A method for preparing quercetin-3-o-glucuronide from cyclocarya paliurus

Abstract

The application discloses a method for preparing quercetin-3-O-glucuronide from Cyclocarya paliurus, and belongs to the technical field of Chinese herbal medicine development. The quercetin-3-O-glucuronide is separated from the Cyclocarya paliurus by utilizing the polar characteristics of the quercetin-3-O-glucuronide, and the purity reaches 98%, which indicates that the method for preparing the quercetin-3-O-glucuronide from the Cyclocarya paliurus has a development prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine active extraction, and particularly relates to a method for preparing quercetin-3-O-glucuronide from Cyclocarya paliurus. BACKGROUND

[0002] Quercetin 3-O-glucuronide (Miquelianin) is a flavonoid chemical, a natural ingredient, has various pharmacological activities such as anti-oxidation and anti-atherosclerosis, has good prospects for medical development, and exists in various plants. At present, there is an existing technology for extracting quercetin 3-O-glucuronide from plants: CN118388565A discloses a preparation method and a detection method of quercetin 3-O-glucuronide. The method takes Polygonum aviculare medicinal materials as raw materials, and the process is ethanol extraction, column chromatography and preparation chromatography separation and purification in sequence. Through specific extraction conditions, elution solvents and preparation chromatography conditions, high-purity quercetin 3-O-glucuronide can be prepared. CN104817603A discloses a method for preparing quercetin 3-O-β-D-glucuronide from lotus seed pods. The method takes lotus seed pods as raw materials, and the lotus seed pods are extracted by reflux extraction with water-containing ethanol. The concentrated solution is preliminarily purified by a macroporous adsorption resin, and the elution part of 10% ethanol is directly purified by a reversed-phase silica gel column to prepare a quercetin 3-O-glucuronide monomer compound.

[0003] However, there is no report on extracting quercetin 3-O-glucuronide from Cyclocarya paliurus. SUMMARY

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

[0005] In order to achieve the above purpose, the present application adopts the following technical means:

[0006] The first aspect of the present application provides a method for preparing quercetin 3-O-glucuronide from Cyclocarya paliurus, comprising the following steps:

[0007] S1, Cyclocarya paliurus is immersed in an organic solvent to obtain a leaching solution by filtration; the weight / volume ratio of 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;

[0008] S2, the leaching solution is concentrated and then water is added to prepare a water suspension, the water suspension is extracted, the extract is concentrated to obtain a leaching extract; the extraction solvent is one of methanol, ethanol, chloroform, ethyl acetate and acetone, and is incompatible with the organic solvent in step S1;

[0009] S3, using normal phase silica gel column chromatography, gradient elution was carried out with eluent, compound quercetin-3-O-glucuronide was separated and purified from the extract.

[0010] In some embodiments of the present application, preferably, the organic solvent is methanol, ethanol or a mixture thereof. The polarity of methanol and ethanol is relatively high, and more low-polarity active ingredients can be obtained by using methanol or ethanol for extraction. During extraction, a solvent that is immiscible with the organic solvent used for extraction is selected; as a preferred, when methanol is used for extraction, ethyl acetate is used for extraction; when ethanol is used for extraction, chloroform is used for extraction. In order to improve the extraction yield, the extract is preferably concentrated first, then water is added to prepare a water suspension, and then the water suspension is extracted.

[0011] In some embodiments of the present application, when the eluent is a mixture of dichloromethane and methanol, the mixing ratio of dichloromethane (CH2Cl2) and methanol (CH3OH) includes 100% CH2Cl2, 100:1, 75:1, 50:1, 25:1, 10:1, 5:1, 3:1 and 1:1, followed by elution with methanol (CH3OH) and dichloromethane (CH2Cl2) in a ratio of 3:1, 5:1 and 100% methanol.

[0012] The eluent is preferably a mixture of petroleum ether and ethyl acetate. Both petroleum ether and ethyl acetate have low polarity, which can fully analyze the target compound and remove all or most impurities. Compared with other organic solvents, petroleum ether and ethyl acetate have lower toxicity, which is beneficial to the health of researchers.

[0013] In some embodiments of the present application, in step S1, the weight-to-volume ratio of Cyclocarya pandurata to organic solvent is (1:2)~(2:1).

[0014] In some embodiments of the present application, the extraction method in step S1 is cold soaking method: Cyclocarya pandurata is added to the organic solvent and soaked for 1.5-2.5 weeks. Specifically, the cold soaking method is to put the leaves, rhizomes and barks of Cyclocarya pandurata into a sealed container, add organic solvent, and soak for about 2 weeks with a weight-to-volume ratio of Cyclocarya pandurata to organic solvent of (1:3)~(3:1). During the soaking process, the mixture is shaken frequently. After the active ingredients of the leaves, rhizomes and barks of Cyclocarya pandurata are fully dissolved in the organic solvent, the extract is obtained by filtration.

