CN114414702B - Preparation method and content measurement method of chebular acid in chebula fruit flesh - Google Patents
Preparation method and content measurement method of chebular acid in chebula fruit flesh Download PDFInfo
- Publication number
- CN114414702B CN114414702B CN202210105897.9A CN202210105897A CN114414702B CN 114414702 B CN114414702 B CN 114414702B CN 202210105897 A CN202210105897 A CN 202210105897A CN 114414702 B CN114414702 B CN 114414702B
- Authority
- CN
- China
- Prior art keywords
- acid
- solution
- chebular
- myrobalan
- water
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8634—Peak quality criteria
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Engineering & Computer Science (AREA)
- Quality & Reliability (AREA)
- Saccharide Compounds (AREA)
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 chebular acid in chebula meat. The method for preparing the chebularetic acid from the chebula fruit flesh provided by the invention has the product purity of more than 99%, the total yield of more than 60%, and the method can be used as a standard or a reference substance for quality control of the chebularetic acid. In addition, the method has simple operation steps, low equipment cost, applicability to industrial production and suitability for the preparation of a large amount of chebular acid reference substances. The method for measuring the content of the myrobalan acid in the myrobalan can accurately detect the content of the myrobalan acid in the myrobalan meat, lays a theoretical foundation and experimental basis for establishing the quality standard of the myrobalan, and has important significance for promoting the industrialized development of the myrobalan.
Description
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 chebular acid in chebula meat.
Background
Fructus Chebulae is fructus Chebulae of CombretaceaeTerminalia chebulaRetz.) or myrobalanTerminalia chebula Retz.var.tomentellaKurt.) has the effects of astringing intestines to check diarrhea, astringing lung to check cough. Modern chemical research shows that the main chemical components of myrobalan are tannins, including hydrolysable myrobalanSub-tannins and ellagitannins. Wherein the component chebulbar acid with higher content is formed by condensing a plurality of gallic acids, and has the following structure:
earlier research shows that myrobalan acid has strong antioxidant and anti-inflammatory capabilities, is a main medicinal component of myrobalan, has the content of 10% in myrobalan meat, and can be used as a quality marker for quality control and evaluation of myrobalan medicinal materials. Therefore, the chebulbar acid reference substance needs to be prepared for reasonably and effectively evaluating the quality of the chebulbar medicinal material.
At present, the separation and preparation of myrobalan acid at home and abroad is still in a starting stage, and the technology is not perfect. CN102020682a,2011, 4 and 20 days disclose a method for preparing chebula retanning acid from chebula leaves, the purity of the obtained chebula retanning acid product can reach more than 98%, but strong alkali and strong acid are needed, and only 35 g products are obtained from 10 kg raw materials, so that the yield is lower; CN104945447a,2015, 9 and 30 days discloses a method for preparing myrobalan ellagic acid from myrobalan leaves, wherein raw materials are subjected to steps of enzymolysis, ultrasonic extraction, ceramic membrane separation, macroporous resin adsorption and the like, the steps are complicated, and the purity of the product is below 90%; CN105949252a, 9 and 21 in 2016 discloses a method for preparing chebulenic acid from chebula medicinal material by high-speed countercurrent chromatography, but the equipment is complex and expensive, the product yield is less than 0.004%, and the purity is only more than 95%. CN109867704a,2019, 6 and 11 disclose a method for preparing chebulic acid by ethanol warm leaching, CN110724172a, and 24, 2020, 1 and 24 disclose a method for preparing chebulic acid by ethanol reflux method, wherein the two methods both adopt phyllanthus emblica as raw materials, and the phyllanthus emblica is separated by macroporous resin column, the purity of the obtained chebulic acid product is above 98%, but 100 g medicinal materials can only obtain about 2 g chebulic acid product.
As can be seen from published documents, in the existing preparation method of the myrobalan acid, the myrobalan fruit, the myrobalan leaf or the phyllanthus emblica are mostly used as raw materials, wherein the content of the myrobalan acid in the phyllanthus emblica is 2-5%, the content of the myrobalan acid in the myrobalan leaf is about 2%, the myrobalan fruit comprises myrobalan pulp and myrobalan kernel, wherein the kernel accounts for 15-30% of the weight of the fruit, and the applicant has found that the content of the myrobalan acid in the myrobalan pulp is about 10%, but the myrobalan kernel hardly contains, so that the method for removing the kernel from the myrobalan fruit and extracting the myrobalan acid from the myrobalan pulp is a path with higher efficiency. In addition, the current method for preparing the chebular acid usually adopts a combination of various column chromatography, high-speed countercurrent 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 first part of the 2020 edition of the current Chinese pharmacopoeia, the quality control method of the myrobalan is limited to qualitative identification and detection of moisture, ash and water-soluble extracts, no provision of a content determination method exists, and the myrobalan rillic acid can be used as a quality marker for quality control of the myrobalan medicinal material. Hu Qiping is equal to "Chinese medicinal materials", and discloses a method for measuring the content of chebular acid in myrobalan, wherein the used sample extraction solvent is methanol, however, the experience of the inventor for studying chebular for a long time discovers that chebular acid is very unstable in methanol, and the higher the content of methanol in the solvent is, the worse the stability is, and the characteristic has a great influence on the accuracy of measuring the content of chebular acid.
The invention aims to develop a preparation method of a chebular acid reference substance with simple operation, high yield and high purity and an accurate and repeatable content determination method for chebular acid in chebula.
Disclosure of Invention
In view of the problems existing in the prior art, the invention aims to provide a method for preparing a chebulbar acid reference substance from chebula fruit flesh. The extraction rate of the chebularetic acid in the chebular meat can reach 85%, the chebularetic acid is extracted by ethyl acetate and then separated by ODS open column, and the total yield can reach more than 60%. The content determination method provided by the invention has the advantages that the specificity, linearity, range, precision, accuracy and solution stability meet the requirements of related guidelines, the operation is simple, and the quality detection can be carried out on the medicinal material of the myrobalan.
In order to achieve the above object, the present invention provides the following technical solutions.
The invention provides a method for extracting chebular acid from chebula fruit flesh, 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, ultrasonic extracting, and collecting extractive solution at 3000 r min -1 Centrifuging at a rotating speed of (1) for 10 min, collecting supernatant, and recovering ethanol under reduced pressure;
(2) Diluting the residue with water, extracting with water-equivalent ethyl acetate for several times, mixing ethyl acetate solutions, recovering ethyl acetate under reduced pressure, and redissolving the residue with a certain amount of methanol water, 10000 r min -1 Centrifuging at the rotating speed for 10 min;
(3) Separating the supernatant by ODS open column, eluting with methanol water solution, collecting eluate, recovering solvent under reduced pressure, standing at 4deg.C until white solid is separated out to obtain chebulenic acid.
Further, in the step (1), the myrobalan pulp is crushed, sieved by a sieve with 65-120 meshes, and the added 70% ethanol water solution is 20-30 times of the medicinal material, and the ultrasonic extraction is carried out for 2-3 times, each time for 10-30 min.
Further, in the step (2), the dilution water adding amount is 2-4 times of the original weighed medicinal material amount, and the extraction times are 6-12 times; the compound solution is a 20% methanol aqueous solution, the dosage is 1:1 with the crude drug, and the concentration of the prepared sample solution is 1.0 g/mL.
