阅读说明：本技术 核桃分心木中酚酸葡萄糖苷类化合物的分离方法及应用 (Separation method and application of phenolic acid glucoside compounds in diaphragma juglandis fructus ) 是由 孙秀兰 徐一达 徐德平 张银志 耿树香 宁德鲁 马婷 于 2020-06-03 设计创作，主要内容包括：本发明公开了核桃分心木中酚酸葡萄糖苷类化合物的分离方法及应用,属于食品技术领域。所述方法包括以下步骤：(1)提取：核桃分心木经粉碎后,采用乙醇提取得到核桃醇提物；(2)分离：将步骤(1)得到的核桃醇提物进行柱层析,并依次采用不同浓度乙醇依次洗脱,反复净化洗脱,分离得到酚酸葡萄糖苷类化合物；所述酚酸葡萄糖苷类化合物为2-羟基-5-甲氧基苯基-O-β-D-吡喃葡萄糖苷,6-羟基萘基-1,2-(2-O-β-D-吡喃葡萄糖苷)和2,6-二羟基萘基-O-β-D-吡喃葡萄糖苷。通过药理实验证实这三种化合物具有良好的抗氧化活性。(The invention discloses a separation method and application of phenolic acid glucoside compounds in diaphragma juglandis, and belongs to the technical field of food. The method comprises the following steps: (1) extraction: crushing walnut diaphragma juglandis, and extracting with ethanol to obtain walnut ethanol extract; (2) separation: performing column chromatography on the walnut alcohol extract obtained in the step (1), sequentially eluting by adopting ethanol with different concentrations, repeatedly purifying and eluting, and separating to obtain phenolic acid glucoside compounds; the phenolic acid glucoside compounds are 2-hydroxy-5-methoxyphenyl-O-beta-D-glucopyranoside, 6-hydroxynaphthyl-1, 2- (2-O-beta-D-glucopyranoside) and 2, 6-dihydroxynaphthyl-O-beta-D-glucopyranoside. Pharmacological experiments prove that the three compounds have good antioxidant activity.)

1. A method for separating phenolic acid glucoside compounds from diaphragma juglandis, which is characterized by comprising the following steps:

(1) extraction: crushing walnut diaphragma juglandis, and extracting with ethanol to obtain walnut ethanol extract;

(2) separation: putting the walnut alcohol extract obtained in the step (1) on an MCI column, eluting with deionized water and 30%, 50% and 70% ethanol in sequence, collecting water elution and 30% ethanol elution parts, and then combining; concentrating the combined eluent under reduced pressure, loading the concentrated eluent into an MCI column, performing gradient elution by using deionized water and ethanol with volume fractions of 10%, 20%, 30% and 50%, and collecting a 10% ethanol elution part; concentrating the 10% ethanol eluate under reduced pressure, loading into ODS column, eluting with deionized water and 5%, 10% and 20% ethanol solution in sequence, collecting, repeatedly purifying and eluting, and separating to obtain phenolic acid glucoside compounds;

the phenolic acid glucoside compound is 2-hydroxy-5-methoxyphenyl-O-beta-D-glucopyranoside, 6-hydroxynaphthyl-1, 2- (2-O-beta-D-glucopyranoside) or 2, 6-dihydroxynaphthyl-O-beta-D-glucopyranoside.

2. The method as claimed in claim 1, wherein the extraction in step (1) is that the walnut is crushed, 75% ethanol is added according to the material-liquid ratio of 1:10-20, the mixture is stirred and extracted for 2-5h at 40-70 ℃, the supernatant is filtered and extracted, the process is repeated for 3 times, and the supernatant obtained after 3 times of decompression and ethanol removal is carried out to obtain the walnut alcohol extract.

3. The method of claim 1, wherein the 2-hydroxy-5-methoxyphenyl-O- β -D-glucopyranoside has the structural formula:

4. the method of claim 1, wherein the 6-hydroxynaphthyl-1, 2- (2-O- β -D-glucopyranoside) has the formula:

5. the method of claim 1, wherein the 2, 6-dihydroxynaphthyl-O- β -D-glucopyranoside has the formula:

6. the application of phenolic acid glucoside compounds in preparing medicaments and health care products for preventing or treating oxidative stress induced diseases is that the phenolic acid glucoside compounds are 2-hydroxy-5-methoxyphenyl-O-beta-D-glucopyranoside, 6-hydroxynaphthyl-1, 2- (2-O-beta-D-glucopyranoside) or 2, 6-dihydroxynaphthyl-O-beta-D-glucopyranoside, and the treatment method of the diseases is not involved.

