A method for separating polyphenolic compounds in a lignin depolymerization product, the polyphenolic compounds and applications thereof

Abstract

The present application belongs to the technical field of depolymerization separation, and particularly relates to a method for separating polyphenolic compounds from lignin depolymerization products, the polyphenolic compounds and applications thereof. The lignin depolymerization products are mixed with an extraction liquid for extraction, the extraction products are mixed with the extraction liquid for extraction, then the obtained upper extraction liquid is cooled for crystallization, and countercurrent chromatography separation is performed to remove phenol in the depolymerization liquid. On this basis, the polyphenolic compounds are obtained through high-pressure preparation separation technology. The polyphenolic compounds obtained by the present application are functional components, and are new antioxidant active compositions. The polyphenolic compounds have high free radical scavenging rate and excellent antioxidant activity. Meanwhile, the polyphenolic compounds are lignin polyphenolic nanomicelles with fluorescence performance. The micelles can obtain strong fluorescence under a relatively short excitation wavelength. The polyphenolic compounds also have excellent anticancer activity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of depolymerization separation, and particularly relates to a separation method of polyphenol compounds in lignin depolymerization products, the polyphenol compounds and application thereof. BACKGROUND

[0002] Lignin is a kind of polyphenol polymer, which can be used as a natural antioxidant. Compared with traditional synthetic antioxidants, lignin has the advantages of rich resources, low toxicity and biodegradability. It is generally believed that lignin has good antioxidant activity, and at the same time, lignin is the main source of plant cell wall fluorescence and a kind of natural high molecular fluorescent material. However, due to the high molecular weight and complex structural characteristics of natural lignin, the activity is low when lignin is applied, which hinders its large-scale application in industry. At present, the research mainly focuses on the research on lignin model compounds, so as to establish the relationship between the antioxidant activity, fluorescent properties and structure of lignin. However, there are few reports on the activity of components separated after lignin depolymerization.

[0003] A process for separating mild acid catalytic lignin depolymerization products is disclosed in Chinese patent No. 201610643766.0. After depolymerization with phenol as a solvent, the above lignin depolymerization products are extracted by using solvent continuous extraction method or simultaneous distillation extraction method. Although the obtained depolymerization product has high yield, the depolymerization products are all polyphenol compounds, which are similar in structure and properties to phenol monomers, thereby limiting the effective separation and performance research of the depolymerization products. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a separation method of polyphenol compounds in lignin depolymerization products, polyphenol compounds and application thereof. In the present application, phenol in lignin depolymerization products is removed first, and then separation is carried out, so that the obtained polyphenol compounds have antioxidant activity, fluorescent properties and anticancer activity.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0006] The present application provides a separation method of polyphenol compounds in lignin depolymerization products, wherein the polyphenol compounds include one or more of components A3, A4, A5 and A6; and the separation method comprises the following steps:

[0007] After the lignin depolymerization products are mixed with the extraction liquid for extraction, the supernatant is concentrated to obtain an extraction product; the lignin depolymerization products are obtained by mixing lignin, phenol and acid catalysts for depolymerization;

[0008] The extraction product is mixed with the extraction liquid for extraction to obtain upper extraction liquid and lower paste sample;

[0009] cooling and crystallizing the upper layer extract, and subjecting the obtained liquid to countercurrent chromatography separation to obtain a phenol-removed extract;

[0010] mixing the phenol-removed extract and the lower layer paste sample, and subjecting the mixture to high-pressure preparative chromatography separation, collecting fractions A1 to A6 in sections, wherein the high-pressure preparative chromatography separation is performed at a flow rate of 70 mL / min and using a mobile phase of a formic acid aqueous solution-methanol, the formic acid aqueous solution having a volume fraction of 0.1%, and the gradient elution program is as shown in the following table:

[0011]

[0012] fractions A1 collected at 3-8 min and A2 collected at 9-16 min are discarded, fraction A3 is collected at 16-23 min, fraction A4 is collected at 23-30 min, fraction A5 is collected at 30-40 min, and fraction A6 is collected at 40-48 min.

[0013] Preferably, the extract solution comprises an organic solvent and water, and the organic solvent comprises one or more of ethyl acetate, tetrahydrofuran, and acetonitrile.

[0014] Preferably, the extract solution is a mixture of petroleum ether and dichloromethane.

[0015] Preferably, the mixture of petroleum ether and dichloromethane has a volume ratio of petroleum ether to dichloromethane of 6-9:1-4.

[0016] Preferably, the cooling and crystallization is performed at a temperature of -20 to -10°C for 1-2 days.

