A green microextraction method for urushiol compounds in lacquer tree

Abstract

The application discloses a green micro-extraction method of urushiol compounds in lacquer tree. The method mainly comprises raw material pretreatment, high-speed grinding of raw materials, additives and stainless steel balls in a vortex instrument, then micro-extraction is carried out by means of ultrasonic cavitation or vortex oscillation, and finally centrifugation and filtration are carried out. The method can effectively extract and enrich urushiol compounds, the operation time is short, the urushiol can be efficiently enriched, and the extraction efficiency is improved. Meanwhile, the operation process is simple, the contact between the sample and the solvent is small, the risk of allergy caused by urushiol is greatly reduced, and the rapid extraction and analysis of urushiol can be realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of separation and enrichment of natural products, and particularly relates to a green microextraction method of urushiol compounds in Toxicodendron succedaneum. BACKGROUND

[0002] Toxicodendron succedaneum is a deciduous tree belonging to the family Anacardiaceae and is widely distributed in Southeast Asia. It is often used as a traditional Chinese medicine in East Asia to treat gastrointestinal inflammation, heart disease, arthritis, hypertension, diabetes, stroke and chronic fatigue syndrome. Laccaic acid is a natural resin coating, known as the "King of Coatings". The seed coat of Toxicodendron succedaneum can be pressed to obtain wax, and the seed core can be pressed to produce oil. The wood is hard. The composition of laccaic acid is urushiol (60-70%), water (20-30%), vegetable gum (4-10%), laccase (1.5-2%) and water-insoluble glycoprotein (3-5%). Urushiol is the main lipid component of laccaic acid and is also the main component of allergic contact dermatitis. Urushiol exists in all parts of Toxicodendron succedaneum, such as leaves, branches and bark.

[0003] Urushiol is a mixture of catechol derivatives, and the side chain contains 15-17 carbons with 0-3 double bonds. It has been reported that urushiol has obvious anti-cancer, anti-fungal, anti-bacterial and anti-oxidation effects. However, urushiol has strong sensitization properties, and about 40% of people have a sensitization level below 2 μg. Urushiol can cause skin inflammation, swelling, and blisters, and can cause acute nephritis in a small number of patients. Therefore, the microextraction and determination of urushiol compounds in Toxicodendron succedaneum have important application value.

[0004] Urushiol is currently mainly characterized by GC-MS and HPLC-MS, and trimethylsilyl silylation is required for GC analysis to achieve the required accuracy and precision. When analyzing urushiol in laccaic acid, tree bark, branches and leaves, extraction and separation processes are required. The extraction methods mainly include traditional extraction techniques such as solution extraction, hot reflux extraction and ultrasonic extraction. These methods usually require a large amount of sample, a large amount of organic solvent and a long completion time. The process is not environmentally friendly, and in the experimental process, the use of a large amount of sample and a long completion time greatly increases the chance of causing skin allergy. Therefore, it is of great significance to establish a simple, rapid and less contact extraction and analysis method for the extraction and quantitative analysis of urushiol in Toxicodendron succedaneum. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the prior art and provide a green microextraction method of urushiol compounds in Toxicodendron succedaneum.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] A green microextraction method of urushiol compounds in Toxicodendron succedaneum, the steps of the method are:

[0008] First step

[0009] The solid raw material is dried, crushed, and sieved through a 40-80 mesh screen to obtain a powder sample; the liquid raw material is directly filtered to remove impurities to obtain a liquid sample.

[0010] Second step

[0011] The powder sample or liquid sample is placed in a 50-100 mL centrifuge tube, an auxiliary agent and 3-5 stainless steel beads of 7-10 mm are added, the ratio of the sample to the auxiliary agent is 1:1-1:10 by g / g, and the cap is tightly closed; then the centrifuge tube is placed in a vortex instrument, the vortex speed is set to 300-1000 rpm, and the vortex grinding time is 1-5 min to uniformly mix the sample; after mixing, the stainless steel beads are removed to obtain a mixed sample; the auxiliary agent is a mixture of one or more of ZSM-5 / Fe3O4, C8, C18, magnesium sulfate, aluminum oxide, Al-SBA-15, Fe-SBA-15, and SBA-15.

[0012] Third step

[0013] The extraction solvent is poured into the centrifuge tube containing the uniformly ground mixed sample, the ratio of the mixed sample to the extraction solvent is 1:2-1:10 by g / mL, microextraction is performed at room temperature, and after extraction is completed, centrifugation and filtration are performed, and the supernatant is analyzed for urushiol compound content by high performance liquid chromatography; the extraction solvent is a mixture of one or more of 0.05-0.5% formic acid / methanol by volume, 0.05-0.2% trifluoroacetic acid / methanol by volume, and 0.1-0.5% acetic acid / methanol by volume.

[0014] The solid raw material in the first step is selected from one of bark, tree, leaf, seed, and lacquer tree extract, and the liquid raw material is raw lacquer.

[0015] The urushiol compounds in the third step mainly include saturated urushiol, monounsaturated urushiol, diunsaturated urushiol, and triunsaturated urushiol.

[0016] The microextraction in the third step is one of ultrasonic treatment at a power of 20-100 W for 5-20 min and vortex oscillation at a speed of 200-800 rpm for 2-10 min.

[0017] The centrifugation condition in the third step is 5000-12000 rpm for 3-10 min.

[0018] The filtration condition in the third step is to use a filter with a pore size of 0.22-0.45 μm.

[0019] The high performance liquid chromatography analysis method in the third step is as follows: a C18 reversed-phase column with a size of 2.1*150 mm and a 5-micron thickness, acetonitrile as phase A, and water with a volume percentage of 0.1-0.5% trifluoroacetic acid as phase B; a flow rate of 0.5-1 mL / min; a column temperature of 40 DEG C; a sample injection amount of 10-20 microliters; a detection wavelength of 277 nm; and a gradient elution program as follows: 0-5 min, 70% B; 5-12 min, from 70% B to 90% B; 12-25 min, 90% B; 25-30 min, 70% B.

[0020] The content of the urushiol compound in the third step is 5-95%.

[0021] The present application has the following beneficial effects.

[0022] The present application has the following beneficial effects.

[0023] (1) The method adopted in the present application can extract and analyze a sample of only 0.1 g, and the process is simple, does not require a large amount of organic solvent, can enrich trace amounts of urushiol in raw materials, and greatly saves time and labor costs.

[0024] (2) The method adopted in the present application can effectively extract and enrich urushiol compounds, has a short operation time, can greatly enrich urushiol, and improves the extraction efficiency.

[0025] (3) The method adopted in the present application greatly reduces the opportunities for human contact with samples and extraction solvents, greatly reduces the risk of urushiol-induced allergies, and can realize rapid extraction and analysis of urushiol. Embodiment

[0026] Example 1

[0027] The bark is dried, crushed, and sieved through a 40-80 mesh screen to obtain a powder sample. The powder sample is placed in a 50-100 mL centrifuge tube, and an additive and 3-5 stainless steel beads of 7-10 mm are added. The ratio of the sample to the additive is 1:1-1:10 by g / g, and the cap is tightly closed. Then the centrifuge tube is placed in a vortex instrument, the vortex speed is set to 300-1000 rpm, and the vortex grinding time is 1-5 min to mix uniformly. After mixing, the stainless steel beads are removed to obtain a mixed sample. The additive is a mixture of one or more of ZSM-5 / Fe3O4, C8, C18, magnesium sulfate, aluminum oxide, Al-SBA-15, Fe-SBA-15, and SBA-15. A volume percentage of 0.05-0.5% formic acid / methanol is poured into the centrifuge tube containing the uniformly ground mixed sample. The ratio of the mixed sample to the volume percentage of 0.05-0.5% formic acid / methanol is 1:2-1:10 by g / mL. Ultrasonic treatment is performed for 5-20 min at an ultrasonic power of 20-100 W and room temperature. After ultrasonic treatment, centrifugation is performed at a centrifugal speed of 5000-12000 rpm for 3-10 min. After centrifugation, the supernatant is filtered using a filter with a pore size of 0.22-0.45 μm. Then the urushiol compound content is analyzed by high performance liquid chromatography.

[0028] Example 2

[0029] The bark is dried, crushed, and sieved through a 40-80 mesh screen to obtain a powder sample. The powder sample is placed in a 50-100 mL centrifuge tube, and an additive and 3-5 stainless steel beads of 7-10 mm are added. The ratio of the sample to the additive is 1:1-1:10 by g / g, and the cap is tightly closed. Then the centrifuge tube is placed in a vortex instrument, the vortex speed is set to 300-1000 rpm, and the vortex grinding time is 1-5 min to mix uniformly. After mixing, the stainless steel beads are removed to obtain a mixed sample. The additive is a mixture of one or more of ZSM-5 / Fe3O4, C8, C18, magnesium sulfate, aluminum oxide, Al-SBA-15, Fe-SBA-15, and SBA-15. A volume percentage of 0.05-0.2% trifluoroacetic acid / methanol is poured into the centrifuge tube containing the uniformly ground mixed sample. The ratio of the mixed sample to the volume percentage of 0.05-0.2% trifluoroacetic acid / methanol is 1:2-1:10 by g / mL. Ultrasonic treatment is performed for 5-20 min at an ultrasonic power of 20-100 W and room temperature. After ultrasonic treatment, centrifugation is performed at a centrifugal speed of 5000-12000 rpm for 3-10 min. After centrifugation, the supernatant is filtered using a filter with a pore size of 0.22-0.45 μm. Then the urushiol compound content is analyzed by high performance liquid chromatography.

[0030] Example 3

[0031] The leaves are dried, crushed, and sieved through a 40-80 mesh screen to obtain a powder sample. The powder sample is placed in a 50-100 mL centrifuge tube, and an additive and 3-5 stainless steel beads of 7-10 mm are added. The ratio of sample to additive is 1:1-1:10 by g / g, and the cap is tightly closed. Then the centrifuge tube is placed in a vortex instrument, and the vortex speed is set to 300-1000 rpm. The vortex grinding time is 1-5 min to mix the sample uniformly. After mixing, the stainless steel beads are removed to obtain the mixed sample. The additive is a mixture of one or more of ZSM-5 / Fe3O4, C8, C18, magnesium sulfate, aluminum oxide, Al-SBA-15, Fe-SBA-15, and SBA-15. A volume percentage of 0.1-0.5% acetic acid / methanol is poured into the centrifuge tube containing the uniformly ground mixed sample. The ratio of mixed sample to volume percentage of 0.1-0.5% acetic acid / methanol is 1:2-1:10 by g / mL. Vortex oscillation is performed at 200-800 rpm for 2-10 min at room temperature. After extraction is completed, centrifugation is performed at a centrifugal speed of 5000-12000 rpm for 3-10 min. After centrifugation, the supernatant is filtered using a filter with a pore size of 0.22-0.45 μm. Then the urushiol compound content is analyzed by high performance liquid chromatography.

[0032] Example 4

[0033] The paint seeds are dried, crushed, and sieved through a 40-80 mesh screen to obtain a powder sample. The powder sample is placed in a 50-100 mL centrifuge tube, and an additive and 3-5 stainless steel beads of 7-10 mm are added. The ratio of sample to additive is 1:1-1:10 by g / g, and the cap is tightly closed. Then the centrifuge tube is placed in a vortex instrument, and the vortex speed is set to 300-1000 rpm. The vortex grinding time is 1-5 min to mix the sample uniformly. After mixing, the stainless steel beads are removed to obtain the mixed sample. The additive is a mixture of one or more of ZSM-5 / Fe3O4, C8, C18, magnesium sulfate, aluminum oxide, Al-SBA-15, Fe-SBA-15, and SBA-15. A volume percentage of 0.05-0.2% trifluoroacetic acid / methanol is poured into the centrifuge tube containing the uniformly ground mixed sample. The ratio of mixed sample to volume percentage of 0.05-0.2% trifluoroacetic acid / methanol is 1:2-1:10 by g / mL. Microextraction is performed by ultrasonic treatment at a power of 20-100 W for 5-20 min or vortex oscillation at 200-800 rpm for 2-10 min at room temperature. After extraction is completed, centrifugation is performed at a centrifugal speed of 5000-12000 rpm for 3-10 min. After centrifugation, the supernatant is filtered using a filter with a pore size of 0.22-0.45 μm. Then the urushiol compound content is analyzed by high performance liquid chromatography.

[0034] Example 5

[0035] The lacquer tree extract is dried, crushed, and passed through a 40-80 mesh screen to obtain a powder sample. The powder sample is placed in a 50-100 mL centrifuge tube, and an auxiliary agent and 3-5 stainless steel beads of 7-10 mm are added. The ratio of the sample to the auxiliary agent is 1:1-1:10 by g / g, and the cap is tightly closed. Then the centrifuge tube is placed in a vortex instrument, the vortex speed is set to 300-1000 rpm, and the vortex grinding time is 1-5 min to mix the sample uniformly. After mixing, the stainless steel beads are removed to obtain a mixed sample. The auxiliary agent is a mixture of one or more of ZSM-5 / Fe3O4, C8, C18, magnesium sulfate, aluminum oxide, Al-SBA-15, Fe-SBA-15, and SBA-15. A volume percentage of 0.05-0.2% trifluoroacetic acid / methanol is poured into the centrifuge tube containing the uniformly ground mixed sample, and the ratio of the mixed sample to the volume percentage of 0.05-0.2% trifluoroacetic acid / methanol is 1:2-1:10 by g / mL. The sample is micro-extracted by ultrasonic treatment at a power of 20-100 W for 5-20 min or vortex oscillation at 200-800 rpm for 2-10 min at room temperature. After extraction, the sample is centrifuged at a centrifugal speed of 5000-12000 rpm for 3-10 min. After centrifugation, the supernatant is filtered using a filter with a pore size of 0.22-0.45 μm. Then the content of urushiol compounds is analyzed by high performance liquid chromatography.

[0036] Example 6

[0037] The raw lacquer material is directly filtered to remove impurities to obtain a liquid sample, the liquid sample is placed in a 50-100 mL centrifuge tube, an auxiliary agent and 3-5 stainless steel beads of 7-10 mm are added, the ratio of the sample to the auxiliary agent is 1:1-1:10 by g / g, and the cap is tightly covered; then the centrifuge tube is placed in a vortex instrument, the vortex speed is set to 300-1000 rpm, and the vortex grinding time is 1-5 min to uniformly mix the sample; after mixing, the stainless steel beads are removed to obtain a mixed sample; the auxiliary agent is one or a mixture of several of ZSM-5 / Fe3O4, C8, C18, magnesium sulfate, aluminum oxide, Al-SBA-15, Fe-SBA-15 and SBA-15; the extraction solvent is poured into the centrifuge tube containing the uniformly ground mixed sample, the ratio of the mixed sample to the extraction solvent is 1:2-1:10 by g / mL, microextraction is performed, the method of microextraction is ultrasonic treatment at a power of 20-100 W for 5-20 min or vortex oscillation at 200-800 rpm for 2-10 min, the temperature is room temperature, after extraction is completed, centrifugation is performed at a centrifugal speed of 5000-12000 rpm for 3-10 min, after centrifugation, the supernatant is filtered using a filter with a pore size of 0.22-0.45 μm. Then the content of urushiol compounds is analyzed by high performance liquid chromatography. The extraction solvent is one or a mixture of several of 0.05-0.5% formic acid / methanol by volume percentage, 0.05-0.2% trifluoroacetic acid / methanol by volume percentage and 0.1-0.5% acetic acid / methanol by volume percentage.

[0038] Example 7

[0039] The urushi lacquer compounds are determined by high performance liquid chromatography analysis, the high performance liquid chromatography analysis method is: a 2.1x150mm, 5μm C18 reversed-phase column, A phase is acetonitrile, B phase is 0.1-0.5% trifluoroacetic acid water by volume percentage; flow rate is 0.5-1 mL / min; column temperature is 40℃; sample amount is 10-20μL; detection wavelength is 277nm; gradient elution program is as follows: 0-5 min, 70% B; 5-12 min, from 70% B to 90% B; 12-25 min, 90% B; 25-30 min, 70% B. The urushi lacquer compounds include saturated urushi lacquer, monounsaturated urushi lacquer, diunsaturated urushi lacquer and triunsaturated urushi lacquer. The content of the urushi lacquer compounds is 5-95%.

Claims

1. A green microextraction method of urushiol compounds in lacquer trees, steps of the method are as follows: Step 1: solid raw materials are dried, crushed, and passed through a 40-80 mesh screen to obtain a powder sample; liquid raw materials are directly filtered to remove impurities to obtain a liquid sample; the solid raw materials are selected from one of bark, leaves, seeds, and lacquer tree extracts, and the liquid raw materials are raw lacquer; Step 2: the powder sample or the liquid sample is placed in a 50-100 mL centrifuge tube, an additive and 3-5 stainless steel beads of 7-10 mm are added, the ratio of the sample to the additive is 1:1-1:10 by g / g, and the cap is tightly covered; then the centrifuge tube is placed in a vortex instrument, the vortex speed is set to 300-1000 rpm, and the vortex grinding time is 1-5 min to uniformly mix the sample, after the mixing is completed, the stainless steel beads are removed to obtain a mixed sample; the additive is one or a mixture of several of ZSM-5 / Fe 3 O 4, C8, C18, magnesium sulfate, aluminum oxide, Al-SBA-15, Fe-SBA-15, and SBA-15; Step 3: an extraction solvent is poured into the centrifuge tube containing the uniformly ground mixed sample, the ratio of the mixed sample to the extraction solvent is 1:2-1:10 by g / mL, microextraction is performed at room temperature, after the extraction is completed, centrifugation and filtration are performed, and the supernatant is analyzed for the content of urushiol compounds by high performance liquid chromatography; the extraction solvent is one or a mixture of several of 0.05-0.5% formic acid / methanol by volume percentage, 0.05-0.2% trifluoroacetic acid / methanol by volume percentage, and 0.1-0.5% acetic acid / methanol by volume percentage; wherein the microextraction is one of ultrasonic treatment at a power of 20-100 W for 5-20 min and vortex oscillation at 200-800 rpm for 2-10 min; the urushiol compounds mainly include saturated urushiol, monounsaturated urushiol, diunsaturated urushiol, and triunsaturated urushiol; the centrifugation conditions are 5000-12000 rpm for 3-10 min; the filtration conditions are filtration using a filter with a pore size of 0.22-0.45 μm; the high performance liquid chromatography analysis method is as follows: a C18 reversed-phase column of 2.1*150 mm and 5 μm, A phase is acetonitrile, B phase is 0.1-0.5% trifluoroacetic acid water by volume percentage; flow rate is 0.5-1 mL / min; column temperature is 40℃; sample injection amount is 10-20 μL; detection wavelength is 277 nm; gradient elution program is as follows: 0-5 min, 70% B; 5-12 min, from 70% B to 90% B; 12-25 min, 90% B; 25-30 min, 70% B.

2. A green microextraction method of urushiol compounds from lacquer tree according to claim 1, characterized by, The content of the urushiol compounds in Step 3 is 5-95%.