Method for preparing quercetin-3-O-glucuronide from cyclocarya paliurus

Abstract

The invention discloses a method for preparing quercetin-3-O-glucuronide from cyclocarya paliurus, and belongs to the technical field of Chinese herbal medicine development. According to the method for preparing the quercetin-3-O-glucuronide from the cyclocarya paliurus, the quercetin-3-O-glucuronide is separated from the cyclocarya paliurus by utilizing the polarity characteristic of the quercetin-3-O-glucuronide, and the purity reaches 98%, so that the method for preparing the quercetin-3-O-glucuronide from the cyclocarya paliurus, provided by the invention, has a development prospect.

Description

Technical Field

[0001] The invention relates to the technical field of active extraction of traditional Chinese medicines, and in particular to a method for preparing quercetin-3-O-glucuronide from Cyclocarya paliurus. Background Art

[0002] Quercetin 3-O-glucuronide (Miquelianin) is a flavonoid chemical and a natural ingredient. It has multiple pharmacological activities such as anti-oxidation and anti-atherosclerosis, has good prospects for pharmaceutical development, and exists in a variety of plants. Currently, there is an existing technology for extracting quercetin 3-O-glucuronide from plants: CN118388565A discloses a preparation method and a detection method for quercetin 3-O-glucuronide, which uses Radix Angelicae Pubescentis as a raw material, and the process is ethanol extraction, column chromatography, and preparative chromatography separation and purification in sequence. High-purity quercetin 3-O-glucuronide can be obtained by specific extraction conditions, elution solvents, and preparative chromatography conditions. CN104817603A discloses a method for preparing quercetin-3-O-β-D-glucuronide from lotus pods. The lotus pods are used as raw materials, extracted by reflux extraction with aqueous ethanol, the concentrated solution is preliminarily purified by macroporous adsorption resin, and the 10% ethanol elution portion is directly purified by reverse phase silica gel column to obtain quercetin-3-O-glucuronide monomer compound.

[0003] However, there is no report on the extraction of quercetin-3-O-glucuronide from Cyclocarya paliurus. Summary of the invention

[0004] In order to solve at least one of the above problems, the present invention provides a method for preparing quercetin-3-O-glucuronide from Cyclocarya paliurus.

[0005] In order to achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides a method for preparing quercetin-3-O-glucuronide from Cyclocarya paliurus, comprising the following steps: S1. Extracting the Cyclocarya paliurus in an organic solvent and filtering to obtain an extract; the weight-to-volume ratio of the Cyclocarya paliurus to the organic solvent is (1:3) to (3:1); the organic solvent is a mixture of one or more of methanol, ethanol, chloroform, ethyl acetate and acetone; S2, concentrating the extract and adding water to prepare a water suspension, extracting the water suspension, concentrating the extract, and obtaining an extract; the extraction solvent is one of methanol, ethanol, chloroform, ethyl acetate and acetone, and is incompatible with the organic solvent in step S1; S3. Using normal phase silica gel column chromatography and gradient elution with an eluent, the compound quercetin-3-O-glucuronide is separated and purified from the extract.

[0006] In some specific embodiments of the present invention, preferably, the organic solvent is methanol, ethanol or a mixture thereof. Methanol and ethanol have relatively high polarity, and more active ingredients with medium and low polarity can be obtained by extracting with methanol or ethanol. During extraction, a solvent that is immiscible with the organic solvent used during extraction is selected; preferably, when methanol is used for extraction, ethyl acetate is used for extraction; when ethanol is used for extraction, chloroform is used for extraction. In addition, in order to improve the extraction yield, it is preferred to first concentrate the extract and then add water to prepare a water suspension, and then extract the water suspension.

[0007] In some specific embodiments of the present invention, when the eluent is a mixed solvent of dichloromethane / methanol, dichloromethane (CH 2 Cl 2 ) and methanol (CH 3 OH) contains 100% CH 2 Cl 2 , 100:1, 75:1, 50:1, 25:1, 10:1, 5:1, 3:1 and 1:1, followed by methanol (CH 3 OH) and dichloromethane (CH 2 Cl 2 ) were eluted with 3:1, 5:1 and 100% methanol ratios.

