CN114414702A - Preparation method and content determination method of chebulagic acid in chebula meat - Google Patents

Preparation method and content determination method of chebulagic acid in chebula meat Download PDF

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CN114414702A
CN114414702A CN202210105897.9A CN202210105897A CN114414702A CN 114414702 A CN114414702 A CN 114414702A CN 202210105897 A CN202210105897 A CN 202210105897A CN 114414702 A CN114414702 A CN 114414702A
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chebulagic acid
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chebulagic
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王巍
鞠成国
张强
陈九妹
胡佳旭
周灵丽
郭美岑
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Liaoning University of Traditional Chinese Medicine
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Abstract

The invention relates to the field of preparation and detection of chemical components of traditional Chinese medicines, in particular to a preparation method and a content determination method of chebulagic acid in chebula meat. The method for preparing the chebulagic acid from the chebula meat provided by the invention has the advantages that the purity of the product can reach more than 99%, the total yield can reach more than 60%, and the product can be used as a standard product or a reference product to be applied to the quality control of the chebulagic acid. And the method has simple operation steps and low equipment cost, can be applied to industrial production, and is suitable for preparing a large amount of myrobalamin reference substances. The content determination method of the chebulagic acid in the chebula fruit provided by the invention can accurately detect the content of the chebulagic acid in the chebula fruit meat, lays theoretical foundation and experimental basis for establishing the quality standard of the chebula fruit, and has important significance for promoting the industrialized development of the chebula fruit.

Description

Preparation method and content determination method of chebulagic acid in chebula meat
Technical Field
The invention relates to the field of preparation and detection of chemical components of traditional Chinese medicines, in particular to a preparation method and a content determination method of chebulagic acid in chebula meat.
Background
Fructus Chebulae is fructus Chebulae (Terminalia chebula Retz. of Combretaceae)Terminalia chebulaRetz.) or Terminalia tomentosa (Terminalia chebula Retz.var.tomentellaKurt.) has effects of relieving diarrhea with astringents, astringing lung, and relieving cough. Modern chemical studies have shown that the main chemical components of myrobalan are tannins, including hydrolysable gallotannins and ellagitannins. Wherein the component chebulagic acid with higher content is prepared by condensing multiple gallic acids, and has the following structure:
Figure DEST_PATH_IMAGE001
early-stage research shows that the chebulagic acid has strong oxidation resistance and anti-inflammatory capability, is a main drug effect component of the chebula fruit, has the content of 10 percent in the chebula fruit meat, and can be used as a quality marker for carrying out quality control and evaluation on a chebula fruit medicinal material. Therefore, the chebulagic acid reference substance needs to be prepared for reasonably and effectively evaluating the quality of the medicine terminalia chebula.
At present, the separation and preparation of the chebulagic acid at home and abroad are still in the initial stage, and the technology is not perfect. CN102020682A, 2011, 4 months and 20 days, discloses a method for preparing chebulagic acid from myrobalan leaves, wherein the purity of the obtained chebulagic acid product can reach more than 98%, strong base strong acid is needed, only 35 g of product is obtained from 10 kg of raw materials, and the yield is low; CN104945447A, discloses a method for preparing chebulariac ellagic acid from myrobalan leaves in 2015, 9 months and 30 days, the raw materials are subjected to enzymolysis, ultrasonic extraction, ceramic membrane separation, macroporous resin adsorption and other steps, the steps are complicated, and the product purity is below 90%; CN105949252A, 2016, 9, 21 disclose a method for preparing chebulagic acid from a chebula fruit medicinal material by high-speed counter-current chromatography, but the used equipment is complex and expensive, the product yield is less than 0.004%, and the purity is only more than 95%. CN109867704A, 11.6.2019 discloses a method for preparing chebulagic acid by an ethanol warm immersion method, CN110724172A, 24.1.2020 discloses a method for preparing chebulagic acid by an ethanol reflux method, the two methods both adopt emblic leafflower fruit as a raw material, and the purity of the obtained chebulagic acid product is more than 98% through macroporous resin column separation, but about 2g of the chebulagic acid product can be obtained by 100 g of the medicinal materials.
The published literature shows that in the prior preparation method of the chebulagic acid, the chebula fruit, the chebula leaf or the phyllanthus emblica are mostly used as raw materials, wherein the content of the chebulagic acid in the phyllanthus emblica is 2-5%, the content of the chebula leaf is about 2%, the chebula fruit comprises chebula fruit and chebula fruit stones, the fruit stones account for 15-30% of the weight of the fruit, and the inventor finds that the content of the chebulagic acid in the chebula fruit is about 10% and the chebula fruit stones almost do not contain, so that the removal of the chebula fruit pits and the extraction of the chebulagic acid from the chebula fruit can be a more efficient way. In addition, the existing preparation method of the chebulagic acid usually adopts combination of various column chromatography, high-speed counter-current chromatography, preparation liquid chromatography and the like, has the defects of high cost, complicated steps and the like, and is not suitable for industrial production.
In the current Chinese pharmacopoeia 2020 edition, the quality control method of the myrobalan is limited to qualitative identification and detection of moisture, ash content and water-soluble extract, no content determination method is specified, and the myrobalan acid can be used as a quality marker to control the quality of the medicine myrobalan. The content determination method of chebulagic acid in chebula is published by the Lemna bicolor et al in traditional Chinese medicines, the used sample extraction solvent is methanol, however, the experience of long-term study on chebula by the inventor finds that chebulagic acid is extremely unstable in methanol, and the higher the content of methanol in the solvent is, the worse the stability is, and the characteristic has great influence on the accuracy of the content determination.
The invention aims to develop a preparation method of a chebulagic acid reference substance with simple operation, high yield and high purity and establish an accurate and repeatable content determination method of chebulagic acid in chebula fruit.
Disclosure of Invention
In view of the problems of the prior art, the invention aims to provide a method for preparing a chebulagic acid reference substance from chebula meat. The extraction rate of the chebulagic acid in the chebular meat by the process for preparing the chebulagic acid from the chebula meat provided by the invention can reach 85%, the total yield can reach more than 60% by extracting with ethyl acetate and separating by an ODS open column. The content determination method provided by the invention has the advantages that the specificity, linearity, range, precision, accuracy and solution stability all meet the requirements of relevant guiding principles, the operation is simple, and the quality detection can be carried out on the medicine terminalia fruit.
In order to achieve the above object, the present invention provides the following technical solutions.
The invention provides a method for extracting chebulagic acid from myrobalan meat, which is characterized by comprising the following specific steps:
(1) removing core of fructus Chebulae, pulverizing pulp, and sieving; weighing a certain amount of fructus Chebulae powder, adding 70% ethanol water solution, performing ultrasonic extraction, and extracting the extractive solution at 3000 r min-1Centrifuging at the rotating speed of (1) for 10 min, and recovering ethanol from the supernatant under reduced pressure;
(2) diluting the above residue with water, extracting with water-equivalent ethyl acetate for several times, mixing ethyl acetate solutions, recovering ethyl acetate under reduced pressure, re-dissolving the residue with methanol water of certain amount, and 10000 r.min-1Centrifuging for 10 min at the rotating speed of (1);
(3) separating the supernatant obtained by centrifuging with ODS open column, eluting with methanol water solution, collecting eluate, recovering solvent under reduced pressure, and standing at 4 deg.C until white solid is separated out to obtain Chebulagic acid.
Further, the myrobalan pulp in the step (1) is crushed, sieved by a sieve with 65-120 meshes, added with 70% ethanol water in an amount which is 20-30 times of the amount of the medicinal materials, and ultrasonically extracted for 2-3 times for 10-30 min each time.
Further, in the step (2), diluting and adding water in an amount which is 2-4 times of the amount of the initially weighed medicinal materials, and extracting for 6-12 times; the compound solution is 20% methanol water solution, and the dosage is 1:1 of the crude drug, thus obtaining the sample solution with the concentration of 1.0 g/mL.