[0015] In some embodiments of the present application, the extraction method in step S1 is hot reflux extraction method: organic solvent is used for heating extraction in a reflux heating device.

[0016] The hot reflux extraction method is more complicated than the cold soaking method, but has higher extraction efficiency.

[0017] In some embodiments of the present application, 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 as follows: the eluent is gradient eluted according to the volume ratio of petroleum ether / ethyl acetate of 9:1, 4:1, 3:1, 1:1, 1:3, and 1:9, and the fraction eluted when the volume ratio of petroleum ether / ethyl acetate is 4:1 is collected and rotary evaporated to dryness.

[0018] In some embodiments of the present application, the method further comprises a step of further purification.

[0019] In some embodiments of the present application, the purification step is recrystallization / reverse phase silica gel column chromatography or high performance liquid chromatography separation.

[0020] In some embodiments of the present application, the eluent for reverse phase silica gel column chromatography separation is a methanol / water mixture, and the gradient elution is performed according to the volume ratio of 9:1, 4:1, 1:1, 1:3, and 1:9.

[0021] Tests show that the Miquelianin has a strong blood vessel repair effect on a zebrafish blood vessel injury model. The Miquelianin can be used in the preparation of an anti-blood vessel injury drug. The Miquelianin is used as a main active ingredient, and a pharmaceutically acceptable excipient is added to prepare a drug preparation. The preparation can be an injection, a drip infusion, a powder injection, a granule, a tablet, a powder, a granule, a liquid for oral use, a sugar-coated tablet, a film-coated tablet, an enteric-coated tablet, a buccal preparation, a granule, a pill, a plaster, a pill, a spray, a dripping pill, a disintegrating agent, a buccal tablet, a pellet, etc.

[0022] Advantages of the present application

[0023] Compared with the prior art, the present application has the following advantages: the Miquelianin is extracted and separated from the leaves, rhizomes, and barks of Cyclocarya paliurus (Batal) Iljinsk by using the difference in polarity of quercetin-3-O-glucuronide, and the method is simple in operation, high in extraction yield, and high in product purity, and is suitable for large-scale production. The present application also proves that the Miquelianin has a wide range of pharmacological activities, and can be developed for potential medical uses, and used for preparing an anti-blood vessel injury / angiogenesis promoting drug or a prophylactic health food. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is the chemical structure of quercetin-3-O-glucuronide;

[0025] Figure 2H NMR spectrum of quercetin-3-O-glucuronide;

[0026] Figure 3 H NMR spectrum of quercetin-3-O-glucuronide;

[0027] Figure 4 H NMR spectrum of quercetin-3-O-glucuronide. DETAILED DESCRIPTION

[0028] The following examples are presented to demonstrate preferred embodiments of the application. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the application, and can be susceptible to modification. It will be recognized that analog and equivalent techniques can be employed in this application without departing from the spirit and scope of the application. Accordingly, the examples are not intended to limit the scope of the application.

[0029] 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 this application belongs. The materials cited herein are incorporated by reference to the extent that they provide exemplary procedural or other details supplementary to those set forth herein. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the application described herein. Such equivalents are intended to be encompassed by the following claims.

[0030] The technical solutions of the present application will be further described in detail below in conjunction with the specific embodiments.

[0031] Example 1 Preparation of quercetin-3-O-glucuronide

[0032] Take 10 kg of Cyclocarya paliurus (Batal) Iljinsk leaves, rhizomes, barks, and immerse in 10 L of methanol for 2 weeks. Chromatographic grade 60-100 M silica gel is used for sample preparation, and 200-300 M silica gel is used for thin layer chromatography detection. The silica gel is from Qingdao Marine Chemical Co., Ltd., Shandong, China. BORO 3.3 column (ϕ 60 x 600 mm, 4 L, Shanghai Minixi Trading Co., Ltd.) is a silica gel-based column chromatography. The sample is prepared with 70 g of 60-100 m silica and 54.5 g of combined fractions, with a cumulative weight of 125 g. The chromatographic column is eluted with various proportions of solvent mixtures containing dichloromethane (CH2Cl2) and methanol (CH3OH), including 100% CH2Cl2, 100:1, 75:1, 50:1, 25:1, 10:1, 5:1, 3:1, and 1:1, followed by methanol (CH3OH) and dichloromethane (CH2Cl2) in proportions of 3:1, 5:1, and 100% methanol to obtain elution fractions. A SHIMADZU LC-20AT liquid chromatograph is used with a YMC-pack ODS a semi-preparative column (12 nm, 5 μm, ϕ 20 x 250 mm, YMC, Japan) to separate quercetin-3-O-glucuronide using 20-100% MeOH.

