A phenolic glycoside compound and its preparation method and application

Abstract

The present invention belongs to the technical field of phenolic glycoside natural organic compounds, and specifically relates to phenolic glycoside compounds extracted and separated from the cones of the genus Conophora, and a preparation method and application thereof, and belongs to the technical field of extraction, separation and purification of plant components. The present invention extracts and separates chinensin D from the leaves of Conophora for the first time, and can obtain a monomer compound with high purity (purity>90%). Phenolic glycoside compounds have large polarity and similar structures, and trace components are difficult to be effectively separated. The preparation method of the present invention is fast and efficient to operate, and a large amount of impurities are removed by cross-repeated use of gel resin materials, and finally two similar components are effectively separated by high-speed countercurrent. The product obtained by this method has high purity, is easy to operate, and is suitable for popularization and application.

Description

Technical Field

[0001] The invention belongs to the technical field of phenolic glycoside natural organic compounds, and in particular relates to a phenolic glycoside compound extracted and separated from the cones of a plant of the genus Conophytum, a preparation method and an application thereof, and belongs to the technical field of extraction, separation and purification of plant components. Background Art

[0002] Glycosides are rich in pharmacological activities, with significant anti-tumor, anti-infection, and immunomodulatory functions. They also have different degrees of therapeutic effects on the cardiovascular system, respiratory system, digestive system, and nervous system. Modern pharmacological studies have shown that some oxygen glycoside compounds connected to aromatic phenol rings also have a variety of pharmacological activities such as inhibiting breast cancer migration and invasion, inhibiting the growth of pancreatic cancer and lung cancer, blocking the signaling pathway of fatty acid synthase, counteracting spasms caused by ACH-induced colon contraction in rats, and protecting focal cerebral ischemia. At present, a variety of natural phenolic glycoside compounds have been used clinically for antibacterial and anti-inflammatory, enhancing the body's immune function, and anti-tumor treatment. Nature is the main raw material library of phenolic glycoside compounds. Extracting and separating new oxygen glycosides from them and discovering potential substances with good pharmacological activity are hot topics in the field of natural product research.

[0003] Phenolic glycoside compounds have complex and variable structures and high polarity, and existing separation methods are difficult to quickly and effectively obtain high-purity monomer compounds. Therefore, conducting research on the chemical composition of natural plants rich in phenolic acid components and discovering an effective separation method for phenolic glycoside compounds is not only an effective way to discover new structural compounds and new functional lead compounds, but also can develop new uses for natural plants and realize the high-value utilization of plant resources. Summary of the invention

[0004] One of the purposes of the present invention is to provide a phenolic glycoside compound with a new structure. The phenolic glycoside compound provided by the present invention (hereinafter referred to as chinensin D) is derived from the leaves of Castanopsis chinensis, and its structure and pharmacological activity have not been reported in the prior art. It has antioxidant activity and provides a new candidate compound for antioxidant products.

[0005] The technical solution of the present invention to solve the above technical problem is as follows: a phenolic glycoside compound chinensin D is a compound shown in the following formula:

[0006]

[0007] The second object of the present invention is to provide a method for preparing the phenolic glycoside compound chinensin D.

[0008] The technical solution of the present invention to solve the above technical problem is as follows: A method for preparing the compound chinensin D comprises the following steps:

[0009] Step 1: Preparation of crude extract of coneflower leaves

[0010] Take the raw material of cone leaf, wash and drain, chop and extract, filter to remove the residue, and concentrate the filtrate under reduced pressure and low temperature to obtain the crude extract of cone leaf;

[0011] Step 2: Preparation of extracts rich in the compound chinensin D

[0012] The crude extract of the cone leaf obtained in step 1 is subjected to column chromatography by gel adsorption, and gradient elution is used to collect the eluate of 60%-80% methanol water eluent, and after low-temperature concentration under reduced pressure, an extract B rich in compound chinensin D is obtained;

[0013] Step 3: Further enrichment of compound chinensin D

[0014] The crude extract B obtained in step 2 is subjected to column chromatography by MCI adsorption, and gradient elution is used to collect the eluate of 80%-100% methanol water eluent, and after low-temperature concentration under reduced pressure, extract C is obtained;

[0015] Step 4: Purification of compound chinensin D

[0016] Extract C obtained in step 3 is subjected to column chromatography by gel adsorption, and reverse gradient elution is used to collect the eluent of 100%-90% ethanol water eluent and the eluate with a specific migration value (Rf) of 0.8 in thin layer chromatography detection, and concentrated under reduced pressure and low temperature to obtain extract D;

[0017] Step 5: High-speed countercurrent chromatography

[0018] The two-phase solvent system is composed of n-hexane, ethyl acetate, methanol and water in a volume ratio of 10:(58-62):30:(38-42), and then the mixture is allowed to stand for separation to obtain an upper and lower phase; the upper phase is used as a stationary phase, and the lower phase is used as a mobile phase; the stationary phase is first injected into a high-speed countercurrent chromatographic column, and after the stationary phase fills the high-speed countercurrent chromatographic column, the rotation speed is adjusted, and then the mobile phase is injected into the high-speed countercurrent chromatographic column; after the whole system establishes a dynamic equilibrium, 200 mg of extract D is taken, dissolved in a mixed solvent consisting of 10 mL of the stationary phase and 10 mL of the mobile phase, and the sample is injected through an injection valve; fractions are collected, and according to the retention time of the high-speed countercurrent chromatogram, a target component with an elution time of 75 min-90 min is received, and the compound chinensin D is obtained after low-temperature concentration under reduced pressure and vacuum drying;

[0019] The principle of the preparation method of chinensin D of the present invention is:

[0020] The inventors of the present application isolated and purified a new compound, chinensin D, from a study of the chemical composition of a cone leaf extract. Therefore, the inventors of the present application continuously improved the extraction and purification methods and experimental parameters to obtain a high-purity chinensin D monomer compound. The details are as follows:

[0021] In step 1 of the present invention, the phenolic acid compounds in the leaves of the coneflower are extracted by using a high concentration of methanol or ethanol solution based on the principle of like dissolves like, and the extraction solvent can extract the target compounds to the greatest extent. The leaves of the coneflower are fresh leaves.

