Method for determining content of trans-emodin dianthrone in polygonum multiflorum

Abstract

The invention belongs to the technical field of traditional Chinese medicine component detection methods, and particularly relates to a method for determining the content of trans-emodin dianthrone in polygonum multiflorum, which comprises the following steps: taking polygonum multiflorum powder, adding a mixture of water and cellulase in a mass ratio of 100: (1-2), carrying out ultrasonic treatment, then adding absolute ethyl alcohol, carrying out ultrasonic treatment, and filtering to obtain filtrate; the method comprises the following steps: extracting components including trans-emodin dianthrone, filtering, taking filtrate, evaporating to dryness, adding an acid solution containing a sulfur element for dissolving, carrying out water bath at 55-65 DEG C, placing to room temperature, adding ethyl acetate for extraction so as to remove impurity components remaining on an acid solution layer, improve the purity of trans-emodin dianthrone and facilitate liquid chromatography separation, adding methanol for dissolving, and purifying to obtain the trans-emodin dianthrone. Obtaining a test solution; and detecting the content of trans-emodin dianthrone in the test solution by liquid chromatography. The method is high in specificity and good in reproducibility, stability and precision, and the content of trans-emodin dianthrone in polygonum multiflorum can be effectively and accurately detected.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine component detection methods, and specifically relates to a method for determining the content of trans-rhein dianthrone in Polygonum multiflorum. Background Art

[0002] Polygonum multiflorum is the dried root of Polygonum multiflorum Thunb, a plant of the Polygonaceae family. It is dug up in autumn and winter when the leaves wither, the two ends are cut off, washed, and the large ones are cut into pieces, dried, with a slight smell and a slightly bitter and sweet taste. It has the effects of detoxification, eliminating carbuncle, stopping malaria, and moistening the intestines and relieving constipation. It is used for carbuncle, scrofula, urticaria itching, long-term malaria physical weakness, dry intestines and constipation. Modern research shows that Polygonum multiflorum contains a variety of anthrone and anthraquinone compounds, and these compounds have certain hepatotoxicity to the human body, but the pharmacopoeia stipulates the content determination method of diphenylethylene glycosides and combined anthraquinones, but does not stipulate the detection of anthrone.

[0003] In recent years, the hepatotoxicity of Polygonum multiflorum has attracted wide attention at home and abroad. Trans-rhizin dianthrone has a CAS number of 61281-20-7 and is one of the components in Polygonum multiflorum that cause hepatotoxicity. Therefore, it is of great significance to determine the content of trans-rhizin dianthrone in Polygonum multiflorum. Although there are detection methods for anthrone substances in the prior art, such as CN112964819A and CN111272908A, they detect a large class of substances and can only reflect the content of a large class of substances. They are mixtures and cannot clearly determine the content of a specific substance, such as trans-rhizin dianthrone. In addition, although the existing technologies such as CN115032310A can clarify the content of some specific compounds, the substances detected are six dianthrone compounds in Polygonum multiflorum, namely, trans-rhizin-rhizin dianthrone, cis-rhizin-rhizin dianthrone, trans-rhizin-rhizin methyl ether dianthrone, cis-rhizin-rhizin methyl ether dianthrone, trans-rhizin methyl ether-rhizin methyl ether dianthrone and cis-rhizin methyl ether-rhizin methyl ether dianthrone, and the content of the specific substance trans-rhizin dianthrone is still not clarified.

[0004] In summary, the prior art lacks a method for determining the content of a specific substance, trans-rhein dianthrone, in Polygonum multiflorum. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for determining the content of trans-rhein dianthrone in Polygonum multiflorum.

[0006] The invention aims to provide a method for determining the content of trans-rheum dianthrone in Polygonum multiflorum. The method comprises the following steps: taking Polygonum multiflorum powder, adding a mixture of water and cellulase in a mass ratio of 100:1-2, ultrasonically treating the mixture to degrade cellulose, then adding anhydrous ethanol, ultrasonically treating the mixture to change the conformation and charge distribution of the cellulase, extracting components including trans-rheum dianthrone, filtering the mixture to remove Polygonum multiflorum residue, taking a filtrate and evaporating it to dryness, adding an acid solution containing sulfur element to dissolve the mixture, bathing the mixture in a water bath at 55-65°C to remove impurities, and increasing the content of trans-rheum dianthrone in a test sample, cooling the mixture to room temperature, and extracting the mixture with ethyl acetate, wherein the cellulase is insoluble in ethyl acetate, and the extraction with ethyl acetate can remove impurity components remaining in the acid solution layer, thereby increasing the purity of trans-rheum dianthrone and facilitating liquid chromatography separation, and dissolving the mixture with methanol to obtain a test sample solution.

[0007] The content of trans-rhaponticin dianthrone in the test solution was determined by liquid chromatography.

[0008] In the present invention, the extraction reagent is composed of anhydrous ethanol, water and cellulase. Note that the three are not directly mixed, but firstly a mixture of water and cellulase in a mass ratio of 100:1-2 is added to the Polygonum multiflorum powder. After ultrasonic treatment, the Polygonum multiflorum is enzymolyzed and the structure is loosened; after adding ethanol and ultrasonic treatment, the conformation and charge distribution of the cellulase are changed, and the dissolution of trans-rheicin dianthrone is promoted; finally, components including trans-rheicin dianthrone are extracted. On the one hand, the cellulase degrades the cellulose in the Polygonum multiflorum and loosens its structure, and on the other hand, it cooperates with ethanol to induce the dissolution of trans-rheicin dianthrone.