Further, in the step (3), the aspect ratio of the ODS column packing is 1:5-1:0.8; the sample loading amount is 1-2 mL per 10 g ODS; the eluting solvent is 20% methanol water solution, 16 column volumes are collected from the 4 th column volume, the solvent is recovered under reduced pressure, and the solution is placed at the temperature of 4 ℃ until white solid is separated out, thus obtaining the chebulbar acid.
Further, the chebular acid extracted by the method is used as a standard or a reference substance to control the quality of the chebular acid.
The invention also provides a method for measuring the content of myrobalan rillic acid in myrobalan, which is characterized by comprising the following steps:
(1) Preparing a reference substance solution: adding acetonitrile solution into chebulbar acid reference substance for dissolution to obtain mixed reference substance solution;
(2) Preparing a test solution: pulverizing myrobalan pulp, and sieving with a 65-120 mesh sieve; 0.1-0.2 g of sample powder, precisely weighing, adding 25-50 mL of acetonitrile water solution, weighing, performing ultrasonic treatment, cooling to extract solvent to complement the lost weight, filtering, diluting the subsequent filtrate by 5-10 times, and filtering the diluted solution by a microporous filter membrane of 0.45 mu m to obtain a sample solution;
(3) Taking the solutions prepared in the steps (1) and (2), and respectively injecting the solutions into a high performance liquid chromatograph for measurement, wherein the high performance liquid chromatograph conditions are as follows: chromatographic column: dikma Platisil column (250 mm ×4.6 mm,5 μm); mobile phase: phase A is water-methanol-formic acid, 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 be not less than 5000 as calculated by the peak of chebulbar acid.
Further, in the step (1), the concentration of chebular acid in the mixed reference substance solution is 0.05-0.1 mg/mL.
Further, in the step (2), the extraction solvent is 30% -70% acetonitrile aqueous solution; the ultrasound conditions: the time is 20-40 min, the power is 250-300W, the frequency is 35-40 kHz, and the temperature is 25-35 ℃.
In the step (3), the sample injection amount of the mixed reference substance solution and the sample solution is 3-7 mu L.
Further, in the step (3), the volume ratio of the mobile phase A phase is 90-95: 5-10: 0.1 to 0.2 of water-methanol-formic acid, wherein the phase B is acetonitrile, and the elution gradient is as follows: 0 to 5 min, B is 2 to 5 percent; 5-10 min, wherein B is 5%; 10-12 min, B is 5% -15%; 12-30 min, and B is 15%; 30-35 min, B is 15% -18%; 35-50 min, wherein B is 18%; 50-55 min, and B is 18% -100%.
Compared with the prior art, the invention has the following beneficial effects.
(1) The current method for separating and preparing the myrobalan acid is mainly low in product yield and purity, and the method adopts the myrobalan meat with high myrobalan acid content as a raw material, and can remove impurities with large polarity by extracting the extracting solution with ethyl acetate, so that the method has an enrichment effect on the myrobalan acid, and ensures the purity of the obtained product. The chebular acid reference substance prepared by the invention has the purity of more than 99 percent and the total yield of more than 60 percent.
(2) The method comprises the steps of extracting chebular acid by ethanol ultrasonic, extracting by ethyl acetate, recovering to dryness, re-dissolving by methanol aqueous solution, loading on an ODS open column, and eluting by 20% methanol aqueous solution. The used reagents can be recycled, and the related ODS filler can be reused for a plurality of times, so that the cost is effectively reduced.
(3) The open column chromatography established by the invention has low requirements on equipment, can be applied to industrial production, and is suitable for preparing a large amount of chebulbar acid reference substances.
(4) According to the method for measuring the content of the chebular acid in the chebular, the chebular acid is in a good linear relationship within the range of 0.0475-1.5200 mu g, the average recovery rate is 100.02%, and the RSD is 0.71%. The RSD of precision, stability and repeatability is less than 2%. Therefore, the content determination method provided by the invention is accurate and precise, has strong specificity and simple operation, can be used for determining the content of the chebular acid in the chebula, provides theoretical basis and experimental basis for establishing the quality standard of the chebula, and has important significance for promoting the industrialized development of the chebula.
Drawings
FIG. 1 shows chebulenic acid 1 H-NMR discrimination chart.
FIG. 2 is chebulenic acid 13 C-NMR discrimination chart.
FIG. 3 is a high performance liquid chromatogram of the chebulenic acid product.
FIG. 4 is a high performance liquid chromatogram of a chebulbar acid control.
FIG. 5 shows full-wavelength scan spectra of chebulbar acid (200-400 nm).
FIG. 6 is a blank solution HPLC chromatogram of the 1.1 specificity test of example 2 of the present invention.
FIG. 7 is a HPLC chromatogram of a mixed control solution for the 1.1 specificity test of example 2 of the present invention.
FIG. 8 is a HPLC chromatogram of a test solution for the 1.1-specific test of example 2 of the present invention.
FIG. 9 is a linear diagram of chebulbar acid with a linear range of 1.2 and a detection limit in example 1 of the present invention.
FIG. 10 is an HPLC chromatogram of a chebulbar acid control dissolved in acetonitrile.
FIG. 11 is an HPLC chromatogram of a chebulbar acid control dissolved in methanol.
Detailed Description
The following is a further description of a method of preparing a chebular acid control from chebula fruit flesh according to the present invention, in conjunction with specific examples, to provide a person skilled in the art with a better understanding of the present invention, but is not intended to limit the invention thereto.
In a first embodiment, a method for extracting chebulenic acid from chebula fruit flesh.
The method for detecting the content of the chebular acid by the high performance liquid chromatography in the first embodiment of the invention is the following method unless specified otherwise:
sample solution preparation: precisely weighing 0.1g of sample, adding 70% methanol of 25 mL, precisely weighing, performing ultrasonic treatment for 30 min, cooling to extract solvent to supplement lost weight, filtering, collecting 2 mL-phase filtrate, fixing volume into 10 mL volumetric flask, and filtering with 0.45 μm microporous membrane to obtain the final product;
Chromatographic conditions: 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, and 48% B; 20-25 min, B is 48% -100%; sample injection amount: 5. mu L; column temperature: 25. the temperature is lower than the temperature; flow rate: 1.0 mL/min; the detection wavelength is 280 nm;
all the raw materials used in the experiment in the embodiment of the invention are in the same batch, and the content of the chebular acid is 140.21 mg/g by the measurement of the method.
Example 1: a method for preparing chebular acid reference substance from chebular fruit flesh, namely an extraction process of chebular acid in chebular fruit, comprises the following steps:
(1) Removing core of fructus Chebulae, pulverizing pulp, and sieving with 65 mesh sieve. Weighing 5g the myrobalan powder is added with 25 times of 70% ethanol water solution for ultrasonic extraction for 2 times, each time for 20 min. The extract is taken for 3000 r min -1 Centrifuging at a rotating speed of (1) for 10 min, collecting supernatant, and recovering ethanol under reduced pressure to dry;
(2) The dry extract corresponding to the raw medicinal material 0.1 and g is taken to prepare a sample solution, and the extraction amount of chebulbar acid is 97.36 mg/g and the extraction rate is 69.44% by HPLC method.
