CN114315931B - Method for preparing quercetin-7-O-L-rhamnoside from phyllanthus emblica - Google Patents

Abstract

The invention discloses a method for preparing quercetin-7-O-L-rhamnoside from phyllanthus emblica, which comprises the following steps: leaching fructus Phyllanthi fruit residue with ethanol to obtain ethanol layer crude extract; dissolving the ethanol layer crude extract with methanol water, separating and purifying with a reversed phase C18 column, and collecting components 77-100 min to obtain component EA-5; dissolving component EA-5 with methanol, separating and purifying with SephadexLH-20, and collecting 3520-4200 min component as component EA-5-3; dissolving component EA-5-3 with methanol, purifying with high performance liquid chromatograph, and collecting 43-45 min component to obtain component A-5-3-10. Therefore, the phyllanthus emblica pomace can be fully recycled, waste is avoided, and the obtained component is A-5-3-10 quercetin-7-O-L-rhamnoside, which is obtained by extracting, separating and purifying phyllanthus emblica pomace for the first time.

Description

Method for preparing quercetin-7-O-L-rhamnoside from phyllanthus emblica

Technical Field

The invention relates to the technical field of active medicines, in particular to a method for preparing quercetin-7-O-L-rhamnoside from phyllanthus emblica.

Background

Emblic leafflower fruit (Phyllanthus emblica l.) is a fruit of the genus Phyllanthus (Phyllanthus) of the family Euphorbiaceae (Euphorbias). Mainly distributed in tropical and subtropical zones; is native to India and distributed in the countries such as Pakistan, malaysia, china, etc.; the yield is the largest in both Fujian and Yunnan provinces at home. The list of "both food and medicine" was put on by the Ministry of health as early as 1998, was received in the pharmacopoeia of the multiple edition of the people's republic of China, and was designated by the United nations health organization as one of 3 health plants promoted and planted worldwide.

The development of phyllanthus emblica mainly comprises the steps of extracting and squeezing fresh fruits, and then processing the fresh fruits into products such as beverage, fine powder, lozenge and the like. During the production process, the waste such as fruit pits, fruit residues or dregs can be generated, and a large amount of sediment, filtering solids and other types of byproducts can be generated during the preparation process of the extract. The data show that only the weight of the phyllanthus emblica fruit residue can account for about 65% of the weight of the original fruit, the drug enterprise can generate nearly hundred tons of phyllanthus emblica drug solid residues each year, if only the solid residues are discarded or buried, the treatment cost is high, the environmental pressure is high, and a large amount of resources are wasted, so that the development of the phyllanthus emblica fruit residue is researched, the environment pollution is prevented, high-added-value products can be produced, the utilization problem of the fruit residue is solved fundamentally, and a sustainable development path for recycling the phyllanthus emblica fruit residue is formed.

Disclosure of Invention

In order to solve the problems, the invention provides a method for preparing quercetin-7-O-L-rhamnoside from phyllanthus emblica, which can fully recycle phyllanthus emblica fruit residues, avoid waste, extract, separate and purify the obtained quercetin-7-O-L-rhamnoside from phyllanthus emblica fruit residues for the first time, and has potential activity and application in the aspects of resisting oxidation, resisting inflammation, resisting aging, protecting liver, eliminating jaundice, resisting hepatitis viruses, promoting blood coagulation and the like.

In order to achieve the above object, an embodiment of the present invention provides a method for preparing quercetin-7-O-L-rhamnoside from phyllanthus emblica, comprising the steps of:

Soaking fructus Phyllanthi fruit residue in 70% ethanol, performing ultrasonic accelerated leaching, repeatedly leaching for 3 times, mixing leaching solutions, and extracting solvent with rotary evaporator to obtain ethanol layer crude extract EA;

dissolving the ethanol layer crude extract EA with 50% methanol water, separating and purifying with a medium-low pressure reverse phase C18 column, setting the flow rate to 30mL/min, eluting with 20% methanol water solution for 60min, changing the mobile phase to 50% methanol water solution, and collecting components 77-100 min to obtain a component EA-5;

Dissolving component EA-5 with methanol, separating and purifying with Sephadex LH-20, setting flow rate to 0.25mL/min, eluting with methanol, and collecting 3520-4200 min component as component EA-5-3;

Dissolving component EA-5-3 with methanol, purifying with high performance liquid chromatograph, and flowing phase: 17% acetonitrile-water mixed solution, and then 0.1% trifluoroacetic acid is added; flow rate: 10mL/min; 200 mu L of sample injection quantity; column temperature 25 ℃; detection wavelength: 254nm and 360nm; collecting the components at 43-45 min to obtain the component A-5-3-10.

