A preparation method of high-purity kaempferol 3-O-(2″-O-apifuranyl)rutin

Abstract

The invention discloses a preparation method of high-purity kaempferol 3-O-(2"-O-apifuranyl)rutin, which comprises the following steps: extracting medicinal materials; injecting the extract into a preparative liquid chromatograph, using acetonitrile-1% glacial acetic acid as a mobile phase, collecting effluent for 10-11 minutes, and obtaining a primary preparative eluent; recovering the solvent from the obtained eluent, dissolving the residue with an appropriate amount of methanol, injecting the obtained solution into a high-performance liquid chromatograph, using acetonitrile-1% glacial acetic acid as a mobile phase, collecting effluent for 5-7 minutes, and obtaining a secondary preparative eluent; recovering the solvent from the eluent, and drying under reduced pressure to obtain a target compound. The method can efficiently, quickly and specifically separate the target compound from senna auriculariae, and the purity is high (>98%), and the yield is 123 times that of the existing method. The target compound can be used as a reference substance for the specific identification of adulterated senna auriculariae in senna, which is of great significance for controlling the quality of senna auriculariae medicinal materials and preparations thereof.

Description

Technical Field

[0001] The invention relates to the technical field of plant extraction, and in particular to a method for preparing high-purity kaempferol 3-O-(2"-O-apifuranyl)rutin. Background Art

[0002] Cassia auriculata L Plant, also known as round-leaved senna, is the dried leaves of Cassia auriculata L., a plant of the leguminous family Lesuminosae, and is often mixed with narrow-leaved senna. The laxative effect and irritation of the dianthrone derivatives sennoside A, B, C, and D in senna are stronger than other laxatives containing anthraquinones, and the toxicity of oral medication is very low. They are often used clinically for acute constipation. The types and contents of dianthrone compounds in senna are much lower than those in senna. Too much addition will affect the quality of senna and affect the effectiveness and safety of medication. Therefore, it is of great significance to find a specific component in senna to identify senna from adulteration. According to mass spectrometry, kaempferol 3-O-(2″-O-apiofuranosyl)rutinoside (see Formula Ⅰ for the structural formula) is a specific component of Senna auriculariae and is not contained in senna leaves. However, there is no commercially available reference substance for this compound.

[0003]

[0004] There is no targeted separation method for the separation and purification of kaempferol 3-O-(2”-O-apiofuranosyl)rutinoside. The existing methods are generally traditional extraction and separation methods, and there are few reports. The compound is often obtained by extensive extraction and separation of plants, without target tracking and extremely low yield. One of the more advanced reports is the use of polysaccharide gel column and semi-preparative liquid phase for separation and purification. The process is to take 0.89 kg of leaves of red melon Cocciniagrandis (L.) Voigt, add 3.25 L of methanol at room temperature, soak for 3 days, and recover the solvent to obtain 27.86 g of crude extract. Take 10 g of the above crude extract, place it on a Sephadex LH-20 column (4×78 cm), and elute with methanol to obtain 22 fractions (F1–F22). Further use semi-preparative C18 HPLC SunFire chromatographic column (19×250mm, 5.0μm) was used to separate all the fractions in the previous step, and three compounds were obtained from the F9 and F10 fractions, one of which was Kaempferol3-O-β-D-apiofuranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→6)]-β-D-glucopyranoside (i.e., kaempferol 3-O-(2″-O-apiofuranosyl)rutinoside, 8.3mg). This method is relatively complicated and uses methanol immersion, which is time-consuming and prone to incomplete extraction. Separation using a dextran gel column requires a large amount of organic solvent, a large workload, and a long time, and there is no clear traceability for the separation of the target component. In addition, the extraction efficiency of this method is extremely low, and the final yield is only 0.0026% (yield % = 8.3 mg / 10 g × 27.86 g / 0.89 kg × 100% = 0.0026%). Summary of the invention

[0005] In order to solve the problems of low yield and complicated process in the existing separation and purification method of kaempferol 3-O-(2"-O-apiofuranosyl)rutinoside, the present invention provides a preparation method of high-purity kaempferol 3-O-(2"-O-apiofuranosyl)rutinoside. The method uses a preparative liquid chromatograph and a high performance liquid chromatograph in combination to separate kaempferol 3-O-(2"-O-apiofuranosyl)rutinoside from a senna auricularia extract, and the recovery rate of each step is as high as more than 80%, and the finally obtained kaempferol 3-O-(2"-O-apiofuranosyl)rutinoside has high purity.

