CN112724419A

CN112724419A - Lignin-carbohydrate and application thereof in inhibiting bisphenol A neurotoxicity

Info

Publication number: CN112724419A
Application number: CN202011451797.9A
Authority: CN
Inventors: 顾杰; 吉贵祥; 黄曹兴; 石利利; 郭敏
Original assignee: Nanjing Forestry University; Nanjing Institute of Environmental Sciences MEE
Current assignee: Nanjing Forestry University; Nanjing Institute of Environmental Sciences MEE
Priority date: 2020-12-09
Filing date: 2020-12-09
Publication date: 2021-04-30
Anticipated expiration: 2040-12-09
Also published as: CN112724419B; AU2021104290A4

Abstract

The invention discloses a lignin-carbohydrate and application thereof in inhibiting bisphenol A neurotoxicity, wherein the preparation method comprises the following steps: grinding wheat straws, extracting with benzene-alcohol to obtain dewaxed powder, and performing ball milling to obtain wheat straw fine powder; extracting with dioxane, extracting the extraction residue with acetic acid, extracting the extractive solution with rotary evaporation to obtain solid, dissolving with dimethyl sulfoxide, adding dichloroethane/ethanol to obtain precipitate, dissolving the precipitate in acetic acid solution, adding acetone to obtain precipitate, and cleaning with acetone, diethyl ether and petroleum ether to obtain lignin-carbohydrate; dispersing the lignin-carbohydrate in distilled water, and quickly passing through a high-temperature reaction tube to obtain another lignin-carbohydrate subjected to hydrothermal treatment. The prepared lignin-carbohydrate has obvious effect on inhibiting BPA neurotoxicity and has great application potential and economic benefit.

Description

Lignin-carbohydrate and application thereof in inhibiting bisphenol A neurotoxicity

Technical Field

The invention relates to the field of lignin materials for inhibiting chemical toxicity, and particularly relates to a lignin-carbohydrate and application thereof in inhibiting bisphenol A neurotoxicity.

Background

Lignin-Carbohydrate (LCC) is a natural polymer consisting of Lignin and carbohydrates through chemical covalent bonds. LCC has many excellent properties due to the structural particularity. The LCC contains a hydrophobic lignin rigid block and a hydrophilic polysaccharide flexible block, and has amphipathy. LCC is formed by connecting polysaccharide and lignin through benzyl ether bond, benzyl ester bond, acetal bond and phenyl glycoside bond, is an amorphous high molecular polymer with physicochemical and biological stability, and can be biodegraded and absorbed. Meanwhile, LCC also has biological activity, such as anti-tumor, antibacterial, antiviral and antioxidant. However, the natural LCC has scarce sources, complex extraction method and serious insufficient yield, and the development and utilization of LCC industrialized products are limited.

Crop straws are quite abundant in China, but the utilization degree of the straws in China is not high, common straw utilization modes mainly include direct burning, returning to the field and serving as animal feed, a small amount of straws serving as a raw material for producing biogas through anaerobic fermentation, and the overall utilization efficiency is still at a lower level. The main component of the straw is lignocellulose, and the LCC prepared from the straw can effectively utilize the lignocellulose in the straw, thereby creating greater utilization value.

Bisphenol a (bpa) is mainly used for synthesizing materials such as Polycarbonate (PC) and epoxy resin, is one of the most widely used industrial compounds in the world, and is widely used in the manufacturing process of food packaging materials, medical instruments, milk bottles, water bottles, spectacle lenses and hundreds of other daily necessities. Therefore, BPA is a substance with which people come into contact in daily life, and as one of environmental hormones (EDCs), safety thereof is also a focus of public attention. A large number of epidemiological and in vitro and in vivo experimental studies show that BPA has neurotoxicity, can affect the development of animal nerve cells, interfere the storage and release of neurotransmitter, cause the abnormality of animal motor ability and social behaviors, and the mechanism of the abnormality can be related to the generation of oxidative stress to cause apoptosis. Therefore, paying attention to and researching the toxic effect brought by BPA exposure, the invention for preparing the medicine or the reagent capable of protecting the neurotoxic effect of BPA has important significance for protecting the health of people.

Disclosure of Invention

In order to solve the problems, the invention researches a method for extracting lignin-carbohydrate (LCC) from wheat straws, and explores the application of the LCC in inhibiting BPA neurotoxicity by utilizing the bioactivity of the LCC. The LCC is prepared by dewaxing, crushing and extracting wheat straws. The preparation method comprises the following steps:

grinding the wheat straws into 20-80 meshes of powder, performing benzene-alcohol extraction on the powder to obtain dewaxed powder, and performing ball milling to obtain fine wheat straw powder.

And step two, extracting the wheat straw fine powder by 96% dioxane, and extracting the extraction residues by acetic acid to obtain an extraction solution.

