A method for extracting tannin fractions from fresh Terminalia chebula Retz., a method for extracting and purifying tannin compounds, and a detection method

Abstract

The present invention relates to the technical field of traditional Chinese medicine extraction and detection, and particularly relates to an extraction method for tannin fractions in fresh Terminalia chebula, an extraction and purification method for tannin compounds, and a detection method. The extraction method for tannin fractions in fresh Terminalia chebula provided by the present invention uses fresh Terminalia chebula fruits as raw materials, and successively performs stone removal, water-added juicing, and ultrasonic extraction, which can not only avoid the degradation of tannic acid, but also obtain an ideal extraction rate, and has a simple process, low energy consumption, and short time consumption. The present invention also provides an extraction and purification method for tannin compounds. Through macroporous adsorption resin elution, alcohol precipitation, octadecylsilyl reverse-phase packing column elution, and preparative chromatography separation, high-purity chebulic acid, gallic acid, 4-galloylshikimic acid, and 5-galloylshikimic acid can be obtained. The detection method provided by the present invention can detect more than ten tannin components, has high efficiency, and helps to provide data support for the in-depth study of tannin components in Terminalia chebula.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine extraction and detection, and particularly relates to an extraction method for tannin parts in fresh Terminalia chebula, an extraction and purification method for tannin compounds, and a detection method. Background Art

[0002] Terminalia chebula is the dried ripe fruit of Terminalia chebula Retz. or Terminalia chebula Retz. var. tomentella Kurt., and has the functions of astringing the intestines to stop diarrhea, astringing the lung to relieve cough, and reducing fire to soothe the throat. Terminalia chebula contains various chemical components such as polyphenols, flavonoids, polysaccharides, and terpenoids, among which the most abundant are tannins, mainly hydrolyzable tannins. Tannins have strong antioxidant ability, and gallic acid, punicalagin, corilagin, and ellagic acid are used as index components. Currently, most common methods for extracting tannins from Terminalia chebula use the powder of dried Terminalia chebula fruits for extraction. However, during the process of drying fresh fruits into dried fruits, tannic acid degrades, and it is energy-consuming, time-consuming, and the operation is cumbersome, with a low extraction rate. Summary of the Invention

[0003] In view of the above technical problems, the present invention provides an extraction method for tannin parts in fresh Terminalia chebula, an extraction and purification method for tannin compounds, and a detection method. The extraction method for tannin parts directly extracts the tannin parts in fresh Terminalia chebula, can reduce the degradation of tannic acid, has low energy consumption, a simple process, and a high extraction rate; the extraction and purification method for tannin compounds can obtain high-purity compounds; the detection method can detect more than a dozen tannin components from the above-mentioned tannin parts.

[0004] To achieve the above invention object, the embodiments of the present invention adopt the following technical solutions:

[0005] In the first aspect, the present invention provides an extraction method for tannin parts in fresh Terminalia chebula, which specifically includes the following operations:

[0006] Remove the pits from fresh Terminalia chebula fruits, add water to extract juice to obtain a fresh Terminalia chebula homogenate;

[0007] Use an ethanol aqueous solution to ultrasonically extract the fresh Terminalia chebula homogenate, perform solid-liquid separation, concentrate and dry the obtained liquid phase, and that's it.

[0008] In the prior art, the methods for extracting tannin components from Terminalia chebula are all from dried ripe fruits of Terminalia chebula, and there is no report on extracting tannin components from fresh Terminalia chebula. In order to extract tannin parts from fresh Terminalia chebula and at the same time obtain an ideal extraction efficiency, the present invention has studied the extraction process. By first preparing a fresh Terminalia chebula homogenate and then extracting with an ethanol aqueous solution, it can not only avoid the degradation of tannic acid caused by making fresh fruits into dried fruits, but also obtain an ideal extraction rate, and the process is simple, with low energy consumption and short time consumption, providing a new method for the extraction of tannin components in Terminalia chebula.

[0009] In combination with the first aspect, the concentration of the aqueous ethanol solution is 60% to 100% v / v. When the ethanol concentration is too low, it is not easy to filter the extract by suction. When the ethanol concentration reaches 60% v / v, this problem can be avoided as much as possible, and at the same time, sugars can be precipitated, the solid content can be reduced, and the hygroscopicity of the extract can be decreased.

[0010] Preferably, the temperature of ultrasonic extraction is 25 to 60 °C, and the power is 500 to 700 W.

[0011] Preferably, the number of times of ultrasonic extraction of the fresh Terminalia chebula homogenate with the aqueous ethanol solution is ≥ 2 times. The number of extraction times has little effect on the extraction rate. After two extractions, the cumulative extraction rates of gallic acid, punicalagin, corilagin, and ellagic acid are all > 78%.

[0012] In the second aspect, the present invention also provides a method for extracting and purifying tannin compounds. The compound is chebulic acid, and the extraction method specifically includes the following steps:

[0013] S1. Extract the tannin part from fresh Terminalia chebula according to the above extraction method;

[0014] S2. Dissolve the tannin part in water and perform solid-liquid separation. Using macroporous adsorption resin as the stationary phase and water as the elution solvent, perform column chromatography on the liquid phase obtained by solid-liquid separation, and collect the eluate of the first 4 to 5 times the column volume;

[0015] S3. Combine the eluates collected in S2, concentrate, add ethanol, let stand for precipitation, and then perform solid-liquid separation;

[0016] S4. Dissolve the solid phase obtained by solid-liquid separation in S3 in water, use octadecylsilane-bonded silica reversed-phase packing (ODS) as the stationary phase, and water as the elution solvent to perform column chromatography, collect the eluate of the first 1 to 2 times the column volume, concentrate, and then separate and purify by preparative chromatography;

[0017] The chromatographic conditions of the preparative chromatography include: mobile phase: mobile phase A is an aqueous solution of 0.095% to 0.105% v / v formic acid, and mobile phase B is methanol, and isocratic elution is performed at a volume ratio of mobile phase A:mobile phase B of 90:10.

