A method for determining the content of trans-emodin dianthrones in radix foetid bupleuri

Abstract

The present application belongs to the technical field of traditional Chinese medicine component detection method, and particularly relates to a content determination method of trans-emodin di-anthrones in Radix Polygoni Multiflori, wherein Radix Polygoni Multiflori powder is taken, a mixture of water and cellulase is added in a mass ratio of 100:1-2, ultrasonic treatment is performed, then anhydrous ethanol is added, ultrasonic treatment is performed, components including trans-emodin di-anthrones are extracted, filtration is performed, the filtrate is evaporated to dryness, an acid solution containing sulfur elements is added for dissolution, a water bath is performed at 55-65 DEG C, it is placed at room temperature, ethyl acetate is added for extraction, so as to remove impurity components remaining in the acid solution layer, improve the purity of trans-emodin di-anthrones, facilitate liquid chromatography separation, methanol is added for dissolution, and a test sample solution is obtained; liquid chromatography is used to detect the content of trans-emodin di-anthrones in the test sample solution. The method has strong specificity, good reproducibility, stability and precision, and can effectively and accurately detect the content of trans-emodin di-anthrones in Radix Polygoni Multiflori.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine component detection methods, and particularly relates to a content determination method of trans-emodin dianthrone in Radix Polygoni Multiflori. BACKGROUND

[0002] Radix Polygoni Multiflori is the dried tuber of Polygonum multiflorum Thunb. It is harvested in autumn and winter when the leaves wither, cut into pieces, washed, dried, with a slight odor and a bitter and astringent taste. It has the effects of detoxification, abscess, malaria, and moistening the intestines. It is used for treating sores, scrofula, itchy skin, chronic malaria, and constipation. Modern research shows that Radix Polygoni Multiflori contains a variety of anthraquinones and anthracene compounds, and such compounds have certain hepatotoxicity to the human body. However, the content determination method of stilbene glucoside and combined anthraquinone is stipulated in the pharmacopoeia, and the detection of anthraquinones is not stipulated.

[0003] In recent years, the hepatotoxicity of Radix Polygoni Multiflori has attracted widespread attention at home and abroad. Trans-emodin dianthrone CAS No. 61281-20-7 is one of the components that cause hepatotoxicity in Radix Polygoni Multiflori. Therefore, it is of great significance to determine the content of trans-emodin dianthrone in Radix Polygoni Multiflori. Although there are some detection methods for anthraquinones in the prior art, such as CN112964819A and CN111272908A, they detect a large class of substances, can only reflect the content of a large class of substances, and are mixtures, and cannot clearly determine the content of trans-emodin dianthrone. In addition, although some specific compounds can be determined by the prior art such as CN115032310A, the detected substances are six dianthrone compounds, namely trans-emodin-emodin dianthrone, cis-emodin-emodin dianthrone, trans-emodin-emodin methyl ether dianthrone, cis-emodin-emodin methyl ether dianthrone, trans-emodin methyl ether-emodin methyl ether dianthrone, and cis-emodin methyl ether-emodin methyl ether dianthrone, and the content of trans-emodin dianthrone is still not determined.

[0004] In summary, the prior art lacks a content determination method for trans-emodin dianthrone in Radix Polygoni Multiflori. SUMMARY

[0005] To solve the above technical problems, the present application provides a content determination method of trans-emodin dianthrone in Radix Polygoni Multiflori.

[0006] The present application aims to provide a method for determining the content of trans-emodin di-anthrones in Radix Polygoni Multiflori, which comprises the following steps: adding a mixture of water and cellulase to Radix Polygoni Multiflori powder according to a mass ratio of 100:1-2, and then performing ultrasonic treatment to degrade cellulose; adding anhydrous ethanol and performing ultrasonic treatment to change the conformation and charge distribution of cellulase, so as to extract components including trans-emodin di-anthrones; filtering to remove Radix Polygoni Multiflori residues; evaporating the filtrate to dryness, dissolving it in an acid solution containing sulfur elements, and then performing water bath treatment at 55-65 ℃ to remove impurities and increase the content of trans-emodin di-anthrones in the test sample; cooling the test sample to room temperature, and then extracting it with ethyl acetate; at this time, the cellulase is insoluble in ethyl acetate, and the extraction operation with ethyl acetate can remove impurities remaining in the acid solution layer, increase the purity of trans-emodin di-anthrones, and facilitate liquid chromatography separation; dissolving the test sample with methanol to obtain a test sample solution.