[0008] The eluent is preferably a petroleum ether / ethyl acetate mixture. Both petroleum ether and ethyl acetate have low polarity and can fully chromatograph the target compound to remove all or most impurities. Compared with other organic solvents, petroleum ether and ethyl acetate have low toxicity and are beneficial to the health of researchers.

[0009] In some specific embodiments of the present invention, in step S1, the weight-to-volume ratio of Cyclocarya paliurus to the organic solvent is (1:2) to (2:1).

[0010] In some specific embodiments of the present invention, the extraction method in step S1 is a cold soaking method: add Cyclocarya paliurus to an organic solvent and soak for 1.5-2.5 weeks. Specifically, the cold soaking method is to put the leaves, rhizomes, and barks of Cyclocarya paliurus into a sealed container, add an organic solvent, and the weight volume ratio of Cyclocarya paliurus to the organic solvent is (1:3) to (3:1), soak for about 2 weeks, shake frequently during the period, and after the active ingredients of the leaves, rhizomes, and barks of Cyclocarya paliurus are fully dissolved in the organic solvent, filter to obtain the extract.

[0011] In some specific embodiments of the present invention, the extraction method in step S1 is a hot reflux extraction method: an organic solvent is used for heating extraction in a reflux heating device.

[0012] The hot reflux extraction method is more complicated than the cold soaking method, but it has high extraction efficiency.

[0013] In some specific embodiments of the present invention, the silica gel column chromatography is normal phase silica gel column chromatography, and when a mixed solvent of petroleum ether / ethyl acetate is used as the eluent, the chromatography method is: the eluent is gradient eluted according to the petroleum ether / ethyl acetate volume ratio of 9:1, 4:1, 3:1, 1:1, 1:3, and 1:9, and the fraction eluted when the petroleum ether / ethyl acetate volume ratio is 4:1 is collected, and the fraction is rotary evaporated to dryness.

[0014] In some specific embodiments of the present invention, a further purification step is also included.

[0015] In some specific embodiments of the present invention, the purification step is: recrystallization / reverse phase silica gel column chromatography or high performance liquid chromatography separation.

[0016] In some specific embodiments of the present invention, the eluent for reverse phase silica gel column chromatography separation is a methanol / water mixture; gradient elution is performed at volume ratios of 9:1, 4:1, 1:1, 1:3, and 1:9.

[0017] After testing, the Miquelianin has a strong vascular repair effect on the zebrafish vascular injury model. The application of Miquelianin in the preparation of anti-vascular injury drugs is made by adding pharmaceutically acceptable excipients with the Miquelianin of the present invention as the main active ingredient, and can be made into a pharmaceutical preparation according to the preparation preparation method recorded in pharmacy. The preparation can be an injection, a drip solution, a powder injection, a granule, a tablet, a granule, a powder, an oral liquid, a sugar-coated tablet, a film-coated tablet, an enteric-coated tablet, a lozenge, a granule, a pill, an ointment, a pill, a spray, a drop pill, a disintegrant, an orally disintegrating tablet, a pellet, etc.

[0018] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: the present invention utilizes the polarity difference of quercetin-3-O-glucuronide to extract and separate Miquelianin from the leaves, rhizomes and barks of Cyclocarya paliurus (Batal) Iljinsk, and the method is simple to operate, has a high extraction yield, and has a high product purity, and is suitable for large-scale production. The present invention also confirms that Miquelianin has a wide range of pharmacological activities, and its potential medical use can be developed for the preparation of anti-vascular injury / angiogenesis-promoting drugs or preventive health foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the chemical structure of quercetin-3-O-glucuronide; Figure 2 is the H NMR spectrum of quercetin-3-O-glucuronide; Figure 3 is the C NMR spectrum of quercetin-3-O-glucuronide; Figure 4 This is the IR spectrum of quercetin-3-O-glucuronide. DETAILED DESCRIPTION

[0020] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0022] The technical solution of the present application is further described in detail below in conjunction with specific implementation methods.