[0018] In this method, the eluate collected by macroporous adsorption resin in S2 contains a high content of the target compound; after alcohol precipitation in S3, chebulic acid can be separated from the precipitate; after separation and purification by preparative chromatography under the above chromatographic conditions in S4, high-purity chebulic acid can be obtained.

[0019] Preferably, the chromatographic conditions of the preparative chromatography further include: the chromatographic column is Cosmosil 5C18-MS-II (20 × 250 mm, 5 μm); the flow rate is 8 ml / min; the detection wavelength is 284 nm.

[0020] In combination with the second aspect, it is preferable to collect the eluate with a volume of 5 times the column volume in S2. At this elution volume, the cumulative elution amount of chebulinic acid is relatively high, and the elution amount of impurities is relatively low.

[0021] In combination with the second aspect, ethanol is added in S3 until the ethanol content reaches 85.7% - 90.0% v / v. At this ethanol content, more precipitation can be obtained, which helps to obtain more target compounds.

[0022] In combination with the second aspect, it is preferable to collect the eluate with a volume of 2 times the column volume in S4. At this elution volume, the cumulative elution amount of chebulinic acid is relatively high.

[0023] In a third aspect, the present invention also provides a method for extracting and purifying tannin compounds, which include gallic acid, 4-galloylshikimic acid, and 5-galloylshikimic acid. The method specifically includes the following steps:

[0024] S1. Extract the tannin part from fresh Terminalia chebula Retz. according to the above extraction method;

[0025] S2. Dissolve the tannin part in water and perform solid-liquid separation. Using macroporous adsorption resin as the stationary phase and water as the elution solvent, perform column chromatography on the liquid phase obtained by solid-liquid separation, and collect the eluate with a volume of 4 - 5 times the column volume;

[0026] S3. Combine the eluates collected in S2, concentrate, add ethanol, let it stand for precipitation, and then perform solid-liquid separation;

[0027] S4. Concentrate the liquid phase obtained by solid-liquid separation in S3, and purify it by preparative chromatography;

[0028] The chromatographic conditions of the preparative chromatography include:

[0029] Mobile phase: Mobile phase A is an aqueous solution of formic acid with a concentration of 0.095% - 0.105% v / v, and mobile phase B is methanol. Perform isocratic elution at a volume ratio of mobile phase A:mobile phase B of (60 - 65):(40 - 35).

[0030] In this method, the eluate collected by macroporous adsorption resin in S2 contains a relatively high content of target compounds; after alcohol precipitation in S3, gallic acid, 4-galloylshikimic acid, and 5-galloylshikimic acid can be separated from the supernatant; after purification by preparative chromatography under the above chromatographic conditions in S4, high-purity gallic acid, 4-galloylshikimic acid, and 5-galloylshikimic acid can be obtained.

[0031] Preferably, the chromatographic conditions of the preparative chromatography further include: the chromatographic column is Cosmosil PBr PackedColumn (20×250mm, 5μm); the flow rate is 8ml / min; the detection wavelength is 270nm.

[0032] In combination with the third aspect, it is preferable to collect 5 column volumes of the eluate in S2. At this elution volume, the cumulative elution amounts of gallic acid, 4-galloylshikimic acid, and 5-galloylshikimic acid are relatively high.

[0033] In combination with the third aspect, ethanol is added in S3 until the ethanol content reaches 85.7% - 90.0% v / v. At this ethanol content, more chebulinic acid precipitates can be removed, thus contributing to fewer interfering substances in the supernatant. Among them, it is preferable to add 8 times the amount of ethanol to the concentrated eluate, that is, the ethanol content is about 88.9% v / v.

[0034] In combination with the third aspect, the ratio of mobile phase A to mobile phase B in S4 is 65:35. At this ratio, the purity of gallic acid is higher.

[0035] In combination with the third aspect, preparative chromatography is used for separation and purification twice in S4. Optionally, when performing the first separation and purification, the ratio of mobile phase A to mobile phase B is 60:40; when performing the second separation and purification, the ratio of mobile phase A to mobile phase B is 65:35; or both are 65:35 to obtain a higher product purity.

[0036] In the fourth aspect, the present invention provides a method for detecting tannin components in Terminalia chebula, and the method is ultra-high performance liquid chromatography. The chromatographic conditions include:

[0037] Chromatographic column: Cosmosil PBr Packed Column (2.1×100mm, 2.6μm);

[0038] Mobile phase A is an aqueous solution of formic acid with a concentration of 0.095% - 0.105% v / v, and mobile phase B is methanol. Linear gradient elution is performed, and the elution program is:

[0039]

[0040]

[0041] Flow rate: 0.28 - 0.32 mL / min.

[0042] The above chromatographic conditions can detect various tannin components in Terminalia chebula, including chebulinic acid, gallic acid, 4-galloylshikimic acid, 5-galloylshikimic acid, punicalagin A, punicalagin B, corilagin, 1,3,6-trigalloylglucose, chebulin, urolithin M5, chebulagic acid, chebulanin, and ellagic acid. It can be used for detecting the content of tannin components in the extraction solutions of each step during the extraction process of tannin components, as well as for performing relevant detections in the research of tannin components in Terminalia chebula.

