonarin, Luteolin 6-C-glucoside-7-O-glucoside, Luteolin 6-C-(6''-O-β-D-glucoside)-glucoside, and 6'''-O-rhamnosyllutonarin. 。 2. The method for the directional separation and preparation of dianthus flavonoid C-glycosides according to claim 1, characterized in that: In steps 1, 2, 3, 4, 5, and 6, the conditions for vacuum drying are: vacuum degree 50~250mbar and temperature 40~60℃.

3. The method for the directional separation and preparation of dianthus flavonoid C-glycosides according to claim 1, characterized in that: In step 2, the separation parameters for the crude fraction in medium-pressure chromatography using microporous resin are as follows: column length 460 mm, diameter 49 mm, stationary phase HP20SS, mobile phase A is water, B is methanol, C is dichloromethane, chromatographic conditions are 0~20 min, 0% B, 20~240 min, 0%~100% B, 240~300 min, 100% B, 300~420 min, 0~100% C, injection volume is 10~80 g, and flow rate is 40~60 mL / min.

4. The method for the directional separation and preparation of dianthus flavonoid C-glycosides according to claim 1, characterized in that: In step 3, the operating parameters for reversed-phase medium-pressure chromatography enrichment are as follows: column length 500 mm, diameter 50 mm, reversed-phase preparative column stationary phase is 50 μm Spherical C18, mobile phase A is water, mobile phase B is methanol, chromatographic conditions are 0~120 min, 20%~65% B, injection volume is 10~30 g, and flow rate is 40~60 mL / min.

5. The method for the directional separation and preparation of dianthus flavonoid C-glycosides according to claim 1, characterized in that: In step 4, the working parameters for preparing the reversed-phase preparation column are as follows: the column size is 250×20 mm, the stationary phase of the reversed-phase preparation column is a 7 μm reversed-phase column 7-X10, and the mobile phase is a 6% methanol-water solution.

6. The method for the directional separation and preparation of dianthus flavonoid C-glycosides according to claim 1, characterized in that: In step 5, the working parameters for reversed-phase preparative liquid chromatography purification are as follows: column length 250 mm, diameter 20 mm, stationary phase of reversed-phase preparative column is 5 μm reversed-phase column SunFire C18, and mobile phase is 20% methanol-water solution.

7. The method for the directional separation and preparation of dianthus flavonoid C-glycosides according to claim 1, characterized in that: In step 6, the working parameters for hydrophilic preparative liquid chromatography purification are as follows: column size is 250×20 mm, stationary phase is 5 μm zwitterionic Click XION column, and mobile phase is 90% acetonitrile-5% trifluoroacetic acid aqueous solution.

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