[0006] The present invention is achieved by adopting the following technical solutions.

[0007] A method for preparing high-purity kaempferol 3-O-(2″-O-apifuranyl)rutin comprises the following steps:

[0008] S1. Extraction of medicinal materials: Add 20-25 times the amount of methanol solution to the auricularia senna, ultrasonically treat for 20-40 minutes, filter to obtain an extract, repeat several times to combine the extracts, recover the solvent under reduced pressure to obtain a solid extract, and dissolve the obtained solid extract with 10-15 times the amount of methanol solution to obtain an extract;

[0009] S2. Primary preparation: The extract obtained in step S1 was subjected to liquid chromatography, and the liquid chromatography conditions were: Agilent preparative liquid chromatograph FL-L100G, chromatographic column Agilent ZORBAX SB-C18 (250×9.4mm, 5μm), acetonitrile-1% glacial acetic acid as mobile phase, flow rate of 2.0ml / min, column temperature of 25°C, detection wavelength of 270nm, collecting 10-11 minutes of effluent to obtain a primary preparation eluent;

[0010] S3. Secondary preparation: recover the solvent from the eluent obtained in step S2 to obtain a solid extract, dissolve the solid extract in 5-10 times the amount of methanol, and perform liquid chromatography on the resulting solution. The liquid chromatography conditions are: Shimadzu high performance liquid chromatograph LC-20AD, chromatographic column Thermo ODS HYPERSIL Dim (50×4.6mm, 5μm), acetonitrile-1% glacial acetic acid as the mobile phase, flow rate of 1.5ml / min, column temperature of 40°C, detection wavelength of 270nm, collect the effluent for 5-7 minutes, and obtain the secondary preparation eluent;

[0011] S4. After the solvent is recovered from the eluate obtained in step S3, it is dried under reduced pressure to obtain a kaempferol 3-O-(2″-O-apifuranyl)rutin extract.

[0012] Furthermore, in step S1, the concentration of the methanol solution is 50%-80%.

[0013] Furthermore, in step S1, the methanol extraction is performed 2-3 times.

[0014] Furthermore, in step S2, the volume ratio of acetonitrile-1% glacial acetic acid is 20:80.

[0015] Furthermore, in step S2, the injection volume is 1-2 ml.

[0016] Furthermore, in step S3, the volume ratio of acetonitrile to 1% glacial acetic acid is 11:89.

[0017] Furthermore, in step S3, the injection volume is 10-40 μl.

[0018] Furthermore, in step S4, the conditions for reduced pressure drying are: temperature of 50-60° C. and vacuum degree of -0.05 to -0.1 MPa.

[0019] This application has the following beneficial effects.

[0020] (1) The present invention separates kaempferol 3-O-(2"-O-apiofuranosyl)rutinoside from the extract of Senna auriculariae for the first time, and separates and purifies it by using a two-step liquid phase preparation method, thereby avoiding the problems of traditional column chromatography separation, which requires the use of a large amount of organic solvent for elution, has high pollution, high cost, long time consumption and poor target tracking, and the yield of each step is higher than 80%;

[0021] (2) The present invention solves the problem that kaempferol 3-O-(2"-O-apiofuranosyl)rutinoside has similar physical and chemical properties to other flavonoid impurities in Senna auriculariae and cannot be characteristically purified using conventional organic solvent extraction methods. It also solves the problems of low organic solvent elution yield, high cost, low resolution and poor use effect in traditional column chromatography separation. The two-step preparative liquid phase separation of the present invention is simple to operate, low in cost and good in separation effect. The high performance liquid chromatography (HPLC) spectrum shows that the purity can reach more than 98%, and the obtained compound can be directly used as a standard substance;

[0022] (3) The extraction method of the present invention has a stable process and strong traceability, which is conducive to small-scale laboratory production and can be industrialized and scaled after subsequent improvement. The present invention has been verified by repeated experiments many times, and the two preparative liquid phase separations can separate and purify kaempferol 3-O-(2"-O-apiofuranosyl)rutinoside well and characteristically, and is easy to scale up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the HPLC spectrum of the eluate after the primary preparation in Example 1 of the present invention;

[0024] Figure 2 This is the HPLC spectrum of the eluate after secondary preparation in Example 1 of the present invention. DETAILED DESCRIPTION

[0025] The present patent application is further described below in conjunction with embodiments.

[0026] Unless otherwise specified, the experimental methods used in the following preparation examples and examples are all conventional methods; the materials, reagents, etc. used in the following preparation examples and examples are all commercially available unless otherwise specified.