[0007] The content of trans-emodin di-anthrones in the test sample solution is detected by liquid chromatography.

[0008] In the present application, the extraction reagent is composed of anhydrous ethanol, water and cellulase. It is noted that the three components are not directly mixed, but a mixture of water and cellulase is first added to Radix Polygoni Multiflori powder according to a mass ratio of 100:1-2, and then ultrasonic treatment is performed to degrade cellulose and loosen the structure of Radix Polygoni Multiflori. Then, ethanol is added and ultrasonic treatment is performed to change the conformation and charge distribution of cellulase, so as to promote the dissolution of trans-emodin di-anthrones. Finally, components including trans-emodin di-anthrones are extracted. Cellulase degrades cellulose in Radix Polygoni Multiflori and loosens its structure, and at the same time, it cooperates with ethanol to induce the dissolution of trans-emodin di-anthrones.

[0009] According to the control experiment of the present application, direct use of cellulase or use of a combination of cellulase and methanol shows that cellulase does not play a role in promoting dissolution, indicating that degradation of cellulose is not a key condition for promoting dissolution. In the present application, cellulase degrades cellulose in Radix Polygoni Multiflori and loosens its structure, and the structure of cellulase cooperates with the polarity of ethanol to induce the dissolution of trans-emodin di-anthrones, which is the key condition for promoting the dissolution of trans-emodin di-anthrones.

[0010] The above extraction reagent (composed of anhydrous ethanol, water and cellulase) is further combined with an acid solution containing sulfur elements, water bath treatment is performed at 55-65 ℃, extraction is performed with ethyl acetate, and dissolution is performed with methanol, and the obtained test sample solution can be well separated by liquid chromatography to obtain complete trans-emodin di-anthrones, and the extracted trans-emodin di-anthrones have high content, high recovery rate and good stability.

[0011] Preferably, in the above method for determining the content of trans-emodin di-anthrones in Radix Polygoni Multiflori, the enzyme activity of cellulase is ≥50000 U / g.

[0012] Preferably, in the content determination method of trans-emodin dianthrones in the above-mentioned Radix Polygoni Multiflori, the amount of the added anhydrous ethanol is equivalent to 90% to 100% of the mass of water.

[0013] Preferably, in the content determination method of trans-emodin dianthrones in the above-mentioned Radix Polygoni Multiflori, the ratio of the Radix Polygoni Multiflori powder to water is 1 kg: 25 L to 35 L.

[0014] Preferably, in the content determination method of trans-emodin dianthrones in the above-mentioned Radix Polygoni Multiflori, the ultrasonic treatment is performed at a power of 100 W, a frequency of 40 kHz, and for 30 min to 40 min.

[0015] Preferably, in the content determination method of trans-emodin dianthrones in the above-mentioned Radix Polygoni Multiflori, the acid solution containing sulfur elements is a 15% to 20% sulfuric acid solution, a 15% to 20% pyrosulfuric acid solution, or a mixture of the two.

[0016] Preferably, in the content determination method of trans-emodin dianthrones in the above-mentioned Radix Polygoni Multiflori, the water bath is performed for 30 min to 50 min.

[0017] Preferably, in the content determination method of trans-emodin dianthrones in the above-mentioned Radix Polygoni Multiflori, the chromatographic column used in the liquid chromatography is an Agilent C 18 .

[0018] Preferably, in the content determination method of trans-emodin dianthrones in the above-mentioned Radix Polygoni Multiflori, acetonitrile is used as the mobile phase A, and a 0.10% to 0.15% phosphoric acid aqueous solution is used as the mobile phase B, and gradient elution is performed.

[0019] Preferably, in the content determination method of trans-emodin dianthrones in the above-mentioned Radix Polygoni Multiflori, in the gradient elution, the volume ratio of the mobile phase A to the mobile phase B is both 50% at 0 min to 10 min, the volume ratio of the mobile phase A is increased from 50% to 80% at 10 min to 30 min, and the volume ratio of the mobile phase B is decreased from 50% to 20% at 10 min to 30 min.