7. The use according to claim 6, wherein the oxidative stress-induced disease is chronic obstructive pulmonary disease, respiratory inflammation, skin lesions, diabetes, neurodegenerative disease or a tumor disease.

8. The use of claim 6, wherein the dosage form for use comprises powder, tablet, injection, oral liquid or injection.

9. Application of phenolic acid glucoside compounds in preparation of foods or cosmetics with antioxidant functions, wherein the phenolic acid glucoside compounds are 2-hydroxy-5-methoxyphenyl-O-beta-D-glucopyranoside, 6-hydroxynaphthyl-1, 2- (2-O-beta-D-glucopyranoside) or 2, 6-dihydroxynaphthyl-O-beta-D-glucopyranoside.

10. A composition with a treatment or health care effect on oxidative stress related diseases is characterized in that an effective component in the composition is a phenolic acid glucoside compound, and the phenolic acid glucoside compound is one or more of 2-hydroxy-5-methoxyphenyl-O-beta-D-glucopyranoside, 6-hydroxynaphthyl-1, 2- (2-O-beta-D-glucopyranoside) and 2, 6-dihydroxynaphthyl-O-beta-D-glucopyranoside.

Technical Field

The invention relates to a separation method and application of phenolic acid glucoside compounds in diaphragma juglandis, and belongs to the technical field of food.

Background

The walnut is the mature seed of walnut (Juglans regia L.), contains abundant unsaturated fatty acid, is called woody oil king, and has the functions of improving cerebral ischemia, resisting fatigue and improving memory. However, the comprehensive utilization and development of walnut products are still insufficient at present, such as the diaphragma juglandis in walnut byproducts. At present, the literature on the walnut diaphragma juglandis reports that the walnut diaphragma juglandis contains flavonoid compounds such as quercetin, catechin and the like, volatile substances such as menthol, cedrol and the like, and phenolic acid compounds such as gallic acid and the like, and has various active functions of resisting bacteria, resisting oxidation, reducing blood sugar, inhibiting brain function decline, resisting tumors and the like. However, which compounds are specific and their corresponding bioactive functions are not clear, and further intensive studies and confirmation are needed. The method is necessary for improving the comprehensive utilization of walnut by-products, increasing the added value of walnut products and clearing the functional components in the walnut diaphragma juglandis.

Disclosure of Invention

The invention separates three phenolic acid glucoside compounds from the diaphragma juglandis of walnut for the first time, wherein the three phenolic acid glucoside compounds are respectively 2-hydroxy-5-methoxyphenyl-O-beta-D-glucopyranoside, 6-hydroxynaphthyl-1, 2- (2-O-beta-D-glucopyranoside) and 2, 6-dihydroxynaphthyl-O-beta-D-glucopyranoside, and pharmacological experiments prove that the phenolic acid glucoside compounds have good antioxidant activity.

The first purpose of the invention is to provide a method for separating phenolic acid glucoside compounds from walnut diaphragma juglandis, which comprises the following steps:

(1) extraction: crushing walnut diaphragma juglandis, and extracting with ethanol to obtain walnut ethanol extract;

(2) separation: performing column chromatography on the walnut alcohol extract obtained in the step (1), sequentially eluting by adopting ethanol with different concentrations, repeatedly purifying and eluting, and separating to obtain phenolic acid glucoside compounds; the phenolic acid glucoside compound is 2-hydroxy-5-methoxyphenyl-O-beta-D-glucopyranoside, 6-hydroxynaphthyl-1, 2- (2-O-beta-D-glucopyranoside) or 2, 6-dihydroxynaphthyl-O-beta-D-glucopyranoside.

In one embodiment of the present invention, the structural formula of the 2-hydroxy-5-methoxyphenyl-O- β -D-glucopyranoside is:

in one embodiment of the invention, the 6-hydroxynaphthyl-1, 2- (2-O- β -D-glucopyranoside) has the structural formula:

in one embodiment of the invention, the 2, 6-dihydroxynaphthyl-O- β -D-glucopyranoside has the structural formula:

in one embodiment of the invention, the material-to-liquid ratio of the walnut wood separation and the ethanol solution in the step (1) is 1:10-20(M/V, g/mL).

In one embodiment of the present invention, the step (1) extraction is: pulverizing semen Juglandis, adding 75% ethanol at a material-to-liquid ratio of 1:10-20(M/V, g/mL), extracting at 40-70 deg.C under stirring for 2-5h, filtering to obtain supernatant, repeating the above steps for 3 times, and concentrating the supernatant under reduced pressure for 3 times to remove ethanol to obtain semen Juglandis alcoholic extract.