[0017] Preferably, the solvent system for the countercurrent chromatography separation comprises n-hexane, ethyl acetate, ethanol, and water, and the n-hexane, ethyl acetate, ethanol, and water have a volume ratio of 0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5.

[0018] Preferably, the countercurrent chromatography separation of the obtained liquid further comprises concentrating the liquid to obtain a concentrated sample, and subjecting the concentrated sample solution obtained by dissolving the concentrated sample to countercurrent chromatography separation, wherein the concentration of the concentrated sample solution is 1-10 mg / mL.

[0019] Preferably, the countercurrent chromatography separation comprises the following steps:

[0020] After mixing the n-hexane, ethyl acetate, ethanol, and water, the mixture is separated into an upper phase and a lower phase, wherein the upper phase is a stationary phase and the lower phase is a mobile phase;

[0021] Pump the mobile phase at a flow rate of 5-30 mL / min, stop pumping after the mobile phase flows out of the stationary phase at the outlet end of the detector for 20-50 mL; rotate the main machine filled with the stationary phase in a positive direction at a speed of 850 rpm, and pump the mobile phase into the separation pipeline at a flow rate of 5-30 mL / min; add 10-50 mL of the concentrated sample solution to the pipeline, and pump the mobile phase at a flow rate of 2-30 mL / min; collect components for 3-40 min, and the collected components are the extract liquid from which phenol is removed.

[0022] The application further provides the polyphenolic compounds obtained by the separation method. 13.52 H 18 O 10.68 The average molecular formula of component A4 is C 17.4 H 19.64 O 6.33 The average molecular formula of components A5 and A6 is C 15 H 14.86 O 3.48 .

[0023] The application further provides application of the polyphenolic compounds obtained by the separation method in antioxidation, preparation of fluorescent substances and preparation of anticancer drugs.

[0024] Compared with the prior art, the application has the following beneficial effects:

[0025] The application provides a separation method of polyphenolic compounds in a lignin depolymerization product.

[0026] The application provides a novel and broad prospect for high-value application of lignin, and provides technical support for mechanism and regulation mechanism research of antioxidation activity, fluorescent properties and anticancer activity of lignin. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description only need to be some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0028] Figure 1 Chromatogram for separating phenol in lignin depolymerization products by counter-current chromatography;

[0029] Figure 2 UV spectrum of A1-A6 components collected by chromatographic separation;

[0030] Figure 3 Particle size and potential of nanomicelles of A1-A6 components;

[0031] Figure 4 Fluorescence spectrum of A1-A6 components at their approximate maximum excitation wavelength;

[0032] Figure 5 Toxic effect of A6 on human breast cancer cells, wherein the left graph is the control and the right graph is the A6 sample;

[0033] Figure 6 Toxic effect of A6 on human esophageal adenocarcinoma cells, wherein the left graph is the control and the right graph is the A6 sample;

[0034] Figure 7 Toxic effect of A6 on human colon cancer cells, wherein the left graph is the control and the right graph is the A6 sample. DETAILED DESCRIPTION

[0035] The present application provides a method for separating polyphenolic compounds in lignin depolymerization products, wherein the polyphenolic compounds include one or more of components A3, A4, A5 and A6; the separation method comprises the following steps:

[0036] After mixing the lignin depolymerization product with the extraction liquid for extraction, the supernatant obtained is concentrated to obtain an extraction product; the lignin depolymerization product is obtained by mixing lignin, phenol and an acid catalyst for depolymerization;

[0037] The extraction product is mixed with the extraction liquid for extraction to obtain an upper extraction liquid and a lower paste sample;

[0038] The upper extraction liquid is cooled to crystallize, and the obtained liquid is subjected to counter-current chromatography to obtain an extraction liquid from which phenol is removed;

[0039] The phenol-removed extract and the lower paste sample were mixed and then subjected to high-pressure preparative chromatography separation, and fractions A1-A6 were collected in stages, the flow rate of the high-pressure preparative chromatography separation was 70 mL / min, the mobile phase was a formic acid aqueous solution-methanol, the volume fraction of the formic acid aqueous solution was 0.1%, and the gradient elution program is shown in Table 1:

[0040] Table 1 Gradient elution program of high-pressure preparative chromatography separation

[0041]

[0042] Fractions A1 collected at 3-8 min and A2 collected at 9-16 min were discarded, fraction A3 was collected at 16-23 min, fraction A4 was collected at 23-30 min, fraction A5 was collected at 30-40 min, and fraction A6 was collected at 40-48 min.

[0043] In the present application, the materials and equipment used are commercially available in the art, unless otherwise specified.

[0044] In the present application, the lignin depolymerization product is mixed with the extractant to perform extraction, and then the supernatant obtained is concentrated to obtain an extraction product; the lignin depolymerization product is obtained by mixing lignin, phenol and an acid catalyst to perform depolymerization.