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

[0021] The content determination method of trans-emodin di-anthrones in the Polygoni Multifidi Radix of the application takes the Polygoni Multifidi Radix powder, adds a mixture of water and cellulase according to a mass ratio of 100:1-2, carries out ultrasonic treatment to degrade cellulose, then adds anhydrous ethanol, carries out ultrasonic treatment to change the conformation and charge distribution of the cellulase, extracts ingredients including trans-emodin di-anthrones, filters to remove the Polygoni Multifidi Radix residue, takes the filtrate to evaporate to dryness, dissolves in an acid solution containing sulfur elements, carries out water bath at 55-65 DEG C to improve the content of trans-emodin di-anthrones, cools to room temperature, extracts with ethyl acetate to remove impurity ingredients remaining in the acid solution layer, dissolves in methanol to obtain a test sample solution; liquid chromatography is used to detect the content of trans-emodin di-anthrones in the test sample solution. The method is targeted to detect the content of trans-emodin di-anthrones in the Polygoni Multifidi Radix, has strong specificity, good reproducibility, stability and precision, can effectively and accurately detect the content of trans-emodin di-anthrones in the Polygoni Multifidi Radix, controls the quality of the Polygoni Multifidi Radix from the source, makes the quality of the Polygoni Multifidi Radix stable, controllable and safe, is the requirement of raw material and quality control of products, overcomes the defects of the prior art, and is helpful to improve the quality of Polygoni Multifidi Radix products. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The chromatogram of the trans-emodin di-anthrones reference substance.

[0023] Figure 2 The chromatogram of the methanol solvent.

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

[0025] Figure 4 The standard curve of Effect Verification 1. DETAILED DESCRIPTION

[0026] In order for those skilled in the art to better understand the technical solutions of the application and to implement them, the application is further described below in conjunction with specific examples and drawings.

[0027] In the description of the application, if not specially stated, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0028] 1 Instruments and reagents

[0029] 1.1 Instruments

[0030] Agilent 1260 type high performance liquid chromatograph (1260 VWD detector, LC 1260 chromatographic work station, Agilent).

[0031] DV215CD type electronic analytical balance (Ohaus).

[0032] SB-120D type ultrasonic cleaning instrument (Ningbo Xinzhi).

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

[0034] 1.2 Reagents:

[0035] The reference substance of trans-emodin di-anthrones (batch number 23103003) was produced by Chengdu Gliplum Biotech Co., Ltd.

[0036] Acetonitrile (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] Water was 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 biological, enzyme activity 50000 U / g).

[0045] The embodiments of the present application are as follows.

[0046] Example 1

[0047] A content determination method of trans-emodin di-anthrones in polygonum multiflorum thunb, comprising the following steps:

[0048] (1) Preparation of reference substance solution: 20.00 mg of the reference substance of trans-emodin di-anthrones was accurately weighed, placed in a 100 mL volumetric flask, dissolved and diluted to the mark with 0.5% methanol solution, shaken well, and 10 mL was taken and diluted to 50 mL with methanol to obtain the reference substance solution.

[0049] Preparation of test solution: 2.00 g of the powdered Radix Polygoni Multiflori which has passed through a No. 4 sieve was precisely weighed into a conical flask with a stopper, 50 mL of a mixture of water and cellulase was added at a mass ratio of 100:1, and ultrasonic treatment was performed for 30 min, wherein the ultrasonic treatment was performed at a power of 100 W and a frequency of 40 kHz; 50 mL of anhydrous ethanol was then added, and ultrasonic treatment was performed for 30 min, wherein the ultrasonic treatment was performed at a power of 100 W and a frequency of 40 kHz; the mixture was then removed, allowed to stand at room temperature, shaken well, filtered through a fast quantitative filter paper to remove the Radix Polygoni Multiflori residue, and the filtrate was evaporated to dryness, 20 mL of a 20% dilute sulfuric acid solution was added, dissolved, and placed in a water bath at 60°C for 30 min; the mixture was then allowed to stand at room temperature, extracted with ethyl acetate in an amount equal to that of the dilute sulfuric acid solution three times, all the ethyl acetate layers were combined, concentrated and evaporated to dryness, dissolved in methanol to a volume of 5 mL, shaken well, filtered through a fast quantitative filter paper, and 10 μL was precisely taken for use as the test solution.

[0050] (2) 10 μL of the control solution and the test solution were precisely taken and injected into the liquid chromatograph for determination.