[0022] In step 2 of the present invention, the crude extract of cone leaf obtained in step 1 contains other phenolic acid compounds and flavonoid components in addition to the target compound. The molecular sieving and adsorption effect of the gel and elution with alcohol-water solution in different proportions can achieve better separation of the target compound from impurities with large differences in molecular weight and polarity.

[0023] In step 3 of the present invention, the extract B obtained in step 2 contains, in addition to the target component, a variety of phenolic acid compounds with similar molecular weights and polarities. Most of the phenolic acid components with different polarities can be removed by resin adsorption elution.

[0024] In step 4 of the present invention, the extract C obtained in step 3 is subjected to gel adsorption and eluted using a reverse gradient elution system to remove impurity compounds with a polarity close to that of the target component.

[0025] In step 5 of the present invention, the extract D obtained in step 4 contains a compound with a retention time extremely close to that of the target component through liquid phase detection, and it is difficult to effectively separate it by column chromatography and high performance liquid phase preparation methods. Through method screening, it is found that the high-speed countercurrent separation method can effectively separate the target compound from similar components. The upper and lower phase system formed by mixing n-hexane, ethyl acetate, methanol and water in a volume ratio of 10:(58-62):30:(38-42) has a good separation effect on the target component.

[0026] High-speed countercurrent separation technology is a continuous and efficient liquid-liquid partition chromatography separation technology. It uses a two-phase solvent system to establish a special unidirectional fluid dynamic equilibrium in a high-speed rotating spiral tube, and separates different substances according to the different distribution coefficients in the two phases. It is particularly suitable for separating active ingredients of natural products. There is no method for separating and preparing new structure phenolic glycoside compounds using high-speed countercurrent chromatography in the prior art reports.

[0027] In summary, the present invention can obtain high-purity (purity>90%) monomer compounds through the above operations, providing a new approach for the comprehensive development and in-depth utilization of cone plant resources.

[0028] The beneficial effects of the preparation method of high-purity chinensin D of the present invention are:

[0029] 1. The present invention uses cone leaves as raw materials to extract and separate chinensin D for the first time, and can obtain a high-purity (purity> 90%) monomer compound. Phenolic glycoside compounds have large polarity and similar structures, and trace components are difficult to be effectively separated. The preparation method of the present invention is fast and efficient to operate. A large amount of impurities are removed by cross-repeated use of gel resin materials, and finally two similar components are effectively separated by high-speed countercurrent. The product obtained by this method has high purity, is easy to operate, and is suitable for promotion and application.

[0030] Based on the above technical solution, the present invention can also be improved as follows.

[0031] Furthermore, in step 1, the width of the chopped pieces is 1-3 cm.

[0032] A further beneficial effect of adopting the above is that adopting the above size is more conducive to the extraction of phenolic glycoside compounds.

[0033] Furthermore, in step 1, the extraction is carried out by any one of leaching and ultrasonic extraction.

[0034] A further beneficial effect of adopting the above method is that the above extraction method is more conducive to the extraction of phenolic glycoside compounds and avoids the destruction of the structure and the reduction of biological activity.

[0035] Furthermore, the specific method of the extraction is: adding 70%-100% alcohol aqueous solution with a volume concentration of 70%-15 times the weight of the raw material to the chopped fresh leaves, extracting at room temperature, each time for 1-2 days, for a total of 2-4 times.

[0036] A further beneficial effect of the above method is that the above extraction method is more conducive to the extraction of the compound chinensin D and maintains the original biological activity.

[0037] Furthermore, the specific method of ultrasonic extraction is: adding 70%-100% alcohol aqueous solution with a volume concentration of 10-20 times the weight of the raw material to the crushed raw material, using a power of 100W and a frequency of 30kHz-80kHz for ultrasonic extraction, each time for 30-60 minutes, for a total of 3 times.

[0038] A further beneficial effect of the above method is that the above extraction method is more conducive to the rapid and effective extraction of chinensin D.

[0039] Furthermore, in step 1, step 2 or step 3, the alcohol aqueous solution is methanol water or ethanol aqueous solution, and the pressure of low-temperature concentration during reduced pressure is 35KPa-85KPa, and the temperature is ≤50°C.

[0040] The above further beneficial effect is that the chinensin D solution is easily denatured at high temperature and easily oxidized when exposed to air for a long time. The possibility of chinensin D denaturation and oxidation can be greatly reduced by using the above parameters for low-temperature concentration under reduced pressure.

[0041] Further, in step 2, the gel is Sephadex LH-20, with a size of 8 cm×80 cm and a filler particle size of 100 μm-163 μm. The eluent is any one of a methanol aqueous solution with a volume concentration of 0%-100% and an ethanol aqueous solution with a volume concentration of 0%-100%. The gradient elution is one gradient per 20% volume concentration, one fraction per 0.2 L, and an elution rate of 2 ml / min.

[0042] A further beneficial effect of the above is that the specifications and filler particle size of the above Sephadex LH-20 are more suitable for the separation of phenolic acids and flavonoids in crude extracts.

[0043] The above-mentioned Sephadex LH-20 can be purchased commercially, for example, from GE Company in the United States.

[0044] Further, in step 3, the MCI is MCI GEL CHP20P, with a specification of 6cm×80cm, and a filler particle size of 37μm-75μm, 75μm-150μm. The eluent is any one of a methanol aqueous solution with a volume concentration of 0%-100% and an ethanol aqueous solution with a volume concentration of 0%-100%. The gradient elution is one gradient per 10% volume concentration, one fraction per 0.01L, and an elution rate of 2ml / min.

[0045] A further beneficial effect of the above method is that the specifications and filler particle size of MCI are more suitable for the separation of medium-polarity flavonoid phenolic acid compounds in natural products.

[0046] The above-mentioned MCI can be purchased commercially, for example, from Mitsubishi Chemical of Japan.