And step three, performing rotary evaporation on the extract to obtain A solid, adding dimethyl sulfoxide to dissolve the solid, adding dichloroethane/ethanol into the dissolved solution to obtain A precipitate, dissolving the precipitate in an acetic acid solution, adding acetone into the dissolved solution to obtain A precipitate, and cleaning the precipitate for 3 times by using acetone, diethyl ether and petroleum ether to obtain lignin-carbohydrate A (LCC-WS-A).

And step four, dispersing the lignin-carbohydrate A in distilled water, and quickly passing through a high-temperature reaction tube to be kept at 180 ℃ for 10 minutes to obtain the hydrothermally treated lignin-carbohydrate B (LCC-WS-B).

Specifically, in the step one, the volume ratio of the benzene to the alcohol is 2:1, and the extraction is carried out for 12 hours.

Specifically, the concentration of dioxane used in the second step is 96%, and the extraction is carried out for 3 times, wherein the interval is 24 hours each time; the acetic acid used was 50% strength and extracted 3 times with 24 hour intervals.

Specifically, the volume ratio of dichloroethane/ethanol used in the third step is 2:1, and the addition amount is 10 times of the volume of the solution; the acetic acid concentration used was 50%.

It is another object of the present invention to provide the use of said lignin-carbohydrate for inhibiting BPA neurotoxicity. The results show that LCC-WS-A and LCC-WS-B have obvious inhibition effect on nerve damage caused by BPA.

The invention has the advantages that: the lignin-carbohydrate provided by the patent is prepared by adopting wheat straws as raw materials, and the provided preparation method can be used for rapidly preparing lignin-carbohydrate with different carbohydrate ratios, so that a new way for utilizing agricultural waste resources is developed. The prepared lignin-carbohydrate has antioxidant activity, can inhibit nerve damage caused by BPA, provides basis for the subsequent research of the structure-activity relationship between the structure of the lignin-carbohydrate and the capability of inhibiting BPA neurotoxicity, and has great application potential.

Drawings

FIG. 1 shows the 2D-HSQC NMR spectra of LCC-WS-A (A) and LCC-WS-B (B).

Figure 2 effect of different treatments on the locomotor behavior of 6dpf zebrafish larvae. (representative motion trajectories and heat map a, average speed b. compare with control group, # P <0.001, # P <0.01, # P <0.05 compare with bisphenol a treated group, # P <0.001, # P <0.01, # P < 0.05.)

FIG. 3 laser scanning confocal microscopy of the expression intensity of the nerve green fluorescent protein of young transgenic Tg (Huc-GFP) fish. (A: green fluorescence image of zebrafish neurogenesis; B: statistical analysis of fluorescence (B) (n 10.) comparison with control group, # P <0.001, # P <0.01, # P <0.05 comparison with bisphenol A treatment group, # P <0.001, # P <0.01, # P < 0.05.)

Detailed Description

In order to better explain the technical scheme of the invention, the invention is further explained by combining the drawings and the specific embodiments.

Example 1 preparation of Lignin-carbohydrates (LCC)

The wheat straw is ground into 20-80 mesh powder, and the powder is extracted for 12 hours by benzene-alcohol (2:1/v: v) to obtain the dewaxing powder. And ball-milling the dewaxed wheat straw powder for 6 hours to obtain wheat straw fine powder.

The wheat straw fine powder is extracted by 96% dioxane for 3 times (each time is 24 hours), and the extraction residue is extracted by 50% acetic acid for 3 times (each time is 24 hours). The extract is rotary evaporated to obtain solid, and then dimethyl sulfoxide is added for dissolution. The dissolved solution was added with 10-fold volume of dichloroethane/ethanol (2:1/v: v) to obtain a precipitated substance. Dissolving the precipitate in 50% acetic acid, adding 10 times volume of acetone into the solution to obtain precipitate, and washing the precipitate with acetone, diethyl ether and petroleum ether for 3 times to obtain LCC-WS-A.

LCC-WS-A is dispersed in distilled water (solid content is 10%), and is quickly heated in A high-temperature reaction tube at 180 ℃ for 10 minutes to obtain the hydrothermally treated LCC-WS-B.

Through solid mass analysis, the recovery rate of LCC-WS-B obtained by a hydrothermal rapid hydrolysis method reaches 81%, which shows that the recovery rate of LCC obtained by the method is higher.

Example 2 compositional and structural characterization of Lignin-carbohydrate (LCC)

The chemical composition of the LCC samples was analyzed according to standard analytical methods proposed by the national renewable energy laboratory. Sugars in the acid hydrolysate during the analysis were determined using HPAEC (Dionex ICS-5000, USA), PA10 chromatography column (2 mm. times.250 mm) and pulsed amperometric detector.

Structural information of LCC was obtained by quantitative 13C-NMR and 2D-HSQC nuclear magnetic resonance using a Bruker Avance 600MHz nuclear magnetic resonance instrument equipped with a 5mm BBO probe. Quantification by binding¹³C-NMR and 2D-HSQC nuclear magnetic resonance spectroscopy, the number of various lignin substructures (. beta. -O-4,. beta. -and. beta. -5) and LCC bonds (benzyl esters, benzyl ethers and. gamma. -esters) were counted.

As can be seen from Table 1, after the LCC-WS-A is subjected to the hydrothermal rapid hydrolysis method, the carbohydrates in the components can be partially degraded, so that the relative proportion of lignin is increased, and the method can obtain the lignin-carbohydrate with low carbohydrate content. In addition, the degradation degree of polyphenol content in the LCC-WS-B obtained by the hydrolysis method is low, and the content is equivalent to that in the original LCC-A.

TABLE 1 compositional analysis and molecular weights of LCC-WS-A and LCC-WS-B

The results of the analysis of the LCC substructure content and the linkage are shown in Table 2 by the quantitative analysis of the individual signal peaks of the LCC spectrum in FIG. 1. As can be seen from Table 2, after LCC-WS-A is rapidly hydrolyzed by hydrothermal reaction, betA-O-4, betA-betA and betA-5 in the substructure are all degraded, and only betA-O-4 is remained. The content of beta-O-4 is reduced, more active functional groups can be exposed, the oxidation resistance of the nano-silver/zinc oxide/. As carbohydrates degrade, the linkages in LCCs are also partially degraded, with the benzyl ethers being the least stable and completely degraded.

TABLE 2 number of lignin substructures (. betA. -O-4,. betA. -and. betA. -5) and lignin-carbohydrate linkages (benzyl ether, benzyl glycoside and. gammA. -ester linkages) (100Ar) in LCC-WS-A and LCC-WS-B

EXAMPLE 3 Lignin-carbohydrate (LCC) recovery of BPA neurotoxicity assay

Effect of LCC on Zebra Fish behavior

To investigate the effect of LCC on the behavior of bisphenol a (bpa) treated zebrafish, 6 days post-fertilization litterns from 4 treatment groups were randomly placed in 24-well plates and in DanioVision chamber (dutch nordst). After acclimation for 10min, free swimming activity was monitored in continuous visible light (25min) and darkness (25 min). The motor behaviour and average speed during movement were analyzed by ethoVision. As shown in fig. 2, zebrafish behavior was significantly inhibited after BPA addition; and meanwhile, LCC and BPA are added, so that the behavior of the zebra fish is basically not influenced.

Neuroprotective Effect of LCC on Zebra Fish

The nerve Green Fluorescent Protein (GFP) expression intensity of the transgenic Tg (Huc-GFP) juvenile fish was measured by a laser scanning confocal microscope (Zeiss 700B ). The fluorescence intensity was then measured using ImageJ (national institute of health, usa, bessel, maryland). FIG. 3 shows that the fluorescence intensity of the group with BPA was weaker than that of the control group and that of the groups with LCC-WS-A and LCC-WS-B, indicating that LCC has antioxidant activity and protective effect on nerve damage caused by BPA.

The above description is only for the specific embodiments of the present invention, and any person skilled in the art should be able to cover the technical scope of the present invention by equivalent replacement or change according to the technical solution of the present invention.

Claims

1. The lignin-carbohydrate is characterized in that the lignin-carbohydrate is prepared by dewaxing, crushing and extracting wheat straws.

2. The lignin-carbohydrate according to claim 1, characterized in that said lignin-carbohydrate is prepared as follows:

grinding wheat straws into 20-80-mesh powder, performing benzene-alcohol extraction on the powder to obtain dewaxed powder, and performing ball milling to obtain fine wheat straw powder;

secondly, extracting the wheat straw fine powder through dioxane, and extracting extraction residues through acetic acid to obtain an extraction solution;

step three, performing rotary evaporation on the extract to obtain a solid, adding dimethyl sulfoxide to dissolve the solid, adding dichloroethane/ethanol into the dissolved solution to obtain a precipitate, dissolving the precipitate in an acetic acid solution, adding acetone to obtain a precipitate, and cleaning for 3 times by using acetone, diethyl ether and petroleum ether to obtain a lignin-carbohydrate A;

dispersing the lignin-carbohydrate A in distilled water, and quickly passing through a high-temperature reaction tube to keep the temperature at 180 ℃ for 10 minutes to obtain the lignin-carbohydrate B subjected to hydrothermal treatment.

3. The lignin-carbohydrate of claim 2, wherein in step one the benzene-alcohol volume ratio is 2:1 and extraction is for 12 hours.

4. The lignin-carbohydrate composition according to claim 2, wherein the concentration of dioxane used in step two is 96%, and the extraction is performed 3 times, each time at 24-hour intervals; the acetic acid used was 50% strength and extracted 3 times with 24 hour intervals.

5. The lignin-carbohydrate composition according to claim 2, wherein dichloroethane/ethanol is used in a volume ratio of 2:1 in step three, and is added in an amount of 10 times the volume of the dissolution solution; the acetic acid concentration used was 50%.

6. Use of a lignin-carbohydrate according to claim 1, wherein the lignin-carbohydrate is used for inhibiting bisphenol a neurotoxicity.