[0045] In the present application, the phenol is a solvent, the acid catalyst is preferably a mixed acid of sulfuric acid and hydrochloric acid, and the amount of the acid catalyst, based on the total amount of H2SO4 and HCl, is preferably 1-6 wt% of the total amount of phenol and lignin; the lignin depolymerization product is preferably prepared by referring to the method disclosed in Chinese Patent Application No. CN201610643766.0, and the obtained lignin mild acid-catalyzed depolymerization product is the lignin depolymerization product.

[0046] In the present application, the content of phenol in the lignin depolymerization product is preferably 40-80 wt%, and more preferably 60-70 wt%.

[0047] In the present application, the extractant preferably includes an organic solvent and water, the organic solvent preferably includes one or more of ethyl acetate, tetrahydrofuran and acetonitrile, and the volume ratio of the organic solvent to water in the extractant is preferably 1:1; the use of the organic solvent and water as the extractant can remove water-soluble impurities, and the water-soluble impurities include undepolymerized lignin sulfonate and catalyst.

[0048] In the present application, the volume ratio of the extractant to the lignin depolymerization product is preferably 10:1. The extraction is preferably performed multiple times until the obtained supernatant is colorless.

[0049] In the present application, the temperature of the extraction is preferably room temperature.

[0050] In the present application, the method of concentration is preferably rotary evaporation, the vacuum degree of the rotary evaporation is preferably 0.06-0.1 MPa, the temperature is preferably 35-65℃, more preferably 40-50℃; the concentration is preferably to the point of product rotary dry, no solvent.

[0051] After obtaining the extraction product, the present application mixes the extraction product with the extraction liquid to perform extraction, obtaining upper layer extraction liquid and lower layer paste sample.

[0052] In the present application, the extraction liquid is preferably a mixture of petroleum ether and dichloromethane, the volume ratio of petroleum ether and dichloromethane in the mixture is preferably 6-9:1-4, more preferably 8:2, the mixture of petroleum ether and dichloromethane is a weak polar solvent system, which can extract a large amount of phenol and weakly polar products in the product.

[0053] In the present application, the usage ratio of the extraction liquid to the extraction product is preferably 10 mL:1 g. The present application preferably performs multiple extractions, the number of extractions is until the obtained upper layer extraction liquid is colorless.

[0054] The present application preferably further comprises separately performing ultra-high performance liquid chromatography analysis on the upper layer extraction liquid and the lower layer paste sample to determine the content of phenol in the upper layer extraction liquid and the lower layer paste sample, the present application does not have special requirements for the method of the ultra-high performance liquid chromatography analysis, and a method commonly used by those skilled in the art can be used.

[0055] After obtaining the upper layer extraction liquid, the present application performs cooling crystallization on the upper layer extraction liquid, and performs counter-current chromatography separation on the obtained liquid to obtain an extraction liquid from which phenol is removed.

[0056] In the present application, the temperature of the cooling crystallization is preferably -20 to -10℃, the time is preferably 1-2 days, and the cooling crystallization is preferably performed in a refrigerator; a large amount of phenol is removed in the form of crystals during the cooling crystallization, and the upper layer liquid is subjected to counter-current chromatography separation to remove the remaining phenol.

[0057] In the present application, before the obtained liquid is subjected to counter-current chromatography separation, the present application further comprises concentrating the liquid to obtain a concentrated sample, the method of concentration is preferably rotary evaporation, the vacuum degree of the rotary evaporation is preferably 0.06-0.1 MPa, and the temperature is preferably 35-65℃; the concentration is preferably to the point of product rotary dry, no solvent.

[0058] The present application performs counter-current chromatography separation on the concentrated sample solution obtained by dissolving the concentrated sample, the dissolving reagent is preferably a mobile phase, and the concentration of the concentrated sample solution is preferably 1-10 mg / mL, more preferably 4-6 mg / mL.

[0059] In the present application, the solvent system for counter-current chromatography separation preferably comprises n-hexane, ethyl acetate, ethanol and water, and the volume ratio of the n-hexane, ethyl acetate, ethanol and water is preferably 0.5-1.5:0.5-1.5:0.5-1.5:0.5-1.5, and more preferably 1:1:1:1.