[0009] According to the control experiment of the present invention, cellulase is directly used, or a combination of cellulase and methanol is used, which shows that cellulase does not promote dissolution, indicating that degradation of cellulose is not a key condition for promoting dissolution. In the present invention, cellulase degrades cellulose in Polygonum multiflorum to loosen its structure, and the cellulase structure cooperates with the polarity of ethanol to induce the dissolution of trans-rheicin dianthrone, which is the key condition for promoting the dissolution of trans-rheicin dianthrone.

[0010] The extraction reagent (composed of anhydrous ethanol, water and cellulase) is combined with an acid solution containing sulfur element, a water bath at 55°C to 65°C, ethyl acetate extraction, methanol dissolution and the like. When the obtained test solution is subjected to liquid chromatography detection, the complete trans-rheic acid dianthrone can be well separated, and the extracted trans-rheic acid dianthrone has a high content, a high recovery rate and good stability.

[0011] Preferably, in the above method for determining the content of trans-rhaponticin dianthrone in Polygonum multiflorum, the enzymatic activity of cellulase is ≥50000 U / g.

[0012] Preferably, in the above method for determining the content of trans-rhodiola rosea dianthrone in Polygonum multiflorum, the amount of anhydrous ethanol added is equivalent to 90% to 100% of the mass of water.

[0013] Preferably, in the above method for determining the content of trans-rhein dianthrone in Polygonum multiflorum, the solid-liquid ratio of Polygonum multiflorum powder to water is 1kg:25L-35L.

[0014] Preferably, in the above method for determining the content of trans-rhein dianthrone in Polygonum multiflorum, the ultrasonic treatment conditions are: power 100 W, frequency 40 kHz, and ultrasonic treatment for 30 min to 40 min.

[0015] Preferably, in the above method for determining the content of trans-rhein dianthrone in Polygonum multiflorum, the sulfur-containing acid solution is a 15% to 20% by volume sulfuric acid solution, a 15% to 20% by volume pyrosulfuric acid solution, or a mixture of the two.

[0016] Preferably, in the above method for determining the content of trans-rhein dianthrone in Polygonum multiflorum, the water bath time is 30 min to 50 min.

[0017] Preferably, the method for determining the content of trans-rhizin dianthrone in Polygonum multiflorum is that the chromatographic column used in the liquid chromatography is an Agilent C-type HPLC column with octadecylsilane bonded silica gel as a filler. 18 .

[0018] Preferably, the method for determining the content of trans-rhein dianthrone in Polygonum multiflorum is to use acetonitrile as mobile phase A and 0.10% to 0.15% by volume aqueous phosphoric acid solution as mobile phase B for gradient elution.

[0019] Preferably, in the above-mentioned method for determining the content of trans-rhexin dianthrone in Polygonum multiflorum, in the 0th to 10th minute of gradient elution, the volume ratio of mobile phase A and mobile phase B is 50%; in the 10th to 30th minute, the volume ratio of mobile phase A increases from 50% to 80%, and the volume of mobile phase B decreases from 50% to 20%.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The method for determining the content of trans-rheum dianthrone in Polygonum multiflorum comprises the following steps: taking Polygonum multiflorum powder, adding a mixture of water and cellulase in a mass ratio of 100:1-2, performing ultrasonic treatment to degrade cellulose, then adding anhydrous ethanol, performing ultrasonic treatment to change the conformation and charge distribution of the cellulase, extracting components including trans-rheum dianthrone, filtering to remove Polygonum multiflorum residue, taking filtrate and evaporating to dryness, adding an acid solution containing sulfur element to dissolve, bathing in water at 55 DEG C-65 DEG C to increase the content of trans-rheum dianthrone, cooling to room temperature, adding ethyl acetate to extract to remove impurity components remaining in the acid solution layer, adding methanol to dissolve, and obtaining a test solution; and detecting the content of trans-rheum dianthrone in the test solution by liquid chromatography. This method specifically detects the content of trans-rhein dianthrone in Polygonum multiflorum, has strong specificity, good reproducibility, stability and precision, and can effectively and accurately detect the content of trans-rhein dianthrone in Polygonum multiflorum, control the quality of Polygonum multiflorum from the source, and make the quality of Polygonum multiflorum stable, controllable and safe. It is a feed requirement and quality control for the product, overcomes the shortcomings of the existing technology, and helps to improve the quality of Polygonum multiflorum products. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the chromatogram of the trans-rhapontin dianthrone reference substance.

[0023] Figure 2 This is the chromatogram of methanol solvent.

[0024] Figure 3 The chromatogram of the test solution of Example 1.

[0025] Figure 4 This is the standard curve for effect verification 1. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0027] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0028] 1. Instruments and reagents

[0029] 1.1 Instrument

[0030] Agilent 1260 high performance liquid chromatograph (1260VWD detector, LC 1260 chromatography workstation, Agilent).

[0031] DV215CD electronic analytical balance (Ohaus).