[0023] Example 1 Preparation of Quercetin-3-O-glucuronide 10 kg of leaves, rhizomes, and barks of Cyclocarya paliurus (Batal) Iljinsk were extracted with 10 L of methanol for 2 weeks. Chromatographic grade 60-100 M silica was used for sample preparation and 200-300 M silica was used for thin layer chromatography. The silica gel was from Qingdao Ocean Chemical Co., Ltd., Shandong, China. BORO 3.3 column (ϕ 60×600 mm, 4L Shanghai Meizhixi Trading Co., Ltd.) was used for silica gel column chromatography. The sample was prepared with 70 g of 60-100 M silica and 54.5 g of combined fractions, with a cumulative weight of 125 g. The column was heated with dichloromethane (CH 2 Cl 2 ) and methanol (CH 3 OH) were eluted with various solvent mixtures, including 100% CH 2 Cl2 , 100:1, 75:1, 50:1, 25:1, 10:1, 5:1, 3:1 and 1:1, followed by methanol (CH 3 OH) and dichloromethane (CH 2 Cl 2 ) was eluted with 3:1, 5:1 and 100% methanol to obtain elution fractions. Quercetin-3-O-glucuronide was separated by SHIMADZU LC-20AT liquid chromatography using a YMC-pack ODS a semi-preparative column (12 nm, 5 μm, ϕ 20×250 mm, YMC, Japan) with 20-100% MeOH.

[0024] Example 2 Preparation of Quercetin-3-O-Glucuronide 10 kg of leaves, rhizomes and barks of Cyclocarya paliurus (Batal) Iljinsk were extracted with 10 L of methanol for 2 weeks. The methanol extract was concentrated and suspended with 1 L of distilled water. The water suspension was extracted three times with 1 L of ethyl acetate. The ethyl acetate extract was concentrated to obtain 23 g of extract. The sample was mixed with silica gel (100 mesh, 100 g) and subjected to normal phase silica gel column chromatography (200-300 mesh, 1 kg; silica gel column size L 500 mm, Ø 120 mm). Gradient elution was performed with a petroleum ether / ethyl acetate mixture with a volume ratio of 9:1, 4:1, 3:1, 1:1, 1:3, and 1:9, respectively, with each gradient elution of 5 L. The fractions were detected by TLC, and the fractions with an elution ratio of 4:1 were collected, combined, concentrated, and recrystallized with acetone at room temperature to obtain quercetin-3-O-glucuronide.

[0025] Example 3 Preparation of Quercetin-3-O-Glucuronide 10 kg of leaves, rhizomes and barks of Cyclocarya paliurus (Batal) Iljinsk were extracted with 5 L of ethanol under reflux. The ethanol extract was concentrated and suspended with 1 L of distilled water. The water suspension was extracted three times with 1 L of chloroform. The chloroform extract was concentrated to obtain 234 g of extract. The sample was mixed with silica gel (100 mesh, 100 g) and subjected to normal phase silica gel column chromatography (200-300 mesh, 1 kg; silica gel column size L 500 mm, Ø 120 mm). Gradient elution was performed with a petroleum ether / ethyl acetate mixture with a volume ratio of 9:1, 4:1, 3:1, 1:1, 1:3, and 1:9, respectively, with each gradient elution of 5 L. The fractions were detected by TLC, and the fractions with an elution ratio of 4:1 were collected, combined, concentrated, and recrystallized with acetone at room temperature to obtain quercetin-3-O-glucuronide.