[0051] The chromatographic conditions of the liquid chromatograph were as follows: Agilent C 18 (4.6 mm x 150 mm, 5 μm) was used as the filler; acetonitrile was used as the mobile phase A, and a 0.1% phosphoric acid aqueous solution was used as the mobile phase B, and gradient elution was performed at a volume ratio specified in Table 1; and the detection wavelength was 280 nm. Figure 1 The chromatogram of the trans-emodin dianthrones control sample is shown in Figure 2 The chromatogram of the methanol solvent is shown in Figure 3 The chromatogram of the test solution of Example 1 is shown in

[0052] Table 1 Volume ratio of the mobile phase for 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 application, the following effect verification was performed.

[0055] Effect verification 1

[0056] Investigation of linear relationship: 4 μL, 8 μL, 12 μL, 16 μL, 20 μL and 24 μL of the control solution of Example 1 were precisely taken and injected into the high performance liquid chromatograph for determination. Linear regression was performed with the injection amount as the abscissa and the peak area as the ordinate, and the regression equation of trans-emodin dianthrones was Y = 1842.7X + 0.9333, R 2 = 0.9999, see Figure 4The results showed that trans-emodin di-anthrones was in good linear relationship at 0.16 μg-0.96 μg. The results are shown in Table 2.

[0057] Table 2 Linear range of trans-emodin di-anthrones

[0058] Serial number Injection volume μL Injection amount 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: The control solution was taken, the sample volume was 10 μL, and the sample was continuously injected for 10 times, and the peak area integral value was recorded. The RSD (relative standard deviation) of the trans-emodin di-anthrones control product concentration was 0.50%, indicating that the method had good precision. The results are shown in Table 3.

[0061] Table 3 Precision determination results of trans-emodin di-anthrones

[0062] Injection times Trans-emodin di-anthrones 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: 10 portions of the same batch of polygonum multiflorum powder were taken, and the sample was prepared according to the method of Example 1, and the sample was injected for 10 μL, the peak area was determined, and the content of trans-emodin di-anthrones in polygonum multiflorum was calculated. The RSD of trans-emodin di-anthrones content was 0.60%, indicating that the method had good repeatability. The results are shown in Table 4.

[0065] Table 4 Repeatability determination results of trans-emodin di-anthrones

[0066]

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

[0068] Effect verification 4

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

[0070] Table 5 Repeatability determination results of trans-emodin di-anthrones

[0071] Number Time h Trans-emodin di-anthrones 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 that there is no such item.

[0073] Effect verification 5

[0074] spiked recovery test

[0075] (5.1) Preparation of control solution: 0.0112 g of trans-emodin dianthrone control sample was precisely weighed into a 100 mL volumetric flask, dissolved with methanol and diluted to the mark, shaken well and ready for use.

[0076] (5.2) Preparation of test solution: about 1.000 g of known content of Radix Polygoni Multiflori powder was precisely weighed, 10 portions were prepared, mixed with 1 mL of the control solution of (5.1) respectively, and the test solution was prepared according to the method of Example 1 to determine the content of trans-emodin dianthrone, and the recovery rate was calculated. The average recovery rate of trans-emodin 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-emodin dianthrone spiked recovery test

[0078]

[0079] Effect verification 6

[0080] Ten batches of Radix Polygoni Multiflori medicinal materials of different batches were purchased, and the content of trans-emodin dianthrone in Radix Polygoni Multiflori was measured according to the above-mentioned measurement conditions of Example 1. The results are shown in Table 7.

[0081] Table 7 Detection results of trans-emodin dianthrone content in Radix Polygoni Multiflori

[0082] Batch number Trans-emodin di-anthrones 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 difference in the production place of Radix Polygoni Multiflori, the content of trans-emodin dianthrone has certain difference.

[0084] In order to prove the advantages of the method of the present application, the following control experiments were carried out.

[0085] Control experiment 1

[0086] This experiment is the influence of different mobile phases and the same mobile phase with different gradient elution on the peak of trans-emodin dianthrone and the test results. Except for the following settings of control 1 to control 5, the rest of the operation is the same as Example 1.