[0043] In combination with the fourth aspect, the flow rate is preferably 0.3 mL / min.

[0044] Preferably, the chromatographic conditions further include: detection wavelength: 210 - 400 nm; injection volume 2 μL; column temperature 30 °C.

[0045] The beneficial effects of the present invention are as follows: The present invention adopts the extraction process of squeezing fresh fruits of Terminalia chebula Retz. to extract juice, which can minimize the loss of active ingredients. Moreover, fresh squeezing can save resources, reduce energy consumption, and the process is simple. The extraction rates of tannin components such as gallic acid, punicalagin, corilagin, and ellagic acid are high. The present invention also provides a method for extracting compounds from the tannin fraction obtained from the above extraction process. This method can obtain high-purity chebulic acid, gallic acid, 4-galloylshikimic acid, and 5-galloylshikimic acid from the above tannin fraction through macroporous adsorption resin elution, alcohol precipitation, octadecylsilyl bonded silica reversed-phase packing column elution, and preparative chromatography separation under specific chromatographic conditions. The obtained compounds can be further used for pharmacological activity research. The present invention also provides a detection method for tannin components, which can detect more than ten kinds of tannin components from the above tannin fraction, helping to provide data support for the in-depth research of tannin components in Terminalia chebula Retz. Description of the Drawings

[0046] The present invention will be further described below in conjunction with the drawings and embodiments. In the drawings:

[0047] Figure 1 is the preparation process of four tannin chemical components of Terminalia chebula Retz. in Example 5 of the present invention;

[0048] Figure 2 is the ultra-high performance liquid chromatogram of 60% v / v ethanol extract of fresh Terminalia chebula Retz. homogenate in Example 5 of the present invention;

[0049] Figure 3 is the ultra-high performance liquid chromatogram of supernatant 1 in Example 5 of the present invention;

[0050] Figure 4 is the ultra-high performance liquid chromatogram of the water eluate of macroporous resin after sample loading in Example 5 of the present invention;

[0051] Figure 5 is the ultra-high performance liquid chromatogram of supernatant 2 in Example 5 of the present invention;

[0052] Figure 6 is the ultra-high performance liquid chromatogram of the solution after precipitation 2 is redissolved in water in Example 5 of the present invention.

[0053] Figures 2 to 6 In, peak 1 in the figure is chebulic acid, peak 2 is gallic acid, peak 3 is 4-galloylshikimic acid, and peak 4 is 5-galloylshikimic acid;

[0054] Figure 7 is the preparative chromatogram of chebulic acid in Example 5 of the present invention;

[0055] Figure 8 is the chebulinic acid in Example 5 of the present invention 1 H NMR spectrum;

[0056] Figure 9 is the chebulinic acid in Example 5 of the present invention 13 C NMR spectrum;

[0057] Figure 10 is the ultra - performance liquid chromatography (UPLC) spectrum of chebulinic acid in Example 5 of the present invention;

[0058] Figure 11 is the preparative chromatography spectrum of gallic acid, 4 - galloylshikimic acid and 5 - galloylshikimic acid in Example 5 of the present invention; Peak 2 in the figure is gallic acid, Peak 3 is 4 - galloylshikimic acid, and Peak 4 is 5 - galloylshikimic acid;

[0059] Figure 12 is the gallic acid in Example 5 of the present invention 1 H NMR spectrum;

[0060] Figure 13 is the gallic acid in Example 5 of the present invention 13 C NMR spectrum;

[0061] Figure 14 is the ultra - performance liquid chromatography (UPLC) spectrum of gallic acid in Example 5 of the present invention;

[0062] Figure 15 is the 4 - galloylshikimic acid in Example 5 of the present invention 1 H NMR spectrum;

[0063] Figure 16 is the 4 - galloylshikimic acid in Example 5 of the present invention 13 C NMR spectrum;

[0064] Figure 17 is the ultra - performance liquid chromatography (UPLC) spectrum of 4 - galloylshikimic acid in Example 5 of the present invention;

[0065] Figure 18 is the 5 - galloylshikimic acid in Example 5 of the present invention 1 H NMR spectrum;

[0066] Figure 19 is the 5 - galloylshikimic acid in Example 5 of the present invention 13 C NMR spectrum;

[0067] Figure 20 is the ultra - performance liquid chromatography (UPLC) spectrum of 5 - galloylshikimic acid in Example 5 of the present invention;

[0068] Figure 21It is the chromatogram of the ethanol extracts with different concentrations in Example 9 of the present invention;

[0069] Figure 22 It is the content (calculated based on fresh fruits) and extraction rate of tannin components in the ethanol extracts with different concentrations in Example 9 of the present invention;

[0070] Figure 23 It is the chromatogram of the extract obtained by extracting fresh Terminalia chebula homogenate four times with 60% v / v ethanol in Example 9 of the present invention;

[0071] Figure 24 It is the extraction rate of tannin components in the extract obtained by extracting fresh Terminalia chebula homogenate four times with 60% v / v ethanol in Example 9 of the present invention. Detailed implementation manners

[0072] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0073] Terminalia chebula contains a variety of chemical components, among which the most abundant are tannins, mainly hydrolyzable tannins. Most of the currently commonly used methods for extracting tannins from Terminalia chebula use the powder of dried Terminalia chebula fruits. However, during the process of drying fresh fruits into dried fruits, tannic acid degrades, and it is energy-consuming, time-consuming, with cumbersome operations and low extraction rates.