Example 2: a method for preparing chebular acid reference substance from chebular fruit flesh, namely an extraction process of chebular acid in chebular fruit, comprises the following steps:
(1) Removing core of fructus Chebulae, pulverizing pulp, and sieving with 120 mesh sieve. Weighing 5 g of the myrobalan powder, adding 25 times of 70% ethanol water solution, and performing ultrasonic extraction for 2 times each for 20 min. The extract is taken for 3000 r min -1 Centrifuging at a rotating speed of (1) for 10 min, collecting supernatant, and recovering ethanol under reduced pressure to dry;
(2) The dry extract equivalent to the raw medicinal material 0.1 g is taken to prepare a sample solution, and the extraction amount of chebular acid is 106.99 mg/g and the extraction rate is 76.31 percent by HPLC method.
Example 3: a method for preparing chebular acid reference substance from chebular fruit flesh, namely an extraction process of chebular acid in chebular fruit, comprises the following steps:
(1) Removing core of fructus Chebulae, pulverizing pulp, and sieving with 120 mesh sieve. Weighing 5 g of the myrobalan powder, adding 20 times of 70% ethanol water solution, and performing ultrasonic extraction for 2 times for 20 min each time. The extract is taken for 3000 r min -1 Centrifuging at a rotating speed of (1) for 10 min, collecting supernatant, and recovering ethanol under reduced pressure to dry;
(2) The dry extract corresponding to the raw medicinal material 0.1 and g is taken to prepare a sample solution, and the extraction amount of myrobalan acid is 102.77 mg/g and the extraction rate is 73.30% by HPLC method.
Example 4: a method for preparing chebular acid reference substance from chebular fruit flesh, namely an extraction process of chebular acid in chebular fruit, comprises the following steps:
(1) Removing core of fructus Chebulae, pulverizing pulp, and sieving with 120 mesh sieve. Weighing 5 g of the myrobalan powder, adding 30 times of 70% ethanol water solution, and performing ultrasonic extraction for 2 times each for 20 min. The extract is taken for 3000 r min -1 Is rotated at a speed of 1Collecting supernatant, recovering ethanol under reduced pressure for 0 min to dry;
(2) The dry extract corresponding to the raw medicinal material 0.1 and g is taken to prepare a sample solution, and the extraction amount of myrobalan acid is 118.86 mg/g and the extraction rate is 84.77% by HPLC method.
Example 5: a method for preparing chebular acid reference substance from chebular fruit flesh, namely an extraction process of chebular acid in chebular fruit, comprises the following steps:
(1) Removing core of fructus Chebulae, pulverizing pulp, and sieving with 120 mesh sieve. Weighing 5 g of the myrobalan powder, adding 30 times of 70% ethanol water solution, and performing ultrasonic extraction for 1 time each for 30 min. The extract is taken for 3000 r min -1 Centrifuging at a rotating speed of (1) for 10 min, collecting supernatant, and recovering ethanol under reduced pressure to dry;
(2) The dry extract corresponding to the raw medicinal material 0.1 and g is taken to prepare a sample solution, and the extraction amount of myrobalan acid is 109.28 mg/g and the extraction rate is 77.94% by HPLC method.
Example 6: a method for preparing chebular acid reference substance from chebular fruit flesh, namely an extraction process of chebular acid in chebular fruit, comprises the following steps:
(1) Removing core of fructus Chebulae, pulverizing pulp, and sieving with 120 mesh sieve. Weighing 5 g of the myrobalan powder, adding 30 times of 70% ethanol water solution, and performing ultrasonic extraction for 3 times each for 10 min. The extract is taken for 3000 r min -1 Centrifuging at a rotating speed of (1) for 10 min, collecting supernatant, and recovering ethanol under reduced pressure to dry;
(2) The dry extract corresponding to the raw medicinal material 0.1 and g is taken to prepare a sample solution, and the extraction amount of myrobalan acid is 121.75 mg/g and the extraction rate is 86.83% by HPLC method.
Example 7: a method for preparing chebulbar acid reference substance from chebulbar flesh comprises the following steps:
(1) Removing core of fructus Chebulae, pulverizing pulp, and sieving with 120 mesh sieve. Weighing 1.0 g myrobalan powder, adding 30 times of 70% ethanol water solution, and extracting with ultrasound for 3 times each for 10 min. The extract is taken for 3000 r min -1 Centrifuging at a rotating speed of (1) for 10 min, collecting supernatant, and recovering ethanol under reduced pressure;
(2) Diluting the residue obtained by recovering ethanol with 2 mL water, extracting with ethyl acetate equivalent to water for 6 timesMixing ethyl acetate solutions, recovering ethyl acetate under reduced pressure, and redissolving the residue with 1 mL% methanol water, 10000 r min -1 Centrifuging at the rotating speed of (2) for 10 min, namely, the concentration of the sample solution is 1.0 g/mL;
(3) Separating the supernatant from the above centrifugation by ODS open column (packing weight 10 g, diameter-to-height ratio 1:5), eluting with 20% methanol water solution for 20 column volumes, and detecting the effluent by HPLC. Collecting the eluent with the volume of 4 th to 20 th columns, recovering the solvent under reduced pressure, and standing at the temperature of 4 ℃ until white solid is precipitated.
The total yield of the obtained product is 73.40 and mg, and the total yield is 52.35%. Warp yarn 1 H-NMR 13 C-NMR spectrum is identified as chebulic acid, HPLC is used for detection, and purity is calculated to be higher than 99% according to peak area normalization method, thus obtaining chebulic acid reference substance.
Example 8: a method for preparing chebulbar acid reference substance from chebulbar flesh comprises the following steps:
(1) Same as in (1);
(2) Diluting the residue with 4 mL water, extracting with water equivalent ethyl acetate for 12 times, mixing ethyl acetate solutions, recovering ethyl acetate under reduced pressure, and redissolving the residue with 1 mL% methanol water, 10000 r min -1 Centrifuging at the rotating speed of (2) for 10 min, namely, the concentration of the sample solution is 1.0 g/mL;
(3) Same as in (3) of example 7.
The total yield of the obtained product 81.71 mg is 58.28%; the purity was higher than 99% as calculated by peak area normalization method by HPLC detection.
Example 9: a method for preparing chebulbar acid reference substance from chebulbar flesh comprises the following steps:
(1) Same as in (1);
(2) Diluting the residue with 2 mL water, extracting with water equivalent ethyl acetate for 10 times, mixing ethyl acetate solutions, recovering ethyl acetate under reduced pressure, and redissolving the residue with 1 mL% methanol water, 10000 r min -1 Centrifuging at the rotating speed of (2) for 10 min, namely, the concentration of the sample solution is 1.0 g/mL;
(3) Same as in (3) of example 7.
The obtained product has 83.44 and mg percent of total yield of 59.51 percent; the purity was higher than 99% as calculated by peak area normalization method by HPLC detection.
Example 10: a method for preparing chebulbar acid reference substance from chebulbar flesh comprises the following steps:
(1) Same as in (1);
(2) Diluting the residue with 4 mL water, extracting with water equivalent ethyl acetate for 10 times, mixing ethyl acetate solutions, recovering ethyl acetate under reduced pressure, and redissolving the residue with 1 mL% methanol water, 10000 r min -1 Centrifuging at the rotating speed of (2) for 10 min, namely, the concentration of the sample solution is 1.0 g/mL;
(3) Separating the supernatant from the above centrifugation by ODS open column (packing weight 10 g, diameter-to-height ratio 1:5), eluting with 20% methanol water solution for 20 column volumes, and detecting the effluent by HPLC. Collecting the eluent with the volume of 6-20 columns, recovering the solvent under reduced pressure, and standing at 4 ℃ until white solid is precipitated.