[0060] In the present application, the counter-current chromatography separation preferably comprises the following steps:

[0061] After mixing the n-hexane, ethyl acetate, ethanol and water, the mixture is separated into upper and lower phases, the upper phase is the stationary phase, and the lower phase is the mobile phase;

[0062] The mobile phase is pumped in at a flow rate of 5-30 mL / min, and the pump is stopped after 20-50 mL of the stationary phase flows out from the detector outlet end. The main machine filled with the stationary phase is rotated forward at a speed of 850 rpm, and the mobile phase is pumped into the separation pipeline at a flow rate of 5-30 mL / min. 10-50 mL of the concentrated sample solution is added to the pipeline, and the mobile phase is pumped in at a flow rate of 2-30 mL / min. The components collected for 3-40 min are the extract liquid from which phenol is removed.

[0063] In the present application, the upper phase is a mixture of n-hexane and ethyl acetate, and the lower phase is a mixture of ethanol and water.

[0064] In the present application, the purpose of counter-current chromatography separation is to remove phenol from the liquid obtained after cooling and crystallization, and the components collected for 40-60 min are phenol.

[0065] After obtaining the extract liquid from which phenol is removed and the lower layer of the paste sample, the extract liquid from which phenol is removed and the lower layer of the paste sample are mixed, and high-pressure preparative chromatography separation is performed, and the components A1-A6 are collected in sections. The flow rate of the high-pressure preparative chromatography separation is 70 mL / min, the mobile phase is formic acid aqueous solution-methanol, the volume fraction of the formic acid aqueous solution is 0.1%, and the gradient elution program is shown in Table 1. The components A1 collected for 3-8 min and the components A2 collected for 9-16 min are discarded, the component A3 is collected for 16-23 min, the component A4 is collected for 23-30 min, the component A5 is collected for 30-40 min, and the component A6 is collected for 40-48 min.

[0066] The phenol-removed extract, the lower layer paste sample and the aqueous methanol solution are mixed and centrifuged to obtain an upper layer depolymerization product to be prepared mother liquor, and the upper layer depolymerization product to be prepared mother liquor is subjected to high-pressure preparative chromatography separation; the volume ratio of methanol to water in the aqueous methanol solution is preferably 1-3:7-9, more preferably 2:8; the use amount ratio of the aqueous methanol solution, the phenol-removed extract and the lower layer paste sample is 7 mL:1 mL:2 g.

[0067] In the present application, the parameters of the high-pressure preparative chromatography separation further include: flow rate accuracy is ±1.0%; delay volume is maximally 350 μL; flow rate precision is 0.3%.

[0068] In the present application, after the liquid components A12, A3, A4, A5 and A6 are obtained by the high-pressure preparative chromatography separation, the components are preferably subjected to concentration and drying, and the component A12 is the component obtained by combining the component A1 and the component A2. In the present application, the components A12, A3, A4, A5 and A6 are preferably identified by Q-TOF / MS, and the Q-TOF / MS preferably includes the following parameters: electrospray ion source (ESI) is used, positive ion scanning (ESI+) is performed under the condition of one-stage mass spectrum; scanning range m / z 100-2000; resolution 22000; scanning time 0-40 min; capillary voltage in positive ion mode is 3 kV; cone voltage is 30 V; source temperature is 120°C, and the desolvation gas is nitrogen (900 L / h); desolvation temperature is 450°C; mode one, collision energy is 6.0 eV, mode two, collision energy is 15-35 eV; MSe (full information tandem mass spectrum) in positive mode is obtained, and the signal strong target compounds in each component are identified. +

[0069] In the present application, the component A1 and the component A2 combined together include 18 target compounds, 7 of which contain sulfur element, and the average molecular formula is C 18.146 H 19.946 O 7.06 S 0.384 ; the component A3 includes 19 target compounds, and the average molecular formula is C 13.52 H 18 O 10.68 ; the component A4 includes 51 target compounds, and the average molecular formula is C 17.4 H 19.64 O 6.33 ; the components A5 and A6 together include 36 target compounds, and the average molecular formula is C 15 H 14.86 O 3.48 ​Compared with lignin sulfonate, the molecular weight of the product after depolymerization is 150-800, while the molecular weight before depolymerization is several thousands to several ten-thousands, and the depolymerization effect is remarkable; the molecular composition of the monomer unit of lignin sulfonate is C 20 H 24 Na2O 10 S2, indicating that the sodium sulfonate is basically removed after depolymerization, and the low molecular compound after depolymerization retains the element composition and organic molecular skeleton structure of the original polyphenol compound of lignin.

[0070] The application further provides the polyphenol compound obtained by the preparation method. 13.52 H 18 O 10.68 The average molecular formula of component A4 is C 17.4 H 19.64 O6.33, and the average molecular formula of components A5 and A6 is C 15 H 14.86 O 3.48 .

[0071] The polyphenol compound obtained by the application has antioxidant activity, fluorescence property and anticancer activity.