[0026] Example 4 Preparation of Quercetin-3-O-Glucuronide 10 kg of crushed leaves, rhizomes and barks of Cyclocarya paliurus (Batal) Iljinsk were extracted with 5 L of methanol / ethanol mixture, and the methanol / ethanol extract was concentrated to obtain 366 g of extract, mixed with 100 g of diatomaceous earth, hot-soaked with 5 L of chloroform for 3 times, and subjected to normal phase silica gel column chromatography (200-300 mesh, 1 kg; silica gel column size L 500 mm, Ø 120 mm), and gradient eluted with petroleum ether / ethyl acetate mixtures in volume ratios of 9:1, 4:1, 3:1, 1:1, 1:3, and 1:9, respectively, with each gradient elution of 5 L; TLC detection fractions, the fractions with an elution ratio of 4:1 were collected, combined, concentrated, and then recrystallized with acetone / n-hexane (volume ratio of 1:1) at room temperature to obtain quercetin-3-O-glucuronide.

[0027] Example 5 Preparation of Quercetin-3-O-Glucuronide 10 kg of crushed leaves, rhizomes and barks of Cyclocarya paliurus (Batal) Iljinsk were extracted with 5 L of methanol / ethanol mixture, and the extract was concentrated to obtain 350 g of extract. The sample was mixed with 100 g of diatomaceous earth, hot-soaked with 5 L of chloroform three times, and subjected to normal phase silica gel column chromatography (200-300 mesh, 1 kg; silica gel column size L 500 mm, Ø 120 mm), and gradient elution was performed with chloroform / methanol mixtures of volume ratios of 9:1, 4:1, 3:1, 1:1, 1:3, and 1:9, respectively, with 5 L of each gradient elution; the fractions were detected by TLC, and the fractions with an elution ratio of 4:1 were collected, combined, and concentrated. The concentrated fractions were subjected to reverse phase silica gel column chromatography. The eluents were methanol / water mixtures with volume ratios of 9:1, 4:1, 1:1, 1:3, and 1:9, respectively. 1 L was used for each gradient elution. Each fraction was detected by TLC. The fractions with an elution ratio of 4:1 were combined and concentrated, and recrystallized with acetone to obtain the target compound.

[0028] The target fraction obtained by normal phase silica gel column chromatography was concentrated and then purified by high performance liquid chromatography: the detection wavelength of the high performance liquid chromatography was 254 nm, the eluent was 40%-100% methanol, and the elution peak with a retention time of 44-47 min was collected. The eluate was combined and concentrated, and recrystallized with acetone to obtain purified quercetin-3-O-glucuronide.

[0029] The separation amount and purity of quercetin-3-O-glucuronide prepared by the methods in Examples 1 to 5 were tested, and the results are shown in Table 1.

[0030] Table 1 The separation amount and purity of quercetin-3-O-glucuronide obtained by different methods in Examples 1-5

[0031] The results showed that when the solvents used in the extraction, elution and other steps in the method were different, the separation amount was different. After the purification step, the purity of the prepared quercetin-3-O-glucuronide could reach 98%.

[0032] Example 6 Structural Identification of Quercetin-3-O-Glucuronide The purity of the prepared compounds was identified by HPLC, and the structures of samples with a purity greater than 98% were identified by mass spectrometry and nuclear magnetic resonance techniques. The nuclear magnetic resonance was determined by Bruker AVANCE DRX-500 NMR Sectrometer with TMS as the internal standard; the high-resolution mass spectrometry FTICRMS was determined by Bruker Apex Spectrometer; and the electrospray mass spectrometry ESI-MS was determined by Bruker Esquire 3000 plus Spectrometer determination.

[0033] The chemical structure of the compound Miquelianin is as follows Figure 1 As shown, the NMR H spectrum of the compound Miquelianin is as follows Figure 2 As shown, the C NMR spectrum of compound Miquelianin is as follows Figure 3 As shown, the IR spectrum of compound Miquelianin is as follows Figure 4 The NMR data are shown in Table 2.