Further, in the step (3), the diameter-height ratio of the ODS column packing is 1: 5-1: 0.8; the sample loading amount is 1-2 mL per 10 g of ODS; eluting with 20% methanol water solution, collecting 16 column volumes from the 4 th column volume, recovering solvent under reduced pressure, and standing at 4 deg.C until white solid is separated out to obtain Chebulagic acid.
Further, the chebulagic acid extracted by the method is used as a standard product or a reference product in the quality control of the chebulagic acid.
The invention also provides a method for measuring the content of chebulagic acid in myrobalan, which is characterized by comprising the following steps:
(1) preparation of control solutions: dissolving Chebulagic acid reference substance in acetonitrile solution to obtain mixed reference substance solution;
(2) preparing a test solution: taking myrobalan pulp, crushing, and screening by a 65-120-mesh sieve; sampling 0.1-0.2 g of sample powder, precisely weighing, adding 25-50 mL of acetonitrile aqueous solution, weighing, performing ultrasonic treatment, cooling, complementing the lost weight with an extraction solvent, filtering, taking a subsequent filtrate for dilution by 5-10 times, and filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution;
(3) respectively injecting the solutions prepared in the steps (1) and (2) into a high performance liquid chromatograph for determination, wherein the high performance liquid chromatograph conditions are as follows: a chromatographic column: a Dikma Platisil column (250 mm × 4.6 mm, 5 μm); mobile phase: the phase A is water-methanol-formic acid, the phase B is acetonitrile, and gradient elution is carried out; column temperature: 25-30 ℃; flow rate: 0.8-1.2 mL/min; the detection wavelength is 280 nm; the number of theoretical plates should not be less than 5000 according to the peak of chebular acid.
Further, in the step (1), the concentration of the chebulagic acid in the mixed control solution is 0.05-0.1 mg/mL.
Further, in the step (2), the extraction solvent is 30-70% acetonitrile water solution; the ultrasonic condition is as follows: the time is 20-40 min, the power is 250-300W, the frequency is 35-40 kHz, and the temperature is 25-35 ℃.
Furthermore, in the step (3), the sample amount of the mixed reference solution and the sample solution is 3-7 muL.
Further, in the step (3), the volume ratio of the mobile phase A phase is 90-95: 5-10: 0.1-0.2 of water-methanol-formic acid, wherein the phase B is acetonitrile, and the elution gradient is as follows: 0-5 min, B is 2% → 5%; 5-10 min, wherein B is 5%; 10-12 min, wherein B is 5% → 15%; 12-30 min, wherein B is 15%; 30-35 min, wherein B is 15% → 18%; 35-50 min, wherein B is 18%; 50-55 min, B is 18% → 100%.
Compared with the prior art, the invention has the following beneficial effects.
(1) The product yield and purity of the traditional method for separating and preparing the chebulagic acid are mostly lower, the invention adopts the chebula meat with higher content of the chebulagic acid as the raw material, and the extraction solution is extracted by ethyl acetate to remove the high-polarity impurities, so that the chebulagic acid is enriched, and the purity of the obtained product is ensured. The purity of the chebulagic acid reference substance prepared by the invention can reach more than 99 percent, and the total yield can reach more than 60 percent.
(2) The invention ultrasonically extracts the chebulagic acid by ethanol, extracts the chebulagic acid by ethyl acetate, recovers the chebulagic acid to be dry, re-dissolves the chebulagic acid by methanol water solution, passes through an ODS open column, and then elutes the chebulagic acid by 20 percent methanol water solution. All used reagents can be recycled, and related ODS fillers can be reused for many times, so that the cost is effectively reduced.
(3) The open column chromatography established by the invention has low equipment requirement, can be applied to industrial production, and is suitable for preparing a large amount of chebulagic acid reference substances.
(4) According to the method for measuring the content of chebulagic acid in the chebula fruit, the chebulagic acid is in a good linear relation within the range of 0.0475-1.5200 mug, the average recovery rate is 100.02%, and the RSD is 0.71%. RSD of precision, stability and repeatability is less than 2%. Therefore, the content determination method is precise and accurate, has strong specificity and simple operation, can be used for determining the content of the chebulagic acid in the chebula fruit, provides theoretical basis and experimental basis for establishing the quality standard of the chebula fruit, and has important significance for promoting the industrialized development of the chebula fruit.
Drawings
FIG. 1 is chebulagic acid1H-NMR identification chart.
FIG. 2 is chebulagic acid13C-NMR identification chart.
FIG. 3 is a high performance liquid chromatogram of a chebulagic acid product.
FIG. 4 is a high performance liquid chromatogram of a chebulagic acid control.
FIG. 5 is a full-wavelength scanning spectrum (200-400 nm) of chebulagic acid.
FIG. 6 is a blank solution HPLC chromatogram of 1.1 specificity test in example 2 of the present invention.
FIG. 7 is an HPLC chromatogram of a mixed control solution of 1.1 specificity test in example 2 of the present invention.
FIG. 8 is an HPLC chromatogram of a test solution for the 1.1 specificity test in example 1 of example 2 of the present invention.
FIG. 9 is a linear graph of Terminalia chebula acid in the 1.2 linear range and detection limit of example 1 of the present invention.
Figure 10 is an HPLC chromatogram of a chebulagic acid control dissolved in acetonitrile.
Figure 11 is an HPLC chromatogram of a chebulagic acid control dissolved in methanol.
Detailed Description
The following method of the present invention for preparing chebulagic acid reference from chebula fruit is further illustrated with reference to the following specific examples, so as to enable those skilled in the art to better understand the present invention, but without limiting the invention thereto.
Example one method of extracting chebulagic acid from chebula meat.
The method for detecting the content of the chebulagic acid by the high performance liquid chromatography in the first embodiment of the invention is the following method if no special description exists:
preparing a test solution: accurately weighing 0.1g of a sample, adding 25 mL of 70% methanol, accurately weighing the weight, carrying out ultrasonic treatment for 30 min, cooling, complementing the loss weight by an extraction solvent, filtering, taking 2 mL of continuous filtrate, fixing the volume to a 10 mL volumetric flask, and filtering through a 0.45 mu m microporous membrane;
chromatographic conditions are as follows: a Dikma Platisil column (250 mm × 4.6 mm, 5 μm); mobile phase: phase A was formic acid-water (0.1: 100 v/v), phase B was methanol, elution gradient: 0-2 min, B is 5% → 38%; 2-12 min, wherein B is 38%; 12-13 min, wherein B is 38% → 48%; 13-20 min, wherein B is 48%; 20-25 min, wherein B is 48% → 100%; sample introduction amount: 5 mu L of the solution; column temperature: 25 ℃; flow rate: 1.0 mL/min; the detection wavelength is 280 nm;
in the first experiment of the invention, the raw materials are all in the same batch, and the content of the chebulagic acid is 140.21 mg/g through the determination of the method.
Example 1: a method for preparing chebulagic acid reference substance from fructus Chebulae meat-extraction process of chebulagic acid from fructus Chebulae comprises the following steps:
(1) removing core of fructus Chebulae, pulverizing pulp, and sieving with 65 mesh sieve. Weighing 5 g of the fructus Chebulae powder, adding 25 times of 70% ethanol water solution, and performing ultrasonic extraction for 2 times, each for 20 min. Extracting at 3000 r min-1Centrifuging at the rotating speed for 10 min, collecting supernatant, and recovering ethanol under reduced pressure to dryness;
(2) taking the dry paste corresponding to 0.1g of the raw medicinal material to prepare a test solution, and detecting the extraction amount of the chebulagic acid by an HPLC method to be 97.36 mg/g, wherein the extraction rate is 69.44%.