[0072] The application further provides the application of the polyphenol compound obtained by the preparation method in antioxidant, preparation of fluorescent substances and preparation of anticancer drugs.

[0073] In the application, the cancer cells resistant to the anticancer drugs include human breast cancer cells, human esophageal adenocarcinoma cells or human colon cancer cells.

[0074] In order to further illustrate the application, the separation method of the polyphenol compound in the lignin depolymerization product, the polyphenol compound and the application thereof are described in detail in combination with the drawings and examples, but they cannot be understood as the limitation on the protection scope of the application.

[0075] Example 1

[0076] A preparation method of a functional composition, comprising the following steps:

[0077] 1) 60.0 g of phenol is added into a reaction kettle as a solvent, under the conditions of oil bath 70 DEG C and magnetic stirring, 7.0 mL of concentrated sulfuric acid with a volume fraction of 60%, 2.5 mL of concentrated hydrochloric acid and 30.0 g of lignin sulfonate are added, 110 DEG C normal pressure reaction is carried out for 1 h, and lignin sulfonate depolymerization liquid (marked as DPL) is obtained, which contains a large amount of phenol, and the phenol content is 60 wt%.

[0078] 2) The lignin sulfonate depolymerization solution was extracted with a mixture of ethyl acetate and ultrapure water (volume ratio of 1:1) for multiple times. The volume ratio of the mixture and the lignin sulfonate depolymerization solution was 10:1 each time. The extraction was stopped until the supernatant was colorless. The supernatant was combined and concentrated by rotary evaporation (temperature 45°C) to obtain the ethyl acetate extraction product (labeled as DPL-EA) 22.5g, with a yield of 75%.

[0079] 3) The ethyl acetate extraction product was repeatedly extracted with a mixed solvent of petroleum ether and dichloromethane (volume ratio of petroleum ether to dichloromethane was 8:2). The extraction was stopped until the supernatant was colorless. The supernatant was combined to obtain the mixed solvent extraction liquid of petroleum ether and dichloromethane (labeled as DPL-UP). The lower layer of the paste sample was labeled as DPL-DW.

[0080] DPL-UP and DPL-DW were analyzed by ultra-high performance liquid chromatography, respectively. The conditions of the ultra-high performance liquid chromatography analysis included: the chromatographic column was CORTECS UPLC C18 column (2.1 mm x 100 mm, 1.6 μm); column temperature: 35°C; volume flow rate: 0.3 mL / min; sample injection volume: 1 μL; ultraviolet detector detection wavelength 275 nm; the mobile phase was 0.1% formic acid aqueous solution (mobile phase A) and methanol (mobile phase B), and the gradient elution program was as shown in Table 2:

[0081] Table 2 Elution gradient of UPLC-Q-TOF / MS

[0082]

[0083]

[0084] The results showed that after steps 2) and 3), the phenol solvent mainly existed in DPL-UP (the retention time of phenol was 3.78 min).

[0085] 4) The DPL-UP liquid was cooled and crystallized in a refrigerator (-10°C) for 1 day. A large amount of phenol solvent was removed in a crystallized manner. The upper liquid was concentrated by rotary evaporation (temperature 45°C) until the solvent was dried to obtain a concentrated sample. Countercurrent chromatography was used to separate the remaining phenol in the upper liquid:

[0086] The n-hexane, ethyl acetate, ethanol and water were mixed in a volume ratio of 1:1:1:1 and shaken well, and after standing and layering, the upper phase was used as the stationary phase and the lower phase was used as the mobile phase. The concentrated sample was dissolved in the mobile phase to prepare a 1 mg / mL test solution. The mobile phase was pumped at a flow rate of 5 mL / min, and the pump was stopped after 20 mL of the mobile phase flowed out of the detector outlet end and entered the stationary phase. The main machine filled with the upper phase was rotated forward at a speed of 850 rpm, and the mobile phase was pumped into the separation pipeline at a flow rate of 5 mL / min. 10 mL of the test solution was added to the pipeline, and the mobile phase was pumped into the pipeline at a flow rate of 5 mL / min. Figure 1 The chromatogram for separating phenol in the lignin depolymerization product by counter-current chromatography is shown in the figure, and the detection wavelength is 275 nm. The components collected from 3 to 40 min and the components after 40 min were collected and analyzed by ultra-high performance liquid chromatography.

[0087] The results show that no absorption peak of phenol is detected in the components collected from 3 to 40 min, and the components after 40 min are relatively pure and are identified as phenol, indicating that the phenol in the depolymerization product has been well removed after the above separation.

