CN115677473A - Phenolic compound, method for extracting and separating phenolic compound from ricepaper pith and application - Google Patents
Phenolic compound, method for extracting and separating phenolic compound from ricepaper pith and application Download PDFInfo
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- CN115677473A CN115677473A CN202211438489.1A CN202211438489A CN115677473A CN 115677473 A CN115677473 A CN 115677473A CN 202211438489 A CN202211438489 A CN 202211438489A CN 115677473 A CN115677473 A CN 115677473A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The application relates to a phenolic compound with a molecular formula of C 10 H 12 O 5 Having the chemical structure shown below:
the compound is a phenol compound with a monophenol structure, has excellent antioxidant activity, and has good application prospect in the fields of cosmetics industry, health care medicines and the like. Furthermore, the phenolic compounds can be obtained from the ricepaperplant pith through a specific extraction and separation step, and the research results on chemical components contained in the ricepaperplant pith are further expanded.
Description
Technical Field
The application relates to the technical field of extraction and separation of traditional Chinese medicinal materials, in particular to a phenolic compound, a method for extracting and separating the phenolic compound from ricepaperplant pith and application.
Background
Medulla Tetrapanacis, tetrapanax Papyrifer (hook.) K.Koch, also known as Datong grass and white medulla Tetrapanacis, is a plant of the genus Tetrapanacis of the family Araliaceae, and is mainly distributed in Guangxi, guizhou, sichuan and Yunnan provinces. Medulla Tetrapanacis is recorded in 2020 edition pharmacopoeia of the people's republic of China, which records that it has the effects of clearing heat, promoting urination, promoting qi circulation and promoting lactation. Furthermore, modern pharmacological studies of the ricepaperplant pith also prove that the ricepaperplant pith has good effects on the aspects of anti-inflammation, diuresis, lactation, oxidation resistance, immune function regulation and the like.
Although the application of the ricepaperplant pith in clinic is wide, the research on the chemical components of the ricepaperplant pith is poor at present, and in the existing research results, the chemical components which are proved to be extracted and separated from the ricepaperplant pith are mainly steroid compounds and ceramide compounds, but the reports on the phenolic compounds in the ricepaperplant pith are less.
Natural phenolic compounds are secondary metabolites of plant tissues and are widely found in plant stems, leaves, fruits and flowers. The phenolic compounds play the roles of oxidation resistance, bacteriostasis and the like in the food industry, reduce the growth of food microorganisms and the oxidative deterioration of products, improve the quality of the products and prolong the shelf life of the products; meanwhile, the phenolic compounds are also used in skin care products, which are helpful for enhancing skin elasticity, reducing pigmentation and inhibiting spot formation; in addition, the antioxidant activity of phenolic compounds is also widely applied to traditional functional foods and medicines. Therefore, if phenolic compounds can be extracted and separated from ricepaper pith, the method has important significance for research and extended application of natural phenolic compounds.
Disclosure of Invention
Based on the above, there is a need for a phenolic compound and a method for extracting and separating the phenolic compound from ricepaper pith.
One embodiment of the present application provides a phenolic compound having a molecular formula of C 10 H 12 O 5 Having the chemical structure shown below:
an embodiment of the present application further provides a method for extracting and separating phenolic compounds from ricepaper pith, which comprises the following steps:
sequentially extracting medulla Tetrapanacis with water and methanol to obtain extract;
and separating the extract by adopting a chromatographic separation method to obtain the phenolic compound.
In one embodiment, the method comprises the steps of:
decocting medulla Tetrapanacis with water, filtering to obtain decoction, concentrating the decoction under reduced pressure, and freeze drying to obtain medulla Tetrapanacis water extract;
ultrasonically extracting the water extract of the ricepaperplant pith with methanol, filtering to obtain filtrate, and concentrating the filtrate to obtain a methanol extract of the ricepaperplant pith;
and eluting the ricepaper pith methanol extract by sequentially adopting a macroporous resin column, an ODS column, a sephadex column and ultra-high performance liquid chromatography to obtain the phenolic compounds.
In one embodiment, the step of eluting with the macroporous resin column comprises:
and (3) performing gradient elution by using ethanol and water as eluent, wherein the volume ratio of the ethanol to the water in the gradient elution process is 10:90 to 100:0;
collecting eluates when the volume ratio of ethanol to water is changed to 20 to 80 in batches, respectively carrying out ultra high performance liquid chromatography detection on the eluates of each batch, and combining the eluates with chromatographic peaks in a detection map within the retention time of 8-9 min; the chromatographic conditions of the ultra-high performance liquid chromatography detection comprise: and (3) performing gradient elution by using methanol and water as eluent, wherein the volume ratio of the methanol to the water is changed from 10.
