CN110590871A

CN110590871A - Double caffeoyl component in walnut green husk and preparation process and anti-inflammatory application thereof

Info

Publication number: CN110590871A
Application number: CN201910951891.1A
Authority: CN
Inventors: 司传领; 韩笑; 刘坤; 许家云; 刘慰; 张淑亚; 陈佳宁; 张筱仪
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2019-10-08
Filing date: 2019-10-08
Publication date: 2019-12-20

Abstract

The invention belongs to the field of application of agricultural and forestry wastes and secondary metabolic activity natural products, and particularly relates to a dicaffeoyl glucoside, namely 1, 6-di-O-trans-caffeoyl-3-O-galloyl-beta-D-glucopyranoside, which has never been reported in documents, a preparation process and anti-inflammatory application thereof. It is prepared from green peel of Juglandis of Juglans of Juglandaceae by extracting and separating. The preparation method provided by the invention has the characteristics of simple process, obvious anti-inflammatory activity of the product, easy control of the process, low cost of raw materials and the like.

Description

Double caffeoyl component in walnut green husk and preparation process and anti-inflammatory application thereof

Technical Field

The invention belongs to the field of application of agricultural and forestry wastes and secondary metabolic activity natural products, and particularly relates to a dicaffeoyl glucoside (1, 6-di-O-trans-caffeoyl-3-O-galloyl-beta-D-glucopyranoside) which is never reported in documents, namely 1, 6-di-O-trans-caffeoyl-3-O-galloyl-beta-D-glucopyranoside, and a preparation process and anti-inflammatory application thereof.

Background

Juglans regia (Juglandoceae) Juglans (Juglans) arbor walnut is also called walnut and walnut, and is also called four well-known dry fruits in the world together with almond, cashew nut and hazelnut. The walnut is called the king of the dry fruit, has high nutritional value, for example, arginine, oleic acid, antioxidant substances and the like contained in the walnut can effectively protect the cardiovascular system and can prevent diseases such as coronary heart disease, stroke, senile dementia and the like. The walnut also contains various phytosterols, and can reduce cholesterol in blood. Besides, the walnuts are rich in cellulose, protein, vitamins and minerals, including folic acid, vitamin E, potassium element, magnesium element and omega-3 fatty acid necessary for human body, and are helpful for skin health (Tong, Chinese fruit industry information, 2017, 34 (5): 59). The walnut tree is suitable for being planted in an environment with moderate temperature, and is a typical 'mountain fruit tree' (Guo Yongsheng, etc., in the biotechnology world, 2013, 12: 19). Walnut cultivation in Tianjin has been carried out for hundreds of years, the production area is mainly in mountain areas of Ji county, about millions of walnut trees are produced, and the walnut trees are one of the areas mainly producing walnuts in China. Juglans regia is a special product in Ji county, Tianjin, and according to records in Ji county, the Juglans regia has a history of over 600 years (Jia Aijun, et al, Tianjin agriculture and forestry science, 2019, (01): 28-31 + 38).

Walnut green husks are immature exocarp of walnuts, a large amount of walnut green husks are generated during the harvesting of the walnuts, but the walnut green husks are generally treated as agricultural and forestry wastes, so that great resource waste is caused (Femadez-Agullo A., et al., Industrial Crops and Products, 2013, 42: 126-132). Walnut green seedcase is also called Qinglongyi, is widely used as a traditional Chinese medicine in Japan, Korea and China, has the effects of clearing away heat and toxic materials, relieving pain, treating skin diseases and the like, and has a certain efficacy on treating cancers (Liu J.X., et al, Journal of Chromatography A, 2008, 1190 (1): 80-85; Tabaraki R., et al, Korean Journal of Chemical Engineering, 2014, 31 (4): 676-683; Jiyuban, and the like, Chinese herbal medicines 2004, 35 (10): 71-73; Yuanyu, and the like, traditional Chinese medicine information 2010, 27 (2): 18-20).