65.61mg of the obtained product is obtained, and the total yield is 46.79%; the purity was higher than 99% as calculated by peak area normalization method by HPLC detection.
Example 11: a method for preparing chebulbar acid reference substance from chebulbar flesh 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 extracting with ultrasound for 3 times each 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 ethanol recovery with 3 mL water, extracting with ethyl acetate equal to water for 10 times, mixing ethyl acetate solutions, recovering ethyl acetate under reduced pressure, re-dissolving the residue with 20% methanol water of 1.5 mL, and centrifuging at 10000 r min-1 for 10 min to obtain a sample solution with a concentration of 1.0 g/mL;
(3) Separating the supernatant from the above centrifugation by ODS open column (packing weight 10 g, diameter-to-height ratio 1:5), eluting with 20% methanol water solution for 20 column volumes, and detecting the effluent by HPLC. Collecting the eluent with the volume of 5 th to 20 th columns, recovering the solvent under reduced pressure, and standing at 4 ℃ until white solid is precipitated.
111.23mg of the obtained product is obtained, and the total yield is 52.89%; the purity was higher than 99% as calculated by peak area normalization method by HPLC detection.
Example 12: a method for preparing chebulbar acid reference substance from chebulbar flesh comprises the following steps:
(1) Same as in (1);
(2) Diluting the residue with 3 mL water, extracting with water equivalent ethyl acetate for 10 times, mixing ethyl acetate solutions, recovering ethyl acetate under reduced pressure, and redissolving the residue with 1.5 mL% methanol water, 10000 r min -1 Centrifuging at the rotating speed for 10 min, wherein the concentration of the sample solution is 1 g/mL;
(3) Separating the supernatant from the above centrifugation by ODS open column (packing weight 10 g, diameter-to-height ratio 1:1.4), eluting with 20% methanol water solution for 20 column volumes, and detecting the effluent by HPLC. Collecting the eluent with the volume of 4 th to 20 th columns, recovering the solvent under reduced pressure, and standing at the temperature of 4 ℃ until white solid is precipitated.
The total yield of the obtained product is 130.3 and mg and is 61.95%; the purity was higher than 99% as calculated by peak area normalization method by HPLC detection.
Example 13: a method for preparing chebulbar acid reference substance from chebulbar flesh comprises the following steps:
(1) Same as in (1);
(2) Diluting the residue obtained after ethanol recovery with 3 mL water, extracting with ethyl acetate equal to water for 10 times, mixing ethyl acetate solutions, recovering ethyl acetate under reduced pressure, re-dissolving the residue with 20% methanol water of 1.5 mL, and centrifuging at 10000 r min-1 for 10 min to obtain a sample solution with a concentration of 1.0 g/mL;
(3) Separating the supernatant from the above centrifugation by ODS open column (packing weight 10 g, diameter-to-height ratio 1:0.8), eluting with 20% methanol water solution for 20 column volumes, and detecting the effluent by HPLC. Collecting the eluent with the volume of 4 th to 20 th columns, recovering the solvent under reduced pressure, and standing at the temperature of 4 ℃ until white solid is precipitated.
The total yield of the obtained product is 57.77% of 121.5 and mg; the purity was higher than 99% as calculated by peak area normalization method by HPLC detection.
The products obtained in examples 7-13 above were subjected to nuclear magnetic resonance spectroscopy to identify the molecular structure, 1 H-NMR 13 The C-NMR spectrum is shown in FIG. 1 and FIG. 2, and the peaks of the structure analysis process are as follows:
1 H NMR（400 MHz，CD 3 OD）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）；
13 C NMR（100 MHz，CD 3 OD）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 experiment result shows that the purity of the obtained product is greater than 99% through HPLC detection, and the product meets the purity requirement of the content measurement reference substance, so the chebularella acid reference substance is prepared from chebula fruit flesh.
In the second embodiment, a method for measuring the content of chebular acid in chebula is provided.
The control in example two of the present invention was prepared according to the method of example 12 in example one and used in the following test.
Example 1: methodology investigation
Test 1.1 specificity test
The method comprises the following steps:
(1) Taking 50% acetonitrile, and filtering the acetonitrile through a microporous membrane with the size of 0.45 mu m to prepare an empty solution;
(2) Adding acetonitrile solution into chebulbar acid reference substance for dissolution to obtain reference substance solution with chebulbar acid of 0.076 mg/mL;
(3) Pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. 0.1g of sample powder is precisely weighed, 50% acetonitrile water solution 25 and mL are added, the weighed weight is adopted, ultrasound is carried out for 30 min (power is 250W, frequency is 40 kHz, temperature is 30 ℃), cooling is carried out, the lost weight is complemented by extracting solvent, filtering is carried out, the subsequent filtrate 2 mL is diluted to 10 mL, and the diluted solution is filtered by a microporous filter membrane of 0.45 mu m, so as to prepare a sample solution;
(4) Taking 5 mu L of each of the blank solution, the mixed reference substance solution and the test substance solution, injecting into a high performance liquid chromatograph, and recording a chromatogram. The chromatographic conditions are as follows: agilent 1100 high performance liquid chromatography system; chromatographic column: 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 is acetonitrile, elution gradient: 0 to 5 min, B is 2 to 5 percent; 5-10 min, wherein B is 5%; 10-12 min, B is 5% -15%; 12-30 min, and B is 15%; 30-35 min, 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 the chromatographic peak with the retention time consistent with that of the chebulbar acid reference substance appears in the chromatogram of the sample solution prepared by the treatment method, and the chromatographic peak is completely separated from the adjacent chromatographic peak. Meanwhile, no chromatographic peak consistent with the retention time of the reference substance appears in the chromatogram of the blank solution, which shows that the method has good specificity for measuring the content of the chebular acid in the chebula, and the chromatograms are shown in figures 6-8.
Test 1.2 Linear Range and detection Limit
The method comprises the following steps:
(1) Dissolving chebulbar acid reference substance in acetonitrile solution, and gradually diluting with acetonitrile to obtain series of reference substance solutions with concentrations of 0.3040, 0.1520, 0.0760, 0.0380, 0.0190 and 0.0095 mg/mL respectively;
(2) 5 mu L of each of the series of control solutions was injected into a high performance liquid chromatograph, and the peak areas were recorded. The chromatographic conditions were the same as in test 1.1;
(3) And (3) carrying out linear regression analysis by taking the sample injection amount of the reference substance as an abscissa (X) and the peak area as an ordinate (Y). And diluting the reference substance solution step by step, respectively injecting the reference substance solution into a liquid chromatograph for analysis under the same chromatographic conditions, and calculating the detection limit by using a signal-to-noise ratio of 3 times.
The result shows that the linear range of the myrobalan acid is good in 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. 0.1g of sample powder is precisely weighed, 50% acetonitrile water solution 25 and mL are added, the weighed weight is adopted, ultrasound is carried out for 30 min (power is 250W, frequency is 40 kHz, temperature is 30 ℃), cooling is carried out, the lost weight is complemented by extracting solvent, filtering is carried out, the subsequent filtrate 2 mL is diluted to 10 mL, and the diluted solution is filtered by a microporous filter membrane of 0.45 mu m, so as to prepare a sample solution;
(2) And 5 mu L of the sample solution is injected into a high performance liquid chromatograph, the measurement is carried out for 6 times in parallel, and the chromatographic peak area of the myrobalan acid is recorded. The chromatographic conditions were the same as in test 1.1.