[0034] Table 2 NMR data of compound quercetin-3-O-glucuronide

[0035] Example 7 Analysis of the angiogenic activity of quercetin-3-O-glucuronide PTK787 (specific VEGF receptor kinase inhibitor), ginsenoside Rg1 and compounds were dissolved in distilled water containing 1% DMSO. After 48 hours, the selected fluorescent zebrafish eggs were placed in a 96-well plate to set up the experimental groups, which were: (1) Blank control group: E3+PTU culture medium supplemented with 1% DMSO; (2) Model injury group and natural repair group: 0.25 μg / mL PTK787 was used for model injury; (3) Positive control group: 0.25 μg / mL PTK787 was used to create injury models; (4) Experimental drug group: 0.25 μg / mL PTK787 was used to create injury models; After 72 h, the above four groups were observed for zebrafish damage under a fluorescence microscope and performed the following operations respectively; (2) Model injury group and natural repair group: only an equal amount of E3 culture medium was added to allow natural repair; (3) Positive control group: Add 100 μg / mL ginsenoside Rg1 for repair; (4) Experimental drug group: Add 200 μL of the experimental drug Miquelianin at a concentration of 40 μg / mL for repair; After 96 hours, methylcellulose was used as a fixative, and each zebrafish was photographed under a fluorescence microscope. The above operation was repeated 6 times to determine and verify the angiogenic activity of these compounds. Image Pro Plus5.0 was used to process the zebrafish vascular images and measure the length of angiogenesis. The results of the 6-repetition measurement and the comparison of the average values ​​of the four groups are shown in Table 3.

[0036] Table 3 Analysis of Miquelianin angiogenic activity in zebrafish model

[0037] The results showed that the positive control group repaired with ginsenoside Rg1 increased the length of vascular ISV by 9.3% compared with the natural repair group of model injury, and the experimental drug group repaired with compound Miquelianin test drug increased the length of vascular ISV by 18.9% compared with the natural repair group of model injury. Compared with the blank control group, the effect of repairing with ginsenoside Rg1 was not as good as the natural growth in the blank control, and the experimental drug group repaired with compound Miquelianin test drug increased the length of vascular ISV by 1.5% compared with the natural growth in the blank control. The results showed that quercetin-3-O-glucuronide (Miquelianin) has pharmacological activity to promote angiogenesis.

[0038] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. A method for preparing quercetin-3-O-glucuronide from Cyclocarya paliurus, characterized in that: The steps include: S1. Extracting the Cyclocarya paliurus in an organic solvent and filtering to obtain an extract; the weight-to-volume ratio of the Cyclocarya paliurus to the organic solvent is (1:3) to (3:1); the organic solvent is a mixture of one or more of methanol, ethanol, chloroform, ethyl acetate and acetone; S2, concentrating the extract and adding water to prepare a water suspension, extracting the water suspension, concentrating the extract, and obtaining an extract; the extraction solvent in the extraction step is one of methanol, ethanol, chloroform, ethyl acetate and acetone, and the extraction solvent is incompatible with the organic solvent in step S1; S3. Using silica gel column chromatography and gradient elution with an eluent, the compound quercetin-3-O-glucuronide is separated and purified from the extract.

2. The method according to claim 1, characterized in that: In step S1, the weight-to-volume ratio of Cyclocarya paliurus to the organic solvent is (1:2) to (2:1).

3. The method according to claim 1, characterized in that: The extraction method in step S1 is a cold soaking method: adding Cyclocarya paliurus to an organic solvent and soaking for 1.5-2.5 weeks.

4. The method according to claim 1, characterized in that: The extraction method in step S1 is a hot reflux extraction method: an organic solvent is used for heating extraction in a reflux heating device.

5. The method according to claim 1, characterized in that: The silica gel column chromatography adopts normal phase silica gel column chromatography; the eluent of normal phase silica gel column chromatography is a mixed solvent of dichloromethane / methanol, or a mixed solvent of petroleum ether / ethyl acetate.

6. The method according to claim 1, characterized in that: Further purification steps may also be included.

7. The method according to claim 6, characterized in that: The purification step is: recrystallization / reverse phase silica gel column chromatography or high performance liquid chromatography separation.

8. The method according to claim 7, characterized in that: The eluent for reverse phase silica gel column chromatography separation was a methanol / water mixture; gradient elution was performed at volume ratios of 9:1, 4:1, 1:1, 1:3, and 1:9.

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