[0032] SB-120D ultrasonic cleaner (Ningbo Xinzhi).

[0033] DXY-100 constant temperature water bath (Shenzhen Dingxinyi Experimental Equipment Co., Ltd.).

[0034] 1.2 Trial drug:

[0035] The reference substance of trans-rhamnolide dianthrone (batch number 23103003) was commissioned to Chengdu Gelipu Biotechnology Co., Ltd. for production.

[0036] Acetonitrile was chromatographically pure (Anhui Tiandi High Purity Solvent Co., Ltd.).

[0037] Formic acid (Chengdu Jinshan Chemical Reagent Co., Ltd.).

[0038] Phosphoric acid (Chengdu Jinshan Chemical Reagent Co., Ltd.).

[0039] Anhydrous ethanol (Tianjin Fuyu Fine Chemical Co., Ltd.).

[0040] The water is deionized water.

[0041] Methanol (Tianjin Fuyu Fine Chemical Co., Ltd.).

[0042] Ethyl acetate (Tianjin Fuyu Fine Chemical Co., Ltd.).

[0043] Sulfuric acid (Chengdu Jinshan Chemical Reagent Co., Ltd.).

[0044] Cellulase (source leaf bio, enzyme activity 50000U / g).

[0045] Examples of the present invention are as follows.

[0046] Example 1

[0047] A method for determining the content of trans-rhein dianthrone in Polygonum multiflorum comprises the following steps:

[0048] (1) Preparation of reference solution: Take 20.00 mg of the reference substance of trans-rhaponticin dianthrone, accurately weigh it, place it in a 100 mL volumetric flask, add 0.5% by volume methanol solution to dissolve it and dilute it to the scale, shake well, measure 10 mL, and dilute it to 50 mL with methanol to obtain the reference solution.

[0049] Preparation of the test solution: take 2.00g of Polygonum multiflorum powder that has passed through a No. 4 sieve, accurately weigh it, put it in a stoppered conical flask, add 50mL of a mixture of water and cellulase in a mass ratio of 100:1, and ultrasonically treat it for 30min, wherein the conditions for the ultrasonic treatment are power 100W and frequency 40kHz; then add 50mL of anhydrous ethanol, and ultrasonically treat it for 30min, wherein the conditions for the ultrasonic treatment are power 100W and frequency 40kHz; take it out, bring it to room temperature, shake it well, filter it with rapid quantitative filter paper to remove Polygonum multiflorum residue, take the filtrate and evaporate it to dryness, add 20mL of dilute sulfuric acid solution with a volume fraction of 20%, dissolve it, bath it in a water bath at 60℃ for 30min, bring it to room temperature, add ethyl acetate with an equal volume of the dilute sulfuric acid solution to extract it 3 times, combine all ethyl acetate layers, concentrate and evaporate to dryness, add methanol to make the volume 5mL, shake it well, filter it with rapid quantitative filter paper, and accurately pipette 10μL to obtain the test solution.

[0050] (2) Accurately pipette 10 μL of the reference solution and the test solution respectively and inject them into the liquid chromatograph for determination.

[0051] The chromatographic conditions of the liquid chromatograph were as follows: octadecylsilane bonded silica gel was used as the filler; Agilent C 18 (4.6mm×150mm, 5μm); acetonitrile was used as mobile phase A, 0.1% by volume phosphoric acid aqueous solution was used as mobile phase B, and volume ratio gradient elution was performed according to the provisions in Table 1; the detection wavelength was 280nm. Figure 1 This is the chromatogram of the trans-rhodiola rosea dianthrone reference substance. Figure 2 is the chromatogram of methanol solvent, Figure 3 This is the chromatogram of the test solution of Example 1, focusing on the peak position of trans-rhaponticin dianthrone after 15 minutes (20.257 minutes).

[0052] Table 1 Mobile phase volume ratio of gradient elution

[0053] Time (min) Mobile phase A (%) Mobile phase B (%) 0～10 50 unchanged 50 unchanged 10～30 From 50 to 80 From 50 to 20

[0054] In order to prove the stability and accuracy of the method of the present invention, the following effect verification was carried out.

[0055] Effect verification 1

[0056] Investigation of linear relationship: 4 μL, 8 μL, 12 μL, 16 μL, 20 μL, and 24 μL of the reference solution of Example 1 were accurately aspirated and injected into the high performance liquid chromatograph for determination. A linear regression was performed with the injection volume as the abscissa and the peak area as the ordinate, and the regression equation of trans-rhein dianthrone was obtained as Y=1842.7X+0.9333, R 2 =0.9999, see Figure 4The results showed that trans-rhodamine dianthrone had a good linear relationship in the range of 0.16 μg to 0.96 μg. The results are shown in Table 2.

[0057] Table 2 Linear range of trans-rhodamine dianthrone

[0058] Serial number Injection volume μL Injection volume μg Peak area 1 4 0.16 295 2 8 0.32 592 3 12 0.48 880 4 16 0.64 1186 5 20 0.80 1478 6 24 0.96 1766

[0059] Effect Verification 2

[0060] Precision test: Take the reference solution, inject 10 μL, inject 10 times continuously, and record the peak area integral value. The RSD (relative standard deviation) of the trans-rhodamine dianthrone reference concentration was 0.50%, indicating that the method has good precision. The results are shown in Table 3.