[0047] Further, in step 4, the gel is Sephadex LH-20, with a size of 4 cm×60 cm and a filler particle size of 100 μm-163 μm. The eluent is an ethanol aqueous solution with a volume concentration of 100%-0%. The gradient elution is one gradient per 10% volume concentration, each gradient is 0.3 L, each 0.008 L is one fraction, and the elution rate is 2 ml / min.

[0048] Further, in step 4, the conditions for the thin layer chromatography detection are: silica gel GF 254 The plate was developed with a volume ratio of toluene: ethyl formate: formic acid of 1:7:1, and the plate was developed in an upright upward direction. The color developer was a 1% FeCl3-ethanol solution, and the elution site with an Rf value of 0.8 showing brown dots was extract D.

[0049] The preparation method of the above-mentioned color developer is: accurately weigh 0.789g of FeCl3 solid, dissolve it in 100ml of anhydrous ethanol, and shake it thoroughly until it is completely dissolved.

[0050] A further beneficial effect of the above is that an extract D containing the target component can be collected through the above detection parameters.

[0051] Further, in step 5, the rotation speed is 800r / min-900r / min.

[0052] A further beneficial effect of adopting the above method is that a suitable rotation speed can improve the retention rate and separation effect of the stationary phase.

[0053] Furthermore, in step 5, the speed of collecting fractions is 1 test tube every 3 minutes.

[0054] A further beneficial effect of adopting the above method is that it helps to improve the purity of the collected chromatographic peaks.

[0055] Furthermore, in step 5, the drying temperature is 45° C. and the drying time is 12 hours.

[0056] The further beneficial effects of the above method are: preventing oxidation of the compound due to high temperature and making the compound dry more thoroughly due to vacuum.

[0057] The third object of the present invention is to provide the use of chinensin D prepared by the above chinensin D preparation method in antioxidant products. Chinensin D prepared by the above preparation method has good antioxidant biological activity, can be used to prepare antioxidant products, and has a broad market space.

[0058] The technical solution of the present invention to solve the above technical problem is as follows: a chinensin D obtained by the preparation method of chinensin D as described above.

[0059] The beneficial effect of the chinensin D preparation method of the present invention is that the chinensin D of the present invention obtained by the above preparation method has good antioxidant biological activity, can be used to prepare antioxidant products, such as medicines or cosmetics, and has a large development space.

[0060] The present invention has conducted a study on the antioxidant biological activity of chinensin D. The results show that the chinensin D prepared by the present invention exhibits good DPPH free radical scavenging ability. Therefore, the chinensin D obtained by the present invention has broad prospects in the development and application of antioxidant products. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is a high-speed countercurrent separation chromatogram of Example 1 of the present invention.

[0062] Figure 2 This is a purity detection chart of chinensin D obtained in Example 1 of the present invention.

[0063] Figure 3 The method for obtaining chinensin D in Example 1 of the present invention 1 H-NMR spectrum.

[0064] Figure 4 The method for obtaining chinensin D in Example 1 of the present invention 13 C-NMR spectrum.

[0065] Figure 5 The LCMS-IT-TOF spectrum of chinensin D obtained in Example 1 of the present invention.

[0066] Figure 6 This is the HPLC preparation diagram of chinensin D obtained in Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0067] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0068] Example 1

[0069] The preparation method of chinensin D in this embodiment comprises the following steps:

[0070] Step 1: Preparation of crude extract of coneflower leaves

[0071] Take 3 kg of fresh cone leaf material, wash and drain, chop into 1-3 cm. Add 90% methanol aqueous solution with a volume concentration of 10 times the weight of the raw material, soak and extract at room temperature for 1 day, filter for the first time, obtain the first filtrate and the first filter residue, the first filtrate is concentrated under reduced pressure at 50 KPa and 50°C to extract; add 90% methanol aqueous solution with a volume concentration of 10 times the weight of the raw material to the first filter residue, soak and extract at room temperature for 1 day, filter for the second time, obtain the second filtrate and the second filter residue, the second filtrate is concentrated under reduced pressure at 50 KPa and 50°C to extract; add 90% methanol aqueous solution with a volume concentration of 10 times the weight of the raw material to the second filter residue, soak and extract at room temperature for 2 days, filter for the third time, obtain the third filtrate, the third filtrate is concentrated under reduced pressure at 50 KPa and 50°C to extract.

[0072] Combine the first extract, the second extract and the third extract, add a small amount of water to dissolve, filter and remove the residue to obtain a crude extract of cone leaf;

[0073] Step 2: Preparation of extracts rich in the compound chinensin D

[0074] The crude extract of cone leaf obtained in step 1 is adsorbed on gel Sephadex LH-20 for column chromatography, and gradient elution is adopted. The specification of gel chromatography column Sephadex LH-20 is 8cm×80cm, and the filler particle size is 100μm-163μm.

[0075] Gradient elution was performed with 2 column volumes of 0%-100% methanol-water solution, with each 20% volume concentration as one gradient, each 0.2L as one fraction, and the elution rate was 2ml / min. The eluate of 60%-80% methanol-water eluent was collected, and after reduced pressure concentration at 50KPa and 50°C, extract B rich in chinensin D was obtained, with a chinensin D content of 0.196%;

[0076] Step 3: Further enrichment of compound chinensin D

[0077] The extract B obtained in step 2 was subjected to column chromatography after MCI adsorption, and gradient elution was adopted. The MCI was MCI GELCHP20P, with a specification of 6cm×80cm and a filler particle size of 37μm-75μm and 75μm-150μm. Gradient elution was performed with a methanol aqueous solution with a volume concentration of 0%-100% for 2 times the column volume, with each 10% volume concentration being a gradient, each 0.01L being a flow fraction, and the elution rate being 2ml / min. The eluate of the 80%-100% methanol aqueous eluent was collected, and after reduced pressure concentration at 50KPa and 50°C, extract C was obtained, and the chinensin D content was 2.32%;