[0074] Through experimental research, the present invention has obtained a method for extracting the tannin part from fresh Terminalia chebula, which can minimize the loss of active ingredients, and has small energy consumption, short time, simple process and high extraction rate. This extraction method includes the following operations:

[0075] Remove the pits from fresh Terminalia chebula fruits, add water and squeeze to obtain fresh Terminalia chebula homogenate;

[0076] Ultrasonically extract the fresh Terminalia chebula homogenate with an ethanol aqueous solution, perform solid-liquid separation, concentrate and dry the obtained liquid phase to obtain the product.

[0077] There are many types of tannin components in Terminalia chebula, and the extraction methods of some tannin components have not been reported. In order to obtain more tannin compounds from Terminalia chebula for further pharmacological research on Terminalia chebula, the embodiments of the present invention also provide a method for extracting and purifying tannin compounds, which specifically includes the following steps:

[0078] S1. Extract the tannin part from fresh Terminalia chebula according to the above extraction method;

[0079] S2. Dissolve the tannin part in water and perform solid-liquid separation. Using macroporous adsorption resin as the stationary phase and water as the elution solvent, perform column chromatography on the liquid phase obtained by solid-liquid separation, and collect the eluate of the first 4-5 times of the column volume;

[0080] S3. Combine the eluates collected in S2, concentrate them, add ethanol, let it stand for precipitation, and then perform solid-liquid separation;

[0081] S4. Dissolve the solid phase obtained from the solid-liquid separation in S3 in water, use octadecylsilyl bonded silica reversed-phase packing (ODS) as the stationary phase, and water as the elution solvent for column chromatography. Collect the eluate of the first 1 - 2 times the column volume, concentrate it, and then separate and purify it by preparative chromatography to obtain chebulinic acid; the chromatographic conditions of the preparative chromatography include: mobile phase: mobile phase A is an aqueous solution of 0.095% - 0.105% v / v formic acid, mobile phase B is methanol, and isocratic elution is performed at a volume ratio of mobile phase A:mobile phase B of 90:10;

[0082] Concentrate the liquid phase obtained from the solid-liquid separation in S3, and then separate and purify it by preparative chromatography to obtain gallic acid, 4-galloylshikimic acid, and 5-galloylshikimic acid; the chromatographic conditions of the preparative chromatography include: mobile phase: mobile phase A is an aqueous solution of 0.095% - 0.105% v / v formic acid, mobile phase B is methanol, and isocratic elution is performed at a volume ratio of mobile phase A:mobile phase B of (60 - 65):(40 - 35).

[0083] To accurately and efficiently detect various tannin components in Terminalia chebula Retz., the present invention also provides a method for detecting tannin components in Terminalia chebula Retz., and the method is ultra-high performance liquid chromatography, and the chromatographic conditions include:

[0084] Chromatographic column: Cosmosil PBr Packed Column (2.1×100mm, 2.6μm);

[0085] Mobile phase A is an aqueous solution of 0.095% - 0.105% v / v formic acid, mobile phase B is methanol, and linear gradient elution is performed. The elution program is:

[0086]

[0087] Flow rate: 0.28 - 0.32 mL / min.

[0088] The following uses specific examples to illustrate the solution of the present invention.

[0089] Other reagents and drugs used in the following examples are all common laboratory reagents and drugs obtained from commercial sources unless otherwise specified.

[0090] The main instruments used in the following examples:

[0091] Ultra-high performance liquid chromatograph (ACQUITY H class plus, Waters, USA); preparative liquid chromatograph (1260 Infinity Ⅱ, Agilent, USA); ten-thousandth balance (ME204, METTLER, Switzerland); intelligent ultrasonic cleaner (DL-720B, Shanghai Zhixin Instrument Co., Ltd.); high-speed refrigerated centrifuge (5430R, Eppendorf, Germany); wall-breaking blender (WS-703, Zhongshan Meisheng Electric Appliance Co., Ltd.).

[0092] The reagents used in the following examples:

[0093] Methanol (chromatographic grade, Sigma-Aldrich Co., Ltd.); absolute ethanol (analytical grade, Tianjin Concord Technology Co., Ltd.); formic acid (chromatographic grade, Shanghai Aladdin Biochemical Technology Co., Ltd.); purified water (Guangzhou Watson's Food and Beverage Co., Ltd.); dimethyl sulfoxide (analytical grade, Tianjin Damao Chemical Reagent Factory).

[0094] The medicinal reagents used in the following examples:

[0095] Fresh Terminalia chebula Retz. (collected from Yongde County, Lincang City, Yunnan Province); gallic acid (batch number C13O9C72105, Shanghai Yuanye Bio-Technology Co., Ltd.); punicalagin (J03HB186918, Shanghai Yuanye Bio-Technology Co., Ltd.); corilagin (P25A11S122217, Shanghai Yuanye Bio-Technology Co., Ltd.); ellagic acid (PJ0707RA14, Shanghai Yuanye Bio-Technology Co., Ltd.).

[0096] Example 1

[0097] This example provides a method for extracting tannin fractions from fresh Terminalia chebula Retz.

[0098] 1. Preparation of fresh Terminalia chebula Retz. homogenate

[0099] Take fresh Terminalia chebula Retz. fruits, remove the cores, weigh 1000.2 g of fresh pulp, add 450 ml of water, place in a juicer to extract juice, and obtain 1420.3 g of fresh Terminalia chebula Retz. homogenate.