Research on chemical components in walnut green seedcase has been partially carried out, and reported components mainly comprise diaryl heptane, naphthoquinone, flavone, phenylpropanoids and the like (Yangyou, and the like, Chinese herbal medicines 2015, 46 (4): 481-485). Qiubeli et al investigated the chemical components of 75% ethanol extract of exocarpium Juglandis Immaturus, and separated 15 compounds, which were identified as epidihydrophaseolic acid, 4-butoxy-5, 8-dihydroxy-3, 4-dihydro-naphthalenone, 4-ethoxy-5, 8-dihydroxy-3, 4-dihydronaphthalenone, myricetin, multisporal ketone, 4S-methoxy-5, 8-dihydroxy-dihydronaphthalenone, 4-methoxy-5-hydroxy-alpha-tetralone, walnut naphthoquinone, 4, 5, 8-trihydroxy-1, 2, 3, 4-tetrahydro-alpha-naphthalenone, malic acid-1-monoethyl ester, malic acid-1-monobutyl ester, Succinic acid, glucoside, 1 alpha, 2 alpha, 4 beta-trihydroxy-1, 2, 3, 4-tetrahydronaphthalene, and L-2-O-methyl-chiral inositol, wherein epidihydrophaseolamic acid, myricetin and L-2-O-methyl-chiral inositol are obtained by separating from walnut green seedcase for the first time (Qiongyingying, and the like, Chinese herbal medicines, 2017, 48 (12): 2385-2389). The sun winter snow and the like are separated and identified 14 compounds from a dichloromethane extraction part of a 95% methanol extract of walnut green peel, wherein the 14 compounds are respectively 5-hydroxy-4-methoxy-1-tetralone, 4, 8-dihydroxy-1-tetralone, 4, 5-dihydroxy-alpha-tetrahydronaphthoquinone, blumenol B, dehydrovomifoliol, megastigm-5-ene-3, 9-diol, juglone B, blumenol C, ryegrass lactone, oleraceone B, syringaresinol, pinoresinol, vanillic acid methyl ester and isovanillic acid. Wherein the compounds 5-hydroxy-4-methoxy-1-tetralone, blumenol B, dehydrovomifoliol, megastigm-5-ene-3, 9-diol, blumenol C, lolium lactone, oleracene B, syringaresinol, pinoresinol and vanillic acid methyl ester are separated from the Qinglongyi for the first time (Sun Dong Xue, etc., Chinese traditional medicine J, 2019, 44 (11): 2278-2282).

However, the chemical components of the walnut green husk, especially the chemical components of the anti-inflammatory secondary metabolism thereof, have not been sufficiently and systematically studied so far. The invention prepares a novel double-trans caffeoyl glucoside compound with obvious anti-inflammatory activity from walnut green seedcase by means of organic solvent extraction and chromatographic separation.

Disclosure of Invention

The invention aims to provide a dicaffeoyl glucoside with remarkable anti-inflammatory activity, namely 1, 6-di-O-trans-caffeoyl-3-O-galloyl-beta-D-glucopyranoside (the compound is correspondingly named as 1, 6-di-O-trans-caffeoyl-3-O-galloyl-beta-D-glucopyranoside in English).

It is another object of the present invention to provide a process for preparing 1, 6-di-O-trans-caffeoyl-3-O-galloyl- β -D-glucopyranoside from green tangerine peel of walnut tree of the genus juglans of the family juglandaceae.

The third purpose of the invention is to provide the anti-inflammatory activity efficacy application of 1, 6-di-O-trans-caffeoyl-3-O-galloyl-beta-D-glucopyranoside.

The technical scheme of the invention is summarized as follows:

a compound of the formula:

the method for preparing 1, 6-di-O-trans caffeoyl-3-O-galloyl-beta-D-glucopyranoside from green peach peel comprises the following steps:

(1) cleaning, drying in the shade, crushing walnut green husks sieved by a 60-mesh sieve, adding an acetone aqueous solution with the volume percentage concentration of 70-85% according to the mass ratio of 1: 1-1: 5, extracting at normal temperature or by heating or microwave-assisted extraction for 1-5 times, each time for 0.5-72 hours, filtering, and concentrating the filtrate under reduced pressure to 2-8% of the original volume to obtain a crude extract;

(2) adding water with the mass 1-4 times of that of the crude extract, stirring, adding n-hexane with the mass 2-5 times of that of the crude extract, extracting for 1-4 times, separating an n-hexane layer, adding ethyl acetate with the mass 2-5 times of that of the crude extract into the residual water layer, extracting for 1-4 times, separating an ethyl acetate layer and a water layer, adding petroleum ether with the mass 2-5 times of that of the crude extract into the residual water layer, extracting for 1-4 times, separating a petroleum ether layer and a final water layer, and concentrating the petroleum ether layer under reduced pressure to obtain a petroleum ether extract;

(3) the petroleum ether extract is subjected to at least one of silica gel column chromatography and vacuum column chromatography by using macroporous resin D101 to prepare the 1, 6-di-O-trans-caffeoyl-3-O-galloyl-beta-D-glucopyranoside.