[0078] Step 4: Purification of compound chinensin D

[0079] Extract C obtained in step 3 was subjected to column chromatography by gel adsorption, and reverse gradient elution was adopted. The gel was Sephadex LH-20, the column size was 4cm×60cm, and the filler particle size was 100μm-163μm. Gradient elution was performed with 100%-0% ethanol aqueous solution with a volume concentration of 2 times the column volume, each 10% volume concentration was a gradient, each 0.008L was a flow fraction, and the elution rate was 2ml / min. The 100%-90% ethanol aqueous eluent portion was collected and placed on silica gel GF 254 In the thin layer chromatography test, the elution site with Rf value of 0.8 showing brown dots is extract D. The developing agent is toluene: ethyl formate: formic acid in a volume ratio of 1:7:1, and the development is vertically ascending. The color developer is a FeCl3-ethanol solution with a mass concentration of 1%. After the eluent is concentrated under reduced pressure at 50KPa and 50℃, extract D is obtained, and the content of chinensin D is 6.44%;

[0080] Step 5: High-speed countercurrent chromatography

[0081] The two-phase solvent system is composed of n-hexane, ethyl acetate, methanol and water in a volume ratio of 1:6:3:4, and then allowed to stand to separate into upper and lower phases;

[0082] The upper phase is used as the stationary phase and the lower phase is used as the mobile phase. The stationary phase is first injected into the high-speed countercurrent chromatographic column at 40 ml / min. After the stationary phase fills the high-speed countercurrent chromatographic column, the rotation speed is adjusted to 860 r / min, and then the mobile phase is injected into the high-speed countercurrent chromatographic column at 2 ml / min.

[0083] After the whole system established dynamic equilibrium, 141 ml of the stationary phase flowed out and the stationary phase retention rate was 53%. Take 200 mg of extract D, dissolve it in a mixed solvent consisting of 10 mL of the stationary phase and 10 mL of the mobile phase, and inject the sample solution through the injection valve;

[0084] The fractions were collected, and the target component with a peak time of 75 min to 90 min was received according to the retention time of the high-speed countercurrent chromatogram. The collected fractions were concentrated under reduced pressure at 50 KPa and 50 ° C, and vacuum dried to obtain the compound chinensin D. The high-speed countercurrent separation spectrum is shown in FIG. Figure 1 shown.

[0085] Purity detection: HPLC analysis was used to detect purity. Chromatographic column: Aiglent ZORBAX SB C18, 4.6 mm × 250 mm, filler particle size 5 μm, column temperature 30 ° C, flow rate 1 mL / min, detection wavelength 227 nm, mobile phase acetonitrile (A) and water (B), gradient elution, 0 min-30 min, 5% A-95% A. The liquid phase spectrum is as follows Figure 2 The results showed that the purity of chinensin D was 93%.

[0086] through 1 H-NMR and 13 C-NMR structural identification showed that the compound was chinensin D, specifically: 1 H-NMR (500MHz, Acetone-d6) δ: 7.64 (1H, d, J = 16.0Hz, 3"'), 7.15 (1H, d, J = 8.2Hz, H- 5), 7.09(1H,d,J=1.5Hz,5"'), 6.99(1H,dd,J=1.5,8.0Hz,H-9"'), 6.85(1H,d,J=1.5 Hz,H-2), 6.81(1H,d,J=8.0Hz,8”'), 6.79(1H,dd,J=1.5,8.2Hz,H-6), 6.38(1H,d,J =16.0Hz,H-2"'), 5.99(1H,m,H-8), 5.17(1H,t,J=8.9Hz,H-3'), 5.06(2H,m,H-9), 5. 02(1H,d,J=7.8Hz,H-1'), 4.30(1H,d,J=6.9Hz,H-1"), 4.12(1H,d,J=12.0Hz,H-6a' ), 3.86(3H,s,H-OCH3), 3.83(1H,m,H-5a”), 3.81(1H,m,H-6b’), 3.80(1H,m,H-4”), 3 .73(1H,m,H-2'), 3.72(1H,m,H-4'), 3.71(1H,m,H-5'), 3.60(1H,dd,J=6.9,8.4Hz, H-2”), 3.50(1H,m,H-3”), 3.45(1H,d,J=12.2Hz,H-5b”), 3.36(2H,d,J=6.7Hz,H-7).1 H-NMR spectrum Figure 3 shown. 13 C-NMR (125MHz, Acetone-d6) δ: 136.6 (C-1), 114.1 (C-2), 150.8 (C-3), 146.1 (C-4), 118.5 (C-5), 122.2 (C-6), 40 .7(C-7), 139.1(C-8), 115.9(C-9), 56.6(C-OCH3), 102.7(C-1'), 77.2(C-2'), 78.4(C-3'), 73.4(C-4'), 69.7(C -5'), 68.9(C-6'), 104.8(C-1"), 72.4(C-2"), 74.1(C-3"), 69.3(C-4"), 66.7(C-5"), 168.9(C-1"'), 115.3(C-2 "'), 147.1(C-3"'), 127.7(C-4"'), 115.1(C-5"'), 149.6(C-6"'), 146.8(C-7"'), 116.5(C-8"'), 122.9(C-9"'). 13 C-NMR spectrum Figure 4 shown.

[0087] LCIT-TOF-MS: m / z 619.2084[MH] - (Calculated value C 30 H 36 O 14 , 620.2157). LCMS-IT-TOF spectrum as shown Figure 5 shown.

[0088] pass 1 H-NMR, 13 The structure of the compound was identified by C-NMR and LCMS-IT-TOF spectral data. The structural formula is shown below:

[0089]

[0090] Example 2

[0091] The preparation method of chinensin D in this embodiment comprises the following steps:

[0092] Step 1: Preparation of crude extract of coneflower leaves

[0093] Take 3 kg of fresh cone leaf material, wash and drain, and chop into 1 cm. Add 90% methanol aqueous solution with a volume concentration of 12 times the weight of the raw material, use 80W power and 40kHz frequency for ultrasonic extraction for 30 minutes, filter for the first time, and obtain the first filtrate and the first filter residue; add 80% methanol aqueous solution with a volume concentration of 12 times the weight of the raw material to the first filter residue, use 80W power and 40kHz frequency for ultrasonic extraction for 30 minutes, filter for the second time, and obtain the second filtrate and the second filter residue; add 70% methanol aqueous solution with a volume concentration of 12 times the weight of the raw material to the second filter residue, use 60W power and 40kHz frequency for ultrasonic extraction for 1 hour, filter for the third time, and obtain the third filtrate.