[0100] 2. Optimization of extraction conditions for tannin components

[0101] Weigh about 100 g of fresh Terminalia chebula Retz. homogenate, add 150 ml of absolute ethanol, then add 250 ml of 60% v / v ethanol, extract ultrasonically (30 °C, 600 W) for 20 min, and filter with a Buchner funnel;

[0102] Take the filtered residue, place it in a 1000 mL beaker, add 400 ml of 60% v / v ethanol, extract ultrasonically (30 °C, 600 W) for 20 min, and filter with a Buchner funnel; combine the filtrates and concentrate them under reduced pressure to an extract in a rotary evaporator.

[0103] Example 2

[0104] This example provides a method for extracting tannin fractions from fresh Terminalia chebula Retz.

[0105] 1. Preparation of fresh Terminalia chebula Retz. homogenate: The same as in Example 1.

[0106] 2. Optimization of extraction conditions for tannin components

[0107] Weigh approximately 100 g of the fresh Terminalia chebula Retz. homogenate, add 400 mL of absolute ethanol, perform ultrasonic extraction (30 °C, 600 W) for 20 min, filter with a Buchner funnel, and concentrate the filtrate to an extract under reduced pressure using a rotary evaporator.

[0108] Example 3

[0109] This example provides a method for extracting tannin fractions from fresh Terminalia chebula Retz.

[0110] 1. Preparation of fresh Terminalia chebula Retz. homogenate: The same as in Example 1.

[0111] 2. Optimization of extraction conditions for tannin components

[0112] Weigh approximately 100 g of the fresh Terminalia chebula Retz. homogenate, add 67 ml of absolute ethanol, then add 333 mL of 40% v / v ethanol, perform ultrasonic extraction (30 °C, 600 W) for 20 min, centrifuge at 25 °C and 12700 rpm for 5 min, and concentrate the supernatant to an extract under reduced pressure using a rotary evaporator.

[0113] Example 4

[0114] This example provides a method for extracting tannin fractions from fresh Terminalia chebula Retz.

[0115] 1. Preparation of fresh Terminalia chebula Retz. homogenate: The same as in Example 1.

[0116] 2. Optimization of extraction conditions for tannin components

[0117] Weigh approximately 100 g of the fresh Terminalia chebula Retz. homogenate, add 25 ml of absolute ethanol, then add 375 ml of 20% v / v ethanol, perform ultrasonic extraction (30 °C, 600 W) for 20 min, centrifuge at 25 °C and 12700 rpm for 5 min, and concentrate the supernatant to an extract under reduced pressure using a rotary evaporator.

[0118] Example 5

[0119] This example provides a method for extracting and purifying tannin compounds. In this example, the preparation process of four tannin chemical components from Terminalia chebula Retz. is as Figure 1 shown.

[0120] 1. Preparation of the extract to be tested and pretreatment of the chromatography column

[0121] 1.1 Preparation of extract to be obtained

[0122] The fresh fruits of Terminalia chebula Retz. were homogenized according to the method of Example 1, and extracted with 60% v / v ethanol. The filtrates obtained from the two extractions were combined (the ultra-high performance liquid chromatography (UHPLC) chromatogram of this ethanol extract is as shown in Figure 2 ). 1 L was taken, dried under reduced pressure in a rotary evaporator, and then redissolved in 100 ml of water. After centrifugation at 12700 rpm for 5 min, supernatant 1 was obtained. The UHPLC chromatogram of this supernatant 1 is as shown in Figure 3 .

[0123] 1.2 Pretreatment of macroporous resin chromatography column

[0124] HP-20 macroporous resin was packed into the column by wet method (1 BV = 100 ml), and eluted with ethanol until the eluate was colorless; then washed with water until there was no alcohol smell; reserved for use.

[0125] 1.3 Pretreatment of octadecylsilyl bonded silica reversed-phase packing (ODS) chromatography column

[0126] 25 g of ODS-C18 packing was packed into the column by wet method (1 BV = 30 ml), eluted with ethanol, and then washed with water until there was no alcohol smell; reserved for use.

[0127] 2. Separation of target compound

[0128] 2.1 Chromatographic column separation

[0129] 100 ml of the supernatant obtained in step 1.1 was loaded onto the macroporous resin chromatography column, eluted with pure water for 5 BV at a flow rate of 10 min / BV, and the eluate of the first 5 column volumes was collected (the UHPLC chromatogram of this eluate is as shown in Figure 4 ).

[0130] The collected eluates were combined, concentrated under reduced pressure to 50 ml in a rotary evaporator, 400 ml of ethanol was added, and left standing in a refrigerator at 4 °C for 24 h. After centrifugation at 12700 rpm for 5 min, the precipitate obtained by ethanol precipitation (i.e., the precipitate 2 in Figure 1 ) and the supernatant obtained by ethanol precipitation (i.e., the supernatant 2 in Figure 1 , the UHPLC chromatogram of this supernatant 2 is as shown in Figure 5 ) were obtained.

[0131] The obtained precipitate by ethanol precipitation was dissolved in 2 ml of water (the UHPLC chromatogram is as shown in Figure 6 ), loaded onto an octadecylsilyl bonded silica reversed-phase packing (ODS) chromatography column, eluted with pure water for 5 BV at a flow rate of 20 min / BV (pressurized by a pressure pump), and eluted with 5 column volumes of water. The eluate of the first 2 column volumes was collected and concentrated under reduced pressure in a rotary evaporator for standby.