In the above preparation process, the plant material of exocarpium Juglandis Immaturus is exocarpium Juglandis Immaturus of any one of Juglans of Juglandaceae.

Experiments prove that the molecular formula of the compound of the invention is C₃₁H₂₈O₁₆The chemical name is 1, 6-di-O-trans-caffeoyl-3-O-galloyl-beta-D-glucopyranoside, and the corresponding English name of the compound is 1, 6-di-O-trans-caffeoyl-3-O-galloyl-beta-D-glucopyranoside. The compound is white amorphous powder, the melting point of the compound is 190-192 ℃, and the optical rotation isPositive FAB-MS m/z[M+H]⁺Is 657, M/z [ M + Na ]]⁺Is 679, M/z [ M + K ]]⁺Is 695.

Experiments prove that 1, 6-di-O-trans-caffeoyl-3-O-galloyl-beta-D-glucopyranoside can cause cells to release NO and TNF-alpha to be obviously reduced after RAW264.7 macrophage is induced by LPS, and the NO release amount under the same conditions is smaller than that of a positive control compound NG-methyl-L-arginine. Test results fully prove that the 1, 6-di-O-trans caffeoyl-3-O-galloyl-beta-D-glucopyranoside has obvious anti-inflammatory activity and can be widely applied to the production of anti-inflammatory foods, medicines, health care products and other products.

The preparation method provided by the invention has the characteristics of simple process, obvious anti-inflammatory activity of the product, easy control of the process, low cost of raw materials and the like.

Detailed Description

The invention will be more fully and more readily understood by reference to the following examples, which are given to illustrate the invention more clearly, and are not intended to limit the invention in any way.

Example 1:

preparation of 1, 6-di-O-trans-caffeoyl-3-O-galloyl-beta-D-glucopyranoside from green walnut peel of common walnut

(1) Cleaning walnut green peel of common walnut (Juglans regia, also known as walnut, Chinese walnut, walnut and the like) which is dried in shade and crushed and sieved by a 60-mesh sieve, adding 75% by volume of acetone aqueous solution according to the mass ratio of 1: 3, extracting for 2 times with the assistance of microwave, each time for 1 hour, filtering, and concentrating the filtrate under reduced pressure to 3% of the original volume to obtain a crude extract;

(2) adding water with the mass 2 times of that of the crude extract, stirring, adding n-hexane with the mass 3 times of that of the crude extract, extracting for 2 times, separating an n-hexane layer, adding ethyl acetate with the mass 3 times of that of the crude extract into the residual water layer, extracting for 2 times, separating an ethyl acetate layer and a water layer, adding petroleum ether with the mass 3 times of that of the crude extract into the residual water layer, extracting for 3 times, separating a petroleum ether layer and a final water layer, and concentrating the petroleum ether layer under reduced pressure to obtain a petroleum ether extract;

(3) the petroleum ether extract is subjected to gel column chromatography and macroporous resin D101 vacuum column chromatography to prepare the 1, 6-di-O-trans-caffeoyl-3-O-galloyl-beta-D-glucopyranoside.

Structural identification of the compound:

the fast atom bombardment mass spectrum data show that the [ M + H ] of the compound]⁺Is M/z 657, [ M + Na ]]⁺Is M/z 679, [ M + K]⁺Is m/z 695, i.e. it has a molecular weight of 656. In combination with the compound¹H and¹³obtaining the molecular formula of C through C nuclear magnetic resonance spectrum data₃₁H₂₈O₁₆. TLC thin-layer chromatography spray color development is carried out on ferric trichloride ethanol solution with the mass percent of 1%, and the reaction is dark green, which shows that the compound contains phenolic hydroxyl (Si, et al, Holzforschung, 2016, 70 (1): 39-45) in the molecule. The infrared spectrum of the compound is 3400cm^-1(hydroxy) 1648cm^-1(isoflavone carbonyl) and 1753cm^-1(Acetocarbonyl) has a strong absorption peak.¹In the H NMR spectrum data (Table 1), the characteristic proton peak [ delta 7.10(2H, s, H-2')&6″)]The compound is shown to contain a galloyl group (Tanaka et al, Phytochemistry, 2005, 66, 675-681). And two ABX-type proton resonancesPeaks [ δ 7.20(1H, d, J ═ 2.1Hz, H-2 '), δ 6.83(1H, d, J ═ 7.8Hz, H-5 '), δ 7.01(1H, dd, J ═ 7.8 ')&2.1Hz，H-6′)]And [ δ 7.14(1H, d, J ═ 1.9Hz, H-2 '"), δ 6.75(1H, d, J ═ 8.1Hz, H-5'"), δ 6.94(1H, dd, J ═ 1.9 '"), δ 6.14 (1H, dd, J ═ 1.9Hz, H-5'")&8.1Hz，H-6″′)]Two AB-type proton resonance peaks [ δ 7.61(1H, d, J ═ 15.9Hz, H-7 '), δ 6.35(1H, d, J ═ 15.9Hz, H-8')]And [ δ 7.56(1H, d, J ═ 16.3Hz, H-7 '"), δ 6.25(1H, d, J ═ 16.3Hz, H-8'")]The Compounds are disclosed to contain two trans-caffeoyl groups (Abbasi et al, Chemistry of Natural Compounds, 2014, 50(5), 836-841). In that¹³On the C NMR spectrum (data in Table 1), with 1, 3, 6-tri-O-galloyl-beta-D-glucopyranose (1, 3, 6-tri-O-galloyl-beta-D-glucoside)¹³The difference in C NMR data is that the two trans-caffeoyl groups in the compound replace one galloyl group in 1, 3, 6-tri-O-galloyl- β -D-glucopyranose (Haddock, E.A., et al, journal of the Chemical Society, Perkin Transactions 1, 1982: 2535-2545). In addition, the HMBC pattern of the compound shows the proton [ delta 5.85(1H, d, J ═ 9.0Hz, H-1) connected with the C-1 position of glucosyl radical of the compound]The existence of a long-range correlation with the C-9 'carbon signal (delta 168.7, C-9') on the trans-caffeoyl group, and the proton connected with the C-6 position [ delta 4.30(1H, dd, J ═ 12.2)&6.2Hz)、δ4.51(1H，dd，J＝2.1&12.2Hz)，H-6_aAnd 6_b]The presence of a remote correlation with the carbon signal at the C-9 'position on the transcaffeoyl group (. delta.168.0, C-9'), confirms that this transcaffeoyl group is linked to the C-1 and C-6 positions of the glucosyl group.

Based on the above information, the chemical structural formula of the compound is determined as 1, 6-di-O-trans-caffeoyl-3-O-galloyl-beta-D-glucopyranoside, namely 1, 6-di-O-trans-caffeoyl-3-O-galloyl-beta-D-glucopyranoside. The compound is a novel dicaffeoyl compound through retrieval. The compound is white amorphous powder, the melting point of the compound is 190-192 ℃, and the optical rotation of the compound isAt-16.6 ° (c, 0.5, MeOH). In thin layer chromatography development test, R of the compound is developed by a solvent system with a volume ratio of tert-butyl alcohol-glacial acetic acid-water of 3: 1_fThe value is about 0.49, and R of the compound when developed in a solvent system having a glacial acetic acid-water volume ratio of 6: 94_fThe value is about 0.66.

TABLE 1 preparation of 1, 6-di-O-trans-caffeoyl-3-O-galloyl-beta-D-glucopyranoside¹H (400MHz) and¹³c NMR (100MHz) data (DMSO-d)₆)

Example 2:

preparation of 1, 6-di-O-trans caffeoyl-3-O-galloyl-beta-D-glucopyranoside from green walnut peel of green Juglans sigillata Dode

(1) Cleaning, drying in the shade, crushing and sieving walnut green husks of a 60-mesh-sieve Yangbi bubble walnut tree (Juglans sigillata, also named three-walnut-bubble, Juglans sigillata, deep-engraved-pattern bubble walnut, tea bubble walnut and the like), adding an acetone aqueous solution with the volume percentage concentration of 80% according to the mass ratio of 1: 4, extracting for 4 times at normal temperature for 48 hours each time, filtering, and concentrating the filtrate under reduced pressure to 5% of the original volume to obtain a crude extract;

(2) adding water with the mass 3 times of that of the crude extract, stirring, adding n-hexane with the mass 4 times of that of the crude extract, extracting for 3 times, separating an n-hexane layer, adding ethyl acetate with the mass 4 times of that of the crude extract into the residual water layer, extracting for 3 times, separating an ethyl acetate layer and a water layer, adding petroleum ether with the mass 4 times of that of the crude extract into the residual water layer, extracting for 3 times, separating a petroleum ether layer and a final water layer, and concentrating the petroleum ether layer under reduced pressure to obtain a petroleum ether extract;

(3) the petroleum ether extract is subjected to silica gel column chromatography and is matched with macroporous resin D101 vacuum column chromatography to prepare the 1, 6-di-O-trans-caffeoyl-3-O-galloyl-beta-D-glucopyranoside.

Example 3:

evaluation of anti-inflammatory Activity of 1, 6-Di-O-Trans-Caffeoyl-3-O-Galloylyl-beta-D-glucopyranoside

1. The sample to be evaluated: the purity of the 1, 6-di-O-trans caffeoyl-3-O-galloyl-beta-D-glucopyranoside prepared above was 99.8% by HPLC.

2. The experimental method comprises the following steps: the anti-inflammatory activity of the samples to be evaluated is evaluated by strictly following the procedures specified in the literature (Hu W.C, et al, Food & Function, 2016, 7: 1002-1013):

3. and (3) test results:

(1) the MTT method was used to evaluate the effect (toxic effect) of 1, 6-di-O-trans-caffeoyl-3-O-galloyl- β -D-glucopyranoside on the growth of RAW264.7 macrophages over 24 hours, and the results are shown in Table 2.

TABLE 2 toxic Effect of 1, 6-di-O-trans-caffeoyl-3-O-galloyl- β -D-glucopyranoside on RAW267.4 cells

(2) LPS at a concentration of 1ug/mL and 1, 6-di-O-trans-caffeoyl-3-O-galloyl- β -D-glucopyranoside were evaluated by Griess method for their effect on NO production by RAW264.7 macrophages over 24 hours, and the results are shown in Table 3 (NG-methyl-L-arginine is a positive control group).

TABLE 3 Effect of 1, 6-di-O-trans-caffeoyl-3-O-galloyl- β -D-glucopyranoside on NO release from LPS-induced RAW264.7 macrophages

(3) The effect of LPS at a concentration of 1ug/mL and 1, 6-di-O-trans-caffeoyl-3-O-galloyl- β -D-glucopyranoside acting together on RAW264.7 macrophages for 24 hours on TNF- α production was evaluated by ELISA method, and the results are shown in Table 4.

TABLE 4 Effect of 1, 6-di-O-trans-caffeoyl-3-O-galloyl- β -D-glucopyranoside on TNF- α production by LPS-induced RAW264.7 macrophages

The results show that the 1, 6-di-O-trans-caffeoyl-3-O-galloyl-beta-D-glucopyranoside has no cytotoxicity at the concentration of 0-100 mu M. In the concentration range, 1, 6-di-O-trans-caffeoyl-3-O-galloyl-beta-D-glucopyranoside can cause the NO and TNF-alpha released by cells to be obviously reduced after the RAW264.7 macrophage is induced by LPS, and the NO release amount is smaller than that of a positive control group reagent NG-methyl-L-arginin under the same condition. Test results fully show that the 1, 6-di-O-trans caffeoyl-3-O-galloyl-beta-D-glucopyranoside has obvious anti-inflammatory activity and can be widely applied to products such as anti-inflammatory food, medicines, health care products and the like.

Claims

1. A preparation method of dicaffeoyl glucoside, namely 1, 6-di-O-trans-caffeoyl-3-O-galloyl-beta-D-glucopyranoside, is characterized by sequentially comprising the following steps:

2. The method as claimed in claim 1, wherein the green walnut peel is a green walnut peel of any one of walnut trees belonging to the genus Juglans of the family Juglandaceae.