[0094] The first filtrate, the second filtrate and the third filtrate were combined, concentrated under reduced pressure at 50 KPa and 50° C., and then filtered to remove the residue to obtain a crude extract of cone leaf;

[0095] Step 2: Preparation of extracts rich in the compound chinensin D

[0096] The crude extract of cone leaf obtained in step 1 is subjected to column chromatography through gel Sephadex LH-20, using gradient elution. The specification of the gel chromatography column Sephadex LH-20 is 8cm×80cm, and the filler particle size is 100μm-163μm.

[0097] Gradient elution was performed with 2 column volumes of 0%-100% methanol-water solution, with each 20% volume concentration as one gradient, each 0.2L as one fraction, and the elution rate was 2ml / min. The eluate of 60%-80% methanol-water eluent was collected, and after reduced pressure concentration at 50KPa and 50°C, extract B rich in compound chinensin D was obtained;

[0098] Step 3: Further enrichment of compound chinensin D

[0099] The extract B obtained in step 2 was subjected to column chromatography by MCI adsorption, and gradient elution was adopted. The MCI was MCI GELCHP20P, with a specification of 6cm×80cm and a filler particle size of 37μm-75μm and 75μm-150μm. Gradient elution was performed with a methanol aqueous solution with a volume concentration of 0%-100% of 2 times the column volume, with each 10% volume concentration being a gradient, each 0.01L being a flow fraction, and the elution rate being 2ml / min. The eluate of the 80%-100% methanol aqueous eluent was collected, and after reduced pressure concentration at 50KPa and 50°C, extract C was obtained;

[0100] Step 4: Purification of compound chinensin D

[0101] Extract C obtained in step 3 was subjected to column chromatography by gel adsorption, and reverse gradient elution was adopted. The gel was Sephadex LH-20, the column size was 4cm×60cm, and the filler particle size was 100μm-163μm. Gradient elution was performed with 100%-0% ethanol aqueous solution with a volume concentration of 2 times the column volume, each 10% volume concentration was a gradient, each 0.008L was a flow fraction, and the elution rate was 2ml / min. The 100%-90% ethanol aqueous eluent portion was collected and placed on silica gel GF 254 In the thin layer chromatography test, the elution part with Rf value of 0.8 showing brown dots is extract D. The developing agent is toluene: ethyl formate: formic acid in a volume ratio of 1:7:1, and the developing agent is FeCl3-ethanol solution with a mass concentration of 1%. After the eluent is concentrated under reduced pressure at 50KPa and 50℃, extract D is obtained;

[0102] Step 5: High-speed countercurrent chromatography

[0103] The two-phase solvent system is composed of n-hexane, ethyl acetate, methanol and water in a volume ratio of 10:62:30:38, and then allowed to stand to separate into upper and lower phases;

[0104] The upper phase is used as the stationary phase and the lower phase is used as the mobile phase. The stationary phase is first injected into the high-speed countercurrent chromatographic column at 40 ml / min. After the stationary phase fills the high-speed countercurrent chromatographic column, the rotation speed is adjusted to 860 r / min, and then the mobile phase is injected into the high-speed countercurrent chromatographic column at 2 ml / min.

[0105] After the whole system establishes dynamic equilibrium, 135 ml of the stationary phase flows out, and the stationary phase retention rate is 55%. Take 200 mg of extract D, dissolve it in a mixed solvent consisting of 10 mL of the stationary phase and 10 mL of the mobile phase, and inject the sample solution through the injection valve;

[0106] The fractions were collected, and the target component with a peak time of 78 min-91 min was received according to the retention time of the high-speed countercurrent chromatogram. The collected fractions were concentrated under reduced pressure at 50 KPa and 50° C., and dried in vacuo to obtain the compound chinensin D.

[0107] The purity test was the same as in Example 1, and the result showed that the purity of chinensin D was 91%.

[0108] The structure identification was the same as in Example 1.

[0109] Example 3

[0110] The preparation method of chinensin D in this embodiment comprises the following steps:

[0111] Step 1: Preparation of crude extract of coneflower leaves

[0112] Take 3.5 kg of fresh cone leaf material, wash and drain, chop into 1-3 cm. Add 90% methanol aqueous solution with a volume concentration of 10 times the weight of the raw material, soak and extract at room temperature for 2 days, filter for the first time, obtain the first filtrate and the first filter residue, the first filtrate is concentrated under reduced pressure at 50 KPa and 50°C to extract; add 90% methanol aqueous solution with a volume concentration of 10 times the weight of the raw material to the first filter residue, soak and extract at room temperature for 2 days, filter for the second time, obtain the second filtrate and the second filter residue, the second filtrate is concentrated under reduced pressure at 50 KPa and 50°C to extract; add 90% methanol aqueous solution with a volume concentration of 10 times the weight of the raw material to the second filter residue, soak and extract at room temperature for 2 days, filter for the third time, obtain the third filtrate, the third filtrate is concentrated under reduced pressure at 50 KPa and 50°C to extract.

[0113] Combine the first extract, the second extract and the third extract, add a small amount of water to filter and remove the residue to obtain a crude extract of cone leaf;

[0114] Step 2: Preparation of extracts rich in the compound chinensin D

[0115] The crude extract of cone leaf obtained in step 1 is adsorbed on gel Sephadex LH-20 for column chromatography, and gradient elution is adopted. The specification of gel chromatography column Sephadex LH-20 is 8cm×80cm, and the filler particle size is 100μm-163μm.