[0033] Example 2 Preparation of quercetin-3-O-glucuronide

[0034] Take 10 kg of Cyclocarya paliurus (Batal) Iljinsk leaves, rhizomes, barks, and immerse in 10 L of methanol for 2 weeks. The methanol extract is concentrated and then suspended in 1 L of distilled water. The water suspension is extracted with 1 L of ethyl acetate three times. The ethyl acetate extract is concentrated to obtain 23 g of extract. The sample is 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 is performed with petroleum ether / ethyl acetate mixtures in the following proportions: 9:1, 4:1, 3:1, 1:1, 1:3, 1:9, with 5 L for each gradient elution. TLC detection is performed on the fractions, and the fractions with an elution ratio of 4:1 are collected and concentrated. Quercetin-3-O-glucuronide is obtained by recrystallization with acetone at room temperature.

[0035] Example 3 Preparation of quercetin-3-O-glucuronide

[0036] Take 10 kg Cyclocarya paliurus (Batal) Iljinsk leaves, rhizome, bark, with 5 L ethanol reflux extraction, ethanol extract was concentrated with 1 L distilled water suspension, water suspension with 1 L chloroform extraction 3 times, chloroform extract was concentrated to get extract 234 g; with silica gel (100 mesh, 100 g) sample, normal phase silica gel column chromatography (200-300 mesh, 1 kg; silica gel column size L 500 mm, 120 mm), with volume ratio 9:1, 4:1, 3:1, 1:1, 1:3, 1:9 of petroleum ether / ethyl acetate mixture gradient elution, each gradient elution 5 L; TLC detection fraction, the elution ratio 4:1 fraction was collected, concentrated, recrystallized with acetone at room temperature, quercetin-3-O-glucuronide was obtained.

[0037] Example 4 Preparation of quercetin-3-O-glucuronide

[0038] Take 10 kg Cyclocarya paliurus (Batal) Iljinsk leaves, rhizome, bark, with 5 L ethanol reflux extraction, ethanol extract was concentrated with 1 L distilled water suspension, water suspension with 1 L chloroform extraction 3 times, chloroform extract was concentrated to get extract 234 g; with silica gel (100 mesh, 100 g) sample, normal phase silica gel column chromatography (200-300 mesh, 1 kg; silica gel column size L 500 mm, 120 mm), with volume ratio 9:1, 4:1, 3:1, 1:1, 1:3, 1:9 of petroleum ether / ethyl acetate mixture gradient elution, each gradient elution 5 L; TLC detection fraction, the elution ratio 4:1 fraction was collected, concentrated, recrystallized with acetone at room temperature, quercetin-3-O-glucuronide was obtained.

[0039] Example 5 Preparation of quercetin-3-O-glucuronide

[0040] Take 10 kg of crushed Cyclocarya paliurus (Batal) Iljinsk leaves, rhizomes, and barks, and immerse them in 5 L of a mixture of methanol and ethanol. Concentrate the extract, and then mix it with 100 g of diatomite. Heat the mixture in 5 L of chloroform for 3 times, and then perform normal-phase silica gel column chromatography (200-300 mesh, 1 kg; silica gel column size L 500 mm, Ø 120 mm). Gradient elution is performed with a mixture of chloroform and methanol in a volume ratio of 9:1, 4:1, 3:1, 1:1, 1:3, and 1:9, respectively, and each gradient elution is performed for 5 L. TLC detection is performed on the fractions, and the fractions with an elution ratio of 4:1 are collected and concentrated. The concentrated fractions are subjected to reversed-phase silica gel column chromatography, and elution is performed with a mixture of methanol and water in a volume ratio of 9:1, 4:1, 1:1, 1:3, and 1:9, respectively, and each gradient elution is performed for 1 L. TLC detection is performed on the fractions, and the fractions with an elution ratio of 4:1 are collected and concentrated. Recrystallization is performed with acetone, and the target compound is obtained.

[0041] The target fractions obtained through normal-phase silica gel column chromatography are concentrated, and then purified through high-performance liquid chromatography. The detection wavelength of the high-performance liquid chromatography is 254 nm, and elution is performed with 40%-100% methanol. The elution peaks with a retention time of 44-47 min are collected, and the eluent is concentrated and recrystallized with acetone, and the purified quercetin-3-O-glucuronide is obtained.

[0042] The separation amount and purity of the quercetin-3-O-glucuronide prepared through the methods in Examples 1-5 are detected, and the results are shown in Table 1.

[0043] Table 1 Separation amount and purity of quercetin-3-O-glucuronide obtained through different methods in Examples 1-5

[0044]

[0045] The results show that the separation amount is different when the solvents used in the extraction, extraction, elution, and other steps in the methods are different. After the purification step, the purity of the prepared quercetin-3-O-glucuronide can reach 98%.