In one embodiment, the step of eluting using the ODS column comprises:
and (3) performing gradient elution by using methanol and water as eluent, wherein the volume ratio of the methanol to the water in the gradient elution process is 5:95 to 50:50;
collecting eluents when the volume ratio of methanol to water is changed to 5; the chromatographic conditions of the ultra-high performance liquid chromatography detection comprise: and (3) performing gradient elution by using methanol and water as eluent, wherein the volume ratio of the methanol to the water is changed from 10.
In one embodiment, the step of eluting with the sephadex column comprises:
eluting by using methanol as eluent, collecting the eluent in batches, respectively carrying out ultra-high performance liquid chromatography detection on the eluents of each batch, and combining the eluents with chromatographic peaks in the retention time of 8-9 min in a detection map; the chromatographic conditions of the ultra-high performance liquid chromatography detection comprise: and (3) performing gradient elution by using methanol and water as eluent, wherein the volume ratio of the methanol to the water is changed from 10.
In one embodiment, the step of eluting with the ultra high performance liquid chromatography comprises:
and (3) performing gradient elution by using methanol and water as eluent, wherein the volume ratio of the methanol to the water in the gradient elution process is 10:90 to 30:70.
in one embodiment, the step of eluting with the ultra high performance liquid chromatography further comprises the following conditions:
the flow rate is 1mL/min; the column temperature is 30 ℃; the elution time is 30min; the chromatographic column is Waters HSS T3; the detection wavelength was 270nm.
In one embodiment, the macroporous resin column is a D101 macroporous resin column.
In one embodiment, in the process of obtaining the ricepaper pith aqueous extract, one or more of the following conditions are satisfied:
(1) The water consumption for each decoction is 28 to 32 times of the mass of the ricepaper pith medicinal material;
(2) The decocting times are 2-4 times;
(3) The time of each decoction is 2 to 4 hours;
(4) The screen mesh for filtering is 300-400 meshes.
In one embodiment, in the process of obtaining the methanol extract of the ricepaper pith, one or more of the following conditions are met:
(1) The dosage of the methanol extracted by ultrasonic every time is 28-32 times of the mass of the ricepaper pith medicinal material;
(2) The times of ultrasonic extraction are 2-4 times;
(3) The time of ultrasonic extraction is 25 min-35 min each time;
(4) The screen mesh for filtering is 300-400 meshes.
An embodiment of the present application further provides an application of the phenolic compound described in one embodiment above or the phenolic compound extracted and separated by the method described in any one embodiment above in the preparation of cosmetics or health products with antioxidant activity.
The compound is a phenol compound with a monophenol structure, has excellent antioxidant activity, and has good application prospect in the fields of cosmetics industry, health care medicines and the like.
Furthermore, the phenolic compounds can be obtained from the ricepaper pith through a specific extraction and separation step, and research results on chemical components contained in the ricepaper pith are further expanded.
Drawings
FIG. 1 is a UV spectrum of phenolic compounds extracted and separated from Tetrapanax papyriferus in example 1;
FIG. 2 is an infrared spectrum of phenolic compounds extracted and separated from medulla Tetrapanacis of example 1;
FIG. 3 is a high resolution mass spectrum of the phenolic compound extracted and separated from Tetrapanax papyriferus in example 1;
FIG. 4 shows the phenolic compounds extracted and separated from medulla Tetrapanacis of example 1 1 H-NMR spectrum chart;
FIG. 5 shows the phenolic compounds extracted and separated from medulla Tetrapanacis of example 1 13 C-NMR spectrum;
FIG. 6 is a nuclear magnetic resonance carbon spectrum (DEPT) spectrum of a phenolic compound extracted and separated from medulla Tetrapanacis of example 1;
FIG. 7 is a spectrum of HSQC spectrum of phenolic compound extracted and separated from medulla Tetrapanacis of example 1;
FIG. 8 is a HMBC spectrum of the phenolic compound isolated from medulla Tetrapanacis of example 1;
FIG. 9 shows NMR of phenolic compounds isolated from medulla Tetrapanacis in example 1 1 H- 1 H COSY spectrogram;
FIG. 10 shows NMR of phenolic compounds isolated from Tetrapanax papyriferus in example 1 1 H- 1 H NOESY spectrogram;
FIG. 11 shows the utilization of the phenolic compounds extracted and separated from Tetrapanax papyriferus in example 1 1 H- 1 H COSY、HMBC、 1 H- 1 Structural analysis diagram of H NOESY;
FIG. 12 is a schematic diagram showing the structure of phenolic compounds extracted and separated from medulla Tetrapanacis in example 1.
Detailed Description
To facilitate an understanding of the present application, the present application will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present application are shown in the drawings. This application may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
In the present application, the technical features described in the open manner include a closed technical solution including the listed features, and also include an open technical solution including the listed features.
One embodiment of the present application provides a phenolic compound having a molecular formula of C 10 H 12 O 5 Having the chemical structure shown below:
the compound is a phenol compound with a monophenol structure, has excellent antioxidant and antibacterial activities, and has good application prospects in the fields of cosmetics industry, health-care medicines and the like.