According to the method for preparing quercetin-7-O-L-rhamnoside from phyllanthus emblica, which is disclosed by the embodiment of the invention, the phyllanthus emblica pomace is taken as a raw material for separation, the raw material is easy to obtain, and the phyllanthus emblica pomace can be fully recycled, so that waste is avoided; the preparation process is simple and easy to operate; the obtained quercetin-7-O-L-rhamnoside is extracted, separated and purified from phyllanthus emblica pomace for the first time, has potential application in the aspects of resisting oxidation, inflammation, aging, protecting liver, removing jaundice, resisting hepatitis virus, promoting blood coagulation and the like, and has very important significance for researching the active ingredients of natural products of Chinese medicinal materials with homology of medicine and food. Provides important reference value for realizing the comprehensive utilization of phyllanthus emblica and the comprehensive development and utilization of active compounds thereof.

Optionally, fresh phyllanthus emblica fruits are taken, and the phyllanthus emblica fruit residues are obtained after stoning and juicing.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

FIG. 1 is a HPLC analysis chart of compound EA-5-3-10 according to an embodiment of the present invention;

FIG. 2 is a ¹ H-NMR chart of compound EA-5-3-10 according to an example of the invention;

FIG. 3 is a ¹³ C-NMR chart of a compound EA-5-3-10 according to an example of the invention.

Detailed Description

The technical scheme of the invention is described below through specific examples. It is to be understood that the mention of one or more method steps of the present invention does not exclude the presence of other method steps before and after the combination step or that other method steps may be interposed between these explicitly mentioned steps; it should also be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the method steps is merely a convenient tool for identifying the method steps and is not intended to limit the order of arrangement of the method steps or to limit the scope of the invention in which the invention may be practiced, as such changes or modifications in their relative relationships may be regarded as within the scope of the invention without substantial modification to the technical matter.

In order to better understand the above technical solution, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the invention are shown, it should be understood that the invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention will now be described with reference to specific examples, which are intended to be illustrative only and not limiting in any way.

Example 1

A method for preparing quercetin-7-O-L-rhamnoside from phyllanthus emblica comprises the following steps:

Step 1, taking 740g of fresh phyllanthus emblica, removing cores and juicing to obtain 234g of pomace. The phyllanthus emblica pomace is placed in a 5000mL beaker, soaked in 2000mL of 70% ethanol and subjected to ultrasonic accelerated leaching for 1h, repeated leaching is carried out for 3 times, the leaching solutions are combined, and a rotary evaporator is used for pumping out the solvent, so that 19.74g of ethanol layer crude Extract (EA) is obtained.

Step 2, taking 19.74g of the ethanol layer crude Extract (EA) obtained in the step 1, dissolving the extract in 50m of 50% methanol water, using A medium-low pressure reverse phase C18 column (packing: YMC-GE ODS-A-HG, s-50 μm; column length: 4.5X35 cm), setting the flow rate to be 30 m/min, eluting the extract with 20% methanol water solution for 60min, changing the mobile phase to 50% methanol water solution, and collecting the 77 th to 100 th min component (EA-5).

And 3, taking 328.8mg of (EA-5) obtained in the step 2, dissolving the 328.8mg with 10mL of 100% methanol, using Sephadex LH-20 (2.5X1170 cm) as an eluent, passing through a column at a flow rate of 0.25mL/min, and collecting 3520-4200 min components (EA-5-3).

Step 4, collecting 81.3mg of (EA-5-3) obtained in the step 3, dissolving with 2mL of 100% methanol, and purifying with a Huideyi preparation type high performance liquid chromatograph; chromatographic column: YMC-Pack ODS-A, 20X 250mm, s-5 μm, mobile phase: 17% acetonitrile-water mixed solution, and then 0.1% trifluoroacetic acid is added; flow rate: 10mL/min; 200 mu L of sample injection quantity; column temperature 25 ℃; detection wavelength: 254nm and 360nm; the 43 th to 45 th min fractions (EA-5-3-10) were collected and concentrated to give approximately 27.2mg of the compound.