Example 2: a method for preparing chebulagic acid reference substance from fructus Chebulae meat-extraction process of chebulagic acid from fructus Chebulae comprises the following steps:
(1) removing core of fructus Chebulae, pulverizing pulp, and sieving with 120 mesh sieve. Weighing 5 g of the fructus Chebulae powder, adding 25 times of 70% ethanol water solution, and performing ultrasonic extraction for 2 times, each for 20 min. Extracting at 3000 r min-1Centrifuging at the rotating speed for 10 min, collecting supernatant, and recovering ethanol under reduced pressure to dryness;
(2) taking the above dry extract equivalent to 0.1g of raw medicinal material to prepare a test solution, and detecting the extraction amount of the chebulagic acid by an HPLC method to be 106.99 mg/g, wherein the extraction rate is 76.31%.
Example 3: a method for preparing chebulagic acid reference substance from fructus Chebulae meat-extraction process of chebulagic acid from fructus Chebulae comprises the following steps:
(1) removing core of fructus Chebulae, pulverizing pulp, and sieving with 120 mesh sieve. Weighing 5 g of the fructus Chebulae powder, adding 20 times of 70% ethanol water solution, and performing ultrasonic extraction for 2 times, each for 20 min. Extracting at 3000 r min-1Centrifuging at the rotating speed for 10 min, collecting supernatant, and recovering ethanol under reduced pressure to dryness;
(2) taking the dry paste corresponding to 0.1g of raw medicinal materials to prepare a test solution, and detecting the extraction amount of the chebulagic acid by an HPLC method to be 102.77 mg/g, wherein the extraction rate is 73.30%.
Example 4: a method for preparing chebulagic acid reference substance from fructus Chebulae meat-extraction process of chebulagic acid from fructus Chebulae comprises the following steps:
(1) removing core of fructus Chebulae, pulverizing pulp, and sieving with 120 mesh sieve. Weighing 5 g of the fructus Chebulae powder, adding 30 times of 70% ethanol water solution, and performing ultrasonic extraction for 2 times, each for 20 min. Extracting at 3000 r min-1Centrifuging at the rotating speed for 10 min, collecting supernatant, and recovering ethanol under reduced pressure to dryness;
(2) taking the above dry extract equivalent to 0.1g of raw medicinal material to prepare a test solution, and detecting the extraction amount of the chebulagic acid by an HPLC method to be 118.86 mg/g, wherein the extraction rate is 84.77%.
Example 5: a method for preparing chebulagic acid reference substance from fructus Chebulae meat-extraction process of chebulagic acid from fructus Chebulae comprises the following steps:
(1) removing core of fructus Chebulae, pulverizing pulp, and sieving with 120 mesh sieve. Weighing 5 g of the fructus Chebulae powder, adding 30 times of 70% ethanol water solution, and ultrasonically extracting for 1 time, each time for 30 min. Extracting at 3000 r min-1Centrifuging at the rotating speed for 10 min, collecting supernatant, and recovering ethanol under reduced pressure to dryness;
(2) taking the dry paste corresponding to 0.1g of raw medicinal materials to prepare a test solution, and detecting the extraction amount of the chebulagic acid by an HPLC method to be 109.28 mg/g, wherein the extraction rate is 77.94%.
Example 6: a method for preparing chebulagic acid reference substance from fructus Chebulae meat-extraction process of chebulagic acid from fructus Chebulae comprises the following steps:
(1) removing core of fructus Chebulae, pulverizing pulp, and sieving with 120 mesh sieve. Weighing 5 g of the fructus Chebulae powder, adding 30 times of 70% ethanol water solution, and performing ultrasonic extraction for 3 times, each time for 10 min. Extracting at 3000 r min-1Centrifuging at the rotating speed for 10 min, collecting supernatant, and recovering ethanol under reduced pressure to dryness;
(2) taking the dry paste corresponding to 0.1g of raw medicinal materials to prepare a test solution, and detecting the extraction amount of the chebulagic acid by an HPLC method to be 121.75 mg/g, wherein the extraction rate is 86.83%.
Example 7: a method for preparing chebulagic acid reference substance from fructus Chebulae meat comprises the following steps:
(1) removing core of fructus Chebulae, pulverizing pulp, and sieving with 120 mesh sieve. Weighing 1.0 g fructus Chebulae powder, adding 30 times of 70% ethanol water solution, and ultrasonic extracting for 3 times, each time for 10 min. Extracting at 3000 r min-1Centrifuging at the rotating speed of (1) for 10 min, and recovering ethanol from the supernatant under reduced pressure;
(2) diluting the above residue with 2 mL of water, extracting with water equivalent amount of ethyl acetate for 6 times, mixing ethyl acetate solutions, recovering ethyl acetate under reduced pressure, re-dissolving the residue with 1 mL of 20% methanol water, 10000 r.min-1Centrifuging at the rotating speed of (1) for 10 min, namely, the concentration of the sample loading solution is 1.0 g/mL;
(3) the supernatant obtained by the above centrifugation was separated by an ODS open column (packing weight 10 g, diameter/height ratio 1: 5), eluted with 20% methanol aqueous solution for 20 column volumes, and the effluent was identified by HPLC. Collecting eluent with the volume of 4-20 columns, recovering the solvent under reduced pressure, and standing at 4 ℃ until white solid is separated out.
The total yield of the obtained product is 73.40 mg, and the total yield is 52.35%. Warp beam1H-NMR and13and identifying the chebulagic acid by using a C-NMR spectrum, and calculating the purity of the chebulagic acid to be higher than 99 percent by using an HPLC (high performance liquid chromatography) identification according to a peak area normalization method, thus obtaining the chebulagic acid reference substance.
Example 8: a method for preparing chebulagic acid reference substance from fructus Chebulae meat comprises the following steps:
(1) same as example 7 (1);
(2) diluting the above residue with 4 mL of water, extracting with water-equivalent ethyl acetate for 12 times, mixing ethyl acetate solutions, recovering ethyl acetate under reduced pressure, re-dissolving the residue with 1 mL of 20% methanol water, 10000 r.min-1Centrifuging at the rotating speed of (1) for 10 min, namely, the concentration of the sample loading solution is 1.0 g/mL;
(3) same as in example 7 (3).
81.71 mg of the obtained product, the total yield is 58.28%; purity was higher than 99% as determined by HPLC, calculated by peak area normalization.
Example 9: a method for preparing chebulagic acid reference substance from fructus Chebulae meat comprises the following steps:
(1) same as example 7 (1);
(2) diluting the above residue with 2 mL of water, extracting with water equivalent ethyl acetate for 10 times, mixing ethyl acetate solutions, recovering ethyl acetate under reduced pressure, re-dissolving the residue with 1 mL of 20% methanol water, 10000 r.min-1Centrifuging at the rotating speed of (1) for 10 min, namely, the concentration of the sample loading solution is 1.0 g/mL;
(3) same as in example 7 (3).
83.44 mg of the obtained product, and the total yield is 59.51%; purity was higher than 99% as determined by HPLC, calculated by peak area normalization.
Example 10: a method for preparing chebulagic acid reference substance from fructus Chebulae meat comprises the following steps:
(1) same as example 7 (1);
(2) diluting the above residue with 4 mL of water, extracting with water equivalent ethyl acetate for 10 times, mixing ethyl acetate solutions, recovering ethyl acetate under reduced pressure, re-dissolving the residue with 1 mL of 20% methanol water, 10000 r.min-1Centrifuging at the rotating speed of (1) for 10 min, namely, the concentration of the sample loading solution is 1.0 g/mL;
(3) the supernatant obtained by the above centrifugation was separated by an ODS open column (packing weight 10 g, diameter/height ratio 1: 5), eluted with 20% methanol aqueous solution for 20 column volumes, and the effluent was identified by HPLC. Collecting eluent with the volume of 6 th to 20 th columns, decompressing and recovering the solvent, and placing the eluent at 4 ℃ until white solid is separated out.
65.61mg of the obtained product, the total yield is 46.79%; purity was higher than 99% as determined by HPLC, calculated by peak area normalization.