[0132] The obtained supernatant after ethanol precipitation was concentrated under reduced pressure using a rotary evaporator for standby.

[0133] 2.2 Preparation of chromatographic separation

[0134] 2.2.1 Preparation chromatographic conditions for chebulinic acid

[0135] Aglient preparative liquid chromatograph;

[0136] Chromatographic column: Cosmosil 5C18-MS-II (20×250 mm, 5 μm);

[0137] Flow rate: 8 ml / min; Detection wavelength: 284 nm;

[0138] Mobile phase: 0.1% formic acid solution (A) – methanol (B), isocratic elution with a volume ratio of mobile phase A:mobile phase B of 90:10.

[0139] The preparation chromatogram of chebulinic acid is as Figure 7 shown.

[0140] The collected chebulinic acid was identified by NMR, and the results are as Figure 8 , Figure 9 shown, and the ultra-high performance liquid chromatogram is as Figure 10 shown.

[0141] 2.2.2 Preparation chromatographic conditions for gallic acid, 4-galloylshikimic acid and 5-galloylshikimic acid Aglient preparative liquid chromatograph;

[0142] Chromatographic column: Cosmosil PBr Packed Column (20×250 mm, 5 μm);

[0143] Flow rate: 8 ml / min; Detection wavelength: 270 nm;

[0144] Mobile phase: 0.1% formic acid solution (A) – methanol (B), isocratic elution with a volume ratio of mobile phase A:mobile phase B of 60:40.

[0145] 2.2.3 Secondary preparation chromatographic conditions for gallic acid, 4-galloylshikimic acid and 5-galloylshikimic acid

[0146] Aglient preparative liquid chromatograph;

[0147] Chromatographic column: Cosmosil PBr Packed Column (20×250 mm, 5 μm);

[0148] Flow rate: 8 ml / min; Detection wavelength: 270 nm;

[0149] Mobile phase: 0.1% formic acid solution (A) – methanol (B), isocratic elution with a volume ratio of mobile phase A: mobile phase B of 65:35.

[0150] The preparative chromatograms of gallic acid, 4-galloylshikimic acid, and 5-galloylshikimic acid are as Figure 11 shown, where peak 2 is gallic acid, peak 3 is 4-galloylshikimic acid, and peak 4 is 5-galloylshikimic acid.

[0151] The collected gallic acid, 4-galloylshikimic acid, and 5-galloylshikimic acid were subjected to NMR identification and ultra-high performance liquid chromatography detection, and the results are as Figures 12 to 20 shown.

[0152] The conditions for the above ultra-high performance liquid chromatography detection were the same as those in Example 9.

[0153] Example 6

[0154] This example provides a method for extracting and purifying tannin compounds.

[0155] 1. Preparation of the extract to be tested and pretreatment of the chromatography column: The same as in Example 5.

[0156] 2. Separation of the target compound

[0157] 2.1 Chromatography column separation

[0158] Take 100 ml of the supernatant obtained in step 1.1 and load it onto a macroporous resin chromatography column. Wash it with pure water for 5 BV at a flow rate of 10 min / BV, and collect the eluate of the first 4 column volumes.

[0159] Combine the collected eluates, concentrate them under reduced pressure to 50 ml using a rotary evaporator, add 450 ml of ethanol, let it stand in a refrigerator at 4 °C for 24 h, and centrifuge at 12700 rpm for 5 min to obtain an alcohol precipitation precipitate and an alcohol precipitation supernatant.

[0160] Dissolve the obtained alcohol precipitation precipitate in 2 ml of water and load it onto an octadecylsilyl bonded silica reversed-phase packing chromatography column. Wash it with pure water for 5 BV at a flow rate of 20 min / BV (pressurized by a pressure pump), collect the eluate of the first 2 column volumes, and concentrate it under reduced pressure in a rotary evaporator for standby.

[0161] Concentrate the obtained alcohol precipitation supernatant under reduced pressure in a rotary evaporator for standby.

[0162] 2.2 Preparative chromatography separation: The same as in Example 5.

[0163] Example 7

[0164] This example provides a method for extracting and purifying tannin compounds.

[0165] 1. Preparation of the extract to be obtained and pretreatment of the chromatography column: The same as in Example 5.

[0166] 2. Separation of the target compound

[0167] 2.1 Chromatography column separation

[0168] Take 100 ml of the supernatant obtained in step 1.1 and load it onto a macroporous resin chromatography column. Elute with pure water for 5 BV at a flow rate of 10 min / BV, and collect the eluate of the first 4 column volumes.

[0169] Combine the collected eluate, concentrate it under reduced pressure to 50 ml using a rotary evaporator, add 350 ml of ethanol, let it stand in a 4°C refrigerator for 24 h, and centrifuge at 12700 rpm for 5 min to obtain the alcohol precipitation and the alcohol precipitation supernatant.

[0170] Dissolve the obtained alcohol precipitation in 2 ml of water and load it onto an octadecylsilyl (ODS) reverse-phase packing chromatography column. Elute with pure water for 5 BV at a flow rate of 20 min / BV (pressurized by a pressure pump), collect the eluate of the first 2 column volumes, and concentrate it under reduced pressure in a rotary evaporator for standby.

[0171] Concentrate the obtained alcohol precipitation supernatant under reduced pressure in a rotary evaporator for standby.

[0172] 2.2 Preparative chromatography separation: The same as in Example 5.

[0173] Example 8

[0174] This example provides a method for extracting and purifying tannin compounds.

[0175] 1. Preparation of the extract to be obtained and pretreatment of the chromatography column: The same as in Example 5.

[0176] 2. Separation of the target compound

[0177] 2.1 Chromatography column separation

[0178] Take 100 ml of the supernatant obtained in step 1.1 and load it onto a macroporous resin chromatography column. Elute with pure water for 5 BV at a flow rate of 10 min / BV, and collect the eluate of the first 5 column volumes.

[0179] Combine the collected eluate, concentrate it under reduced pressure to 50 ml using a rotary evaporator, add 300 ml of ethanol, let it stand in a 4°C refrigerator for 24 h, and centrifuge at 12700 rpm for 5 min to obtain the alcohol precipitation and the alcohol precipitation supernatant.

[0180] Dissolve the obtained alcohol precipitation in 2 ml of water and load it onto an octadecylsilyl (ODS) reverse-phase packing chromatography column. Elute with pure water for 5 BV at a flow rate of 20 min / BV (pressurized by a pressure pump), collect the eluate of the first 2 column volumes, and concentrate it under reduced pressure in a rotary evaporator for standby.

[0181] The obtained supernatant after alcohol precipitation was concentrated under reduced pressure using a rotary evaporator for later use.

[0182] 2.2 Preparation of chromatographic separation: The same as in Example 5.

[0183] Example 9

[0184] This example provides a method for detecting the tannin fraction in fresh Terminalia chebula Retz.

[0185] 1. Chromatographic conditions

[0186] Chromatograph: ACQUITY H class plus;

[0187] Chromatographic column: Cosmosil PBr Packed Column (2.1×250 mm, 2.6 μm);

[0188] Mobile phase: 0.1% v / v formic acid aqueous solution (A) – methanol (B), gradient elution program is shown in Table 1;

[0189] Detection wavelength: 210 - 400 nm; injection volume 2 μL; flow rate 0.3 mL / min; column temperature 30 °C.

[0190] Table 1 Mobile phase gradient elution conditions

[0191]

[0192]

[0193] 2. Preparation of the standard curve

[0194] Weigh 2 mg, 5 mg, 4 mg, and 3 mg of gallic acid, punicalagin, corilagin, and ellagic acid reference substances respectively and place them in 10 mL volumetric flasks. Add 50% v / v methanol or dimethyl sulfoxide to dissolve (ellagic acid is dissolved with dimethyl sulfoxide) and make up to the mark, shake well, and obtain reference substance stock solutions with concentrations of 0.2 mg / mL, 0.5 mg / mL, 0.4 mg / mL, and 0.3 mg / mL respectively; Take appropriate amounts of the gallic acid, punicalagin, corilagin, and ellagic acid reference substance stock solutions and place them in 10 mL volumetric flasks, and make up the volume with 50% v / v methanol to prepare mixed reference substance solutions with concentrations of 7.600 μg / mL, 79.218 μg / mL, 30.000 μg / mL, and 22.638 μg / mL respectively; Gradually dilute the solutions by 2, 4, 8, 16, 32, 64, and 128 times with 50% v / v methanol to obtain a series of mixed reference substance solutions with different concentrations. Measure according to the above chromatographic conditions. Take the reference substance concentration x (μg / mL) as the abscissa and the peak area y of each reference substance compound as the ordinate to draw the standard curve. The linear regression equations are shown in Table 2, and all r values are greater than 0.999.

[0195] Table 2 Regression equations and linear ranges of each compound

[0196]

[0197] 3. Detection of tannin components

[0198] 3.1 Detection of tannin components in fresh Terminalia chebula homogenate

[0199] Take 10 g of the fresh Terminalia chebula homogenate obtained in Example 1 (equivalent to about 7 g of fresh pulp), accurately weigh it, place it in a 1000 mL beaker, add 500 ml of methanol, extract by ultrasound (30 °C, 600 W) for 20 min, centrifuge at 25 °C and 12700 rpm for 5 min, and take the supernatant as the test solution. Prepare two portions in parallel. Inject the test solution and measure it in parallel 2 times respectively according to the above chromatographic conditions. The results are shown in Table 3.

[0200] Table 3 Contents of 4 tannin components in fresh Terminalia chebula homogenate (calculated based on fresh fruit, mg / g, n = 2)

[0201]

[0202] 3.2 Detection of tannin components in the ethanol extract of fresh Terminalia chebula homogenate

[0203] Take an appropriate amount of the filtrate obtained by the first suction filtration through a Buchner funnel in Examples 1 and 2 and the supernatant in Examples 3 and 4 (equivalent to 0.7 g of fresh pulp), accurately measure it, place it in a 50 mL volumetric flask respectively, add methanol to the scale, shake well, centrifuge at 12700 rpm for 5 min, and take the supernatant as the test solution. Prepare three portions in parallel. Inject the test solution and measure it in parallel 2 times respectively according to the above chromatographic conditions. The obtained chromatograms are as Figure 21 shown, and the contents and extraction rates of tannin components in each extract are as Figure 22 shown. Since enzymatic catalytic hydrolysis will occur during the extraction with 20% and 40% ethanol, and some components will be converted into gallic acid, resulting in the extraction rate of gallic acid > 100%.

[0204] Take an appropriate amount of the filtrate obtained by the second suction filtration through a Buchner funnel in Example 1 (equivalent to 0.7 g of fresh pulp), accurately measure it, place it in a 50 mL volumetric flask, add methanol to the scale, shake well, centrifuge at 25 °C and 12700 rpm for 5 min, and take the supernatant as the test solution. Prepare three portions in parallel. Inject the test solution and measure it in parallel 2 times respectively according to the above chromatographic conditions.

[0205] The filter residue obtained after the second suction filtration in Example 1 was placed in a 1000 mL beaker, 400 mL of 60% v / v ethanol was added, and ultrasonic extraction (30 °C, 600 W) was carried out for 20 min. Then, suction filtration was performed using a Buchner funnel. An appropriate amount of the filtrate (equivalent to 0.7 g of fresh pulp) was precisely measured and placed in a 50 mL volumetric flask. Methanol was added to the scale, shaken well, centrifuged at 12700 rpm for 5 min at 25 °C, and the supernatant was taken as the test solution. Three portions were prepared in parallel. The test solution was separately determined in parallel 2 times according to the above chromatographic conditions. The fresh Terminalia chebula homogenate in Example 1 was extracted with 60% v / v ethanol four times, and the chromatograms of the filtrates obtained after filtration each time were as Figure 23 shown, and the extraction rates of the tannin components in each filtrate were as Figure 24 shown. From Figure 23 , 24 it can be seen that the number of extraction times has little effect on the extraction rate of the target components. After two extractions, the cumulative extraction rates of gallic acid, punicalagin, corilagin, and ellagic acid are all > 78%.

[0206] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for extracting and purifying a tannin compound, characterized in that, The compound is chebulinic acid, and the extraction method specifically includes the following steps: S1. Extract the tannin part from fresh Terminalia chebula Retz.; the extraction method specifically includes the following operations: Remove the pits from fresh Terminalia chebula Retz. fruits, add water to extract juice to obtain a fresh Terminalia chebula Retz. homogenate; perform ultrasonic extraction on the fresh Terminalia chebula Retz. homogenate with an ethanol aqueous solution having a concentration of 60% - 100% v / v, wherein the temperature of the ultrasonic extraction is 25 - 60 °C, the power is 500 - 700 W, and the number of times is ≥ 2 times; perform solid-liquid separation, concentrate and dry the obtained liquid phase to obtain it; S2. Dissolve the tannin part in water and then perform solid-liquid separation. Use HP-20 macroporous adsorption resin as the stationary phase and water as the elution solvent, perform column chromatography on the liquid phase obtained by solid-liquid separation, and collect the eluate of the first 4 - 5 times the column volume; S3. Combine the eluates collected in S2, concentrate, add ethanol, let it stand for precipitation, and then perform solid-liquid separation; S4. Dissolve the solid phase obtained by solid-liquid separation in S3 in water, use octadecylsilyl silica gel reversed-phase packing as the stationary phase and water as the elution solvent to perform column chromatography, collect the eluate of the first 1 - 2 times the column volume, concentrate, and then separate and purify by preparative chromatography; The chromatographic conditions of the preparative chromatography include: the chromatographic column is Cosmosil 5C18-MS-II, with a specification of 20×250 mm, 5 μm; mobile phase: mobile phase A is a 0.095% - 0.105% v / v formic acid aqueous solution, mobile phase B is methanol, and isocratic elution is performed at a volume ratio of mobile phase A:mobile phase B of 90:10; the flow rate is 8 ml / min; the detection wavelength is 284 nm.

2. The extraction and purification method of the tannin compound according to claim 1, characterized in that, Collect the eluate of 5 times the column volume in S2; and / or Add ethanol in S3 until the ethanol content is 85.7% - 90.0% v / v; and / or Collect the eluate of the first 2 times the column volume in S4.

3. A method for extracting a tannin compound, characterized in that, The compound includes gallic acid, 4-galloylshikimic acid, and 5-galloylshikimic acid, and the extraction method specifically includes the following steps: S1. Extract the tannin part from fresh Terminalia chebula Retz.; the extraction method specifically includes the following operations: Remove the pits from fresh Terminalia chebula Retz. fruits, add water to extract juice to obtain a fresh Terminalia chebula Retz. homogenate; perform ultrasonic extraction on the fresh Terminalia chebula Retz. homogenate with an ethanol aqueous solution having a concentration of 60% - 100% v / v, wherein the temperature of the ultrasonic extraction is 25 - 60 °C, the power is 500 - 700 W, and the number of times is ≥ 2 times; perform solid-liquid separation, concentrate and dry the obtained liquid phase to obtain it; S2. Dissolve the tannin part in water and then perform solid-liquid separation. Use HP-20 macroporous adsorption resin as the stationary phase and water as the elution solvent, perform column chromatography on the liquid phase obtained by solid-liquid separation, and collect the eluate of the first 4 - 5 times the column volume; S3. Combine the eluates collected in S2, concentrate, add ethanol, let it stand for precipitation, and then perform solid-liquid separation; S4. Concentrate the liquid phase obtained by solid-liquid separation in S3, and then separate and purify by preparative chromatography; The chromatographic conditions of the preparative chromatography include: the chromatographic column is Cosmosil Pbr Packed Column, with a specification of 20×250 mm, 5 μm; the mobile phase: mobile phase A is an aqueous formic acid solution with a concentration of 0.095% - 0.105% v / v, and mobile phase B is methanol, and isocratic elution is performed at a volume ratio of mobile phase A:mobile phase B of (60 - 65):(40 - 35); the flow rate is 8 ml / min; the detection wavelength is 270 nm.

4. The extraction method of the tannin compound according to claim 3, wherein Collect the eluate of 5 column volumes in S2; and / or Add ethanol in S3 until the ethanol content is 85.7% - 90.0% v / v.

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