The compound EA-5-3-10 prepared in this example was yellow powder, which was analyzed by HPLC with the following parameters: HPLC model: agilent Technologiees; a detector: 1260 InfinityII; chromatographic column: welch-LP-C18 column, 4.6X250 mm, s-5 μm; liquid phase conditions: mobile phase: acetonitrile-water mixed solution, 5% acetonitrile to 60% acetonitrile, for 35min; flow rate: 1mL/min, column temperature: 35 ℃, sample injection amount: 20.0. Mu.L; detection wavelength: 360nm.

The results are shown in FIG. 1, and FIG. 1 shows a high performance liquid chromatogram of compound EA-5-3-10 at a detection wavelength of 360nm, with a retention time of 16.767min.

Mass spectrometry was performed on compound EA-5-3-10: NMR instrument: bruker Avance II-500 nuclear magnetic resonance apparatus, wherein the ion source is ESI source, positive ion mode, and emission current is 34.6 μA; air curtain gas (CUR) 20.0psi; ion source voltage (IS) 2500V; ion source Temperature (TEM) 420 ℃; interface temperature 260 ℃; atomizing gas (GS 1) 40psi; assist gas (GS 2) 40psi; the mass range is 100-1000m/z.

The results are shown in FIGS. 2 and 3. ¹H-NMR(500MHz,CD₃ OD) data is ：δ:7.36(1H,s,H-2′),7.31(1H,dd,J＝8.3,2.1Hz,H-6′),6.93(1H,d,J＝8.3Hz,H-5′),6.39(1H,s,H-8),6.21(1H,s,H-6),5.36(1H,s,H-1″),3.76(1H,m,H-5″),3.65(1H,m,H-3″),3.43(1H,m,H-2″),3.21(1H,s,H-4″),0.94(3H,d,J＝5.2Hz,Rha-CH₃); as follows

¹³C-NMR(125MHz,CD₃ OD) data is ：δ:150.2(C-2),136.7(C-3),180.1(C-4),163.2(C-5),97.7(C-6),164.1(C-7),95.2(C-8),159.8(C-9),103.9(C-10),123.4(C-1′),116.9(C-2′),146.8(C-3′),146.9(C-4′),115.1(C-5′),122.0(C-6′),100.3(C-1″),72.5(C-2″),72.6(C-3″),73.7(C-4″),72.4(C-5″),18.1(C-6″). as follows

The nuclear magnetic data and mass spectrum data ESI-MS (m/z) of the compound EA-5-3-10 are synthesized, namely 471.7924[ M+Na ] ⁺,449.7961[M+H]⁺ is compared with the literature, and the compound EA-5-3-10 is presumed to be quercetin-7-O-L-rhamnoside; english name: quercetin-7-O-L-rhamnoside; the molecular formula: c ₂₁H₂₀O₁₁; relative molecular weight: 448.1006; the structure is as follows:

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms should not be understood as necessarily being directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Further, one skilled in the art can engage and combine the different embodiments or examples described in this specification.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims (2)

1. A method for preparing quercetin-7-O-L-rhamnoside from phyllanthus emblica, which is characterized by comprising the following steps:

Soaking fructus Phyllanthi fruit residue in 70% ethanol, performing ultrasonic accelerated leaching, repeatedly leaching for 3 times, mixing leaching solutions, and extracting solvent with rotary evaporator to obtain ethanol layer crude extract EA;

dissolving the ethanol layer crude extract EA with 50% methanol water, separating and purifying with a medium-low pressure reverse phase C18 column, setting the flow rate to 30mL/min, eluting with 20% methanol water solution for 60min, changing the mobile phase to 50% methanol water solution, and collecting components 77-100 min to obtain a component EA-5;

Dissolving component EA-5 with methanol, separating and purifying with Sephadex LH-20, setting flow rate to 0.25mL/min, eluting with methanol, and collecting 3520-4200 min component as component EA-5-3;

Dissolving component EA-5-3 with methanol, purifying with high performance liquid chromatograph, and flowing phase: 17% acetonitrile-water mixed solution, and then 0.1% trifluoroacetic acid is added; flow rate: 10mL/min; 200 mu L of sample injection quantity; column temperature 25 ℃; detection wavelength: 254nm and 360nm; collecting the components at 43-45 min to obtain the component A-5-3-10.

2. The method for preparing quercetin-7-O-L-rhamnoside from phyllanthus emblica according to claim 1, wherein fresh phyllanthus emblica fruit is taken, and after stoning and juicing, phyllanthus emblica fruit residues are obtained.