[0116] Gradient elution was performed with 2 column volumes of 0%-100% methanol-water solution, with each 20% volume concentration as one gradient, each 0.2L as one fraction, and the elution rate was 2ml / min. The eluate of 60%-80% methanol-water eluent was collected, and after reduced pressure concentration at 50KPa and 50°C, extract B rich in compound chinensin D was obtained;

[0117] Step 3: Further enrichment of compound chinensin D

[0118] The extract B obtained in step 2 was subjected to column chromatography by MCI adsorption, and gradient elution was adopted. The MCI was MCI GELCHP20P, with a specification of 6cm×80cm and a filler particle size of 37μm-75μm and 75μm-150μm. Gradient elution was performed with a methanol aqueous solution with a volume concentration of 0%-100% of 2 times the column volume, with each 10% volume concentration being a gradient, each 0.01L being a flow fraction, and the elution rate being 2ml / min. The eluate of the 80%-100% methanol aqueous eluent was collected, and after reduced pressure concentration at 50KPa and 50°C, extract C was obtained;

[0119] Step 4: Purification of compound chinensin D

[0120] Extract C obtained in step 3 was subjected to column chromatography by gel adsorption, and reverse gradient elution was adopted. The gel was Sephadex LH-20, the column size was 4cm×60cm, and the filler particle size was 100μm-163μm. Gradient elution was performed with 100%-0% ethanol aqueous solution with a volume concentration of 2 times the column volume, each 10% volume concentration was a gradient, each 0.008L was a flow fraction, and the elution rate was 2ml / min. The 100%-90% ethanol aqueous eluent portion was collected and placed on silica gel GF 254 In the thin layer chromatography test, the elution part with Rf value of 0.8 showing brown dots is extract D. The developing agent is toluene: ethyl formate: formic acid in a volume ratio of 1:7:1, and the developing agent is FeCl3-ethanol solution with a mass concentration of 1%. After the eluent is concentrated under reduced pressure at 50KPa and 50℃, extract D is obtained;

[0121] Step 5: High-speed countercurrent chromatography

[0122] The two-phase solvent system is composed of n-hexane, ethyl acetate, methanol and water in a volume ratio of 10:58:30:42, and then allowed to stand to separate into upper and lower phases;

[0123] The upper phase is used as the stationary phase and the lower phase is used as the mobile phase. The stationary phase is first injected into the high-speed countercurrent chromatographic column at 40 ml / min. After the stationary phase fills the high-speed countercurrent chromatographic column, the speed is adjusted to 800 r / min, and then the mobile phase is injected into the high-speed countercurrent chromatographic column at 2 ml / min.

[0124] After the whole system establishes dynamic equilibrium, 145 ml of the stationary phase flows out, and the stationary phase retention rate is 52%. Take 200 mg of extract D, dissolve it in a mixed solvent consisting of 10 mL of the stationary phase and 10 mL of the mobile phase, and inject the sample solution through the injection valve;

[0125] The fractions were collected, and the target component with a peak time of 69 min-87 min was received according to the retention time of the high-speed countercurrent chromatogram. The collected fractions were concentrated under reduced pressure at 50 KPa and 50° C., and dried in vacuo to obtain the compound chinensin D.

[0126] The purity test was the same as in Example 1, and the result showed that the purity of chinensin D was 90%.

[0127] The structure identification was the same as in Example 1.

[0128] Comparative experiment 1

[0129] The extract C in step 4 of Example 1 was subjected to gel adsorption and column chromatography, using a positive gradient elution. The specific steps are:

[0130] Step 1: Preparation of crude extract of coneflower leaves

[0131] Take 2 kg of fresh cone leaf material, wash and drain, chop into 1-3 cm. Add 90% methanol aqueous solution with a volume concentration of 10 times the weight of the raw material, soak and extract at room temperature for 2 days, filter for the first time, obtain the first filtrate and the first filter residue, the first filtrate is concentrated under reduced pressure at 50 KPa and 50°C to extract; add 90% methanol aqueous solution with a volume concentration of 10 times the weight of the raw material to the first filter residue, soak and extract at room temperature for 2 days, filter for the second time, obtain the second filtrate and the second filter residue, the second filtrate is concentrated under reduced pressure at 50 KPa and 50°C to extract; add 90% methanol aqueous solution with a volume concentration of 10 times the weight of the raw material to the second filter residue, soak and extract at room temperature for 2 days, filter for the third time, obtain the third filtrate, the third filtrate is concentrated under reduced pressure at 50 KPa and 50°C to extract.

[0132] Combine the first extract, the second extract and the third extract, add a small amount of water to dissolve, filter and remove the residue to obtain a crude extract of cone leaf;

[0133] Step 2: Preparation of extracts rich in the compound chinensin D

[0134] The crude extract of cone leaf obtained in step 1 is subjected to column chromatography through gel Sephadex LH-20, using gradient elution. The specification of the gel chromatography column Sephadex LH-20 is 8cm×80cm, and the filler particle size is 100μm-163μm.

[0135] Gradient elution was performed with 2 column volumes of 0%-100% methanol-water solution, with each 20% volume concentration as one gradient, each 0.2L as one fraction, and the elution rate was 2ml / min. The eluate of 60%-80% methanol-water eluent was collected, and after reduced pressure concentration at 50KPa and 50°C, extract B rich in compound chinensin D was obtained;

[0136] Step 3: Further enrichment of compound chinensin D

[0137] The extract B obtained in step 2 was subjected to column chromatography by MCI adsorption, and gradient elution was adopted. The MCI was MCI GELCHP20P, with a specification of 6cm×80cm and a filler particle size of 37μm-75μm and 75μm-150μm. Gradient elution was performed with a methanol aqueous solution with a volume concentration of 0%-100% of 2 times the column volume, with each 10% volume concentration being a gradient, each 0.01L being a flow fraction, and the elution rate being 2ml / min. The eluate of the 80%-100% methanol aqueous eluent was collected, and after reduced pressure concentration at 50KPa and 50°C, extract C was obtained;

[0138] Step 4: Purification of compound chinensin D

[0139] Extract C obtained in step 3 was subjected to column chromatography by gel adsorption, using a positive gradient elution, the gel was Sephadex LH-20, the column size was 4cm×60cm, and the filler particle size was 100μm-163μm. Gradient elution was performed with 0%-100% ethanol aqueous solution with a volume concentration of 2 times the column volume, each 10% volume concentration was a gradient, each 0.008L was a flow fraction, and the elution rate was 2ml / min. The 80%-100% ethanol aqueous eluent portion was collected, and this portion was on the silica gel GF 254 In the thin layer chromatography test, there is no obvious brown spot with Rf value of 0.8 and silica gel GF 254 There are obvious impurities in the middle and lower part of the thin layer plate. The fractions in this part were detected by HPLC, and there were many impurities at different retention times, and there was no obvious chromatographic peak in the target retention time area. It can be seen that the use of column chromatography forward gradient elution in step 4 did not further purify the compound chinensin D. The silica gel GF 254 It is difficult to detect the target compound by plate thin layer chromatography detection, and it is impossible to continue high-speed countercurrent separation to obtain the target compound.