The results showed that the myrtillin acid peak area RSD was 0.74%. Indicating that the instrument has good precision.
Test 1.4 stability test
The method comprises the following steps:
(1) Pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. 0.1g of sample powder is precisely weighed, 50% acetonitrile water solution 25 and mL are added, the weighed weight is adopted, ultrasound is carried out for 30 min (power is 250W, frequency is 40 kHz, temperature is 30 ℃), cooling is carried out, the lost weight is complemented by extracting solvent, filtering is carried out, the subsequent filtrate 2 mL is diluted to 10 mL, and the diluted solution is filtered by a microporous filter membrane of 0.45 mu m, so as to prepare a sample solution;
(2) Taking the sample solution, respectively standing at room temperature for 0, 2, 4, 8, 12, 24 and h, taking 5 μl, and injecting into high performance liquid chromatograph, and recording the peak area of chebulbar acid chromatograph. The chromatographic conditions were the same as in test 1.1.
The results showed that the myrtillin acid peak area RSD was 0.84%. The test solution is shown to have good stability in 24 h at room temperature.
Test 1.5 repeatability test
The method comprises the following steps:
(1) Dissolving chebulbar 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. 0.1g of sample powder is precisely weighed, 50% acetonitrile water solution 25 mL is added, the weighed weight is adopted, ultrasound is carried out for 30 min (power is 250W, frequency is 40 kHz, temperature is 30 ℃), cooling is carried out, the lost weight is complemented by extracting solvent, filtering is carried out, the subsequent filtrate 2 mL is diluted to 10 mL, the diluted solution is filtered by a microporous filter membrane with the size of 0.45 mu m, the sample solution is prepared, and 6 parts of sample solution is prepared in parallel;
(3) Taking 5 mu L of each of the reference substance solution and 6 parts of the sample solution, injecting into a high performance liquid chromatograph, recording the chromatographic peak area of the myrobalan acid, and calculating the content. The chromatographic conditions were the same as in test 1.1.
The results showed that the average chebular acid content was 140 mg/g and the RSD was 2.6% in 6 samples. Indicating that the method is well reproducible.
Test 1.6 sample recovery test
The method comprises the following steps:
(1) Pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. Sample powder 0.05 g, precisely weighing, adding a reference substance which is 100% of chebular acid content in the sample, adding 50% acetonitrile water solution 25 mL, weighing, performing ultrasonic treatment for 30 min (power 250W, frequency 40 kHz, temperature 30 ℃), cooling to extract the solvent to complement the lost weight, filtering, diluting the subsequent filtrate 2 mL to 10 mL, filtering the diluent with a microporous filter membrane of 0.45 mu m to obtain a sample solution, and preparing 6 parts in parallel;
(2) Taking 6 parts of sample solution, 5 mu L of each sample solution, injecting into a high performance liquid chromatograph, recording the area of a chebulic acid peak, and calculating the content; the chromatographic conditions were the same as in test 1.1.
The results show that the average sample recovery rate of chebular acid is 100.02%, the RSD is 0.71%, and the results are shown in Table 1.
TABLE 1 sample recovery results
Example 2: a method for measuring the content of chebular acid in myrobalan by adopting high performance liquid chromatography comprises the following steps:
(1) Dissolving chebulbar 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. 0.1g of sample powder is precisely weighed, 50% acetonitrile water solution 25 and mL are added, the weighed weight is adopted, ultrasound is carried out for 30 min (power is 250W, frequency is 40 kHz, temperature is 30 ℃), cooling is carried out, the lost weight is complemented by extracting solvent, filtering is carried out, the subsequent filtrate 2 mL is diluted to 10 mL, and the diluted solution is filtered by a microporous filter membrane of 0.45 mu m, so as to prepare a sample solution;
(3) Taking 5 mu L of each of the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph, and recording each parameter of chebular acid. The chromatographic conditions are as follows: agilent 1100 high performance liquid chromatography system; chromatographic column: 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 is acetonitrile, elution gradient: 0 to 5 min, B is 2 to 5 percent; 5-10 min, wherein B is 5%; 10-12 min, B is 5% -15%; 12-30 min, and B is 15%; 30-35 min, 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 the content of chebular acid in myrobalan by adopting high performance liquid chromatography comprises the following steps:
(1) In the same manner as in (1) of example 2;
(2) Pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. 0.1g of sample powder is precisely weighed, 30% acetonitrile water solution 25 and mL are added, the weighed weight is adopted, ultrasound is carried out for 30 min (power is 250W, frequency is 40 kHz, temperature is 30 ℃), cooling is carried out, the lost weight is complemented by extracting solvent, filtering is carried out, the subsequent filtrate 2 mL is diluted to 10 mL, and the diluted solution is filtered by a microporous filter membrane of 0.45 mu m, so as to prepare a sample solution;
(3) Taking 5 mu L of each of the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph, and recording each parameter of chebular acid. The chromatographic conditions are as follows: agilent 1100 high performance liquid chromatography system; chromatographic column: 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 is acetonitrile, elution gradient: 0 to 5 min, B is 2 to 5 percent; 5-10 min, wherein B is 5%; 10-12 min, B is 5% -15%; 12-30 min, and B is 15%; 30-35 min, 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 the content of chebular acid in myrobalan by adopting high performance liquid chromatography comprises the following steps:
(1) In the same manner as in (1) of example 2;
(2) Pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. 0.1g of sample powder is precisely weighed, a 70% acetonitrile water solution 25 and mL are added, the weighed weight is adopted, ultrasound is carried out for 30 min (power is 250W, frequency is 40 kHz, temperature is 30 ℃), cooling is carried out, the lost weight is complemented by extracting solvent, filtering is carried out, the subsequent filtrate 2 mL is diluted to 10 mL, and the diluted solution is filtered by a microporous filter membrane of 0.45 mu m, so as to prepare a sample solution;
(3) Taking 5 mu L of each of the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph, and recording each parameter of chebular acid. The chromatographic conditions are as follows: agilent 1100 high performance liquid chromatography system; chromatographic column: 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 is acetonitrile, elution gradient: 0 to 5 min, B is 2 to 5 percent; 5-10 min, wherein B is 5%; 10-12 min, B is 5% -15%; 12-30 min, and B is 15%; 30-35 min, 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 the content of chebular acid in myrobalan by adopting high performance liquid chromatography comprises the following steps:
(1) In the same manner as in (1) of example 2;
(2) Pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. 0.1g of sample powder is precisely weighed, 50% acetonitrile water solution 25 and mL are added, the weighed weight is adopted, ultrasound is carried out for 30 min (power is 250W, frequency is 40 kHz, temperature is 30 ℃), cooling is carried out, the lost weight is complemented by extracting solvent, filtering is carried out, the subsequent filtrate 2 mL is diluted to 10 mL, and the diluted solution is filtered by a microporous filter membrane of 0.45 mu m, so as to prepare a sample solution;
(3) Taking 3 μl of each of the control solution and the sample solution, and injecting into high performance liquid chromatograph, and recording parameters of chebular acid. The chromatographic conditions are as follows: agilent 1100 high performance liquid chromatography system; chromatographic column: 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 is acetonitrile, elution gradient: 0 to 5 min, B is 2 to 5 percent; 5-10 min, wherein B is 5%; 10-12 min, B is 5% -15%; 12-30 min, and B is 15%; 30-35 min, 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 the content of chebular acid in myrobalan by adopting high performance liquid chromatography comprises the following steps:
(1) In the same manner as in (1) of example 2;