[0061] Table 3 Trans-rhubarb dianthrone precision determination results

[0062] Injection times Trans-rhodamine dianthrone peak area 1 744 2 735 3 733 4 740 5 735 6 732 7 736 8 738 9 735 10 733 average value 736 RSD / % 0.50

[0063] Effect Verification 3

[0064] Repeatability test: Take 10 portions of the same batch of Polygonum multiflorum powder, and repeat the preparation of samples according to the method of Example 1, and inject 10 μL of each sample, measure the peak area, and calculate the content of trans-rhizin dianthrone in Polygonum multiflorum. The RSD of the content of trans-rhizin dianthrone is 0.60%, indicating that the method has good repeatability. The results are shown in Table 4.

[0065] Table 4 Repeatability determination results of trans-rhodamine dianthrone

[0066]

[0067] Note: “ / ” in Table 4 means there is no such item.

[0068] Effect verification 4

[0069] Stability test: The test solution of Example 1 was selected, and the injection volume was 10 μL. The peak area of ​​trans-rhizoma dianthrone was measured at 0h, 2h, 4h, 6h, 12h, 18h, 24h, 30h, 36h, and 42h after preparation. The RSD of the trans-rhizoma dianthrone area was 0.27%, indicating that the test solution remained stable within 42h. The results are shown in Table 5.

[0070] Table 5 Repeatability determination results of trans-rhodiola rosea dianthrone

[0071] serial number Time Trans-rhodamine dianthrone peak area 1 0 287 2 2 288 3 4 287 4 6 288 5 12 287 6 18 288 7 24 286 8 30 288 9 36 287 10 42 286 Average peak area / 287 RSD% / 0.27

[0072] Note: “ / ” in Table 5 means there is no such item.

[0073] Effect verification 5

[0074] Spike recovery test

[0075] (5.1) Preparation of reference solution: Accurately weigh 0.0112 g of trans-rhodamine dianthrone reference substance into a 100 mL volumetric flask, add methanol to dissolve and make up to volume, shake well and set aside.

[0076] (5.2) Preparation of test solution: About 1.000 g of Polygonum multiflorum powder with known content was accurately weighed, and a total of 10 portions were mixed with 1 mL of the reference solution of (5.1) respectively. The test solution was prepared according to the method of Example 1, and the content of trans-rhizoma dianthrone was determined. The recovery rate was calculated. The average recovery rate of trans-rhizoma dianthrone was 99.60%, and the RSD was 0.20%, indicating that the method had good accuracy. The results are shown in Table 6.

[0077] Table 6 Trans-rhodiola rosea dianthrone sample recovery test

[0078]

[0079] Effect verification 6

[0080] Ten batches of Radix Polygoni Multiflori with different batch numbers were purchased, and the content of trans-rhein dianthrone in Radix Polygoni Multiflori was measured according to the measurement conditions in Example 1. The results are shown in Table 7.

[0081] Table 7 Detection results of trans-rhizin dianthrone content in Polygonum multiflorum

[0082] batch number Trans-rhodamine dianthrone content, g / 00g 231102 0.00385 231105 0.00393 231206 0.00396 231209 0.00397 240103 0.00389 240306 0.00315 240307 0.00311 240309 0.00345 240401 0.00312 240405 0.00366

[0083] Due to the different origins of Polygonum multiflorum, the content of trans-rhexin dianthrone in its Polygonum multiflorum varies to a certain extent.

[0084] In order to demonstrate the advantages of the method of the present invention, the following control experiments were performed.

[0085] Control experiment 1

[0086] This experiment is about the influence of different mobile phases and different gradient elution of the same mobile phase on the peak of trans-rhexin dianthrone and the test results. Except for the differences of the following control 1 to control 5, the rest of the operations are the same as in Example 1.

[0087] Control 1: The mobile phase was acetonitrile-water-glacial acetic acid solution, and the volume ratio of acetonitrile, water, and glacial acetic acid was 60:40:1.

[0088] Control 2: The mobile phase was methanol-water solution, and the volume ratio of methanol to water was 80:20.

[0089] Control 3: acetonitrile (mobile phase A), 0.1% by volume formic acid aqueous solution (mobile phase B), gradient elution according to the mobile phase volume ratio in Table 1.

[0090] Control 4: acetonitrile (mobile phase A), 0.1% by volume phosphoric acid aqueous solution (mobile phase B), gradient elution according to the mobile phase volume ratio in Table 1.

[0091] Control 5: acetonitrile (mobile phase A), 0.1% by volume phosphoric acid aqueous solution (mobile phase B), from 0 min to 30 min, the volume ratio of mobile phase A increased from 50% to 80%, and the volume ratio of mobile phase B decreased from 50% to 20%.

[0092] The results showed that when eluting with control 1 and control 2, the target peaks could not be completely separated and the peak shape was poor, and tailing peaks and wrapped peaks appeared, indicating that these eluents and elution methods were ineffective. After comparing the different gradient elution programs of different mobile phases of control 3, control 4 and control 5, the results showed that the mobile phase acetonitrile-0.1 phosphoric acid aqueous solution (control 4), gradient elution (0-10min, 50% A, 10-30min, 50%-80% A) had a suitable peak elution time, relatively good chromatographic peak shape, and good separation, while the target peaks of control 3 and control 5 could not be completely separated and the peak shape was poor, and wrapped peaks appeared. The above results show that both the mobile phase and the elution conditions have a great influence on the peak of trans-rheicin dianthrone. Even the control 5, which is very close to the control 4, still cannot successfully separate the peak of trans-rheicin dianthrone, resulting in the inability to quantitatively detect trans-rheicin dianthrone. It can be seen that the peak of trans-rheicin dianthrone is unpredictable due to the different mobile phases and elution procedures, and there is no regularity to follow. The present invention finds for the first time that the conditions of control 4 can accurately and completely separate the peak of trans-rheicin dianthrone, which is convenient for accurate quantitative detection.

[0093] Control experiment 2

[0094] This control experiment is to explore the influence of different chromatographic columns on the peak of trans-rhaponticin dianthrone and the test results. Except for the difference in the chromatographic columns set in Table 8, the rest of the operations are the same as in Example 1.

[0095] Table 8 Inspection table of different chromatographic columns

[0096]

[0097] The results in Table 8 show that Agilent C 18 The peak quality of the chromatographic column is the best, and other types of chromatographic columns cannot completely separate the peak of trans-rhubarb dianthrone. The prior art usually optimizes the peak quality by adjusting the chromatographic column material, but the present application selects C 18 The results show that although the same C 18However, when the manufacturer's model is different, the peak quality of trans-rheic acid dianthrone is also significantly different. It can be seen that the peak of trans-rheic acid dianthrone is unpredictable due to different chromatographic columns, and there is no regularity to follow. The present invention first discovered that Agilent C 18 The high-quality peak of trans-rhaponticin dianthrone can be separated accurately and completely.

[0098] Control experiment 3

[0099] This control experiment is based on the method of Example 1 to explore the effects of different detection wavelengths on the peak of trans-rhaponticin dianthrone and the test results.

[0100] Preparation of reference solution: Take 20.00 mg of trans-rhaponticin dianthrone reference substance, accurately weigh it, place it in a 100 mL volumetric flask, add 0.5% by volume methanol solution to dissolve and dilute to the scale, shake well, measure 10 mL, and dilute to 50 mL with methanol to obtain the reference solution.

[0101] Using methanol as blank reagent, full wavelength scanning was performed on a UV spectrophotometer. The results showed that the reference solution had a maximum absorption peak at 280nm, so the detection wavelength could be set at 280nm.

[0102] Control experiment 4

[0103] This control experiment compares the effects of different extraction methods on the content determination results of trans-rhamnolide dianthrone. Except for the differences in the following extraction methods 1 to 10, the rest of the operations are the same as in Example 1.

[0104] Extraction method 1: Take 2.00g of Polygonum multiflorum powder that has passed through a No. 4 sieve, weigh it accurately, put it in a stoppered conical flask, add 50mL of a mixture of water and cellulase in a mass ratio of 100:1, and ultrasonically treat it for 30 minutes, wherein the conditions for ultrasonic treatment are power 100W and frequency 40kHz; then add 50mL of anhydrous ethanol, and ultrasonically treat it for 30 minutes, wherein the conditions for ultrasonic treatment are power 100W and frequency 40kHz; take it out, put it to room temperature, shake it well, filter it with rapid quantitative filter paper to remove Polygonum multiflorum residue, take the filtrate and evaporate it to dryness, add 20mL of dilute sulfuric acid solution with a volume fraction of 20%, dissolve it, bath it in a water bath at 60℃ for 30min, put it to room temperature, add ethyl acetate with an equal volume of the dilute sulfuric acid solution to extract it 3 times, combine all ethyl acetate layers, concentrate and evaporate to dryness, add methanol to make the volume to 5mL, shake it well, filter it, and accurately pipette 10μL to obtain the test solution.

[0105] Extraction method 2: Take 2.00g of Polygonum multiflorum powder that has passed through a No. 4 sieve, weigh it accurately, put it in a stoppered conical flask, add 50mL of a mixture of water and cellulase in a mass ratio of 100:1, and heat to reflux at 50℃ for 1.5h; then add 50mL of anhydrous ethanol, heat to reflux at 50℃ for 1.5h; take it out, bring it to room temperature, shake it well, filter it with rapid quantitative filter paper to remove the Polygonum multiflorum residue, take the filtrate and evaporate it to dryness, add 20mL of a 20% volume fraction dilute sulfuric acid solution, dissolve it, bath it in a 60℃ water bath for 30min, bring it to room temperature, add ethyl acetate with an equal volume of the dilute sulfuric acid solution to extract it 3 times, combine all ethyl acetate layers, concentrate and evaporate to dryness, add methanol to make the volume to 5mL, shake it well, filter it, and accurately pipette 10μL to obtain the test solution.

[0106] Extraction method 3: Take 2.00g of Polygonum multiflorum powder that has passed through a No. 4 sieve, weigh it accurately, put it in a stoppered conical flask, add 50mL of a mixture of water and cellulase in a mass ratio of 100:1, and let it stand and soak for 24 hours; then add 50mL of anhydrous ethanol, let it stand and soak for 24 hours; take it out, bring it to room temperature, shake it well, filter it with rapid quantitative filter paper to remove the Polygonum multiflorum residue, take the filtrate and evaporate it to dryness, add 20mL of a 20% volume fraction dilute sulfuric acid solution, dissolve it, bath it in a 60℃ water bath for 30min, bring it to room temperature, add ethyl acetate with an equal volume of the dilute sulfuric acid solution to extract 3 times, combine all ethyl acetate layers, concentrate and evaporate to dryness, add methanol to make the volume to 5mL, shake it well, filter it, and accurately pipette 10μL to obtain the test solution.

[0107] Extraction method 4: Take 2.00g of Polygonum multiflorum powder that has passed through a No. 4 sieve, weigh it accurately, put it in a stoppered conical bottle, add 50mL of water, and ultrasonically treat it for 30min, wherein the conditions for ultrasonic treatment are power 100W and frequency 40kHz; then add 50mL of anhydrous ethanol, and ultrasonically treat it for 30min, wherein the conditions for ultrasonic treatment are power 100W and frequency 40kHz; take it out, put it to room temperature, shake it well, filter it with a rapid quantitative filter paper to remove the Polygonum multiflorum residue, take the filtrate and evaporate it to dryness, add 20mL of dilute sulfuric acid solution with a volume fraction of 20%, dissolve it, bath it in a water bath at 60℃ for 30min, put it to room temperature, add ethyl acetate with an equal volume of dilute sulfuric acid solution to extract it 3 times, combine all ethyl acetate layers, concentrate and evaporate, add methanol to make the volume 5mL, shake it well, filter it, and accurately draw 10μL to obtain the test solution. Compared with extraction method 1, no enzyme is added.

[0108] Extraction method 5: Take 2.00g of Polygonum multiflorum powder that has passed through a No. 4 sieve, weigh it accurately, put it in a stoppered conical flask, add 100mL of a mixture of water, cellulase, and anhydrous ethanol in a mass ratio of 100:1:50, and ultrasonically treat it for 30 minutes, wherein the conditions for ultrasonic treatment are power 100W and frequency 40kHz; take it out, put it to room temperature, shake it well, filter it with a rapid quantitative filter paper to remove the Polygonum multiflorum residue, take the filtrate and evaporate it to dryness, add 20mL of a 20% volume fraction dilute sulfuric acid solution, dissolve it, water bath it at 60℃ for 30 minutes, put it to room temperature, add ethyl acetate equal to the volume of the dilute sulfuric acid solution to extract it 3 times, combine all ethyl acetate layers, concentrate and evaporate, add methanol to make the volume 5mL, shake it well, filter it, and accurately draw 10μL to obtain the test solution. Compared with extraction method 1, all substances added to the extraction reagent are mixed.

[0109] Extraction method 6 (also recorded as Example 2): Take 2.00g of Polygonum multiflorum powder that has passed through a No. 4 sieve, accurately weigh it, place it in a stoppered conical flask, add 50mL of a mixture of water and cellulase in a mass ratio of 100:2, and ultrasonically treat it for 30min, wherein the conditions for the ultrasonic treatment are power 100W and frequency 40kHz; then add 50mL of anhydrous ethanol, and ultrasonically treat it for 30min, wherein the conditions for the ultrasonic treatment are power 100W and frequency 40kHz; take it out, bring it to room temperature, shake it well, filter it with a rapid quantitative filter paper to remove the Polygonum multiflorum residue, take the filtrate and evaporate it to dryness, add 20mL of a 20% by volume dilute sulfuric acid solution, dissolve it, bath it in a 60°C water bath for 30min, bring it to room temperature, add ethyl acetate equal to the volume of the dilute sulfuric acid solution, extract it 3 times, combine all ethyl acetate layers, concentrate and evaporate to dryness, add methanol to make the volume 5mL, shake it well, filter it, and accurately pipette 10μL to obtain the test solution.

[0110] Extraction method 7 (also recorded as Example 3): Take 2.00g of Polygonum multiflorum powder that has passed through a No. 4 sieve, accurately weigh it, place it in a stoppered conical flask, add 50mL of a mixture of water and cellulase in a mass ratio of 100:1, and ultrasonically treat it for 30min, wherein the conditions for the ultrasonic treatment are power 100W and frequency 40kHz; then add 50mL of anhydrous ethanol, and ultrasonically treat it for 30min, wherein the conditions for the ultrasonic treatment are power 100W and frequency 40kHz; take it out, bring it to room temperature, shake it well, filter it with a rapid quantitative filter paper to remove the Polygonum multiflorum residue, take the filtrate and evaporate it to dryness, add 20mL of a 20% by volume dilute sulfuric acid solution, dissolve it, bath it in a 65°C water bath for 30min, bring it to room temperature, add ethyl acetate with an equal volume of the dilute sulfuric acid solution to extract it 3 times, combine all ethyl acetate layers, concentrate and evaporate to dryness, add methanol to make the volume 5mL, shake it well, filter it, and accurately pipette 10μL to obtain the test solution.

[0111] Extraction method 8 (also recorded as Example 4): Take 2.00g of Polygonum multiflorum powder that has passed through a No. 4 sieve, accurately weigh it, place it in a stoppered conical flask, add 50mL of a mixture of water and cellulase in a mass ratio of 100:1, and ultrasonically treat it for 30min, wherein the conditions for the ultrasonic treatment are power 100W and frequency 40kHz; then add 50mL of anhydrous ethanol, and ultrasonically treat it for 30min, wherein the conditions for the ultrasonic treatment are power 100W and frequency 40kHz; take it out, bring it to room temperature, shake it well, filter it with rapid quantitative filter paper to remove Polygonum multiflorum residue, take the filtrate and evaporate it to dryness, add 20mL of dilute sulfuric acid solution with a volume fraction of 20%, dissolve it, bath it in a water bath at 55°C for 40min, bring it to room temperature, add ethyl acetate with an equal volume of the dilute sulfuric acid solution to extract it 3 times, combine all ethyl acetate layers, concentrate and evaporate to dryness, add methanol to make the volume 5mL, shake it well, filter it, and accurately pipette 10μL to obtain the test solution.

[0112] Extraction method 9 (also recorded as Example 5): Take 2.00g of Polygonum multiflorum powder that has passed through a No. 4 sieve, accurately weigh it, place it in a stoppered conical flask, add 70mL of a mixture of water and cellulase in a mass ratio of 100:1, and ultrasonically treat it for 30min, wherein the conditions for the ultrasonic treatment are power 100W and frequency 40kHz; then add 63mL of anhydrous ethanol, and ultrasonically treat it for 30min, wherein the conditions for the ultrasonic treatment are power 100W and frequency 40kHz; take it out, bring it to room temperature, shake it well, filter it with a rapid quantitative filter paper to remove the Polygonum multiflorum residue, take the filtrate and evaporate it to dryness, add 20mL of a 20% volume fraction dilute sulfuric acid solution, dissolve it, bath it in a 60℃ water bath for 30min, bring it to room temperature, add ethyl acetate with an equal volume of the dilute sulfuric acid solution to extract it 3 times, combine all ethyl acetate layers, concentrate and evaporate to dryness, add methanol to make the volume up to 5mL, shake it well, filter it, and accurately pipette 10μL to obtain the test solution.

[0113] Extraction method 10 (also recorded as Example 6): Take 2.00g of Polygonum multiflorum powder that has passed through a No. 4 sieve, accurately weigh it, put it in a stoppered conical flask, add 50mL of a mixture of water and cellulase in a mass ratio of 100:1, and ultrasonically treat it for 30min, wherein the conditions for the ultrasonic treatment are power 100W and frequency 40kHz; then add 50mL of anhydrous ethanol, and ultrasonically treat it for 30min, wherein the conditions for the ultrasonic treatment are power 100W and frequency 40kHz; take it out, bring it to room temperature, shake it well, filter it with a rapid quantitative filter paper to remove the Polygonum multiflorum residue, take the filtrate and evaporate it to dryness, add 20mL of a 15% volume fraction pyrosulfuric acid solution, dissolve it, bath it in a 60℃ water bath for 30min, bring it to room temperature, add ethyl acetate with an equal volume to the dilute sulfuric acid solution, extract it 3 times, combine all ethyl acetate layers, concentrate and evaporate to dryness, add methanol to make the volume 5mL, shake it well, filter it, and accurately pipette 10μL to obtain the test solution.

[0114] The differences in extraction processes and content test results of extraction methods 1 to 10 are shown in Table 9.

[0115] Table 9 Differences in extraction processes and content test results

[0116]

[0117] The results in Table 9 show that among the three extraction methods, the content of "static immersion for 24 hours" is lower, and the extraction method of "ultrasonic treatment for 30 minutes, wherein the ultrasonic treatment conditions are power 100W and frequency 40kHz" has the best detection effect.

[0118] Control experiment 5

[0119] This control experiment studies the effect of different ultrasonic times on the content of trans-rhamnolide dianthrone. Except for the differences in the settings in Table 10 below, the other operations are the same as those in Example 1. The results are shown in Table 10.

[0120] Table 10 Test results of different ultrasonic time and content

[0121] Ultrasonic time (min) Trans-rhodamine dianthrone content 20 0.00312g / 100g 30 0.00393g / 100g 40 0.00389g / 100g 50 0.00388g / 100g

[0122] Table 10 investigated the ultrasonic treatment for 20 min, 30 min, 40 min and 50 min respectively. The results showed that after 30 min of ultrasonic treatment, the content hardly increased. Therefore, 30 min of ultrasonic treatment was selected as the ultrasonic time, which not only retained the efficient extraction amount of trans-rhamnolide dianthrone but also saved time.

[0123] Control experiment 6

[0124] This control experiment studies the effect of different extraction solvents on the content of trans-rhaponticin dianthrone. Except for the difference in the settings of Table 11 below, the other operations are the same as those in Example 1. The results are shown in Table 11.

[0125] Table 11 Investigation table of different extraction solvent amounts

[0126]

[0127] Table 11 investigates the effects of different solid-liquid ratios of Polygonum multiflorum powder to water on the content of trans-rhizoma dianthrone. The results show that the content hardly increases when the solid-liquid ratio is 1kg:50L, so 1kg:50L is selected as the extraction solvent dosage.