[0140] Comparative experiment 2

[0141] The high-speed countercurrent chromatography separation in step 5 of Example 1 is replaced by high-performance liquid separation, and the specific steps are as follows:

[0142] Step 1: Preparation of crude extract of coneflower leaves

[0143] Take 2 kg of fresh cone leaf material, wash and drain, chop into 1-3 cm. Add 90% methanol aqueous solution with a volume concentration of 10 times the weight of the raw material, soak and extract at room temperature for 2 days, filter for the first time, obtain the first filtrate and the first filter residue, the first filtrate is concentrated under reduced pressure at 50 KPa and 50°C to extract; add 90% methanol aqueous solution with a volume concentration of 10 times the weight of the raw material to the first filter residue, soak and extract at room temperature for 2 days, filter for the second time, obtain the second filtrate and the second filter residue, the second filtrate is concentrated under reduced pressure at 50 KPa and 50°C to extract; add 90% methanol aqueous solution with a volume concentration of 10 times the weight of the raw material to the second filter residue, soak and extract at room temperature for 2 days, filter for the third time, obtain the third filtrate, the third filtrate is concentrated under reduced pressure at 50 KPa and 50°C to extract.

[0144] Combine the first extract, the second extract and the third extract, add a small amount of water to dissolve, filter and remove the residue to obtain a crude extract of cone leaf;

[0145] Step 2: Preparation of extracts rich in the compound chinensin D

[0146] The crude extract of cone leaf obtained in step 1 is adsorbed on gel Sephadex LH-20 for column chromatography, and gradient elution is adopted. The specification of gel chromatography column Sephadex LH-20 is 8cm×80cm, and the filler particle size is 100μm-163μm.

[0147] Gradient elution was performed with 2 column volumes of 0%-100% methanol-water solution, with each 20% volume concentration as one gradient, each 0.2L as one fraction, and the elution rate was 2ml / min. The eluate of 60%-80% methanol-water eluent was collected, and after reduced pressure concentration at 50KPa and 50°C, extract B rich in compound chinensin D was obtained;

[0148] Step 3: Further enrichment of compound chinensin D

[0149] The extract B obtained in step 2 was subjected to column chromatography by MCI adsorption, and gradient elution was adopted. The MCI was MCI GELCHP20P, with a specification of 6cm×80cm and a filler particle size of 37μm-75μm and 75μm-150μm. Gradient elution was performed with a methanol aqueous solution with a volume concentration of 0%-100% of 2 times the column volume, with each 10% volume concentration being a gradient, each 0.01L being a flow fraction, and the elution rate being 2ml / min. The eluate of the 80%-100% methanol aqueous eluent was collected, and after reduced pressure concentration at 50KPa and 50°C, extract C was obtained;

[0150] Step 4: Purification of compound chinensin D

[0151] Extract C obtained in step 3 was subjected to column chromatography by gel adsorption, and reverse gradient elution was adopted. The gel was Sephadex LH-20, the column size was 4cm×60cm, and the filler particle size was 100μm-163μm. Gradient elution was performed with 100%-0% ethanol aqueous solution with a volume concentration of 2 times the column volume, each 10% volume concentration was a gradient, each 0.008L was a flow fraction, and the elution rate was 2ml / min. The 100%-90% ethanol aqueous eluent portion was collected and placed on silica gel GF 254 In the thin layer chromatography test, the elution part with Rf value of 0.8 showing brown dots is extract D. The developing agent is toluene: ethyl formate: formic acid in a volume ratio of 1:7:1, and the developing agent is FeCl3-ethanol solution with a mass concentration of 1%. After the eluent is concentrated under reduced pressure at 50KPa and 50℃, extract D is obtained;

[0152] Step 5: HPLC separation

[0153] The extract D obtained in step 4 was added to 80% methanol aqueous solution for dissolution and filtration. 50 μL of the sample solution to be prepared was taken and injected into the HPLC column for preparation. The preparation parameters of the HPLC were: 9.4 mm × 250 mm Agilent Eclipse XDB C18 chromatographic column, the particle size of the filler was 5 μm, the column temperature was 30 ° C, the mobile phase flow rate was 2.0 mL / min, and the detection wavelength was 227 nm. After repeated conditions exploration, the target compound and the impurities could not be effectively separated. The preparation chromatogram is shown in the figure below. Figure 6 shown.

[0154] Experimental example: Experimental study on the scavenging effect of chinensin D on DPPH free radicals.

[0155] The antioxidant biological activity of the compounds was determined by DPPH free radical scavenging activity assay with Vc as the positive control.

[0156] Weigh appropriate amounts of chinensin D and positive control obtained in Example 1, dissolve them in 95% ethanol, dilute to a series of sample solutions, and set aside. Accurately weigh 2 mg of DPPH powder and dissolve it in 24 ml of 95% ethanol, shake it thoroughly, and obtain a 0.211 mmol / L DPPH solution, store it away from light, and set aside.

[0157] Add 100 μL of sample solution to a 96-well plate, then add 300 μL of the above DPPH solution, shake and mix, react in the dark for 30 minutes, and measure the absorbance at a wavelength of 517 nm. Use 95% ethanol instead of the sample as a blank, and the other steps and reagents are the same as above. Perform the above operation with the same series of Vc concentrations as a positive control experiment.

[0158] Calculate the DPPH free radical scavenging rate at each sample concentration series, and then calculate the IC 50 value.

[0159] Clearance rate = (A blank - (A sample - A background)) / A blank × 100%, wherein A blank is the absorbance value when 95% ethanol is used instead of the sample to be tested; A sample is the absorbance value after adding the sample to be tested; A background is the absorbance value when 95% ethanol is used instead of DPPH solution.

[0160] The experimental results are as follows: IC of Vc in scavenging DPPH free radicals 50 The IC value of chinensin D for scavenging DPPH free radicals is 0.015 mg / ml. 50 It is 0.0375mg / ml.

[0161] The experimental results show that the compound chinensin D can effectively scavenge DPPH free radicals, and its scavenging activity is equivalent to that of the positive control Vc, and it has good antioxidant activity.

[0162] The biological activity of the new compound chinensin D provided by the present invention has not been developed and applied yet, and the present invention provides a new candidate compound for antioxidant products.

[0163] It can be seen that the chinensin D obtained in Example 1 of the present invention has broad application prospects in the development and application of antioxidant products.

[0164] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A phenolic glycoside compound, which is a compound represented by the following formula: 。 2. A method for preparing a phenolic glycoside compound according to claim 1, characterized in that: The following steps are involved: Step 1: Preparation of crude extract of coneflower leaves Take the raw material of cone leaf, wash and drain, chop and extract, filter to remove the residue, and concentrate the filtrate under reduced pressure and low temperature to obtain the crude extract of cone leaf; Step 2: preparing an extract rich in the phenolic glycoside compound chinensin D according to claim 1 The crude extract of the cone leaf obtained in step 1 is subjected to column chromatography by gel adsorption, and gradient elution is used to collect the eluate of 60%-80% alcohol water eluent, and after low-temperature concentration under reduced pressure, an extract B rich in compound chinensin D is obtained; Step 3: Further enrichment of compound chinensinD The extract B obtained in step 2 is subjected to column chromatography after MCI adsorption, and gradient elution is used to collect the eluate of 80%-100% alcohol water eluent, and after low-temperature concentration under reduced pressure, extract C is obtained; Step 4: Purification of compound chinensinD Extract C obtained in step 3 is subjected to column chromatography by gel adsorption, and reverse gradient elution is used to collect the eluent of 100%-90% alcohol water eluent and the eluate with a specific transfer value Rf of 0.8 in thin layer chromatography detection, and concentrated under reduced pressure and low temperature to obtain extract D; Step 5: High-speed countercurrent chromatography The two-phase solvent system is composed of n-hexane, ethyl acetate, methanol and water in a volume ratio of 10:(58-62):30:(38-42), and then the mixture is allowed to stand for separation to obtain an upper and lower phase; the upper phase is used as a stationary phase, and the lower phase is used as a mobile phase; the stationary phase is first injected into a high-speed countercurrent chromatographic column, and after the stationary phase fills the high-speed countercurrent chromatographic column, the rotation speed is adjusted, and the mobile phase is then injected into the high-speed countercurrent chromatographic column; after the whole system establishes a dynamic equilibrium, 200 mg of an extract D is dissolved in a mixed solvent consisting of 10 mL of the stationary phase and 10 mL of the mobile phase, and the mixture is injected through an injection valve; fractions are collected, and according to the retention time of the high-speed countercurrent chromatogram, a target component with an elution time of 75 min-90 min is received, and the compound chinensin D is obtained after low-temperature concentration under reduced pressure and vacuum drying.

3. The preparation method according to claim 2, characterized in that: In step 1, the extraction is carried out by either leaching or ultrasonic extraction.

4. The preparation method according to claim 2, characterized in that: In step 2: The gel is Sephadex LH-20, with a size of 8 cm×80 cm and a filler particle size of 100 μm-163 μm; The eluent is any one of a methanol aqueous solution with a volume concentration of 0%-100% or an ethanol aqueous solution with a volume concentration of 0%-100%; The gradient elution was performed with a gradient of every 20% volume concentration, a fraction of every 0.2 L, and an elution rate of 2 ml / min.

5. The preparation method according to claim 2, characterized in that: In step 3: The MCI is MCI GEL CHP20P, with a specification of 6cm×80cm and a filler particle size of 37μm-75μm, 75μm-150μm; The eluent is any one of a methanol aqueous solution with a volume concentration of 0%-100% and an ethanol aqueous solution with a volume concentration of 0%-100%; The gradient elution was performed with a gradient of every 10% volume concentration, a fraction of every 0.01 L, and an elution rate of 2 ml / min.

6. The preparation method according to claim 2, characterized in that: In step 4: The gel is Sephadex LH-20, with a size of 4 cm×60 cm and a filler particle size of 100 μm-163 μm; The eluent is an ethanol aqueous solution with a volume concentration of 100%-0%; The gradient elution is performed with a gradient of 10% volume concentration, each gradient is 0.3 L, each 0.008 L is a fraction, and the elution rate is 2 ml / min.

7. The preparation method according to claim 2, characterized in that: The conditions for the thin layer chromatography detection are as follows: in step 4, silica gel GF 254 The plate was developed with a volume ratio of toluene: ethyl formate: formic acid of 1:7:1, and the plate was developed in an upright upward direction. The color developer was a 1% FeCl3-ethanol solution, and the elution site with an Rf value of 0.8 showing brown dots was extract D.

8. The preparation method according to claim 2, characterized in that: In steps 1-3, the alcohol aqueous solution used in each step is methanol water or ethanol aqueous solution, and the pressure of the low-temperature concentration under reduced pressure in each step is 35KPa-85KPa, and the temperature is ≤50°C.

9. The preparation method according to claim 2, characterized in that: In step 5: The rotating speed is 800r / min-900r / min; The speed of collecting fractions is to collect one test tube every 3 minutes; The drying temperature is 45° C. and the drying time is 12 h.

10. A use of the phenolic glycoside compound according to claim 1, characterized in that: The application is the application of the phenolic glycoside compound according to claim 1 in the preparation of antioxidant medicines or cosmetics.

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