[0088] 5) After the removal of the phenol solvent (3-40 min components), DPL-UP and DPL-DW were combined and dissolved in methanol water solution with a volume ratio of methanol to water of 1:9. The amount ratio of methanol water solution, phenol-removed extract and lower layer paste sample was 7 mL:1 mL:2 g, and centrifugation was performed to obtain the upper layer depolymerization product preparation mother liquor. High pressure preparation chromatography was used for separation: the flow rate was set to 70 mL / min; the flow rate accuracy was ±1.0%; the delay volume was 350 μL; the flow rate precision was 0.3%; and the mobile phase was 0.1% formic acid aqueous solution (mobile phase A)-methanol (mobile phase B), and the gradient elution program is shown in Table 1.

[0089] According to the ultraviolet spectrum signal at 275 nm and the retention time, the depolymerization product preparation mother liquor was fractionated into six components: A1 was collected from 3 to 8 min, A2 was collected from 9 to 16 min, A3 was collected from 16 to 23 min, A4 was collected from 23 to 30 min, A5 was collected from 30 to 40 min, and A6 was collected from 40 to 48 min. A1 sample has an early peak time, and the peak time is close to that of A2, so A1 and A2 are combined and recorded as A12. After rotary evaporation and drying of A1-A6, 0.5 g of A12 component, 4.5 g of A3 component, 5.2 g of A4 component, 5.0 g of A5 component and 4.2 g of A6 component were obtained.

[0090] Figure 2 The ultraviolet spectrum of the A1-A6 components collected by the preparation chromatography is shown in the figure. It can be seen that the components are preliminarily separated, and the active substances are mainly concentrated in A3, A4, A5 and A6, and no obvious ultraviolet absorption signal is observed in A12.

[0091] The components of A12, A3, A4, A5 and A6 were identified by Q-TOF / MS, and the positive ion scanning (ESI + ) was carried out under the first mass spectrum by using an electric spray ion source (ESI); the scanning range was m / z 100-2000; the resolution was 22000; the scanning time was 0-40 min; the capillary voltage was 3 kV in the positive ion mode; the cone hole voltage was 30 V; the source temperature was 120°C; the desolvation gas was nitrogen (900 L / h); the desolvation temperature was 450°C; the mode was one, the collision energy was 6.0 eV, the mode was two, the collision energy was 15-35 eV; the MSE (full information tandem mass spectrum) in the positive mode was obtained, the target compounds with strong signals in each component were identified, and the element composition and average molecular weight of each component were as follows:

[0092] A total of 18 target compounds were identified in the A12 sample, 7 of which contained sulfur element, and the average molecular formula was C 18.146 H 19.946 O 7.06 S 0.384 , as shown in Table 3.

[0093] A total of 19 target compounds were identified in the A3 sample, and the average molecular formula was C 13.52 H 18 O 10.68 , as shown in Table 4.

[0094] A total of 51 target compounds were identified in the A4 sample, and the average molecular formula was C 17.4 H 19.64 O 6.33 , as shown in Table 5.

[0095] A total of 36 target compounds were identified in the A5 and A6 samples, and the average molecular formula was C 15 H 14.86 O 3.48 , as shown in Table 6.

[0096] Table 3 Identification table of A12 sample under first mass spectrum

[0097]

[0098]

[0099] Table 4 Identification table of A3 sample under first mass spectrum

[0100]

[0101]

[0102] Table 5 Identification table of A4 sample under first mass spectrum

[0103]

[0104]

[0105]

[0106]

[0107]

[0108] Table 6A5, A6 sample primary mass spectrum identification table

[0109]

[0110]

[0111]

[0112] Compared with lignin sulfonate, the molecular weight of the product after depolymerization is 150-800, while the molecular weight before depolymerization is several thousand to tens of thousands, and the depolymerization effect is remarkable; the molecular composition of lignin sulfonate monomer unit is C 20 H 24 Na2O 10 S2, indicating that the sodium sulfonate is basically removed after depolymerization, and the low molecular compound after depolymerization better retains the element composition and organic molecular skeleton structure of the original polyphenol compound of lignin.

[0113] Example 2

[0114] 1. Antioxidant activity of functional components

[0115] Methanol was used as a solvent to prepare 1 mg / mL mother liquor of each component A3-A6 obtained by separation in Example 1, and the sample liquid was prepared by dilution. A 1 mg / mL DPPH solution was also prepared. The sample liquid with a concentration of 20 μg / mL was reacted with the DPPH solution with a concentration of 20 μg / mL at a volume ratio of 1:1. The ultraviolet absorption spectrum of the DPPH control solution without each component, the solution after reaction of each component with DPPH, the same amount of sample without DPPH as a blank sample of each component, and the undepolymerized lignin sample were tested by ultraviolet spectrometer. According to the DPPH standard curve obtained by experiment, the absorbance A at 517 nm was calculated, and the DPPH clearance rate of each sample was calculated according to the following clearance rate formula:

[0116]

[0117] Wherein: Ac- the absorbance of DPPH solution without antioxidant; Ai- the absorbance of solution after adding separated components; Aj- the absorbance of solution only with sample;

[0118] The DPPH scavenging rate of each component at the concentration of 10 mg / L was calculated, and the results are shown in Table 7:

[0119] Table 7 Antioxidant activity of each component

[0120] Component name DPPH scavenging rate A12 43.4869％ A3 65.1306％ A4 64.3286％ A5 85.3707％ A6 80.3587％ Lignin (comparative sample) 13.4269％

[0121] The results show that the undegraded lignin, A12, A3, A4, A5 and A6 have certain scavenging effect on DPPH, and the scavenging rates are 13.4269%, 43.4869%, 65.1306%, 64.3286%, 85.3707% and 80.3587%, respectively. Among them, A5 and A6 have the most outstanding scavenging effect on DPPH free radicals and the best antioxidant activity. The comparative experiment shows that the antioxidant activity of undegraded lignin is low, indicating that the low molecular weight products obtained by depolymerization of lignin have more significant antioxidant activity than lignin.

[0122] 2. Nanomicelles and fluorescence performance of functional components

[0123] The prepared separated samples were dissolved in tetrahydrofuran to prepare a 1 mg / mL solution, water was added to the solution to prepare a 1:19 (volume ratio of tetrahydrofuran to water) solution, and the solution was left to stand for 10 min. After centrifugal separation at 10000 rpm, the supernatant was taken, and the particle size and potential of the prepared supernatant were tested by a (Malvern Nano-ZS90) nanoparticle size analyzer. Potential measurement: measurement temperature 25℃, measurement times 3, cycle 1, sample cell type DTS0012, dispersant water, equilibrium time 120s, calculation model General Purpose. Particle size measurement: measurement temperature 25℃, measurement times 1, cycle 1, sample cell type DTS0012, dispersant water, equilibrium time 120s, calculation model General Purpose.

[0124] Figure 3 The particle size and potential of the nanomicelles of A1-A6 components can be seen that the nanomicelles exist in each component sample, and the formed nanomicelles have relatively uniform particle size, and the particle size of each nanomicelle is between 70-260 nm. Except A12, each component nanomicelle has a high negative charge and good stability.

[0125] The nanomicelles prepared above were tested by F-4500 fluorescence spectrophotometer, starting from excitation wavelength 260 nm, each scan adding 20 nm to the end of excitation wavelength 420 nm, to determine the fluorescence interval and approximate maximum excitation wavelength of each component nanomicelle, and test the fluorescence performance of each nanomicelle at the excitation wavelength.

[0126] Figure 4 The fluorescence spectrum of each component at its approximate maximum excitation wavelength was analyzed, and it can be seen that the micelles of each sample have a strong fluorescence emission peak, with an emission wavelength of about 320 nm, and have significant fluorescence performance. The maximum emission wavelength is shorter than that of common fluorescent compounds in plants (generally 400-700 nm), indicating that the structure of each component nanomicelle has a high degree of conjugation.

[0127] 3. Anti-cancer activity of the functional component

[0128] The CCK-8 method was used to detect the cell proliferation inhibition rate. The water-soluble tetrazolium salt WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazole monosodium salt) was used for rapid detection, and the number of viable cells was quantified dynamically by colorimetry, thereby detecting cell proliferation or drug toxicity. When the 96-well plate was plated: 100 μL per well, cell density 8000 cells / well, after incubation of the cells for 12 h, 10 μL of CCK-8 was added to each well, and attention was paid to avoid light during the process. After adding, it was wrapped with aluminum foil paper and placed in a 37°C shaking bed at 70 rpm for 1 h. Then, the absorbance at 450 nm was measured by a microplate reader as the OD value of the control group. At the same time, a blank group was prepared and tested by adding only medium without cells. Sample groups A12, A3, A4, A5, and A6 were selected, with specific concentrations of 1000, 285.7, 81.6, 23.3, 6.7, 1.9, 0.5, and 0.17 μg / mL, respectively. The dilution was 2 / 7 times (i.e., 0.2857 times), i.e., 2 mL of 1000 μg / mL sample was added to 5 mL of medium and mixed to 7 mL of solution. After incubation for 24 h, the cells in each group were observed under a microscope and photographed, and CCK-8 was added. At the same time, a sample blank group was prepared and tested.

[0129] The cell survival rate after adding each sample was calculated using the following formula:

[0130] Cell viability % = (sample group OD value - sample blank group OD value) / (control group OD value - blank group OD value) x 100%;

[0131] In the formula: sample blank group OD value: without cells, only with culture medium containing samples, sample concentration is the same as that of the same column sample group; control group OD value: cells without drugs; blank group OD value: without cells, only with culture medium;

[0132] The CCK-8 method was used to detect the inhibition rate of the proliferation of three kinds of cancer cells, and the results are shown in Table 8. Figure 5 A6 on human breast cancer cell toxicity effect diagram, wherein the left graph is the control, and the right graph is the A6 sample; Figure 6 A6 on human esophageal adenocarcinoma cell toxicity effect diagram, wherein the left graph is the control, and the right graph is the A6 sample; Figure 7 A6 on human colon cancer cell toxicity effect diagram, wherein the left graph is the control, and the right graph is the A6 sample. Figures 5-7 The scale in the figure is the same, and is 200 μm.

[0133] Table 8: Inhibition rate (IC 50 Value, μg / mL)

[0134]

[0135]

[0136] The results show that the A3, A4, A5 and A6 components have cytotoxic inhibition effect on human colon cancer cells, showing an increasing trend. Among them, the A6 component has a significant inhibitory effect on human breast cancer cells, human esophageal adenocarcinoma cells and human colon cancer cells, especially on human colon cancer cells, with an IC 50 value of only 16.43 μg / mL, while the IC 50 value of the lignin control sample on human colon cancer cells is 743.32 μg / mL.

[0137] Although the above examples make a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and people can also obtain other embodiments according to the embodiments of the present application without creative labor, which all belong to the protection scope of the present application.

Claims

1. A method for separating polyphenolic compounds in a lignin depolymerization product, characterized by, The polyphenols are component A6; the separation method comprises the following steps: After mixing the lignin depolymerization product with the extraction liquid for extraction, the supernatant obtained is concentrated to obtain an extraction product; the lignin depolymerization product is obtained by mixing lignin, phenol and an acid catalyst for depolymerization, the acid catalyst is a mixed acid of sulfuric acid and hydrochloric acid, and the amount of the acid catalyst is 1-6 wt% of the total amount of phenol and lignin; the extraction liquid is an organic solvent and water, and the organic solvent is one or more of ethyl acetate, tetrahydrofuran and acetonitrile; The extraction product is mixed with the extraction liquid for extraction to obtain an upper layer extraction liquid and a lower layer paste sample; the extraction liquid is a mixed liquid of petroleum ether and dichloromethane, and the volume ratio of petroleum ether to dichloromethane in the mixed liquid is 8:2; The upper layer extraction liquid is cooled for crystallization, and the obtained liquid is subjected to countercurrent chromatography separation to obtain a phenol-removed extraction liquid; the solvent system for the countercurrent chromatography separation is n-hexane, ethyl acetate, ethanol and water, and the volume ratio of n-hexane, ethyl acetate, ethanol and water is 1:1:1:1; Before the obtained liquid is subjected to countercurrent chromatography separation, the liquid is concentrated to obtain a concentrated sample, and the concentrated sample solution obtained by dissolving the concentrated sample is subjected to countercurrent chromatography separation, and the concentration of the concentrated sample solution is 1-10 mg / mL; The countercurrent chromatography separation comprises the following steps: After mixing the n-hexane, ethyl acetate, ethanol and water, the upper phase and the lower phase are obtained, the upper phase is the stationary phase, and the lower phase is the mobile phase; The mobile phase is pumped in at a flow rate of 5-30 mL / min, the detector outlet end flows out of the stationary phase for 20-50 mL, and then the pump is stopped; the main machine filled with the stationary phase is rotated forward at a speed of 850 rpm, and the mobile phase is pumped into the separation pipeline at a flow rate of 5-30 mL / min; 10-50 mL of the concentrated sample solution is added to the pipeline, and the mobile phase is pumped in at a flow rate of 2-30 mL / min; the components collected for 3-40 min are the phenol-removed extraction liquid; The phenol-removed extraction liquid and the lower layer paste sample are mixed and subjected to high-pressure preparative chromatography separation, and components A1-A6 are collected in sections, the flow rate of the high-pressure preparative chromatography separation is 70 mL / min, the mobile phase is formic acid aqueous solution-methanol, and the volume fraction of the formic acid aqueous solution is 0.1%, and the gradient elution program is as follows: Components A1 collected for 3-8 min and component A2 collected for 9-16 min are discarded, component A3 is collected for 16-23 min, component A4 is collected for 23-30 min, component A5 is collected for 30-40 min, and component A6 is collected for 40-48 min.

2. The separation method of claim 1, wherein, The temperature for the cooling crystallization is -20--10℃, and the time is 1-2 days.

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