[0046] Example 6 Structure identification of quercetin-3-O-glucuronide

[0047] The purity of the prepared compound was identified by HPLC, and the sample with purity greater than 98% was subjected to structure identification by mass spectrometry and nuclear magnetic resonance technology. The nuclear magnetic resonance was determined by Bruker AVANCE DRX-500 NMR Sectrometer, and TMS was used as an internal standard; high-resolution mass spectrometry FTICRMS was determined by Bruker Apex Spectrometer; and electrospray mass spectrometry ESI-MS was determined by Bruker Esquire 3000 plus Spectrometer.

[0048] The chemical structure of the compound Miquelianin is shown in Figure 1 The nuclear magnetic resonance H spectrum of the compound Miquelianin is shown in Figure 2 The nuclear magnetic resonance C spectrum of the compound Miquelianin is shown in Figure 3 The IR spectrum of the compound Miquelianin is shown in Figure 4 The NMR data are shown in Table 2.

[0049] Table 2 NMR data of the compound Quercetin-3-O-glucuronide

[0050]

[0051] Example 7 Analysis of the pro-angiogenic activity of Quercetin-3-O-glucuronide

[0052] PTK787 (a specific vascular endothelial growth factor VEGF receptor kinase inhibitor), ginsenoside Rg1 and the compound were dissolved in distilled water containing 1% DMSO. After 48 h, the selected fluorescent zebrafish eggs were subjected to experimental group setting in a 96-well plate, and the experimental groups were as follows:

[0053] (1) Blank control group: adding 1% DMSO E3 + PTU culture solution;

[0054] (2) Modeling damage group natural repair group: using 0.25 μg / mL PTK787 for modeling damage;

[0055] (3) Positive control group: using 0.25 μg / mL PTK787 for modeling damage;

[0056] (4) Test drug group: using 0.25 μg / mL PTK787 for modeling damage;

[0057] After 72 h, the above 4 groups were observed under a fluorescence microscope for zebrafish damage and the following operations were performed, respectively.

[0058] (2) Modeling damage group natural repair group: only an equal amount of E3 culture medium was added for natural repair;

[0059] (3) Positive control group: 100 μg / mL ginsenoside Rg1 was added for repair;

[0060] (4) Test drug group: 200 μL of the test drug Miquelianin at a concentration of 40 μg / mL was added for repair;

[0061] After 96 hours, each zebrafish was photographed under a fluorescence microscope using methylcellulose as a fixative. This process was repeated six times to verify the angiogenesis activity of these compounds. Image Pro Plus 5.0 was used to process the zebrafish vascular images and measure the angiogenesis length. The results of the six repeated measurements and the comparison of the average values ​​of the four groups are shown in Table 3.

[0062] Table 3. Analysis of the pro-angiogenic activity of Miquelianin zebrafish model

[0063]

[0064] The results showed that, compared with the natural repair group, the ISV length of the blood vessels in the positive control group treated with ginsenoside Rg1 increased by 9.3%. The ISV length of the experimental drug group treated with the compound Miquelianin increased by 18.9% compared with the natural repair group. Compared with the blank control group, the repair effect of ginsenoside Rg1 was not as good as the natural growth in the blank control. The ISV length of the experimental drug group treated with the compound Miquelianin increased by 1.5% compared with the natural growth in the blank control. These results indicate that quercetin-3-O-glucuronide (Miquelianin) has pro-angiogenic pharmacological activity.

[0065] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by this application.

Claims

1. A method for preparing quercetin-3-O-glucuronide from *Cyclocarya paliurus*, characterized in that, Includes the following steps: S1. Extract the Eucommia ulmoides in an organic solvent and filter to obtain the extract; the weight-to-volume ratio of Eucommia ulmoides to the organic solvent is (1:3) to (3:1); the organic solvent is one or a mixture of methanol and ethanol. S2. Concentrate the extract and add water to prepare an aqueous suspension. Extract the aqueous suspension and concentrate the extract to obtain an extract. The extraction solvent in the extraction step is one of chloroform and ethyl acetate. S3. The compound quercetin-3-O-glucuronide was isolated and purified from the extract by silica gel column chromatography with gradient elution using eluent. The silica gel column chromatography used was normal phase silica gel column chromatography; the eluent for normal phase silica gel column chromatography was a mixed solvent of petroleum ether / ethyl acetate; the eluent was used for gradient elution at volume ratios of 9:1, 4:1, 3:1, 1:1, 1:3, and 1:

9. It also includes a further purification step; The purification step is: recrystallization or reversed-phase silica gel column chromatography; The eluent for reversed-phase silica gel column chromatography was a methanol / water mixture; gradient elution was performed at volume ratios of 9:1, 4:1, 1:1, 1:3, and 1:

9.

2. The method according to claim 1, characterized in that: In step S1, the weight-volume ratio of Eucommia ulmoides to 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 cold maceration: add Cyclocarya paliurus to an organic solvent and soak for 1.5-2.5 weeks.

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

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