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

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

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

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

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

[0092] The results show that when control 1 and control 2 are eluted, the target peak cannot be completely separated, the peak shape is poor, and tailing peaks and wrapped peaks appear, indicating that these eluents and elution methods are poor in effect. After comparing the different gradient elution procedures of different mobile phases of control 3, control 4 and control 5, the results show that using the mobile phase acetonitrile-0.1 phosphoric acid aqueous solution (control 4), gradient elution (0-10 min, 50% A, 10-30 min, 50%-80% A) has appropriate peak time, relatively good chromatographic peak shape, and good separation degree, while control 3 and control 5 cannot completely separate the target peak and have poor peak shape, and wrapped peaks appear. The above results show that the mobile phase and elution conditions have a great influence on the peak situation of trans-emodin dianthrones, and even control 5 which is very close to the conditions of control 4 still cannot successfully separate the peak of trans-emodin dianthrones, resulting in the inability to quantitatively detect trans-emodin dianthrones; it can be seen that the peak situation of trans-emodin dianthrones is unpredictable and has no regularity due to the difference of mobile phase and elution procedure, and the present application first discovers that the conditions of control 4 can accurately and completely separate the peak of trans-emodin dianthrones, facilitating accurate quantitative detection.

[0093] Control experiment 2

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

[0095] Table 8 Different chromatographic column investigation table

[0096]

[0097] The results of Table 8 show that the peak situation of Agilent C 18 is the best, and the peaks of trans-emodin dianthrones cannot be completely separated by other types of chromatographic columns. The prior art usually optimizes the peak quality by adjusting the material of the chromatographic column, but the present application chooses C 18 columns, and the results show that although they are C 18column, but when the manufacturer's model is different, the peak mass of trans-emodin di-anthrones also differs significantly, which shows that the peak of trans-emodin di-anthrones is unpredictable and has no regularity due to different chromatographic columns. The present application first discovers that Agilent C 18 The peak of high-quality trans-emodin di-anthrones can be separated accurately and completely.

[0098] Control experiment 3

[0099] The present control experiment is referenced to the method of Example 1 to explore the influence of different detection wavelengths on the peak of trans-emodin di-anthrones and test results.

[0100] Preparation of the control solution: 20.00 mg of the control of trans-emodin di-anthrones was precisely weighed, placed in a 100-mL volumetric flask, dissolved and diluted to the mark with a methanol solution with a volume fraction of 0.5%, shaken, 10 mL was taken, and diluted to 50 mL with methanol to obtain the control solution.

[0101] Methanol was used as a blank reagent, and full-wavelength scanning was determined on a UV spectrophotometer. The results show that the control solution has a maximum absorption peak at 280 nm, so the detection wavelength can be set to 280 nm.

[0102] Control experiment 4

[0103] The present control experiment compares the influence of different extraction methods on the determination results of the content of trans-emodin di-anthrones. Except for the differences between extraction method 1 to extraction method 10 as described below, the rest of the operations are the same as Example 1.

[0104] Extraction method 1: 2.00 g of the polygonum multiflorum powder passed through a No. 4 sieve was precisely weighed, placed in a conical flask with a plug, 50 mL of a mixture of water and cellulase at a mass ratio of 100:1 was added, and ultrasonic treatment was performed for 30 min, wherein the ultrasonic treatment conditions were power 100 W and frequency 40 kHz; 50 mL of anhydrous ethanol was further added, and ultrasonic treatment was performed for 30 min, wherein the ultrasonic treatment conditions were power 100 W and frequency 40 kHz; then it was taken out and placed at room temperature, shaken, filtered with a quantitative filter paper to remove the polygonum multiflorum residue, the filtrate was taken and evaporated, 20 mL of a dilute sulfuric acid solution with a volume fraction of 20% was added, dissolved, and placed in a 60℃ water bath for 30 min, then it was placed at room temperature, extracted with ethyl acetate for 3 times with an equal volume of the dilute sulfuric acid solution, all ethyl acetate layers were combined, concentrated and evaporated, diluted to 5 mL with methanol, shaken, filtered, and 10 μL was precisely taken to obtain the test solution.

[0105] Extraction method 2: take the radices polygonati powder 2.00 g, precision weighing, placed in a conical flask with a plug, add water and cellulase according to the mass ratio of 100:1 mixture of 50 mL, 50 ℃ heating reflux 1.5 h; add anhydrous ethanol 50 mL, 50 ℃ heating reflux 1.5 h; take out, put into room temperature, shake, quickly quantitative filter paper filter to remove the radices polygonati residue, take the filtrate evaporated, add volume fraction 20% dilute sulfuric acid solution 20 mL, dissolve, 60 ℃ water bath 30 min, put into room temperature, add with dilute sulfuric acid solution equal volume of ethyl acetate extraction 3 times, combine all the ethyl acetate layer, concentrated dry, add methanol constant volume to 5 mL, shake, filter, precision 10 μL, namely the test solution.

[0106] Extraction method 3: take the radices polygonati powder 2.00 g, precision weighing, placed in a conical flask with a plug, add water and cellulase according to the mass ratio of 100:1 mixture of 50 mL, static immersion 24 h; add anhydrous ethanol 50 mL, static immersion 24 h; take out, put into room temperature, shake, quickly quantitative filter paper filter to remove the radices polygonati residue, take the filtrate evaporated, add volume fraction 20% dilute sulfuric acid solution 20 mL, dissolve, 60 ℃ water bath 30 min, put into room temperature, add with dilute sulfuric acid solution equal volume of ethyl acetate extraction 3 times, combine all the ethyl acetate layer, concentrated dry, add methanol constant volume to 5 mL, shake, filter, precision 10 μL, namely the test solution.

[0107] Extraction method 4: take the radices polygonati powder 2.00 g, precision weighing, placed in a conical flask with a plug, add water 50 mL, ultrasonic treatment 30 min, wherein, the ultrasonic treatment conditions are power 100 W, frequency 40 kHz; add anhydrous ethanol 50 mL, ultrasonic treatment 30 min, wherein, the ultrasonic treatment conditions are power 100 W, frequency 40 kHz; take out, put into room temperature, shake, quickly quantitative filter paper filter to remove the radices polygonati residue, take the filtrate evaporated, add volume fraction 20% dilute sulfuric acid solution 20 mL, dissolve, 60 ℃ water bath 30 min, put into room temperature, add with dilute sulfuric acid solution equal volume of ethyl acetate extraction 3 times, combine all the ethyl acetate layer, concentrated dry, add methanol constant volume to 5 mL, shake, filter, precision 10 μL, namely the test solution. Compared with extraction method 1, no enzyme.

[0108] Extraction method 5: take the radices polygonati powder 2.00 g, precision weighing, placed in a conical flask with a plug, add water, cellulase, anhydrous ethanol according to the mass ratio of 100:1:50 mixture 100 mL, ultrasonic treatment for 30 min, wherein the ultrasonic treatment conditions are power 100 W, frequency 40 kHz; take out, put into room temperature, shake up, fast quantitative filter paper filter to remove the radices polygonati residue, take the filtrate to evaporate dry, add 20% dilute sulfuric acid solution 20 mL, dissolve, 60 ℃ water bath for 30 min, put into room temperature, add the same volume of ethyl acetate as the dilute sulfuric acid solution to extract 3 times, combine all the ethyl acetate layers, concentrated to dry, add methanol to constant volume to 5 mL, shake up, filter, precision 10 μL, namely the test sample solution. Compared with extraction method 1, all the substances are mixed and added to the extraction reagent.

[0109] Extraction method 6 (also recorded as example 2): take the radices polygonati powder 2.00 g, precision weighing, placed in a conical flask with a plug, add water and cellulase according to the mass ratio of 100:2 mixture 50 mL, ultrasonic treatment for 30 min, wherein the ultrasonic treatment conditions are power 100 W, frequency 40 kHz; then add anhydrous ethanol 50 mL, ultrasonic treatment for 30 min, wherein the ultrasonic treatment conditions are power 100 W, frequency 40 kHz; take out, put into room temperature, shake up, fast quantitative filter paper filter to remove the radices polygonati residue, take the filtrate to evaporate dry, add 20% dilute sulfuric acid solution 20 mL, dissolve, 60 ℃ water bath for 30 min, put into room temperature, add the same volume of ethyl acetate as the dilute sulfuric acid solution to extract 3 times, combine all the ethyl acetate layers, concentrated to dry, add methanol to constant volume to 5 mL, shake up, filter, precision 10 μL, namely the test sample solution.