[0128] Control experiment 7

[0129] This control experiment studies the effect of different water bath temperatures on the content of trans-rhaponticin dianthrone. Except for the difference in the settings in Table 12 below, the other operations are the same as those in Example 1. See Table 12 for the results.

[0130] Table 12 Investigation table of different water bath temperatures

[0131] Temperature(℃) Trans-rhodamine dianthrone content 40 0.00363g / 100g 60 0.00393g / 100g 80 0.00323g / 100g 100 0.00310g / 100g

[0132] Table 12 examines the effects of different water bath temperatures on the content of trans-rheic acid dianthrone. The results show that as the temperature increases, the content of trans-rheic acid dianthrone decreases, and the content is highest at 60°C water bath, so the water bath temperature is selected to be 60°C.

[0133] Control experiment 8

[0134] This control experiment studies the effect of different water bath time on the content of trans-rhodiola rosea dianthrone. Except for the difference in the settings in Table 12 below, the other operations are the same as those in Example 1. The results are shown in Table 13.

[0135] Table 13 Investigation table of different water bath time with the same water bath temperature

[0136] Time (min) Trans-rhodamine dianthrone content 20 0.00344g / 100g 30 0.00393g / 100g 40 0.00391g / 100g

[0137] The effects of different water bath times at the same water bath temperature on the content of trans-rhaponticin dianthrone were investigated. The results showed that the content hardly increased after 30 minutes of water bath time, so 30 minutes was selected as the water bath time.

[0138] Control experiment 9

[0139] This control experiment studies the effect of different acids on the content of trans-rhaponticin dianthrone. Except for the difference in the settings of Table 14 below, the other operations are the same as those of Example 1. The results are shown in Table 14.

[0140] Table 14 Different acid investigation table

[0141] acid Trans-rhodamine dianthrone content sulfuric acid 0.00390g / 100g Pyrosulfate 0.00393g / 100g hydrochloric acid 0.00305g / 100g

[0142] The results showed that sulfuric acid and pyrosulfuric acid had the largest extraction amounts.

[0143] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, once those skilled in the art know the basic inventive concept, they can make other changes and modifications to these embodiments, and these changes and modifications all fall within the scope of the present invention.

[0144] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. If these modifications and variations of the present invention fall within the scope of the equivalent technology of the present invention, the present invention is also intended to include these modifications and variations.

Claims

1. A method for determining the content of trans-rhodiola rosea in Radix Polygoni Multiflori, characterized in that: Take Polygonum multiflorum powder, add a mixture of water and cellulase in a mass ratio of 100:1-2, perform ultrasonic treatment to degrade cellulose, then add anhydrous ethanol, perform ultrasonic treatment to change the conformation and charge distribution of cellulase, extract components including trans-rhizoma dianthrone, filter to remove Polygonum multiflorum residue, take the filtrate and evaporate to dryness, add sulfur-containing acid solution to dissolve, water bath at 55°C-65°C to increase the content of trans-rhizoma dianthrone, cool to room temperature, add ethyl acetate to extract to remove impurities remaining in the acid solution layer, add methanol to dissolve, and obtain a test solution; The content of trans-rhaponticin dianthrone in the test solution was determined by liquid chromatography.

2. The method for determining the content of trans-rhodiola rosea in Polygonum multiflorum according to claim 1, characterized in that: The enzymatic activity of cellulase is ≥50000U / g.

3. The method for determining the content of trans-rhodiola rosea in Polygonum multiflorum according to claim 1, characterized in that: The amount of anhydrous ethanol added is equivalent to 90% to 100% of the mass of water.

4. The method for determining the content of trans-rhodiola rosea in Polygonum multiflorum according to claim 1, characterized in that: The solid-liquid ratio of Polygonum multiflorum powder to water is 1kg:25L~35L.

5. The method for determining the content of trans-rhodiola rosea in Polygonum multiflorum according to claim 1, characterized in that: The conditions of ultrasonic treatment are power 100 W, frequency 40 kHz, and ultrasonic treatment for 30 min to 50 min.

6. The method for determining the content of trans-rhodiola rosea in Polygonum multiflorum according to claim 1, characterized in that: The acid solution containing sulfur element is a sulfuric acid solution with a volume fraction of 15% to 20%, a pyrosulfuric acid solution with a volume fraction of 15% to 20%, or a mixture of the two.

7. The method for determining the content of trans-rhodiola rosea in Polygonum multiflorum according to claim 1, characterized in that: The water bath time is 30min to 40min.

8. The method for determining the content of trans-rhodiola rosea in Polygonum multiflorum according to claim 1, characterized in that: The chromatographic column used in the liquid chromatography method is an Agilent C 18 .

9. The method for determining the content of trans-rhodiola rosea in Polygonum multiflorum according to claim 8, characterized in that: Acetonitrile was used as mobile phase A and 0.10% to 0.15% by volume aqueous phosphoric acid solution was used as mobile phase B for gradient elution.

10. The method for determining the content of trans-rhodiola rosea in Polygonum multiflorum according to claim 9, characterized in that: In the 0th to 10th minute of gradient elution, the volume ratio of mobile phase A and mobile phase B is 50%; in the 10th to 30th minute, the volume ratio of mobile phase A increases from 50% to 80%, and the volume of mobile phase B decreases from 50% to 20%.

Images