[0027] Unless otherwise specified, "%" in the present invention refers to volume percentage.

[0028] Example 1

[0029] A method for preparing high-purity kaempferol 3-O-(2″-O-apifuranyl)rutin comprises the following steps:

[0030] (1) Extraction of medicinal materials: 10 g of senna auriculariae medicinal materials (wherein the content of kaempferol3-O-(2"-O-apiofuranosyl)rutinoside is 5.09 mg / g), 20 times (g / mL) of 50% methanol solution is added, ultrasonic extraction is performed for 30 min, and then the extract is filtered to obtain an extract. 20 times (g / mL) of 50% methanol solution is added to the medicinal residue again, and the above steps are repeated twice. The three extracts are combined, the solvent is recovered under reduced pressure to dryness, and 10 times (g / mL) of 50% methanol solution is added to dissolve to obtain a medicinal material extract;

[0031] (2) Primary preparation: The extract obtained in step (1) was injected into an Agilent preparative liquid chromatograph FL-L100G, the chromatographic column was Agilent ZORBAX SB-C18 (250×9.4 mm, 5 μm), acetonitrile-1% glacial acetic acid (20:80) was used as the mobile phase, the flow rate was 2.0 ml / min, the column temperature was 25° C., the detection wavelength was 270 nm, the injection volume was 1 ml, and the effluent was collected for 10-11 minutes to obtain a primary preparation eluent. The HPLC detection spectrum of the primary preparation eluent is shown in Figure 1 (The box shows the chromatographic peak of kaempferol 3-O-(2”-O-apiofuranosyl)rutinoside);

[0032] (3) Secondary preparation: The solvent of the eluate obtained in step (2) was recovered to obtain a solid extract, which was dissolved in 5 times the amount of pure methanol. The solution was then injected into a Shimadzu high performance liquid chromatograph LC-20AD, the chromatographic column was Thermo ODS HYPERSILDim (50×4.6 mm, 5 μm), acetonitrile-1% glacial acetic acid (11:89) was used as the mobile phase, the flow rate was 1.5 ml / min, the column temperature was 40°C, the detection wavelength was 270 nm, the injection volume was 40 μl, and the effluent was collected for 5-7 minutes to obtain a secondary preparation eluate. The HPLC detection spectrum of the secondary preparation eluate is shown in Figure 2 (The box shows the chromatographic peak of kaempferol3-O-(2”-O-apiofuranosyl)rutinoside);

[0033] (4) After the solvent is recovered from the eluate obtained in step (3), the eluate is dried under reduced pressure at 60°C and a vacuum degree of -0.05 to -0.1 MPa to obtain a powdered extract (32.4 mg) of kaempferol 3-O-(2"-O-apiofuranosyl)rutinoside.

[0034] (5) Samples were taken for each of the above steps and the content was tested. The experimental data is shown in Table 1.

[0035] Table 1

[0036]

[0037]

[0038] Note: The content of medicinal materials is calculated based on 10g of extraction; yield % = content in this step / content in the previous step × 100%

[0039] (6) Calculate the yield: Yield % = 32.4 mg / 10 g × 100% = 0.32%.

[0040] Example 2

[0041] A method for preparing high-purity kaempferol 3-O-(2″-O-apifuranyl)rutin comprises the following steps:

[0042] (1) Extraction of medicinal materials: 10 g of senna auriculariae medicinal materials (wherein the content of kaempferol3-O-(2"-O-apiofuranosyl)rutinoside is 5.09 mg / g), 25 times (g / mL) of 50% methanol solution is added, ultrasonic extraction is performed for 40 min, and then the extract is filtered to obtain an extract. 25 times (g / mL) of 50% methanol solution is added to the medicinal residue again, and the above steps are repeated once. The two extracts are combined, the solvent is recovered under reduced pressure to dryness, and 10 times (g / mL) of 50% methanol solution is added to dissolve to obtain a medicinal material extract;

[0043] (2) Primary preparation: The extract obtained in step (1) was injected into an Agilent preparative liquid chromatograph FL-L100G, the chromatographic column was Agilent ZORBAX SB-C18 (250×9.4 mm, 5 μm), acetonitrile-1% glacial acetic acid (20:80) was used as the mobile phase, the flow rate was 2.0 ml / min, the column temperature was 25° C., the detection wavelength was 270 nm, the injection volume was 1 ml, and the effluent was collected for 10-11 minutes to obtain a primary preparation eluent;

[0044] (3) Secondary preparation: The solvent of the eluate obtained in step (2) was recovered to obtain a solid extract, which was dissolved in 5 times the amount of pure methanol. The solution was then injected into a Shimadzu high performance liquid chromatograph LC-20AD, the chromatographic column was Thermo ODS HYPERSILDim (50×4.6 mm, 5 μm), acetonitrile-1% glacial acetic acid (11:89) was used as the mobile phase, the flow rate was 1.5 ml / min, the column temperature was 40°C, the detection wavelength was 270 nm, the injection volume was 20 μl, and the effluent was collected for 5-7 minutes to obtain a secondary preparation eluate;

[0045] (4) After the solvent is recovered from the eluate obtained in step (3), the eluate is dried under reduced pressure at 50°C and a vacuum degree of -0.05 to -0.1 MPa to obtain a powdered extract (29.4 mg) of kaempferol 3-O-(2"-O-apiofuranosyl)rutinoside.

[0046] (5) Samples were taken for each of the above steps and the content was tested. The experimental data is shown in Table 1.

[0047] Table 1

[0048]

[0049]

[0050] Note: The content of medicinal materials is calculated based on 10g of extraction; yield % = content in this step / content in the previous step × 100%

[0051] (6) Calculate the yield: Yield % = 29.4 mg / 10 g × 100% = 0.29%.

[0052] Example 3

[0053] A method for preparing high-purity kaempferol 3-O-(2″-O-apifuranyl)rutin comprises the following steps:

[0054] (1) Extraction of medicinal materials: 10 g of senna auriculariae medicinal materials (wherein the content of kaempferol3-O-(2"-O-apiofuranosyl)rutinoside is 5.09 mg / g), 20 times (g / mL) of 80% methanol solution is added, ultrasonic extraction is performed for 30 min, and then the extract is filtered to obtain an extract. 20 times (g / mL) of 80% methanol solution is added to the medicinal residue again, and the above steps are repeated twice. The three extracts are combined, the solvent is recovered under reduced pressure to dryness, and 10 times (g / mL) of 50% methanol solution is added to dissolve to obtain a medicinal material extract;

[0055] (2) Primary preparation: The extract obtained in step (1) was injected into an Agilent preparative liquid chromatograph FL-L100G, the chromatographic column was Agilent ZORBAX SB-C18 (250×9.4 mm, 5 μm), acetonitrile-1% glacial acetic acid (20:80) was used as the mobile phase, the flow rate was 2.0 ml / min, the column temperature was 25° C., the detection wavelength was 270 nm, the injection volume was 1 ml, and the effluent was collected for 10-11 minutes to obtain a primary preparation eluent;

[0056] (3) Secondary preparation: The solvent of the eluate obtained in step (2) was recovered to obtain a solid extract, which was dissolved in 5 times the amount of pure methanol. The solution was then injected into a Shimadzu high performance liquid chromatograph LC-20AD, the chromatographic column was Thermo ODS HYPERSILDim (50×4.6 mm, 5 μm), acetonitrile-1% glacial acetic acid (11:89) was used as the mobile phase, the flow rate was 1.5 ml / min, the column temperature was 40°C, the detection wavelength was 270 nm, the injection volume was 20 μl, and the effluent was collected for 5-7 minutes;

[0057] (4) After the solvent is recovered from the eluate obtained in step (3), the eluate is dried under reduced pressure at 60°C and a vacuum degree of -0.05 to -0.1 MPa to obtain a powdered extract (30.7 mg) of kaempferol 3-O-(2"-O-apiofuranosyl)rutinoside.

[0058] (5) Samples were taken for each of the above steps and the content was tested. The experimental data is shown in Table 1.

[0059] Table 1

[0060]

[0061]

[0062] Note: The content of medicinal materials is calculated based on 10g of extraction; yield % = content in this step / content in the previous step × 100%

[0063] (6) Calculate the yield: Yield % = 30.7 mg / 10 g × 100% = 0.31%.

[0064] Comparative Example 1

[0065] A method for preparing kaempferol 3-O-(2"-O-apifuranyl)rutin comprises the following steps:

[0066] (1) Extraction of medicinal materials: 0.89 kg of leaves of Cocciniagrandis (L.) Voigt were taken, 3.25 L of methanol was added at room temperature, and the mixture was soaked for 3 days. The solvent was recovered under reduced pressure until dryness, and 27.86 g of crude extract was obtained;

[0067] (2) LH-20 column separation: 10 g of the crude extract was placed on a Sephadex LH-20 column (4 × 78 cm) and eluted with methanol to obtain 22 fractions (F1–F22);

[0068] (3) Semi-preparative liquid phase separation: Further purification was performed using a semi-preparative C18 HPLC SunFire column (19×250 mm, 5.0 μm). Acetonitrile:water (20:80) was used for isocratic elution at a flow rate of 10 mL / min and a total run time of 30 min. The F9 and F10 fractions from the previous step were separated to obtain three compounds, one of which was kaempferol 3-O-(2″-O-apiofuranosyl)rutinoside (8.3 mg).

[0069] (4) Calculate the yield: Yield % = 8.3 mg / 10 g × 27.86 g / 0.89 kg × 100% = 0.0026%.

[0070] For the specific experimental process and results, please refer to LC-QTOF-MS / MS Based Molecular Networking Approach for the Isolation of α-Glucosidase Inhibitors and Virucidal Agents from Coccinia grandis (L.) Voigt, Maharani A. Astiti. et al, Foods 2021, 10, 3041.

[0071] The embodiments of this specific implementation method are all preferred embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Therefore, any equivalent changes made based on the structure, shape, and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing high-purity kaempferol 3-O-(2''-O-apifuranyl)rutin, characterized in that: The following steps are involved: S1. Extraction of medicinal materials: Add 20-25 times the amount of methanol solution to the auricularia senna, ultrasonically treat for 20-40 minutes, filter to obtain an extract, repeat several times to combine the extracts, recover the solvent under reduced pressure to obtain a solid extract, and dissolve the obtained solid extract with 10-15 times the amount of methanol solution to obtain an extract; S2. One-time preparation: The extract obtained in step S1 was subjected to liquid chromatography, and the liquid chromatography conditions were: Agilent preparative liquid chromatograph FL-L100G, the chromatographic column was Agilent ZORBAX SB-C18, the specification was 250×9.4mm, 5μm, acetonitrile-1% glacial acetic acid was used as the mobile phase, the flow rate was 2.0ml / min, the column temperature was 25°C, the detection wavelength was 270nm, and the effluent was collected for 10-11 minutes to obtain a one-time preparation eluent; S3. Secondary preparation: The solvent of the eluent obtained in step S2 is recovered to obtain a solid extract, and then 5-10 times the amount of methanol is used to dissolve the solid extract, and the resulting solution is subjected to liquid chromatography. The liquid chromatography conditions are: Shimadzu high performance liquid chromatograph LC-20AD, the chromatographic column is Thermo ODS HYPERSIL Dim, the specification is 50×4.6mm, 5μm, acetonitrile-1% glacial acetic acid as the mobile phase, the flow rate is 1.5ml / min, the column temperature is 40°C, the detection wavelength is 270nm, and the effluent is collected for 5-7 minutes to obtain the secondary preparation eluent; S4. After the solvent is recovered from the eluate obtained in step S3, it is dried under reduced pressure to obtain a kaempferol 3-O-(2''-O-apifuranyl)rutin extract.

2. The method for preparing high-purity kaempferol 3-O-(2''-O-apifuranyl)rutin according to claim 1, characterized in that: In step S1, the concentration of the methanol solution is 50%-80%.

3. The method for preparing high-purity kaempferol 3-O-(2''-O-apifuranyl)rutin according to claim 1, characterized in that: In step S1, the methanol extraction is performed 2-3 times.

4. The method for preparing high-purity kaempferol 3-O-(2''-O-apifuranyl)rutin according to claim 1, characterized in that: In step S2, the volume ratio of acetonitrile-1% glacial acetic acid is 20:

80.

5. The method for preparing high-purity kaempferol 3-O-(2''-O-apifuranyl)rutin according to claim 1, characterized in that: In step S2, the injection volume is 1-2 ml.

6. The method for preparing high-purity kaempferol 3-O-(2''-O-apifuranyl)rutin according to claim 1, characterized in that: In step S3, the volume ratio of acetonitrile-1% glacial acetic acid is 11:

89.

7. The method for preparing high-purity kaempferol 3-O-(2''-O-apifuranyl)rutin according to claim 1, characterized in that: In step S3, the injection volume is 10-40 μl.

8. The method for preparing high-purity kaempferol 3-O-(2''-O-apifuranyl)rutin according to claim 1, characterized in that: In step S4, the conditions for reduced pressure drying are: temperature of 50-60° C. and vacuum degree of -0.05 to -0.1 MPa.

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