It is to be understood that the phenolic compound in the above embodiment can be obtained by any method, and the present application does not limit the obtaining method of the phenolic compound in the above embodiment.
One embodiment of the present application provides a method for extracting and separating phenolic compounds from ricepaper pith, comprising the following steps:
sequentially extracting medulla Tetrapanacis with water and methanol to obtain extract;
separating the extract by chromatography to obtain phenolic compounds.
In one embodiment, the method for extracting and separating phenolic compounds from medulla Tetrapanacis comprises the following steps S110 to S130.
Step S110: decocting medulla Tetrapanacis in water, filtering, concentrating under reduced pressure, and freeze drying to obtain medulla Tetrapanacis water extract.
Step S120: ultrasonically extracting the medulla Tetrapanacis water extract with methanol, filtering to obtain filtrate, and concentrating the filtrate to obtain medulla Tetrapanacis methanol extract.
Step S130: and eluting the methanol extract of the ricepaper pith by sequentially adopting a macroporous resin column, an ODS column, a sephadex column and ultra-high performance liquid chromatography to obtain the phenolic compounds.
According to the method, the ricepaperplant pith is taken as a raw material source, and the phenolic compound is successfully extracted and separated by sequentially adopting water decoction, methanol ultrasonic extraction, macroporous resin column chromatography, ODS (ozone depleting substance) chromatography, sephadex column chromatography and ultra-high performance liquid chromatography for separation and purification.
It is understood that the above embodiment provides a method for extracting and separating phenolic compound from medulla Tetrapanacis, wherein the molecular formula of the extracted and separated phenolic compound is C 10 H 12 O 5 Having the chemical structure shown below:
the following describes the extraction and separation steps from step S110 to step S130 in detail:
step S110: decocting medulla Tetrapanacis in water, filtering, concentrating under reduced pressure, and freeze drying to obtain medulla Tetrapanacis water extract.
In one embodiment, the water consumption for each decoction is 28-32 times of the mass of the ricepaper pith medicinal material. It is understood that the amount of water used for each decoction can be, but not limited to, 28 times, 29 times, 30 times, 31 times, 32 times, etc. of the mass of the ricepaperplant pith. Furthermore, the water consumption for each decoction is 30 times of the mass of the ricepaper pith medicinal material.
In one embodiment, the time for each decoction is 2 to 4 hours. It is understood that the time of each decoction may be, for example, but not limited to, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, and the like. Further, the time for each decoction was 3 hours.
In one embodiment, the number of times of decoction is 2 to 4 times. It is understood that the number of times of decoction may be, for example, but not limited to, 2 times, 3 times, 4 times, etc. Further, the number of times of decoction was 3.
In one embodiment, the screen for filtration is 300 to 400 mesh. It is understood that the screen mesh for filtration may be, for example, but not limited to, 300 mesh, 310 mesh, 320 mesh, 330 mesh, 340 mesh, 350 mesh, 360 mesh, 370 mesh, 380 mesh, 390 mesh, 400 mesh, and the like. Further, the mesh for filtration was 350 mesh.
Understandably, the medulla Tetrapanacis can be sliced and decocted, which is more beneficial to extraction.
Step S120: ultrasonically extracting the medulla Tetrapanacis water extract with methanol, filtering to obtain filtrate, and concentrating the filtrate to obtain medulla Tetrapanacis methanol extract.
In one embodiment, the amount of methanol extracted by ultrasonic extraction is 28-32 times of the mass of the medulla Tetrapanacis medicinal material. It is understood that the amount of methanol used in each ultrasonic extraction can be, but is not limited to, 28 times, 29 times, 30 times, 31 times, 32 times, etc. of the mass of the ricepaperplant pith. Further, the amount of methanol extracted by ultrasonic every time is 30 times of the mass of the ricepaper pith medicinal material.
In one embodiment, the time for each ultrasonic extraction is 25min to 35min. It is understood that the time of each ultrasonic extraction can be, for example, but not limited to, 25min, 26min, 27min, 28min, 29min, 30min, 31min, 32min, 33min, 34min, 35min, and the like. Further, the time for each ultrasonic extraction was 30min.
In one embodiment, the number of ultrasound extractions is 2 to 4. It is understood that the number of ultrasound extractions may be, for example, but not limited to, 2, 3, 4, and so on. Further, the number of times of ultrasonic extraction was 3.
In one embodiment, the screen for filtration is 300 to 400 mesh. It is understood that the screen mesh for filtration can be, for example, but not limited to, 300 mesh, 310 mesh, 320 mesh, 330 mesh, 340 mesh, 350 mesh, 360 mesh, 370 mesh, 380 mesh, 390 mesh, 400 mesh, etc. Further, the mesh for filtration was 350 mesh.
Step S130: and eluting the methanol extract of the ricepaper pith by sequentially adopting a macroporous resin column, an ODS column, a sephadex column and ultra-high performance liquid chromatography to obtain the phenolic compounds.