[0110] Extraction method 7 (also recorded as example 3): take the radices polygonati powder 2.00 g, precision weighing, placed in a conical flask with a plug, add water and cellulase according to the mass ratio of 100:1 mixture 50 mL, ultrasonic treatment for 30 min, wherein the ultrasonic treatment conditions are power 100 W, frequency 40 kHz; then add anhydrous ethanol 50 mL, ultrasonic treatment for 30 min, wherein the ultrasonic treatment conditions are power 100 W, frequency 40 kHz; take out, put into room temperature, shake up, fast quantitative filter paper filter to remove the radices polygonati residue, take the filtrate to evaporate dry, add 20% dilute sulfuric acid solution 20 mL, dissolve, 65 ℃ water bath for 30 min, put into room temperature, add the same volume of ethyl acetate as the dilute sulfuric acid solution to extract 3 times, combine all the ethyl acetate layers, concentrated to dry, add methanol to constant volume to 5 mL, shake up, filter, precision 10 μL, namely the test sample solution.

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

[0112] Extraction method 9 (also recorded as example 5): take the powder of Radix Polygoni Multiflori 2.00 g which has passed through the No. 4 sieve, accurately weigh, place in a conical flask with a stopper, add a mixture of water and cellulase according to a mass ratio of 100:1 70 mL, ultrasonic treatment for 30 min, wherein the ultrasonic treatment conditions are power 100 W and frequency 40 kHz; then add anhydrous ethanol 63 mL, ultrasonic treatment for 30 min, wherein the ultrasonic treatment conditions are power 100 W and frequency 40 kHz; take out, place at room temperature, shake well, quickly filter through quantitative filter paper to remove the residue of Radix Polygoni Multiflori, take the filtrate, evaporate to dryness, add a dilute sulfuric acid solution with a volume fraction of 20% 20 mL, dissolve, 60°C water bath for 30 min, place at room temperature, extract with ethyl acetate in an equal volume to the dilute sulfuric acid solution for 3 times, combine all the ethyl acetate layers, concentrate to dryness, add methanol to constant volume to 5 mL, shake well, filter, accurately take 10 μL, and the test sample solution is obtained.

[0113] Extraction method 10 (also recorded as example 6): take the powder of Radix Polygoni Multiflori 2.00 g which has passed through the No. 4 sieve, accurately weigh, place in a conical flask with a stopper, add a mixture of water and cellulase according to a mass ratio of 100:1 50 mL, ultrasonic treatment for 30 min, wherein the ultrasonic treatment conditions are power 100 W and frequency 40 kHz; then add anhydrous ethanol 50 mL, ultrasonic treatment for 30 min, wherein the ultrasonic treatment conditions are power 100 W and frequency 40 kHz; take out, place at room temperature, shake well, quickly filter through quantitative filter paper to remove the residue of Radix Polygoni Multiflori, take the filtrate, evaporate to dryness, add a dilute sulfuric acid solution with a volume fraction of 20% 20 mL, dissolve, 60°C water bath for 30 min, place at room temperature, extract with ethyl acetate in an equal volume to the dilute sulfuric acid solution for 3 times, combine all the ethyl acetate layers, concentrate to dryness, add methanol to constant volume to 5 mL, shake well, filter, accurately take 10 μL, and the test sample solution is obtained.

[0114] The extraction process differences of extraction method 1-extraction method 10 and the content test results are shown in Table 9.

[0115] Table 9 extraction process differences and content test results

[0116]

[0117] The results of Table 9 show that among the three extraction methods, the "standing immersion for 24h" has the lowest content, and the extraction method of "ultrasonic treatment for 30min, 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-emodin dianthrone. Except for the differences set in Table 10 below, the rest of the operations are the same as in Example 1. The results are shown in Table 10.

[0120] Table 10 different ultrasonic times and content test results

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

[0122] Table 10 respectively investigates ultrasonic treatment for 20min, 30min, 40min, and 50min. The results show that after ultrasonic treatment for 30min, the content almost does not increase, so ultrasonic treatment for 30min is selected as the ultrasonic time, which not only retains the high extraction amount of trans-emodin dianthrone, but also saves time.

[0123] Control experiment 6

[0124] This control experiment studies the effect of different extraction solvents on the content of trans-emodin dianthrone. Except for the differences set in Table 11 below, the rest of the operations are the same as in Example 1. The results are shown in Table 11.

[0125] Table 11 different extraction solvent amount investigation table

[0126]

[0127] Table 11 respectively investigates the effect of different material-to-liquid ratios of polygonum multiflorum powder to water on the content of trans-emodin dianthrone. The results show that when the material-to-liquid ratio is 1kg:50L, the content almost does not increase, so 1kg:50L is selected as the extraction solvent amount.