Example 11: a method for preparing chebulagic acid reference substance from fructus Chebulae meat comprises the following steps:
(1) removing core of fructus Chebulae, pulverizing pulp, and sieving with 120 mesh sieve. Weighing 1.5 g fructus Chebulae powder, adding 30 times of 70% ethanol water solution, and ultrasonic extracting for 3 times, each time for 10 min. Centrifuging the extractive solution at 3000 r min-1 for 10 min, collecting supernatant, and recovering ethanol under reduced pressure;
(2) diluting the residue obtained after recovering ethanol with 3 mL of water, extracting with ethyl acetate equivalent to water for 10 times, mixing ethyl acetate solutions, recovering ethyl acetate under reduced pressure, re-dissolving the residue with 1.5 mL of 20% methanol water, and centrifuging at 10000 r min-1 for 10 min to obtain a sample solution with a concentration of 1.0 g/mL;
(3) the supernatant obtained by the above centrifugation was separated by an ODS open column (packing weight 10 g, diameter/height ratio 1: 5), eluted with 20% methanol aqueous solution for 20 column volumes, and the effluent was identified by HPLC. Collecting eluent with the volume of 5-20 columns, recovering the solvent under reduced pressure, and standing at 4 ℃ until white solid is separated out.
111.23mg of the obtained product, the total yield is 52.89%; purity was higher than 99% as determined by HPLC, calculated by peak area normalization.
Example 12: a method for preparing chebulagic acid reference substance from fructus Chebulae meat comprises the following steps:
(1) same as example 11 (1);
(2) diluting the above residue with 3 mL of water, extracting with water equivalent ethyl acetate for 10 times, mixing ethyl acetate solutions, recovering ethyl acetate under reduced pressure, re-dissolving the residue with 1.5 mL of 20% methanol water, 10000 r.min-1Centrifuging at the rotating speed of (1) for 10 min, namely, the concentration of the sample loading solution is 1 g/mL;
(3) the supernatant obtained by the above centrifugation was separated by an ODS open column (packing weight 10 g, diameter/height ratio 1: 1.4), eluted with 20% methanol aqueous solution for 20 column volumes, and the effluent was identified by HPLC. Collecting eluent with the volume of 4-20 columns, recovering the solvent under reduced pressure, and standing at 4 ℃ until white solid is separated out.
130.3 mg of the obtained product, and the total yield is 61.95%; purity was higher than 99% as determined by HPLC, calculated by peak area normalization.
Example 13: a method for preparing chebulagic acid reference substance from fructus Chebulae meat comprises the following steps:
(1) same as example 11 (1);
(2) diluting the residue obtained after recovering ethanol with 3 mL of water, extracting with ethyl acetate equivalent to water for 10 times, mixing ethyl acetate solutions, recovering ethyl acetate under reduced pressure, re-dissolving the residue with 1.5 mL of 20% methanol water, and centrifuging at 10000 r min-1 for 10 min to obtain a sample solution with a concentration of 1.0 g/mL;
(3) the supernatant obtained by the above centrifugation was separated by an ODS open column (packing weight 10 g, diameter/height ratio 1: 0.8), eluted with 20% methanol aqueous solution for 20 column volumes, and the effluent was identified by HPLC. Collecting eluent with the volume of 4-20 columns, recovering the solvent under reduced pressure, and standing at 4 ℃ until white solid is separated out.
121.5 mg of the obtained product, wherein the total yield is 57.77%; purity was higher than 99% as determined by HPLC, calculated by peak area normalization.
The molecular structures of the products obtained in the above examples 7 to 13 were identified by NMR spectroscopy,1H-NMR and13the C-NMR spectrum is shown in figure 1 and figure 2, and the peak attribution in the structure analysis process is as follows:
Figure 909573DEST_PATH_IMAGE001
1H NMR（400 MHz，CD3OD）δ：6.50（1H，d，J = 1.3 Hz，H-1），5.39（1H，brs，H-2），5.82（1H，brs，H-3），5.22（1H，d，J = 3.5 Hz，H-4），4.81（1H，d，J = 5.4 Hz，H-5），4.37（1H，dd，J= 10.5，7.6 Hz，H-6a），4.92（1H，d，J = 10.2 Hz，H-6b），4.82（1H，s，H-2'），5.05（1H，dd，J= 7.2，1.6 Hz，H-3'），3.80（1H，ddd，J = 11.6，3.7，1.6 Hz，H-4'），2.19（1H，dd，J = 17.1，3.8 Hz，H-5'a），2.11（1H，dd，J = 17.0，11.5 Hz，H-5'b），7.48（1H，s，H-2''），7.07（2H，s，H-2'''，6'''），6.84（1H，s，H-2''''），6.64（1H，s，H-2'''''），3.80（1H，s）；
13C NMR（100 MHz，CD3OD）δ：92.59（C-1），71.16（C-2），62.47（C-3），67.06（C-4），74.33（C-5），64.79（C-6），170.83（C-1'），66.85（C-2'），41.79（C-3'），40.06（C-4'），30.55（C-5'），175.05（C-6'），174.48（C-7'），116.29（C-1''），119.09（C-2''），117.69（C-3''），147.46（C-4''），140.45（C-5''），141.47（C-6''），166.47（C-7''），120.15（C-1'''），110.98（C-2'''，6'''），146.62（C-3'''，5'''），140.93（C-4'''），166.34（C-7'''），116.03（C-1''''），125.65（C-2''''），108.26（C-3''''），146.22（C-4''''），137.63（C-5''''），145.41（C-6''''），170.20（C-7''''），117.72（C-1'''''），124.59（C-2'''''），110.52（C-3'''''），145.67（C-4'''''），138.74（C-5'''''），145.62（C-6'''''），167.56（C-7'''''）。
the experimental result shows that the obtained product is the chebulagic acid, the purity of the chebulagic acid is more than 99% through HPLC detection, and the chebulagic acid reference substance is shown in figures 3 and 4 and meets the purity requirement of a content determination reference substance, so that the chebulagic acid reference substance is prepared from the chebula fruit meat.
Example two, a method for determining chebulagic acid content in chebula.
The control described in example two of the present invention was prepared according to the method of example 12 of example one and used in the following experiments.
Example 1: methodology investigation
Test 1.1 specificity test
The method comprises the following steps:
(1) taking 50% acetonitrile, and filtering the acetonitrile by a 0.45 mu m microporous filter membrane to obtain a blank solution;
(2) dissolving Chebulagic acid reference substance in acetonitrile solution to obtain reference substance solution with Chebulagic acid content of 0.076 mg/mL;
(3) pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sampling 0.1g of sample powder, precisely weighing, adding 25 mL of 50% acetonitrile aqueous solution, weighing, carrying out ultrasonic treatment for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling, complementing the lost weight with an extraction solvent, filtering, taking 2 mL of subsequent filtrate to dilute to 10 mL, and filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution;
(4) injecting 5 μ L of each of the blank solution, the mixed reference solution and the sample solution into a high performance liquid chromatograph, and recording the chromatogram. The chromatographic conditions are as follows: an Agilent 1100 high performance liquid chromatography system; a chromatographic column: a Dikma Platisil column (250 mm × 4.6 mm, 5 μm); mobile phase: phase A is water-methanol-formic acid (95: 5:0.1 v/v/v); phase B was acetonitrile, elution gradient: 0-5 min, B is 2% → 5%; 5-10 min, wherein B is 5%; 10-12 min, wherein B is 5% → 15%; 12-30 min, wherein B is 15%; 30-35 min, wherein B is 15% → 18%; 35-50 min, wherein B is 18%; 50-55 min, wherein B is 18% → 100%; column temperature: 25 ℃; flow rate: 1.0 mL/min; the detection wavelength was 280 nm.
The result shows that chromatographic peaks consistent with the retention time of the chebulariac acid reference substance appear in the chromatogram of the test solution prepared by the treatment method, and the chromatographic peaks are completely separated from adjacent chromatographic peaks. Meanwhile, no chromatographic peak consistent with the retention time of a reference substance appears in the chromatogram of the blank solution, which shows that the method has good specificity for measuring the content of chebulagic acid in the myrobalan, and the chromatogram is shown in fig. 6-8.
Test 1.2 Linear Range and detection limits
The method comprises the following steps:
(1) dissolving Chebulagic acid reference substance in acetonitrile solution, diluting with acetonitrile step by step to obtain serial reference substance solutions with concentrations of 0.3040, 0.1520, 0.0760, 0.0380, 0.0190 and 0.0095 mg/mL;
(2) injecting 5 μ L of each series of control solutions into high performance liquid chromatograph, and recording peak area. The chromatographic conditions were the same as in test 1.1;
(3) and taking the sample amount of the reference substance as a horizontal coordinate (X) and the peak area as a vertical coordinate (Y) to perform linear regression analysis. And diluting the reference substance solution step by step under the same chromatographic conditions, respectively injecting the reference substance solution into a liquid chromatograph for analysis, and calculating the detection limit by 3 times of the signal-to-noise ratio.
The result shows that the linear range of the chebulagic acid is good within the mass range of 0.04750-1.520 mug, and the detection limit is 0.3800 mug/mL. The linear results are shown in FIG. 9.
Test 1.3 precision test
The method comprises the following steps:
(1) pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sampling 0.1g of sample powder, precisely weighing, adding 25 mL of 50% acetonitrile aqueous solution, weighing, carrying out ultrasonic treatment for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling, complementing the lost weight with an extraction solvent, filtering, taking 2 mL of subsequent filtrate to dilute to 10 mL, and filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution;
(2) injecting 5 μ L of the sample solution into high performance liquid chromatograph, measuring for 6 times, and recording the peak area of Chebular acid chromatogram. The chromatographic conditions were the same as in test 1.1.
The results showed 0.74% RSD for chebulagic acid peak area. Indicating that the precision of the instrument is good.
Test 1.4 stability test
The method comprises the following steps:
(1) pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sampling 0.1g of sample powder, precisely weighing, adding 25 mL of 50% acetonitrile aqueous solution, weighing, carrying out ultrasonic treatment for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling, complementing the lost weight with an extraction solvent, filtering, taking 2 mL of subsequent filtrate to dilute to 10 mL, and filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution;
(2) placing the sample solution at room temperature for 0, 2, 4, 8, 12, and 24 hr respectively, injecting 5 μ L into high performance liquid chromatograph, and recording the peak area of Chebular acid chromatogram. The chromatographic conditions were the same as in test 1.1.
The results showed 0.84% RSD for chebulagic acid peak area. The test solution is proved to have good stability within 24 hours of standing at room temperature.
Test 1.5 repeatability test
The method comprises the following steps:
(1) dissolving Chebulagic acid reference substance in acetonitrile solution to obtain reference substance solution with concentration of 0.076 mg/mL;
(2) pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sampling 0.1g of sample powder, precisely weighing, adding 25 mL of 50% acetonitrile aqueous solution, weighing, carrying out ultrasonic treatment for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling, complementing the lost weight with an extraction solvent, filtering, taking 2 mL of subsequent filtrate to dilute to 10 mL, filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution, and preparing 6 parts in parallel;
(3) injecting 5 μ L of each of the reference solution and 6 parts of the sample solution into a high performance liquid chromatograph, recording the peak area of the chebulagic acid chromatographic peak, and calculating the content. The chromatographic conditions were the same as in test 1.1.
The results showed that the mean chebulagic acid content was 140 mg/g and the RSD was 2.6% in 6 samples. The method is shown to have good repeatability.
Test 1.6 sample recovery test
The method comprises the following steps:
(1) pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sampling 0.05 g of sample powder, precisely weighing, adding a reference substance with the content of 100% of chebulagic acid in the sample, adding 25 mL of 50% acetonitrile aqueous solution, weighing, carrying out ultrasound for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling, complementing the lost weight of an extraction solvent, filtering, taking 2 mL of subsequent filtrate to dilute to 10 mL, filtering the diluent through a 0.45 mu m microporous membrane to prepare a sample solution, and preparing 6 parts in parallel;
(2) injecting 5 mu L of each 6 parts of test solution into a high performance liquid chromatograph, recording the peak area of the chebulagic acid, and calculating the content; the chromatographic conditions were the same as in test 1.1.
The result shows that the average sample recovery rate of the chebulagic acid is 100.02 percent, the RSD is 0.71 percent, the requirement is met, and the result is shown in the table 1.
TABLE 1 sample recovery results
Figure 967659DEST_PATH_IMAGE002
。
Example 2: a method for measuring content of chebulagic acid in fructus Chebulae by high performance liquid chromatography comprises the following steps:
(1) dissolving Chebulagic acid reference substance in acetonitrile solution to obtain reference substance solution with concentration of 0.076 mg/mL;
(2) pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sampling 0.1g of sample powder, precisely weighing, adding 25 mL of 50% acetonitrile aqueous solution, weighing, carrying out ultrasonic treatment for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling, complementing the lost weight with an extraction solvent, filtering, taking 2 mL of subsequent filtrate to dilute to 10 mL, and filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution;
(3) injecting 5 μ L of each of the reference solution and the sample solution into high performance liquid chromatograph, and recording each parameter of Chebulagic acid. The chromatographic conditions are as follows: an Agilent 1100 high performance liquid chromatography system; a chromatographic column: a Dikma Platisil column (250 mm × 4.6 mm, 5 μm); mobile phase: phase A is water-methanol-formic acid (95: 5:0.1 v/v/v); phase B was acetonitrile, elution gradient: 0-5 min, B is 2% → 5%; 5-10 min, wherein B is 5%; 10-12 min, wherein B is 5% → 15%; 12-30 min, wherein B is 15%; 30-35 min, wherein B is 15% → 18%; 35-50 min, wherein B is 18%; 50-55 min, wherein B is 18% → 100%; column temperature: 25 ℃; flow rate: 1.0 mL/min; the detection wavelength was 280 nm.
Example 3: a method for measuring content of chebulagic acid in fructus Chebulae by high performance liquid chromatography comprises the following steps:
(1) the same as in (1) of example 2;
(2) pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sampling 0.1g of sample powder, precisely weighing, adding 25 mL of 30% acetonitrile aqueous solution, weighing, carrying out ultrasonic treatment for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling, supplementing lost weight with an extraction solvent, filtering, taking 2 mL of subsequent filtrate to dilute to 10 mL, and filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution;
(3) injecting 5 μ L of each of the reference solution and the sample solution into high performance liquid chromatograph, and recording each parameter of Chebulagic acid. The chromatographic conditions are as follows: an Agilent 1100 high performance liquid chromatography system; a chromatographic column: a Dikma Platisil column (250 mm × 4.6 mm, 5 μm); mobile phase: phase A is water-methanol-formic acid (95: 5:0.1 v/v/v); phase B was acetonitrile, elution gradient: 0-5 min, B is 2% → 5%; 5-10 min, wherein B is 5%; 10-12 min, wherein B is 5% → 15%; 12-30 min, wherein B is 15%; 30-35 min, wherein B is 15% → 18%; 35-50 min, wherein B is 18%; 50-55 min, wherein B is 18% → 100%; column temperature: 25 ℃; flow rate: 1.0 mL/min; the detection wavelength was 280 nm.
Example 4: a method for measuring content of chebulagic acid in fructus Chebulae by high performance liquid chromatography comprises the following steps:
(1) the same as in (1) of example 2;
(2) pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sampling 0.1g of sample powder, precisely weighing, adding 25 mL of 70% acetonitrile aqueous solution, weighing, carrying out ultrasonic treatment for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling, complementing the lost weight with an extraction solvent, filtering, taking 2 mL of subsequent filtrate to dilute to 10 mL, and filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution;
(3) injecting 5 μ L of each of the reference solution and the sample solution into high performance liquid chromatograph, and recording each parameter of Chebulagic acid. The chromatographic conditions are as follows: an Agilent 1100 high performance liquid chromatography system; a chromatographic column: a Dikma Platisil column (250 mm × 4.6 mm, 5 μm); mobile phase: phase A is water-methanol-formic acid (95: 5:0.1 v/v/v); phase B was acetonitrile, elution gradient: 0-5 min, B is 2% → 5%; 5-10 min, wherein B is 5%; 10-12 min, wherein B is 5% → 15%; 12-30 min, wherein B is 15%; 30-35 min, wherein B is 15% → 18%; 35-50 min, wherein B is 18%; 50-55 min, wherein B is 18% → 100%; column temperature: 25 ℃; flow rate: 1.0 mL/min; the detection wavelength was 280 nm.
Example 5: a method for measuring content of chebulagic acid in fructus Chebulae by high performance liquid chromatography comprises the following steps:
(1) the same as in (1) of example 2;
(2) pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sampling 0.1g of sample powder, precisely weighing, adding 25 mL of 50% acetonitrile aqueous solution, weighing, carrying out ultrasonic treatment for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling, complementing the lost weight with an extraction solvent, filtering, taking 2 mL of subsequent filtrate to dilute to 10 mL, and filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution;
(3) injecting the reference solution and the sample solution into high performance liquid chromatograph at 3 μ L, and recording each parameter of Chebulagic acid. The chromatographic conditions are as follows: an Agilent 1100 high performance liquid chromatography system; a chromatographic column: a Dikma Platisil column (250 mm × 4.6 mm, 5 μm); mobile phase: phase A is water-methanol-formic acid (95: 5:0.1 v/v/v); phase B was acetonitrile, elution gradient: 0-5 min, B is 2% → 5%; 5-10 min, wherein B is 5%; 10-12 min, wherein B is 5% → 15%; 12-30 min, wherein B is 15%; 30-35 min, wherein B is 15% → 18%; 35-50 min, wherein B is 18%; 50-55 min, wherein B is 18% → 100%; column temperature: 25 ℃; flow rate: 1.0 mL/min; the detection wavelength was 280 nm.
Example 6: a method for measuring content of chebulagic acid in fructus Chebulae by high performance liquid chromatography comprises the following steps:
(1) the same as in (1) of example 2;
(2) pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sampling 0.1g of sample powder, precisely weighing, adding 25 mL of 50% acetonitrile aqueous solution, weighing, carrying out ultrasonic treatment for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling, complementing the lost weight with an extraction solvent, filtering, taking 2 mL of subsequent filtrate to dilute to 10 mL, and filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution;
(3) injecting 7 μ L of each of the reference solution and the sample solution into high performance liquid chromatograph, and recording each parameter of Chebulagic acid. The chromatographic conditions are as follows: an Agilent 1100 high performance liquid chromatography system; a chromatographic column: a Dikma Platisil column (250 mm × 4.6 mm, 5 μm); mobile phase: phase A is water-methanol-formic acid (95: 5:0.1 v/v/v); phase B was acetonitrile, elution gradient: 0-5 min, B is 2% → 5%; 5-10 min, wherein B is 5%; 10-12 min, wherein B is 5% → 15%; 12-30 min, wherein B is 15%; 30-35 min, wherein B is 15% → 18%; 35-50 min, wherein B is 18%; 50-55 min, wherein B is 18% → 100%; column temperature: 25 ℃; flow rate: 1.0 mL/min; the detection wavelength was 280 nm.
Example 7: a method for measuring content of chebulagic acid in fructus Chebulae by high performance liquid chromatography comprises the following steps:
(1) the same as in (1) of example 2;
(2) pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sampling 0.1g of sample powder, precisely weighing, adding 25 mL of 50% acetonitrile aqueous solution, weighing, carrying out ultrasonic treatment for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling, complementing the lost weight with an extraction solvent, filtering, taking 2 mL of subsequent filtrate to dilute to 10 mL, and filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution;
(3) injecting 5 μ L of each of the reference solution and the sample solution into high performance liquid chromatograph, and recording each parameter of Chebulagic acid. The chromatographic conditions are as follows: an Agilent 1100 high performance liquid chromatography system; a chromatographic column: diamonsil Plus C18 column (250 mm. times.4.6 mm, 5 μm); mobile phase: phase A is water-methanol-formic acid (95: 5:0.1 v/v/v); phase B was acetonitrile, elution gradient: 0-5 min, B is 2% → 5%; 5-10 min, wherein B is 5%; 10-12 min, wherein B is 5% → 15%; 12-30 min, wherein B is 15%; 30-35 min, wherein B is 15% → 18%; 35-50 min, wherein B is 18%; 50-55 min, wherein B is 18% → 100%; column temperature: 25 ℃; flow rate: 1.0 mL/min; the detection wavelength was 280 nm.
Example 8: a method for measuring content of chebulagic acid in fructus Chebulae by high performance liquid chromatography comprises the following steps:
(1) the same as in (1) of example 2;
(2) pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sampling 0.1g of sample powder, precisely weighing, adding 25 mL of 50% acetonitrile aqueous solution, weighing, carrying out ultrasonic treatment for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling, complementing the lost weight with an extraction solvent, filtering, taking 2 mL of subsequent filtrate to dilute to 10 mL, and filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution;
(3) injecting 5 μ L of each of the reference solution and the sample solution into high performance liquid chromatograph, and recording Chebulagic acid and various parameters. The chromatographic conditions are as follows: an Agilent 1100 high performance liquid chromatography system; a chromatographic column: an illite SinoChrom ODS-BP chromatography column (250 mm. times.4.6 mm, 5 μm); mobile phase: phase A is water-methanol-formic acid (95: 5:0.1 v/v/v); phase B was acetonitrile, elution gradient: 0-5 min, B is 2% → 5%; 5-10 min, wherein B is 5%; 10-12 min, wherein B is 5% → 15%; 12-30 min, wherein B is 15%; 30-35 min, wherein B is 15% → 18%; 35-50 min, wherein B is 18%; 50-55 min, wherein B is 18% → 100%; column temperature: 25 ℃; flow rate: 1.0 mL/min; the detection wavelength was 280 nm.
Example 9: a method for measuring content of chebulagic acid in fructus Chebulae by high performance liquid chromatography comprises the following steps:
(1) the same as in (1) of example 2;
(2) pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sampling 0.1g of sample powder, precisely weighing, adding 25 mL of 50% acetonitrile aqueous solution, weighing, carrying out ultrasonic treatment for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling, complementing the lost weight with an extraction solvent, filtering, taking 2 mL of subsequent filtrate to dilute to 10 mL, and filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution;
(3) injecting 5 μ L of each of the reference solution and the sample solution into high performance liquid chromatograph, and recording each parameter of Chebulagic acid. The chromatographic conditions are as follows: an Agilent 1100 high performance liquid chromatography system; a chromatographic column: a Dikma Platisil column (250 mm × 4.6 mm, 5 μm); mobile phase: the phase A is water-methanol-formic acid (90: 10:0.2 v/v/v); phase B was acetonitrile, elution gradient: 0-5 min, B is 2% → 5%; 5-10 min, wherein B is 5%; 10-12 min, wherein B is 5% → 15%; 12-30 min, wherein B is 15%; 30-35 min, wherein B is 15% → 18%; 35-50 min, wherein B is 18%; 50-55 min, wherein B is 18% → 100%; column temperature: 25 ℃; flow rate: 1.0 mL/min; the detection wavelength was 280 nm.
Example 10: a method for measuring content of chebulagic acid in fructus Chebulae by high performance liquid chromatography comprises the following steps:
(1) the same as in (1) of example 2;
(2) pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sampling 0.1g of sample powder, precisely weighing, adding 25 mL of 50% acetonitrile aqueous solution, weighing, carrying out ultrasonic treatment for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling, complementing the lost weight with an extraction solvent, filtering, taking 2 mL of subsequent filtrate to dilute to 10 mL, and filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution;
(3) injecting 5 μ L of each of the reference solution and the sample solution into high performance liquid chromatograph, and recording each parameter of Chebulagic acid. The chromatographic conditions are as follows: an Agilent 1100 high performance liquid chromatography system; a chromatographic column: a Dikma Platisil column (250 mm × 4.6 mm, 5 μm); mobile phase: phase A is water-methanol-formic acid (95: 5:0.1 v/v/v); phase B was acetonitrile, elution gradient: 0-5 min, B is 2% → 5%; 5-10 min, wherein B is 5%; 10-12 min, wherein B is 5% → 15%; 12-30 min, wherein B is 15%; 30-35 min, wherein B is 15% → 18%; 35-50 min, wherein B is 18%; 50-55 min, wherein B is 18% → 100%; column temperature: 25 ℃; flow rate: 1.0 mL/min; the detection wavelength was 270 nm.
Example 11: a method for measuring content of chebulagic acid in fructus Chebulae by high performance liquid chromatography comprises the following steps:
(1) the same as in (1) of example 2;
(2) pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sampling 0.1g of sample powder, precisely weighing, adding 25 mL of 50% acetonitrile aqueous solution, weighing, carrying out ultrasonic treatment for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling, complementing the lost weight with an extraction solvent, filtering, taking 2 mL of subsequent filtrate to dilute to 10 mL, and filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution;
(3) injecting 5 μ L of each of the reference solution and the sample solution into high performance liquid chromatograph, and recording each parameter of Chebulagic acid. The chromatographic conditions are as follows: an Agilent 1100 high performance liquid chromatography system; a chromatographic column: a Dikma Platisil column (250 mm × 4.6 mm, 5 μm); mobile phase: phase A is water-methanol-formic acid (95: 5:0.1 v/v/v); phase B was acetonitrile, elution gradient: 0-5 min, B is 2% → 5%; 5-10 min, wherein B is 5%; 10-12 min, B is 5% → 14%; 12-30 min, wherein B is 14%; 30-35 min, wherein B is 14% → 17%; 35-50 min, wherein B is 17%; 50-55 min, wherein B is 17% → 100%; column temperature: 25 ℃; flow rate: 1.0 mL/min; the detection wavelength was 280 nm.
Example 12: a method for measuring content of chebulagic acid in fructus Chebulae by high performance liquid chromatography comprises the following steps:
(1) the same as in (1) of example 2;
(2) pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sampling 0.1g of sample powder, precisely weighing, adding 25 mL of 50% acetonitrile aqueous solution, weighing, carrying out ultrasonic treatment for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling, complementing the lost weight with an extraction solvent, filtering, taking 2 mL of subsequent filtrate to dilute to 10 mL, and filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution;
(3) injecting 5 μ L of each of the reference solution and the sample solution into high performance liquid chromatograph, and recording each parameter of Chebulagic acid. The chromatographic conditions are as follows: shimadzu LC-16 high performance liquid chromatography system; a chromatographic column: a Dikma Platisil column (250 mm × 4.6 mm, 5 μm); mobile phase: phase A is water-methanol-formic acid (95: 5:0.1 v/v/v); phase B was acetonitrile, elution gradient: 0-5 min, B is 2% → 5%; 5-10 min, wherein B is 5%; 10-12 min, wherein B is 5% → 15%; 12-30 min, wherein B is 15%; 30-35 min, wherein B is 15% → 18%; 35-50 min, wherein B is 18%; 50-55 min, wherein B is 18% → 100%; column temperature: 25 ℃; flow rate: 1.0 mL/min; the detection wavelength was 280 nm.
Comparative example 1: a method for measuring content of chebulagic acid in fructus Chebulae by high performance liquid chromatography comprises the following steps:
(1) dissolving Chebulagic acid reference substance in methanol solution to obtain mixed reference substance solution with Chebulagic acid content of 0.068 mg/mL;
(2) pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sampling 0.1g of sample powder, precisely weighing, adding 25 mL of 70% methanol aqueous solution, weighing, carrying out ultrasonic treatment for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling, supplementing lost weight with an extraction solvent, filtering, taking 2 mL of subsequent filtrate to dilute to 10 mL, and filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution;
(3) injecting 5 μ L of each of the reference solution and the sample solution into high performance liquid chromatograph, and recording each parameter of Chebulagic acid. The chromatographic conditions are as follows: an Agilent 1100 high performance liquid chromatography system; a chromatographic column: a Dikma Platisil column (250 mm × 4.6 mm, 5 μm); mobile phase: the phase A is water-formic acid (100: 0.1 v/v); the phase B is methanol; elution gradient: 0-8 min, B is 5% → 10%; 8-15 min, wherein B is 10% → 25%; 15-25 min, wherein B is 25%; 25-30 min, B is 25% → 30%; 30-50 min, wherein B is 30% → 45%; 50-55 min, wherein B is 45%; 55-60 min, B is 45% → 100%. Column temperature: 25 ℃; flow rate: 1.0 mL/min; the detection wavelength is 280 nm;
comparative example 2: a method for measuring content of chebulagic acid in fructus Chebulae by high performance liquid chromatography comprises the following steps:
(1) the same as in (1) of example 2;
(2) pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sampling 0.1g of sample powder, precisely weighing, adding 25 mL of 70% methanol aqueous solution, weighing, carrying out ultrasonic treatment for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling, supplementing lost weight with an extraction solvent, filtering, taking 2 mL of subsequent filtrate to dilute to 10 mL, and filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution;
(3) injecting 5 μ L of each of the reference solution and the sample solution into high performance liquid chromatograph, and recording each parameter of Chebulagic acid. The chromatographic conditions are as follows: an Agilent 1100 high performance liquid chromatography system; a chromatographic column: a Dikma Platisil column (250 mm × 4.6 mm, 5 μm); mobile phase: phase A is water-methanol-formic acid (95: 5:0.1 v/v/v); phase B was acetonitrile, elution gradient: 0-5 min, B is 2% → 5%; 5-10 min, wherein B is 5%; 10-12 min, B is 5% → 14%; 12-30 min, wherein B is 14%; 30-35 min, wherein B is 14% → 17%; 35-50 min, wherein B is 17%; 50-55 min, wherein B is 17% → 100%; column temperature: 25 ℃; flow rate: 1.0 mL/min; the detection wavelength was 280 nm.
Comparative example 3: a method for measuring content of chebulagic acid in fructus Chebulae by high performance liquid chromatography comprises the following steps:
(1) the same as comparative example 1 (1);
(2) pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sampling 0.1g of sample powder, precisely weighing, adding 25 mL of 70% methanol aqueous solution, weighing, carrying out ultrasonic treatment for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling, supplementing lost weight with an extraction solvent, filtering, taking 2 mL of subsequent filtrate to dilute to 10 mL, and filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution;
(3) injecting 5 μ L of each of the reference solution and the sample solution into high performance liquid chromatograph, and recording each parameter of Chebulagic acid. The chromatographic conditions are as follows: an Agilent 1100 high performance liquid chromatography system; a chromatographic column: a Dikma Platisil column (250 mm × 4.6 mm, 5 μm); mobile phase: phase A is water-methanol-formic acid (95: 5:0.1 v/v/v); phase B was acetonitrile, elution gradient: 0-5 min, B is 2% → 5%; 5-10 min, wherein B is 5%; 10-12 min, wherein B is 5% → 15%; 12-30 min, wherein B is 15%; 30-35 min, wherein B is 15% → 18%; 35-50 min, wherein B is 18%; 50-55 min, wherein B is 18% → 100%; column temperature: 25 ℃; flow rate: 1.0 mL/min; the detection wavelength was 280 nm.
Comparative example 4: a method for measuring content of chebulagic acid by high performance liquid chromatography comprises the following steps:
(1) dissolving Chebulagic acid reference substance in acetonitrile solution to obtain 0.078 mg/mL Chebulagic acid reference substance solution;
(2) injecting 5 μ L of the above control solution into high performance liquid chromatograph, and recording each parameter of Chebulagic acid. The chromatographic conditions are as follows: shimadzu LC-16 high performance liquid chromatography system; a chromatographic column: a Dikma Platisil column (250 mm × 4.6 mm, 5 μm); mobile phase: the phase A is water-methanol-formic acid (90: 10:0.1 v/v/v); phase B was acetonitrile, elution gradient: 0-2 min, B is 5% → 13%; 2-20 min, wherein B is 13%; column temperature: 25 ℃; flow rate: 1.0 mL/min; the detection wavelength was 278 nm.
Comparative example 5: a method for measuring content of chebulagic acid by high performance liquid chromatography comprises the following steps:
(1) dissolving Chebulagic acid reference substance in methanol solution to obtain 0.082 mg/mL Chebulagic acid reference substance solution;
(2) injecting 5 μ L of the above control solution into high performance liquid chromatograph, and recording each parameter of Chebulagic acid. The chromatographic conditions are as follows: shimadzu LC-16 high performance liquid chromatography system; a chromatographic column: a Dikma Platisil column (250 mm × 4.6 mm, 5 μm); mobile phase: the phase A is water-methanol-formic acid (90: 10:0.1 v/v/v); phase B was acetonitrile, elution gradient: 0-2 min, B is 5% → 13%; 2-20 min, wherein B is 13%; column temperature: 25 ℃; flow rate: 1.0 mL/min; the detection wavelength was 278 nm.
The results of the above-described tests of examples 2-12 and comparative examples 1-5 show the range of durability and the system applicability of the method of the present invention. However, the results show that the peak sequences of the chemical components in the myrobalan are inconsistent under the condition that methanol and acetonitrile are used as mobile phases, and direct comparison cannot be carried out, so that the stability of the myrobalan in methanol and acetonitrile is examined by using a myrobalan acid reference substance. The examination conditions and results are shown in tables 2-4.
TABLE 2 durability examination conditions
Figure 198263DEST_PATH_IMAGE003
。
TABLE 3 chebulagic acid stability examination conditions
Figure 796734DEST_PATH_IMAGE004
。
TABLE 4 System suitability results
Figure 876686DEST_PATH_IMAGE005
。
The results of the determinations of the chebulagic acid in the examples 2 to 12 show that the sample peak and the adjacent peaks can be completely separated by the content determination method, the retention time, the theoretical plate number, the tailing factor and the separation degree all meet the requirements, the tailing factor and the separation degree of the determination method in the comparative example 1 do not meet the requirements, the peak area is larger than that of the example, the result of the determination is inaccurate, and the comparison between the comparative examples 1 to 3 and the example 2 shows that the influence of selecting methanol as a solvent during the preparation of a sample solution on the chebulagic acid content determination is larger, and the influence of selecting methanol as a mobile phase is also certain.
The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims (10)

1. A method for extracting chebulagic acid from chebula meat is characterized by comprising the following specific steps:
(1) removing core of fructus Chebulae, pulverizing pulp, and sieving; weighing a certain amount of fructus Chebulae powder, adding 70% ethanol water solution, performing ultrasonic extraction, and extracting the extractive solution at 3000 r min-1Centrifuging at the rotating speed of (1) for 10 min, and recovering ethanol from the supernatant under reduced pressure;
(2) diluting the residue obtained after recovering ethanol with water,extracting with water-equivalent ethyl acetate for several times, mixing ethyl acetate solutions, recovering ethyl acetate under reduced pressure, re-dissolving the residue with methanol water at 10000 r.min-1Centrifuging for 10 min at the rotating speed of (1);
(3) separating the supernatant obtained by centrifuging with ODS open column, eluting with methanol water solution, collecting eluate, recovering solvent under reduced pressure, and standing at 4 deg.C until white solid is separated out to obtain Chebulagic acid.
2. The method for extracting chebulagic acid from myrobalan pulp according to claim 1, wherein the chebulagic acid pulp in the step (1) is crushed, sieved by a 65-120 mesh sieve, added with 70% ethanol water in an amount of 20-30 times of the amount of the medicinal materials, and ultrasonically extracted for 2-3 times, 10-30 min each time.
3. The method for extracting chebulagic acid from myrobalan meat according to claim 1, wherein in the step (2), the dilution water addition amount is 2-4 times of the amount of the initially weighed medicinal materials, and the extraction times are 6-12 times; the compound solution is 20% methanol water solution, and the dosage is 1:1 of the crude drug, thus obtaining the sample solution with the concentration of 1.0 g/mL.
4. The method for extracting the chebulagic acid from the myrobalan meat according to claim 1, wherein in the step (3), the ratio of the diameter to the height of the ODS column packing is 1: 5-1: 0.8; the sample loading amount is 1-2 mL per 10 g of ODS; eluting with 20% methanol water solution, collecting 16 column volumes from the 4 th column volume, recovering solvent under reduced pressure, and standing at 4 deg.C until white solid is separated out to obtain Chebulagic acid.
5. The method for extracting chebulagic acid from chebula meat as claimed in claim 1, wherein the chebulagic acid extracted by the method is used as standard or control in quality control of chebula.
6. A determination method for extracting chebulagic acid content from myrobalan is characterized by comprising the following steps:
(1) preparation of control solutions: dissolving Chebulagic acid reference substance in acetonitrile solution to obtain mixed reference substance solution;
(2) preparing a test solution: taking myrobalan pulp, crushing, and screening by a 65-120-mesh sieve; sampling 0.1-0.2 g of sample powder, precisely weighing, adding 25-50 mL of acetonitrile aqueous solution, weighing, performing ultrasonic treatment, cooling, complementing the lost weight with an extraction solvent, filtering, taking a subsequent filtrate for dilution by 5-10 times, and filtering the diluent through a 0.45 mu m microporous membrane to obtain a sample solution;
(3) respectively injecting the solutions prepared in the steps (1) and (2) into a high performance liquid chromatograph for determination, wherein the high performance liquid chromatograph conditions are as follows: a chromatographic column: a Dikma Platisil column (250 mm × 4.6 mm, 5 μm); mobile phase: the phase A is water-methanol-formic acid, the phase B is acetonitrile, and gradient elution is carried out; column temperature: 25-30 ℃; flow rate: 0.8-1.2 mL/min; the detection wavelength is 280 nm; the number of theoretical plates should not be less than 5000 based on the chebulagic acid peak.
7. The method for determining the content of the chebulagic acid extracted from the myrobalan according to claim 6, wherein in the step (1), the concentration of the chebulagic acid in the mixed control solution is 0.05-0.1 mg/mL.
8. The method for determining the content of the chebulagic acid extracted from the myrobalan according to claim 6, wherein in the step (2), the extraction solvent is 30-70% acetonitrile water solution; the ultrasonic condition is as follows: the time is 20-40 min, the power is 250-300W, the frequency is 35-40 kHz, and the temperature is 25-35 ℃.
9. The method for determining the content of the chebulagic acid extracted from the myrobalan according to claim 6, wherein in the step (3), the sample amount of the mixed control solution and the sample solution is 3-7 μ L.
10. The method for determining the content of the chebulagic acid extracted from the myrobalan according to claim 6, wherein in the step (3), the volume ratio of the mobile phase A to the mobile phase A is 90-95: 5-10: 0.1-0.2 of water-methanol-formic acid, wherein the phase B is acetonitrile, and the elution gradient is as follows: 0-5 min, B is 2% → 5%; 5-10 min, wherein B is 5%; 10-12 min, wherein B is 5% → 15%; 12-30 min, wherein B is 15%; 30-35 min, wherein B is 15% → 18%; 35-50 min, wherein B is 18%; 50-55 min, B is 18% → 100%.
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