In one embodiment of the invention, in the step (2), the walnut alcohol extract obtained in the step (1) is loaded on an MCI column, and is sequentially eluted by deionized water and 30%, 50% and 70% of ethanol, the water elution part and the 30% ethanol elution part are collected and then combined, the mixture is loaded on the MCI column after being concentrated under reduced pressure, gradient elution is sequentially carried out by deionized water and ethanol with volume fractions of 10%, 20%, 30% and 50%, the 10% ethanol elution part is collected, the mixture is loaded on an ODS column after being concentrated under reduced pressure, elution and collection is sequentially carried out by deionized water and ethanol with volume fractions of 5%, 10% and 20%, and then the phenolic acid glucoside compound is obtained by repeated purification and elution.

The second purpose of the invention is to provide an application of phenolic acid glucoside compounds in preparing medicines and health products for preventing or treating oxidative stress induced diseases, wherein the phenolic acid glucoside compounds are 2-hydroxy-5-methoxyphenyl-O-beta-D-glucopyranoside, 6-hydroxynaphthyl-1, 2- (2-O-beta-D-glucopyranoside) or 2, 6-dihydroxynaphthyl-O-beta-D-glucopyranoside.

In one embodiment of the present invention, the oxidative stress-induced disease is chronic obstructive pulmonary disease, respiratory inflammation, skin lesions, diabetes, neurodegenerative disease or tumor disease.

In one embodiment of the present invention, the phenolic acid glucoside compound is 2-hydroxy-5-methoxyphenyl-O- β -D-glucopyranoside, and has a structural formula:

in one embodiment of the present invention, the phenolic acid glucoside compound is 6-hydroxynaphthyl-1, 2- (2-O- β -D-glucopyranoside), and has a structural formula:

in one embodiment of the present invention, the phenolic acid glucoside compound is 2, 6-dihydroxynaphthyl-O- β -D-glucopyranoside, and has a structural formula:

in one embodiment of the invention, the application is the application of improving the activity of superoxide dismutase SOD and reducing the MDA level of liver malondialdehyde.

The third purpose of the invention is to provide a composition with a treatment or health care effect on oxidative stress related diseases, wherein the effective component of the composition is phenolic acid glucoside compounds, and the composition contains one or more of 2-hydroxy-5-methoxyphenyl-O-beta-D-glucopyranoside, 6-hydroxynaphthyl-1, 2- (2-O-beta-D-glucopyranoside) and 2, 6-dihydroxynaphthyl-O-beta-D-glucopyranoside.

In one embodiment of the present invention, the composition further comprises a carrier and/or an adjuvant.

In one embodiment of the present invention, the dosage form of the composition is any pharmaceutically acceptable dosage form, and the dosage form includes powder, tablet, injection, oral liquid or injection.

The fourth purpose of the invention is to provide an application of phenolic acid glucoside compounds in preparing foods or cosmetics with antioxidant functions, wherein the phenolic acid glucoside compounds are 2-hydroxy-5-methoxyphenyl-O-beta-D-glucopyranoside, 6-hydroxynaphthyl-1, 2- (2-O-beta-D-glucopyranoside) or 2, 6-dihydroxynaphthyl-O-beta-D-glucopyranoside.

The invention has the beneficial effects that:

in the research process of the walnut diaphragma juglandis extract, three phenolic acid glucosides are found in the walnut diaphragma juglandis for the first time, a method for preparing the three compounds from walnuts is provided, and pharmacological experiments prove that the three compounds can improve SOD activity and reduce MDA level and have good antioxidant activity.

Drawings

FIG. 1 is a drawing of Compound 1¹H-NMR spectrum.

FIG. 2 is a drawing of Compound 1¹³C-NMR spectrum.

FIG. 3 is a drawing of Compound 1¹³⁵DEPT-NMR spectrum.

FIG. 4 shows the structural formula of compound 1.

FIG. 5 is a drawing of Compound 2¹H-NMR spectrum

FIG. 6 is a drawing of Compound 2¹³C-NMR spectrum

FIG. 7 is a drawing of Compound 2¹³⁵DEPT-NMR Spectrum

FIG. 8 shows the structural formula of Compound 2.

FIG. 9 is a drawing of Compound 3¹H-NMR spectrum.

FIG. 10 shows Compound 3¹³C-NMR spectrum.

FIG. 11 is a drawing of Compound 3¹³⁵DEPT-NMR spectrum.

FIG. 12 shows the structural formula of compound 3.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.