[0128] Control experiment 7

[0129] This control experiment studies the effect of different water bath temperatures on the content of trans-emodin dianthrone. Except for the differences set in Table 12 below, the rest of the operations are the same as in Example 1. The results are shown in Table 12.

[0130] Table 12 different water bath temperature investigation table

[0131] Temperature (°C) Trans-emodin di-anthrones content 40 0.00363 g / 100g 60 0.00393 g / 100g 80 0.00323 g / 100g 100 0.00310 g / 100g

[0132] Table 12 respectively investigates the influence of different water bath temperatures on the content of trans-emodin dianthrone, and the results show that with the increase of temperature, the content of trans-emodin dianthrone decreases, and the content is the highest at 60℃ water bath, so the water bath temperature is selected as 60℃.

[0133] Control experiment 8

[0134] This control experiment studies the influence of different water bath times on the content of trans-emodin dianthrone, and except for the following Table 12 setting differences, the rest of the operation is the same as Example 1. The results are shown in Table 13.

[0135] Table 13 same water bath temperature different water bath time investigation table

[0136] Time (min) Trans-emodin di-anthrones content 20 0.00344 g / 100g 30 0.00393 g / 100g 40 0.00391 g / 100g

[0137] The influence of the same water bath temperature and different water bath time on the content of trans-emodin dianthrone is investigated respectively, and the results show that after 30min of water bath time, the content almost does not increase, so 30min is selected as the water bath time.

[0138] Control experiment 9

[0139] This control experiment studies the influence of different acids on the content of trans-emodin dianthrone, and except for the following Table 14 setting differences, the rest of the operation is the same as Example 1. The results are shown in Table 14.

[0140] Table 14 different acid investigation table

[0141] Acid Trans-emodin di-anthrones content Sulfuric acid 0.00390 g / 100g Pyrosulfuric acid 0.00393 g / 100g Hydrochloric acid 0.00305 g / 100g

[0142] The results show that the extraction amount of sulfuric acid and pyrosulfuric acid is the most.

[0143] It should be noted that when the numerical range is involved in the present application, it should be understood that each numerical range of two endpoints and any number between the two endpoints can be selected. Since the same steps and examples are adopted, in order to prevent repetition, the preferred examples are described in the present application. Although the preferred examples of the present application have been described, those skilled in the art can make additional changes and modifications to these examples once they know the basic inventive concept, and these changes and modifications all fall within the scope of the present application.

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

Claims

1. A method for determining the content of trans-emodin dianthrone in Polygonum multiflorum, characterized in that, Take Polygonum multiflorum powder, add water and cellulase in a mass ratio of 100:1~2, sonicate to degrade cellulose, then add anhydrous ethanol and sonicate again to change the conformation and charge distribution of cellulase, extracting components including trans-emodin dianthrone, filter to remove Polygonum multiflorum residue, take the filtrate, evaporate to dryness, add sulfur-containing acid solution to dissolve, water bath at 55℃~65℃ to increase the content of trans-emodin dianthrone, let it stand at room temperature, add ethyl acetate to extract to remove impurities remaining in the acid solution layer, add methanol to dissolve, and obtain the test solution. Detection of trans-emodin dianthrone content in the test sample solution by liquid chromatography; The sulfur-containing acid solution is a 15%~20% sulfuric acid solution, a 15%~20% pyrosulfuric acid solution, or a mixture of the two. The liquid chromatography method uses an Agilent C10 column packed with octadecylsilane-bonded silica gel. 18 ; Gradient elution was performed using acetonitrile as mobile phase A and 0.10%~0.15% (v / v) phosphoric acid aqueous solution as mobile phase B. During gradient elution, the volume ratio of mobile phase A to mobile phase B was 50% for the first 0-10 minutes; from the 10th to the 30th minute, the volume ratio of mobile phase A increased from 50% to 80%, while the volume ratio of mobile phase B decreased from 50% to 20%.

2. The method for determining the content of trans-emodin dianthrone in Polygonum multiflorum according to claim 1, characterized in that, Cellulase activity ≥50000U / g.

3. The method for determining the content of trans-emodin dianthrone 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-emodin dianthrone in Polygonum multiflorum according to claim 1, characterized in that, The ratio of He Shou Wu powder to water is 1kg:25L~35L.

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

6. The method for determining the content of trans-emodin dianthrone in Polygonum multiflorum according to claim 1, characterized in that, The water bath should last for 30 to 40 minutes.

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