(2) Pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. 0.1g of sample powder is precisely weighed, 50% acetonitrile water solution 25 and mL are added, the weighed weight is adopted, ultrasound is carried out for 30 min (power is 250W, frequency is 40 kHz, temperature is 30 ℃), cooling is carried out, the lost weight is complemented by extracting solvent, filtering is carried out, the subsequent filtrate 2 mL is diluted to 10 mL, and the diluted solution is filtered by a microporous filter membrane of 0.45 mu m, so as to prepare a sample solution;
(3) Taking 7 mu L of each of the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph, and recording each parameter of chebular acid. The chromatographic conditions are as follows: agilent 1100 high performance liquid chromatography system; chromatographic column: 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 is acetonitrile, elution gradient: 0 to 5 min, B is 2 to 5 percent; 5-10 min, wherein B is 5%; 10-12 min, B is 5% -15%; 12-30 min, and B is 15%; 30-35 min, 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 the content of chebular acid in myrobalan by adopting high performance liquid chromatography comprises the following steps:
(1) In the same manner as in (1) of example 2;
(2) Pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. 0.1g of sample powder is precisely weighed, 50% acetonitrile water solution 25 and mL are added, the weighed weight is adopted, ultrasound is carried out for 30 min (power is 250W, frequency is 40 kHz, temperature is 30 ℃), cooling is carried out, the lost weight is complemented by extracting solvent, filtering is carried out, the subsequent filtrate 2 mL is diluted to 10 mL, and the diluted solution is filtered by a microporous filter membrane of 0.45 mu m, so as to prepare a sample solution;
(3) Taking 5 mu L of each of the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph, and recording each parameter of chebular acid. The chromatographic conditions are as follows: agilent 1100 high performance liquid chromatography system; chromatographic column: diamond Plus C18 column (250 mm X4.6 mm,5 μm); mobile phase: phase A is water-methanol-formic acid (95:5:0.1 v/v/v); phase B is acetonitrile, elution gradient: 0 to 5 min, B is 2 to 5 percent; 5-10 min, wherein B is 5%; 10-12 min, B is 5% -15%; 12-30 min, and B is 15%; 30-35 min, 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 the content of chebular acid in myrobalan by adopting high performance liquid chromatography comprises the following steps:
(1) In the same manner as in (1) of example 2;
(2) Pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. 0.1g of sample powder is precisely weighed, 50% acetonitrile water solution 25 and mL are added, the weighed weight is adopted, ultrasound is carried out for 30 min (power is 250W, frequency is 40 kHz, temperature is 30 ℃), cooling is carried out, the lost weight is complemented by extracting solvent, filtering is carried out, the subsequent filtrate 2 mL is diluted to 10 mL, and the diluted solution is filtered by a microporous filter membrane of 0.45 mu m, so as to prepare a sample solution;
(3) Taking 5 μl of each of the reference solution and the sample solution, and injecting into high performance liquid chromatograph, and recording chebulbar acid and various parameters. The chromatographic conditions are as follows: agilent 1100 high performance liquid chromatography system; chromatographic column: illite SinoChrom ODS-BP 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 is acetonitrile, elution gradient: 0 to 5 min, B is 2 to 5 percent; 5-10 min, wherein B is 5%; 10-12 min, B is 5% -15%; 12-30 min, and B is 15%; 30-35 min, 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 the content of chebular acid in myrobalan by adopting high performance liquid chromatography comprises the following steps:
(1) In the same manner as in (1) of example 2;
(2) Pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. 0.1g of sample powder is precisely weighed, 50% acetonitrile water solution 25 and mL are added, the weighed weight is adopted, ultrasound is carried out for 30 min (power is 250W, frequency is 40 kHz, temperature is 30 ℃), cooling is carried out, the lost weight is complemented by extracting solvent, filtering is carried out, the subsequent filtrate 2 mL is diluted to 10 mL, and the diluted solution is filtered by a microporous filter membrane of 0.45 mu m, so as to prepare a sample solution;
(3) Taking 5 mu L of each of the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph, and recording each parameter of chebular acid. The chromatographic conditions are as follows: agilent 1100 high performance liquid chromatography system; chromatographic column: dikma Platisil column (250 mm ×4.6 mm,5 μm); mobile phase: phase A is water-methanol-formic acid (90:10:0.2 v/v/v); phase B is acetonitrile, elution gradient: 0 to 5 min, B is 2 to 5 percent; 5-10 min, wherein B is 5%; 10-12 min, B is 5% -15%; 12-30 min, and B is 15%; 30-35 min, 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 the content of chebular acid in myrobalan by adopting high performance liquid chromatography comprises the following steps:
(1) In the same manner as in (1) of example 2;
(2) Pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. 0.1g of sample powder is precisely weighed, 50% acetonitrile water solution 25 and mL are added, the weighed weight is adopted, ultrasound is carried out for 30 min (power is 250W, frequency is 40 kHz, temperature is 30 ℃), cooling is carried out, the lost weight is complemented by extracting solvent, filtering is carried out, the subsequent filtrate 2 mL is diluted to 10 mL, and the diluted solution is filtered by a microporous filter membrane of 0.45 mu m, so as to prepare a sample solution;
(3) Taking 5 mu L of each of the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph, and recording each parameter of chebular acid. The chromatographic conditions are as follows: agilent 1100 high performance liquid chromatography system; chromatographic column: 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 is acetonitrile, elution gradient: 0 to 5 min, B is 2 to 5 percent; 5-10 min, wherein B is 5%; 10-12 min, B is 5% -15%; 12-30 min, and B is 15%; 30-35 min, 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 the content of chebular acid in myrobalan by adopting high performance liquid chromatography comprises the following steps:
(1) In the same manner as in (1) of example 2;
(2) Pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. 0.1g of sample powder is precisely weighed, 50% acetonitrile water solution 25 and mL are added, the weighed weight is adopted, ultrasound is carried out for 30 min (power is 250W, frequency is 40 kHz, temperature is 30 ℃), cooling is carried out, the lost weight is complemented by extracting solvent, filtering is carried out, the subsequent filtrate 2 mL is diluted to 10 mL, and the diluted solution is filtered by a microporous filter membrane of 0.45 mu m, so as to prepare a sample solution;
(3) Taking 5 mu L of each of the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph, and recording each parameter of chebular acid. The chromatographic conditions are as follows: agilent 1100 high performance liquid chromatography system; chromatographic column: 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 is acetonitrile, elution gradient: 0 to 5 min, B is 2 to 5 percent; 5-10 min, wherein B is 5%; 10-12 min, B is 5% -14%; 12-30 min, and B is 14%; 30-35 min, wherein B is 14% -17%; 35-50 min, wherein B is 17%; 50-55 min, 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 the content of chebular acid in myrobalan by adopting high performance liquid chromatography comprises the following steps:
(1) In the same manner as in (1) of example 2;
(2) Pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. 0.1g of sample powder is precisely weighed, 50% acetonitrile water solution 25 and mL are added, the weighed weight is adopted, ultrasound is carried out for 30 min (power is 250W, frequency is 40 kHz, temperature is 30 ℃), cooling is carried out, the lost weight is complemented by extracting solvent, filtering is carried out, the subsequent filtrate 2 mL is diluted to 10 mL, and the diluted solution is filtered by a microporous filter membrane of 0.45 mu m, so as to prepare a sample solution;
(3) Taking 5 mu L of each of the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph, and recording each parameter of chebular acid. The chromatographic conditions are as follows: an Shimadzu LC-16 high performance liquid chromatography system; chromatographic column: 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 is acetonitrile, elution gradient: 0 to 5 min, B is 2 to 5 percent; 5-10 min, wherein B is 5%; 10-12 min, B is 5% -15%; 12-30 min, and B is 15%; 30-35 min, 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 the content of chebular acid in myrobalan by adopting high performance liquid chromatography comprises the following steps:
(1) Adding the myrobalan acid reference substance into methanol solution for dissolution to obtain a mixed reference substance solution with the myrobalan acid of 0.068 mg/mL;
(2) Pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. 0.1g of sample powder is precisely weighed, a 70% methanol aqueous solution 25 and mL are added, the weight is weighed, ultrasound is carried out for 30 min (power is 250W, frequency is 40 kHz, temperature is 30 ℃), cooling is carried out, the lost weight is complemented by extracting solvent, filtering is carried out, the subsequent filtrate 2 mL is diluted to 10 mL, and the diluted solution is filtered by a microporous filter membrane of 0.45 mu m, so as to prepare a sample solution;
(3) Taking 5 mu L of each of the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph, and recording each parameter of chebular acid. The chromatographic conditions are as follows: agilent 1100 high performance liquid chromatography system; chromatographic column: dikma Platisil column (250 mm ×4.6 mm,5 μm); mobile phase: 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, B is 10% -25%; 15-25 min, wherein B is 25%; 25-30 min, B is 25% -30%; 30-50 min, B is 30% -45%; 50-55 min, 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 the content of chebular acid in myrobalan by adopting high performance liquid chromatography comprises the following steps:
(1) In the same manner as in (1) of example 2;
(2) Pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. 0.1g of sample powder is precisely weighed, a 70% methanol aqueous solution 25 and mL are added, the weight is weighed, ultrasound is carried out for 30 min (power is 250W, frequency is 40 kHz, temperature is 30 ℃), cooling is carried out, the lost weight is complemented by extracting solvent, filtering is carried out, the subsequent filtrate 2 mL is diluted to 10 mL, and the diluted solution is filtered by a microporous filter membrane of 0.45 mu m, so as to prepare a sample solution;
(3) Taking 5 mu L of each of the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph, and recording each parameter of chebular acid. The chromatographic conditions are as follows: agilent 1100 high performance liquid chromatography system; chromatographic column: 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 is acetonitrile, elution gradient: 0 to 5 min, B is 2 to 5 percent; 5-10 min, wherein B is 5%; 10-12 min, B is 5% -14%; 12-30 min, and B is 14%; 30-35 min, wherein B is 14% -17%; 35-50 min, wherein B is 17%; 50-55 min, 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 the content of chebular acid in myrobalan by adopting high performance liquid chromatography comprises the following steps:
(1) Same as comparative example 1 (1);
(2) Pulverizing fructus Chebulae pulp, and sieving with 65 mesh sieve. 0.1g of sample powder is precisely weighed, a 70% methanol aqueous solution 25 and mL are added, the weight is weighed, ultrasound is carried out for 30 min (power is 250W, frequency is 40 kHz, temperature is 30 ℃), cooling is carried out, the lost weight is complemented by extracting solvent, filtering is carried out, the subsequent filtrate 2 mL is diluted to 10 mL, and the diluted solution is filtered by a microporous filter membrane of 0.45 mu m, so as to prepare a sample solution;
(3) Taking 5 mu L of each of the reference substance solution and the sample solution, injecting into a high performance liquid chromatograph, and recording each parameter of chebular acid. The chromatographic conditions are as follows: agilent 1100 high performance liquid chromatography system; chromatographic column: 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 is acetonitrile, elution gradient: 0 to 5 min, B is 2 to 5 percent; 5-10 min, wherein B is 5%; 10-12 min, B is 5% -15%; 12-30 min, and B is 15%; 30-35 min, 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 the content of myrobalan acid by adopting high performance liquid chromatography comprises the following steps:
(1) Dissolving chebulbar acid reference substance in acetonitrile solution to obtain chebulbar acid reference substance solution with concentration of 0.078 mg/mL;
(2) Taking 5 μl of the reference solution, injecting into high performance liquid chromatograph, and recording parameters of chebulenic acid. The chromatographic conditions are as follows: an Shimadzu LC-16 high performance liquid chromatography system; chromatographic column: dikma Platisil column (250 mm ×4.6 mm,5 μm); mobile phase: phase A is water-methanol-formic acid (90:10:0.1 v/v/v); phase B is 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 the content of myrobalan acid by adopting high performance liquid chromatography comprises the following steps:
(1) Dissolving chebulbar acid reference substance in methanol solution to obtain chebulbar acid reference substance solution with concentration of 0.082, 0.082 mg/mL;
(2) Taking 5 μl of the reference solution, injecting into high performance liquid chromatograph, and recording parameters of chebulenic acid. The chromatographic conditions are as follows: an Shimadzu LC-16 high performance liquid chromatography system; chromatographic column: dikma Platisil column (250 mm ×4.6 mm,5 μm); mobile phase: phase A is water-methanol-formic acid (90:10:0.1 v/v/v); phase B is 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 durability range of the method of the present invention and the system applicability test results are shown by the above-described test results of examples 2 to 12 and comparative examples 1 to 5. However, the results show that the peak sequence of the chemical components in the myrobalan is inconsistent under the system with methanol and acetonitrile as mobile phases, and the chemical components cannot be directly compared, so that the stability of the myrobalan in methanol and acetonitrile is examined by using a myrobalan acid reference substance. The examination conditions and the results are shown in tables 2 to 4.
Table 2 durability test conditions
TABLE 3 examination conditions for stability of chebulic acid
Table 4 system applicability results
The measurement results of the above examples 2 to 12 chebulic acid show that the sample peak and the adjacent peak of the content measurement method can be completely separated, the retention time, the theoretical plate number, the tailing factor and the separation degree are all in accordance with the requirements, the tailing factor and the separation degree of the measurement method of comparative example 1 are not in accordance with the requirements, the peak area is larger in comparison with the examples, the measurement results are inaccurate, and the comparison of comparative examples 1 to 3 and example 2 shows that the influence of the selection of methanol as a solvent on the chebulic acid content measurement is larger when the sample solution is prepared, and the selection of methanol as a mobile phase also has a certain influence.
The foregoing description of the preferred embodiments of the present application is not intended to limit the invention to the particular embodiments of the present application, but to limit the scope of the invention to the particular embodiments of the present application.
Claims (4)
1. A method for extracting chebular acid from chebula fruit flesh, 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, ultrasonic extracting, and collecting extractive solution at 3000 r min -1 Centrifuging at a rotating speed of (1) for 10 min, collecting supernatant, and recovering ethanol under reduced pressure;
(2) Diluting the residue with water, extracting with water-equivalent ethyl acetate for several times, mixing ethyl acetate solutions, recovering ethyl acetate under reduced pressure, and redissolving the residue with a certain amount of methanol water, 10000 r min -1 Centrifuging at the rotating speed for 10 min;
(3) Separating supernatant obtained by centrifuging with ODS open column: the diameter-to-height ratio of the ODS column packing is 1:5-1:0.8; the sample loading amount is 1-2 mL per 10 g ODS; the eluting solvent is 20% methanol water solution, and from the 4 th column volume, the eluting solution of 16 column volumes is collected, the solvent is recovered under reduced pressure, and the solution is placed at 4 ℃ until white solid is separated out, thus obtaining the chebulbar acid.