In one embodiment, the step S130 may include the following steps S131 to S134.
Step S131: separating the methanol extract of the ricepaperplant pith by adopting a macroporous resin column to obtain a ricepaperplant pith macroporous resin column separation product.
Further, in step S131, gradient elution is performed with ethanol and water as eluents, and the volume ratio of ethanol to water in the gradient elution process is from 10:90 to 100:0.
further, in step S131, the eluates when the volume ratio of ethanol to water is changed to 20; the chromatographic conditions of the ultra-high performance liquid chromatography detection comprise: and (3) performing gradient elution by using methanol and water as eluent, wherein the volume ratio of the methanol to the water is changed from 10. Further, the chromatographic conditions of the ultra-high performance liquid chromatography detection also comprise: the elution time was 30 minutes; the flow rate is 0.2mL/min; the column temperature is 30 ℃; the chromatographic column is Waters CORTECS T3; the detection wavelength was 270nm.
Further, the macroporous resin column is a D101 macroporous resin column.
Further, after step S131 and before step S132, the method may further include the steps of concentrating the product of the separation by the medulla Tetrapanacis macroporous resin column under reduced pressure, drying, etc.
Step S132: separating the separated product of the medulla Tetrapanacis macroporous resin column by using an ODS column to obtain the separated product of the medulla Tetrapanacis ODS column.
It is understood that the packing of the ODS column includes octadecylsilane bonded silica packing.
Further, in step S132, gradient elution is performed with methanol and water as an eluent, and the volume ratio of methanol to water in the gradient elution process is from 5:95 to 50:50.
further, in step S132, the eluates when the volume ratio of methanol to water is changed to 5; the chromatographic conditions of the ultra-high performance liquid chromatography detection comprise: and (3) performing gradient elution by using methanol and water as eluent, wherein the volume ratio of the methanol to the water is changed from 10. Further, the chromatographic conditions of the ultra-high performance liquid chromatography detection also comprise: the elution time was 30 minutes; the flow rate is 0.2mL/min; the column temperature is 30 ℃; the chromatographic column is Waters CORTECS T3; the detection wavelength was 270nm.
Further, after step S132 and before step S133, the method may further comprise the steps of concentrating the separated product of the ODS column of medulla Tetrapanacis under reduced pressure, drying, etc.
Step S133: separating the separated product of the medulla Tetrapanacis ODS column by using a sephadex column to obtain a medulla Tetrapanacis sephadex column separated product.
Further, in step S133, eluting with methanol as an eluent, collecting the eluates in batches, performing ultra high performance liquid chromatography on the eluates of each batch, and combining the eluates having a chromatographic peak in a detection pattern within a retention time of 8min to 9 min; the chromatographic conditions of the ultra-high performance liquid chromatography detection comprise: and (3) performing gradient elution by using methanol and water as eluent, wherein the volume ratio of the methanol to the water in the gradient elution process is changed from 10. Further, the chromatographic conditions of the ultra-high performance liquid chromatography detection also comprise: the elution time was 30 minutes; the flow rate is 0.2mL/min; the column temperature is 30 ℃; the chromatographic column is Waters CORTECS T3; the detection wavelength was 270nm.
Further, in step S133, the end point of the elution is that the collected eluate is detected by ultra high performance liquid chromatography, and the detection profile has only a chromatographic peak of the eluate methanol.
Further, after step S133 and before step S134, the method may further include the steps of concentrating the separated product of the sephadex column under reduced pressure, drying, and the like.
Step S134: separating the product of the Tetrapanax papyriferus sephadex column separation by adopting ultra-high performance liquid chromatography to obtain the phenolic compounds.
Further, in step S134, gradient elution is performed with methanol and water as an eluent, and the volume ratio of methanol to water in the gradient elution process is from 10:90 to 30:70.
further, in step S134, the flow rate is 0.8mL/min to 1.2mL/min. Further, in step S134, the flow rate is 1mL/min.
Further, in step S134, the column temperature is 28 ℃ to 32 ℃. Further, in step S134, the column temperature was 30 ℃.
Further, in step S134, the elution time is 20 to 40min. Further, in step S134, the elution time is 30min.
Further, in step S134, the column is a C18 column. Further, in step S134, the chromatography column is Waters HSS T3.
Further, in step S134, the detection wavelength is 265nm to 275nm. Further, in step S134, the detection wavelength is 270nm.
In one embodiment, the stationary phase material in the ODS column and the sephadex column is pretreated by a process comprising: soaking the fixed phase material ODS or sephadex in methanol for more than 24 hr, loading into column, washing with methanol until the effluent liquid from the column is not turbid, and balancing with initial mobile phase of ODS column or sephadex column.
An embodiment of the present application further provides an application of the phenolic compound according to the above embodiment or the phenolic compound extracted and separated by the method according to any of the above embodiments in preparing cosmetics or health products with antioxidant activity.
The phenolic compound has good antioxidant and antibacterial activities, and has good application prospects in the fields of cosmetics or health-care products.
Furthermore, the phenolic compounds can be obtained from the ricepaper pith through a specific extraction and separation step, and research results on chemical components contained in the ricepaper pith are further expanded.
The phenolic compounds of the present application, and the method and application of extracting and separating phenolic compounds from medulla Tetrapanacis are described in further detail below with reference to specific examples. It is to be understood that the phenolic compounds provided in the embodiments of the present application are not limited to those obtained by the following examples.
Example 1
1. Apparatus and materials
Thermo Vanqish Flex binary ultra high performance liquid chromatograph, thermo Fisher Q active high resolution mass spectrometer, waters H-class ultra high performance liquid chromatograph (Watts, quaternary Pump, online degasser, autosampler, column oven, TUV Detector), nuclear magnetic resonance spectrometer AVANCE IIITM HD MHz (BRUKER, germany), UV-26001 ultraviolet visible Spectrophotometer (Shimadzu instruments Co., ltd.), RE-3000A rotary evaporator (Shanghai subsumonbo Biochemical Instrument Co., ltd.), SHZ-D III circulating water vacuum pump (Chengyu instruments Co., ltd.), DLSB-5120 circulating water pump of cryogenic cooling liquid (Zhengchangcheng Ke trades Co., ltd.), KQ-500DE type digital ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.), quartz microcuvette (Nanjing Kelvin Crystal science Co., ltd.), D101 macroporous adsorbent resin (Tianjin Shuichi Shuzo science Co., ltd.), ODS column chromatography (Octadecylsilylsilyl, octadecylsilane bonded silica gel filler) (Merck Co., ltd.), sephadex LH-20 column chromatography (Amersham Biosciences Swede), CDCl3 deuterated reagent (CIL Co., USA), 1,1-diphenyl-2-picrylhydrazine free radical (DPPH) (Fuzhou fly Bio-Tech Biotech Co., ltd.), 2,6-di-tert-butyl-p-cresol (BHT) (Shanghai Micheln Biochemical Co., ltd.).
2. Extracting and separating phenolic compounds from medulla Tetrapanacis
Step 1: cutting 8Kg of ricepaperplant pith into thick pieces, decocting with water for 3 times, each time for 3 hours, the water consumption for each time of decoction is 30 times of the mass of the ricepaperplant pith, filtering with a 350-mesh screen after each time of decoction, combining water decoctions, concentrating under reduced pressure, and freeze-drying to obtain 220g of ricepaperplant pith water extract.
Step 2: and (2) ultrasonically extracting the medulla Tetrapanacis water extract obtained in the step (1) for 3 times by using methanol, ultrasonically extracting for 30min each time, wherein the amount of the methanol extracted by each time is 30 times of the mass of the medulla Tetrapanacis medicinal material, filtering by using a 350-mesh screen after each time of ultrasonic extraction is finished, combining filtrates, and concentrating under reduced pressure to obtain 156.4g of medulla Tetrapanacis methanol extract.
And step 3: separating the methanol extract of the ricepaper pith obtained in the step 2 by using a D101 macroporous resin column, and performing gradient elution by using ethanol and water as eluent, wherein the volume ratio of the ethanol to the water in the gradient elution process is 10:90 to 100:0. collecting eluates (totally collecting 16 erlenmeyer flasks, wherein 200mL of each erlenmeyer flask) when the volume ratio of ethanol to water is changed to 20; the chromatographic conditions of the ultra-high performance liquid chromatography detection comprise: performing gradient elution by using methanol and water as eluent, wherein the volume ratio of the methanol to the water in the gradient elution process is changed from 10 to 90 to 30; the flow rate is 0.2mL/min; the column temperature is 30 ℃; the chromatographic column is Waters CORTECS T3; the detection wavelength was 270nm. And concentrating the combined eluent under reduced pressure until the eluent is dried to obtain a ricepaperplant pith macroporous resin column separation product.
And 4, step 4: and (3) subjecting the separated product of the medulla Tetrapanacis macroporous resin column obtained in the step (3) to chromatographic separation by a pretreated ODS column (octadecylsilane chemically bonded silica filler), and performing gradient elution by using methanol and water as eluent, wherein the volume ratio of methanol to water in the gradient elution process is 5:95 to 50:50. collecting eluates when the volume ratio of methanol to water is changed to 5 (10 erlenmeyer flasks are collected in total, and each erlenmeyer flask is 100 mL) in batches, respectively carrying out ultra performance liquid chromatography detection on the eluates in each batch, and combining the eluates with chromatographic peaks in the detection pattern within the retention time of 8-9 min; the chromatographic conditions of the ultra-high performance liquid chromatography detection comprise: performing gradient elution by using methanol and water as eluent, wherein the volume ratio of the methanol to the water in the gradient elution process is changed from 10 to 90 to 30; the flow rate is 0.2mL/min; the column temperature is 30 ℃; the chromatographic column is Waters CORTECS T3; the detection wavelength was 270nm. And concentrating the combined elution parts under reduced pressure until the elution parts are dried to obtain a ricepaper pith ODS column separation product.
The pretreatment process of the ODS column comprises the following steps: soaking ODS in methanol for more than 24 hr, loading into the column, washing ODS column with methanol until the effluent liquid from the washing column is dripped into water without turbidity, and balancing with initial mobile phase of ODS column.
And 5: carrying out chromatographic separation on the separated product of the medulla Tetrapanacis ODS column obtained in the step 4 through a pretreated Sephadex LH-20 column, eluting by using methanol as eluent, collecting the eluent in batches (totally collecting 18 penicillin bottles, wherein each penicillin bottle is 3 mL), respectively carrying out ultra-high performance liquid chromatography detection on the eluents of the batches, and combining the eluents with chromatographic peaks in the retention time of 8-9 min in a detection map; the chromatographic conditions of the ultra-high performance liquid chromatography detection comprise: performing gradient elution by using methanol and water as eluent, wherein the volume ratio of the methanol to the water in the gradient elution process is changed from 10 to 90 to 30; the flow rate is 0.2mL/min; the column temperature is 30 ℃; the chromatographic column is Waters CORTECS T3; the detection wavelength was 270nm. Concentrating the combined elution parts under reduced pressure to dryness to obtain a Tetrapanax papyriferus sephadex column separation product.
The pretreatment process of the sephadex column comprises the following steps: soaking the sephadex in methanol for more than 24 hours, loading on the column, washing the sephadex column with methanol until the liquid flowing out of the column is not turbid when dropped into water, and then balancing with the initial mobile phase of the sephadex column.
Step 6: separating and preparing the separated product of the ricepaperplant hyssop gel column obtained in the step 5 by ultra-high performance liquid chromatography, performing gradient elution by taking methanol and water as eluent, wherein the volume ratio of the methanol to the water in the gradient elution process is 10:90 to 30:70, the flow rate is 1mL/min, and the column temperature is 30 ℃; the elution time is 30min; the chromatographic column is Waters HSS T3; the detection wavelength was 270nm, and 3.1mg of the phenolic compound was finally obtained.
3. Performing structural identification on phenolic compounds extracted and separated from medulla Tetrapanacis
The phenolic compound extracted from medulla Tetrapanacis is brown powder, is easily dissolved in organic solvent such as methanol, and is sprayed with FeCl 3 The test solution appeared cyan, indicating that the compound may contain a phenolic hydroxyl structure.
As shown in FIG. 1, methanol (CH) is added to phenolic compounds extracted and separated from medulla Tetrapanacis 3 OH) as solvent, ultraviolet spectrum test shows that the visible lambda is max 231nm, 279nm and 308nm ultraviolet absorption peaks.
As shown in fig. 2, the infrared spectrum test of the phenolic compound extracted and separated from the ricepaper pith is carried out, and the IR (KBr) test result is that: v is max 3379cm -1 、2927cm -1 、1665cm -1 、1593cm -1 、1120cm -1 ，777cm -1 (ν OH 3379cm -1 ，ν CH 2927cm -1 ，ν C＝O 1665cm -1 ，ν C-O 1120cm -1 )。
As shown in fig. 3, the phenolic compound extracted and separated from the ricepaper pith is subjected to high resolution mass spectrometry, and the ESI-MS test result is m/z:211.0605, M-H-, the relative molecular mass of the compound is 212.0684.
As shown in fig. 4, the extraction and separation of phenolic compounds from medulla Tetrapanacis are performed 1 And (3) performing H-NMR (hydrogen-nuclear magnetic resonance) testing, 1 H-NMR(600MHz，CD 3 OD) spectrum shows that the low field region has 3 benzene ring proton signals delta H 6.98(1H，d，J＝8.28Hz，H-3′)，δ H 7.49(1H，dd，J＝8.22，1.98Hz，H-5′)，δ H 7.53 (1H, d, J=1.98Hz, H-6'), 1 methine H signal delta H 5.12(1H，t，J＝45Hz, H-2), 1 methylene H signal delta H 4.01 (1H, dd, J =11.70Hz, H-3 a), δ H3.73 (1H, dd, J =5.28Hz, H-3 b), 1 methoxy H signal δ H 3.73(3H，dd，J＝5.34Hz，H-1″)。
As shown in fig. 5, the extraction and separation of phenolic compounds from medulla Tetrapanacis are performed 13 And (3) C-NMR (nuclear magnetic resonance) testing, 13 C-NMR(150MHz，CD 3 OD) spectrum shows 11 carbon signals, and shows that 1 methoxyl C signal delta exists in the compound C 56.09(2″-OCH 3 ) 1 carbonyl C signal delta C 197.40 (1-C = O), 1 methine C signal δ C 73.91 (C-2) and 1 methylene C Signal. Delta C 65.85 (C-3), and the C signal δ C 73.91 and the C signal delta C 65.85 is located in the low field region, and presumably both are connected oxygen C signals, delta C 114.20～δ C 151.58 is the 6C signals (C-1 'to 6') on the benzene ring.
The analysis of the test results can determine that the molecular formula of the phenolic compound extracted and separated from the ricepaper pith is C 10 H 12 O 5 The unsaturation degree was 6.
As shown in FIGS. 6 to 12, the phenolic compounds extracted and separated from medulla Tetrapanacis were subjected to nuclear magnetic resonance carbon spectrum (DEPT) test, HSQC test, HMBC test, and the like, 1 H- 1 H COSY test, 1 H- 1 H NOESY test, nuclear magnetic resonance carbon spectrum (DEPT) DEPT 135 spectrum finding 1 CH 3 Signal, 1 CH 2 Signal, 4 CH signals and 4C signals. To determine the position and the connection mode of each substituent in the molecular structure, the method is firstly carried out 1 H- 1 Three spin systems, including delta, were found in the H COSY spectra H 6.98(H-3′)、δ H 7.49(H-5′)、δ H 7.53 (H-6') and is defined as 1,2,4-trisubstituted benzene ring, and then the relevant H signal directly linked to C was determined from HSQC spectra (see Table 1). The aromatic H signal delta is seen from the 2D-NMR spectrum, i.e. HMBC spectrum H 6.98 (H-3') with the C signal delta C 151.58(C-4′)、δ C 147.04 (C-2') and delta C 197.40 (1-C = O) correlation, fragrance H signal δ H 7.49 (H-5') with the C signal delta C 110.37(C-6′)、δ C 151.58 (C-4') and delta C 197.40 (1-C = O) correlation, fragrance H signal δ H 7.53 (H-6') with the C signal delta C 125.98(C-5′)、δ C 151.58(C-4′)、δ C 197.40 (1-C = O) and δ C 147.04 (C-2') so that the carbonyl group is attached to the 1-position, the methoxy group is attached to the 2-position, and the hydroxy group is attached to the 4-position on the phenyl ring; h signal delta H 5.12 (H-2) and H Signal δ H 4.01 (H-3 a) both of the C signal and the H signal are correlated with each other and with the carbonyl C signal delta C 197.40 (1-C = O), C-2 and C-3 are assumed to be side chains connected to the carbonyl group as benzene rings. According to 1 H- 1 H and H signals delta on hydroxyl (2-OH) in H NOESY spectra H 4.01 (1H, dd, J =11.70Hz, H-3 a) and the substitution of the hydroxyl group at C-2 is presumed.
TABLE 1 determination from HSQC spectra 1 H-NMR and 13 C-NMR data
Based on the above test results and analysis, it can be determined that the phenolic compound extracted and separated from medulla Tetrapanacis has a molecular formula of C 10 H 12 O 5 Has a chemical structure of
4. Evaluation of antioxidant ability of phenolic Compounds
The test method comprises the following steps: and evaluating the antioxidant capacity of the extracted and separated phenolic compound by a DPPH spectrophotometry.
(1) Preparing a solution: taking a phenolic compound and using methanol as a solvent to prepare sample solutions with 7 series concentrations from high concentration to low concentration, wherein the 7 series concentrations are 2.50 mu M, 5.00 mu M, 10.00 mu M, 20.00 mu M, 40.00 mu M, 80.00 mu M and 120.00 mu M, and using BHT and ethanol as a solvent to prepare the sample solutions with the same concentration as a positive control; the DPPH solution with the preparation concentration of 0.08mmol/L is prepared just before the experiment.
(2) The test method comprises the following steps: the experiment is divided into three groups, 1mL of methanol is added into 1mL of the LDPPH solution to serve as a blank group of the phenolic compound, and 1mL of ethanol is added into the 1mL of the LDPPH solution to serve as a blank group of BHT; adding 1mL of phenolic compound sample solution into 1mL of the LDPPH solution to serve as a phenolic compound sample group, and adding 1mLBHT sample solution into 1mLDPPH solution to serve as a BHT sample group; 1mL of the methanol solution was added to 1mL of the phenolic compound sample solution as a phenolic compound control, and 1mL of the ethanol solution was added to 1mLBHT sample solution as a BHT control. After each set of solutions was mixed, it was left to react well for 30 minutes at room temperature in the dark, and each sample was measured three times at 517 nm.
The DPPH clearance is calculated as follows:
the test results are shown in tables 2 and 3 below.
TABLE 2 DPPH radical scavenging Rate of phenolic Compounds
TABLE 3 DPPH radical clearance of BHT
As can be seen from tables 2 and 3, the phenolic compounds extracted and separated from the ricepaper pith have strong antioxidant capacity, and the antioxidant capacity has concentration dependence, and the higher the concentration is, the stronger the antioxidant capacity is.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent application shall be subject to the appended claims.
Claims (12)
1. A phenolic compound characterized by having a molecular formula of C 10 H 12 O 5 Having the chemical structure shown below:
2. a method for extracting and separating phenolic compounds from ricepaperplant pith is characterized by comprising the following steps:
sequentially extracting medulla Tetrapanacis with water and methanol to obtain extract;
and separating the extract by adopting a chromatographic separation method to obtain the phenolic compounds.
3. The method for extracting and separating phenolic compounds from ricepaper pith as claimed in claim 2, wherein the method comprises the steps of:
decocting the ricepaperplant pith medicinal material with water, filtering the decoction, concentrating the decoction under reduced pressure, and freeze-drying to obtain a ricepaperplant pith aqueous extract;
ultrasonically extracting the water extract of the ricepaperplant pith with methanol, filtering to obtain filtrate, and concentrating the filtrate to obtain a ricepaperplant pith methanol extract;
and eluting the ricepaper pith methanol extract by sequentially adopting a macroporous resin column, an ODS column, a sephadex column and ultra-high performance liquid chromatography to obtain the phenolic compounds.
4. The method for extracting and separating phenolic compounds from ricepaperplant pith as claimed in claim 3, wherein the step of eluting with the macroporous resin column comprises:
and (3) performing gradient elution by using ethanol and water as eluent, wherein the volume ratio of the ethanol to the water in the gradient elution process is 10:90 to 100:0;
collecting eluates when the volume ratio of ethanol to water is changed to 20 to 80 in batches, respectively carrying out ultra high performance liquid chromatography detection on the eluates of each batch, and combining the eluates with chromatographic peaks in the detection map within the retention time of 8-9 min; the chromatographic conditions of the ultra-high performance liquid chromatography detection comprise: and (3) performing gradient elution by using methanol and water as eluent, wherein the volume ratio of the methanol to the water in the gradient elution process is changed from 10.
5. The method of claim 3, wherein the step of eluting with said ODS column comprises:
and (3) performing gradient elution by using methanol and water as eluent, wherein the volume ratio of methanol to water in the gradient elution process is 5:95 to 50:50;
collecting eluates when the volume ratio of methanol to water is changed to 5; the chromatographic conditions of the ultra-high performance liquid chromatography detection comprise: and (3) performing gradient elution by using methanol and water as eluent, wherein the volume ratio of the methanol to the water is changed from 10.
6. The method for extracting and separating phenolic compounds from ricepaperplant pith as claimed in claim 3, wherein the step of eluting with said sephadex column comprises:
eluting by using methanol as eluent, collecting the eluent in batches, respectively carrying out ultra-high performance liquid chromatography detection on the eluents of each batch, and combining the eluents with chromatographic peaks in the retention time of 8-9 min in a detection map; the chromatographic conditions of the ultra-high performance liquid chromatography detection comprise: and (3) performing gradient elution by using methanol and water as eluent, wherein the volume ratio of the methanol to the water is changed from 10.
7. The method for extracting and separating phenolic compounds from ricepaperplant pith as claimed in claim 3, wherein the step of eluting by using said ultra high performance liquid chromatography comprises:
and (3) performing gradient elution by using methanol and water as eluent, wherein the volume ratio of methanol to water in the gradient elution process is 10:90 to 30:70.
8. the method for extracting and separating phenolic compounds from ricepaper pith as claimed in claim 7, wherein the step of eluting with said ultra high performance liquid chromatography further comprises the following conditions:
the flow rate is 1mL/min; the column temperature is 30 ℃; the elution time is 30min; the chromatographic column is Waters HSS T3; the detection wavelength was 270nm.
9. The method for extracting and separating phenolic compounds from ricepaperplant pith as claimed in claim 3, wherein the macroporous resin column is a D101 macroporous resin column.
10. The method for extracting and separating phenolic compounds from ricepaperplant pith as claimed in any one of claims 3 to 9, wherein one or more of the following conditions are satisfied in the process of obtaining the ricepaperplant pith aqueous extract:
(1) The water consumption for each decoction is 28 to 32 times of the mass of the ricepaper pith medicinal material;
(2) The decocting times are 2 to 4 times;
(3) The time of each decoction is 2 to 4 hours;
(4) The screen mesh for filtering is 300-400 meshes.
11. The method for extracting and separating phenolic compounds from ricepaperplant pith as claimed in any one of claims 3 to 9, wherein in the process of obtaining the ricepaperplant pith methanol extract, one or more of the following conditions are satisfied:
(1) The dosage of the methanol extracted by each ultrasonic is 28 to 32 times of the mass of the ricepaper pith medicinal material;
(2) The times of ultrasonic extraction are 2-4 times;
(3) The time of ultrasonic extraction is 25 min-35 min each time;
(4) The screen mesh for filtering is 300-400 meshes.
12. Use of a phenolic compound according to claim 1 or a phenolic compound isolated by extraction according to any one of claims 2 to 11 in the preparation of a cosmetic or health product having antioxidant activity.
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