2. The method for extracting chebular acid from chebula fruit flesh according to claim 1, wherein the chebula fruit flesh in the step (1) is crushed, sieved by a sieve of 65-120 meshes, and added 70% ethanol water solution is 20-30 times of the medicinal material amount, and the ultrasonic extraction is carried out for 2-3 times for 10-30 min each time.
3. The method for extracting chebular acid from chebula fruit flesh according to claim 1, wherein in the step (2), the dilution water addition amount is 2-4 times of the initial weighed medicinal material amount, and the extraction times are 6-12 times; the compound solution is a 20% methanol aqueous solution, the dosage is 1:1 with the crude drug, and the concentration of the prepared sample solution is 1.0 g/mL.
4. The method for extracting chebular acid from chebula fruit flesh according to claim 1, wherein the chebular acid extracted by the method is used as a standard or a reference substance in the quality control of chebula fruit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210105897.9A CN114414702B (en) | 2022-01-28 | 2022-01-28 | Preparation method and content measurement method of chebular acid in chebula fruit flesh |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210105897.9A CN114414702B (en) | 2022-01-28 | 2022-01-28 | Preparation method and content measurement method of chebular acid in chebula fruit flesh |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114414702A CN114414702A (en) | 2022-04-29 |
| CN114414702B true CN114414702B (en) | 2023-06-13 |
Family
ID=81279972
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210105897.9A Active CN114414702B (en) | 2022-01-28 | 2022-01-28 | Preparation method and content measurement method of chebular acid in chebula fruit flesh |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114414702B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117646004B (en) * | 2024-01-29 | 2024-04-05 | 中国医学科学院药用植物研究所 | Label-free biosensor for myrobalan acid aptamer |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021073175A1 (en) * | 2019-10-16 | 2021-04-22 | 石家庄以岭药业股份有限公司 | Method for identifying various ingredients in traditional chinese medicine composition and measuring contents |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07138165A (en) * | 1993-09-24 | 1995-05-30 | Shionogi & Co Ltd | Anticancer agent |
| JP2006335728A (en) * | 2005-06-03 | 2006-12-14 | Taiyo Kagaku Co Ltd | Blood coagulation-suppressing composition |
| JP4986413B2 (en) * | 2005-06-03 | 2012-07-25 | 太陽化学株式会社 | Composition for improving blood circulation |
| CN101428043B (en) * | 2007-11-06 | 2012-04-18 | 天津天士力制药股份有限公司 | Effective component of chebula fruit, preparation method and use thereof |
| CN101391961A (en) * | 2008-10-28 | 2009-03-25 | 广西中医学院 | Phyllanthus emblica leaf or fruit anticancer Chinese medicine and preparation method and use thereof |
| US20200221740A1 (en) * | 2017-02-08 | 2020-07-16 | Rising Phoenix Industries Pty Ltd | Terminalia ferdinandiana leaf extract and products containing extract of terminalia ferdinandiana leaf |
| CN108467417B (en) * | 2018-04-23 | 2021-07-06 | 中国科学院成都生物研究所 | Fructus chebulae immaturus extract, fructus chebulae immaturus extract monomer compound and application of fructus chebulae immaturus extract monomer compound in inhibition of monoamine oxidase B activity |
| CN110794064A (en) * | 2019-11-23 | 2020-02-14 | 北京中医药大学 | One-test-multiple-evaluation content detection method for nine components of emblic leafflower fruit medicinal material and tannin part |
| CN110772550B (en) * | 2019-12-06 | 2021-12-14 | 辽宁中医药大学 | Method for preparing ellagic acid from scalded myrobalan meat and application |
| CN111272904B (en) * | 2020-03-23 | 2022-04-05 | 广东一方制药有限公司 | Construction method and application of characteristic spectrum of medicine terminalia chebula and fructus chebulae tomentosa |
-
2022
- 2022-01-28 CN CN202210105897.9A patent/CN114414702B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021073175A1 (en) * | 2019-10-16 | 2021-04-22 | 石家庄以岭药业股份有限公司 | Method for identifying various ingredients in traditional chinese medicine composition and measuring contents |
Non-Patent Citations (2)
| Title |
|---|
| RP-HPLC法测定余甘子中诃黎勒酸、没食子酸、粘酸-2-O-没食子酸酯的含量;沙磊;张兰珍;徐义侠;郭亚健;;药物分析杂志(02);293-295\n * |
| 诃子及其易混淆品中二种丹宁成分的高效毛细管电泳分析;丁岗,陆蕴如,冀春茹,刘延泽;药学学报(04);292-295\n * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114414702A (en) | 2022-04-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101891750A (en) | Preparation method of stephanine and hydrochloride thereof | |
| CN114414702B (en) | Preparation method and content measurement method of chebular acid in chebula fruit flesh | |
| Wu et al. | Determination of caffeoylquinic acid derivatives in Azolla imbricata by chitosan-based matrix solid-phase dispersion coupled with HPLC–PDA | |
| CN114414701B (en) | Preparation method of myrobalan acid in myrobalan meat and content determination method thereof | |
| CN1709900A (en) | Comprehensive extracting mehtod for balsam pear saponin and balsam pear polysaccharide | |
| CN1919256A (en) | Scutellaria root extract quality standard and content measuring method thereof | |
| CN108840845A (en) | The method of Xanthatin is extracted from Siberian cocklebur | |
| CN101961405A (en) | Method for testing content of pinoresinol diglucoside in compound eucommia bark tablet | |
| CN111747914B (en) | Compound separated from ginkgo root bark and application thereof | |
| CN115368359A (en) | Alkaloid antioxidant and preparation method and application thereof | |
| CN109917045B (en) | HPLC method for simultaneously measuring contents of 5 components in prepared rhizoma cibotii decoction pieces | |
| CN108218935B (en) | Method for extracting myricitrin, quercitrin and afzelin from Chinese redbud leaves by using ionic liquid | |
| CN107064334A (en) | The method of quality control of anthracene shellfish element | |
| CN102584779B (en) | Preparation method for biphenyl cyclooctene lignin compound | |
| CN109142571B (en) | Method for measuring content of anthraquinone components | |
| CN111346195A (en) | Preparation method of tsaoko total flavonoids | |
| LU502858B1 (en) | Alkaloid antioxidant, preparation method and application thereof | |
| CN103923138A (en) | Preparation method and application of nicotiflorin | |
| CN111175416A (en) | Method for simultaneously detecting 7 components in dogwood | |
| CN114605423B (en) | Method for preparing alkaloids in gelsemium elegans | |
| CN113461747B (en) | 2 compounds with hypoglycemic activity in rose hip | |
| CN111363000B (en) | Method for separating and preparing two trace components from sodium aescinate | |
| LU501405B1 (en) | Method for preparing acacetin-7-o-glucuronide | |
| CN109498686B (en) | Preparation method of triterpenic acid in jujube material | |
| CN113521803A (en) | Eutectic solvent, preparation method thereof and application of eutectic solvent in extraction of isofraxidin in acanthopanax senticosus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |