Multi-index content determination method of fructus aurantii immaturus and rhizoma atractylodis bowel-relaxing granules

Abstract

The invention discloses a multi-index content determination method of hovenia acerba and rhizoma atractylodis bowel-relaxing granules, and belongs to the technical field of traditional Chinese medicine detection. The invention establishes a high performance liquid chromatography method for simultaneously determining nine components including caffeic acid, chicoric acid, naringin, naringin, hesperidin, neohesperidin, chrysophanol, aurantio-obtusin and atractylenolide I in a preparation, and the content of the components is calculated through a relative correction factor by taking caffeic acid as an internal reference by adopting a quantitative analysis of multi-components by single marker. According to the method, effective detection of the immature bitter orange medicinal material and the immature bitter orange base source is realized, and the immature bitter orange and the sweet orange base source of the traditional Chinese medicine immature bitter orange can be accurately distinguished, so that more comprehensive and accurate quality control of the traditional Chinese medicine is realized, and the stability and consistency of clinical efficacy of the traditional Chinese medicine are guaranteed.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine detection, and relates to a multi-index content determination method of Zhishu Tongbian granules. BACKGROUND

[0002] Zhishu Tongbian granules are a class 1 new drug of traditional Chinese medicine researched by Jianmin Pharmaceutical Group Co., Ltd. The product has been started for phase III clinical research. The product is made of five traditional Chinese medicinal materials of white atractylodes rhizome, dandelion, bitter orange fruit, cassia seed and bitter almond, and is used for treating infantile constipation due to heat. CN 115754061 A discloses a method for simultaneously detecting the contents of five components of caffeic acid, chicoric acid, atractylodes lactone I, orange yellow cassia element and chrysophanol in the product, but there is no index component of bitter orange fruit in the five components, and the quality control of bitter orange fruit medicinal material cannot be realized.

[0003] The origin of traditional Chinese medicinal materials directly affects the quality thereof. Different plants, animals or mineral sources may lead to significant differences in chemical components and physiological activities. Ensuring the true origin of traditional Chinese medicinal materials is crucial for ensuring the efficacy and safety of traditional Chinese medicine. Bitter orange fruit is a multi-origin medicinal material, which is derived from the dried young fruits of Citrus aurantium L. Citrus aurantium and cultivated varieties or sweet oranges Citrus sinensis of Citrus sinensis Osbeck. Bitter orange fruit mainly contains flavonoids, alkaloids and volatile oil components. The Chinese Pharmacopoeia only detects the content of the alkaloid component synephrine, but according to the literature report, there is no significant difference in the content of synephrine between C. aurantium and C. sinensis, and the bitter orange fruit origin cannot be distinguished. Flavonoids are the main effective components of bitter orange fruit, and play an important role in the function and indication of bitter orange fruit. Studies have confirmed that flavonoids in bitter orange fruit have an inhibitory effect on the contraction of isolated intestinal smooth muscle in rats, which is significantly related to the efficacy of the product. It has been confirmed that the difference in bitter orange fruit varieties leads to a large difference in the types and contents of flavonoids.

[0004] Traditional Chinese medicinal materials are the source of traditional Chinese medicine, and their detection is the key to the quality control of traditional Chinese medicine. The prescribed source of bitter orange fruit in Zhishu Tongbian granules is C. aurantium, and the main flavonoids of C. aurantium are hesperidin, naringin, neohesperidin and naringin. The main flavonoids of C. sinensis are hesperidin and naringin. According to the literature report, the lack of naringin and neohesperidin in C. sinensis can improve the gastric emptying rate. Compared with C. aurantium Zhishu pills, C. sinensis Zhishu pills have a weaker effect on gastrointestinal function due to the lack of neohesperidin and naringin, which reveals that the efficacy difference between the two bitter orange fruit origins is related to the difference in flavonoid components (Comparison of chemical components and gastrointestinal function of C. aurantium Zhishu pills and C. sinensis Zhishu pills, Du Zhiyong, 2015). Therefore, the detection of flavonoids in bitter orange fruit is beneficial to the precise control of the origin of bitter orange fruit and the stability of the quality of the product, and is of great significance to the safety and effectiveness of clinical medication. SUMMARY

[0005] The present application aims at the defects of the prior art, and provides a multi-index content determination method of Zhishu Tongbian granules.

[0006] To achieve the above-mentioned purpose, the technical scheme of the present application is as follows: A multi-index content determination method of Zhishu Tongbian granules, the Zhishu Tongbian granules being made of five traditional Chinese medicinal materials of Baizhu, Pugongying, Zhishi, Juemingzi and Kuaxingren, the determination method comprising the following steps: (1) extracting the sample to be tested with methanol, filtering, collecting the extract, adding methanol and pectinase to the residue, performing flash extraction, centrifuging, collecting the supernatant and combining with the aforementioned methanol extract, passing the combined solution through a silica gel column, eluting with a cyclohexane-ethyl acetate mixed solution, dissolving and constant-volumeing the eluate after being evaporated to dryness with methanol, to obtain a test solution; (2) detecting the test solution on a high performance liquid chromatograph, the high performance liquid chromatograph comprising an ultraviolet detector, the ultraviolet detector being set at a detection wavelength of 270-290 nm; the high performance liquid chromatograph having an octadecylsilane-bonded silica gel as a filler; a mobile phase A being a phosphate buffer with a pH of 2.0-2.5, and a mobile phase B being acetonitrile; gradient elution, a column temperature being 25-35℃, and a flow rate being 0.9-1.1 mL / min; (3) adopting a one measurement multiple evaluation method, taking caffeic acid as an internal reference, and calculating the contents of chicoric acid, naringin, naringin, hesperidin, neohesperidin, emodin, orange yellow semen citrulli and atractylodes lactone I through a relative correction factor.

[0007] Preferably, the concentration of the pectinase is 1-3 mg / ml.

[0008] Preferably, the volume ratio of the cyclohexane-ethyl acetate mixed solution is 1:1.

[0009] Preferably, the model of the chromatographic column is NanoChrom ChromCore-C18, 4.6x250mm, 5μm.

[0010] Preferably, the phosphate buffer is a potassium dihydrogen phosphate buffer with a pH of 2.1.

[0011] Preferably, the gradient elution program is as follows:

[0012] Preferably, the medicinal material source of the Zhishi is Citrus aurantium L. Citrus aurantium L. ).

[0013] The beneficial effects of the present application are: The present application solves the problem that the prior art cannot detect the key flavonoid components in Zhishu Tongbian granules, fills in the blank of origin differentiation and quality control, and the four newly added flavonoid components are all characteristic components of Zhishi (Citrus aurantium), which are closely related to efficacy and have clear biological and pharmaceutical basis. The present application also solves the problem of expensive and difficult-to-obtain reference substances, uses caffeic acid as an internal reference, establishes a relative correction factor, and realizes simultaneous determination of multiple components. The method is economical and efficient, and the established determination method has the advantages of high precision, accuracy, and good repeatability. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 : HPLC chromatogram of the test solution obtained by the method of the present application.

[0015] Figure 2 : HPLC chromatogram of the mixed reference solution obtained by the method of the present application.

[0016] Figure 3 : HPLC chromatogram of the test solution obtained by the method of CN 115754061 A.

[0017] Figure 4 : HPLC chromatogram of the mixed reference solution obtained by the method of CN 115754061 A. DETAILED DESCRIPTION

[0018] The present application will be described in detail below through specific examples.

[0019] The Zhishu Tongbian granules used in the following examples were provided by Jianmin Pharmaceutical Group Co., Ltd.

[0020] Prescription: Dandelion 400g, Zhishi 266.67g, Baizhu 1000g, Kuaxingren 200g, Juemingzi 266.67g Preparation method: The above 5 ingredients are added with water for decoction and extraction, the extract is concentrated to a relative density of 1.06-1.08, cooled to room temperature, alcohol is added for alcohol precipitation, the supernatant after alcohol precipitation is reduced pressure concentrated into a thick paste, sugar powder, dextrin, fruit flavor, sucralose are added to the thick paste, mixed, granulated, and 1000 grams of granules are prepared.

[0021] Instruments and reagents Agilent 1260 high performance liquid chromatograph, four-element pump, online vacuum degassing system, automatic sampler, column oven, ultraviolet detector; AB204-E electronic balance (Shanghai Mettler-Toledo Instrument Co., Ltd.). LC-350A ultrasonic wave traditional Chinese medicine processing machine (Jining Lu Chao Ultrasonic Equipment Co., Ltd. in Shandong); JHBE-50T flash extractor (Shanghai Vanli Precision Equipment Co., Ltd.).

[0022] Zhi Shu Tong Bian Granules (10 batches of 250701-04, 250801-03, 250901-03); caffeic acid reference substance (for content determination, China Institute for Drug Control, batch number: 110885-201703, purity: 99.7%); chicoric acid reference substance (for content determination, China Institute for Drug Control, batch number: 111752-202506, purity: 98.7%); naringin reference substance (for characteristic spectrum, China Institute for Drug Control, batch number: 112080-202201); naringin reference substance (for content determination, China Institute for Drug Control, batch number: 110722-202417, purity: 96.1%); hesperidin reference substance (for content determination, China Institute for Drug Control, batch number: 110721-202220, purity: 97.2%); neohesperidin reference substance (for content determination, China Institute for Drug Control, batch number: 111857-202305, purity: 99.6%); chrysophanol reference substance (for content determination, China Institute for Drug Control, batch number: 110796-202423, purity: 99.8%); aurantiamarin reference substance (for content determination, China Institute for Drug Control, batch number: 111900-202407, purity: 98.0%); atractylodin reference substance (for content determination, China Institute for Drug Control, batch number: 111975-201501, purity: 99.9%). Acetonitrile is chromatographic grade, and water is purified water.

[0023] Example 1: Establishment of detection method 1. Preparation of test solution Grind the Zhi Shu Tong Bian Granules to be tested, take about 2.0 g, accurately weigh, add 50 ml of methanol, weigh the weight, ultrasonic for 40 minutes (power 250 W, frequency 40 kHz), shake well, filter, add 25 ml of methanol to the filter residue, add appropriate amount of pectinase (2 mg / ml) into the flash extractor for flash extraction, then centrifuge at 5000 r / min for 15 min, collect the supernatant, combine with the methanol extract, pass through a silica gel column, elute with cyclohexane-ethyl acetate (1:1), evaporate to dryness, dissolve with appropriate amount of methanol and dilute to 50 ml, to obtain the test sample extract.

[0024] 2. Preparation of mixed reference solution Accurately weigh the control samples of caffeic acid, chicoric acid, quercitrin, naringin, hesperidin, neohesperidin, chrysophanol, aurantiamarin and atractylodes lactone I into 50 ml volumetric flask, dissolve in methanol, and prepare a mixed solution containing 7.6 ug, 15.8 ug, 15.6 ug, 65.6 ug, 15.2 ug, 82.8 ug, 5.6 ug, 3.2 ug and 1.8 ug of caffeic acid, chicoric acid, quercitrin, naringin, hesperidin, neohesperidin, chrysophanol, aurantiamarin and atractylodes lactone I per 1 ml, respectively.

[0025] 3. Chromatographic conditions Accurately pipette the test sample solution and the mixed control sample solution into the high performance liquid chromatograph for detection, respectively. The chromatographic column is NanoChrom ChromCore-C 18 , 4.6x250mm, 5um; mobile phase A: potassium dihydrogen phosphate buffer (2.0g of potassium dihydrogen phosphate is dissolved and diluted to 1000ml with water, and the pH is adjusted to 2.1 with appropriate amount of glacial acetic acid); mobile phase B: acetonitrile; injection volume: 10ul; gradient elution; detection wavelength: 280nm; flow rate: 1ml / min; column temperature: 30℃.

[0026] Table 1: Gradient elution program

[0027] 4. Peak identification and separation effect analysis The analysis program of one sample is completed, and the recording time is 0-160min. The chromatograms of the test sample solution and the mixed control sample solution are shown in Figure 1 and Figure 2 respectively. According to the single control sample positioning and peak sequence, the peaks 1-9 in the mixed control sample solution are identified as caffeic acid, chicoric acid, quercitrin, naringin, hesperidin, neohesperidin, chrysophanol, aurantiamarin and atractylodes lactone I, respectively.

[0028] According to the chromatogram of the mixed control sample solution, the peaks 1-9 in the test sample solution are identified as the corresponding components to be tested. The peak time, separation degree and peak area of each component in the test sample solution are shown in the following table 2.

[0029] Table 2: Separation degree and peak area of test sample solution

[0030] The results show that the peak separation degree of the 9 components to be tested is greater than 1.5, and the 9 components can be completely separated, indicating that the detection method is feasible.

[0031] 5. Calculation of relative correction factor (f) and performance analysis (1) Calculation of relative correction factor Prepare a series of mixed reference solution according to the preparation method of the mixed reference solution in Example 1, and accurately take the mixed reference solution of a series of concentrations for injection determination under the above chromatographic conditions, and record the peak area. Take caffeic acid as the internal reference, and calculate the relative correction factors f of chicoric acid, naringin, naringin, hesperidin, neohesperidin, chrysophanol and aurantosid, and atractylodes lactone I, respectively k / s , the formula is f k / s = f k / f s = (C k *A s ) / (Cs*A k ), wherein C k is the concentration of other components, A k is the peak area of other components, Cs is the concentration of the internal reference, and As is the peak area of the internal reference. The results are shown in Table 3.

[0032] Table 3: Relative correction factors of each component

[0033] Note: subscript S is the internal reference caffeic acid, and subscripts 1-8 are chicoric acid, naringin, naringin, hesperidin, neohesperidin, chrysophanol and aurantosid, and atractylodes lactone I.

[0034] (2) Durability test Examine the effects of Waters ARC, Agilent 1260 type high performance liquid chromatograph and Agilent Eclipse XDB-C18, Kromasil 100-5-C18, NanoChrom ChromCore-C18(5μm, 4.6mm×250mm) chromatographic column on f, respectively. The results are shown in Table 4, RSD<2.00%, indicating that different instruments and chromatographic columns have no significant effect on f, and the durability is good.

[0035] Table 4: Effect of instrument and chromatographic column on relative correction factor

[0036] Note: subscript S is the internal reference caffeic acid, and subscripts 1-8 are chicoric acid, naringin, naringin, hesperidin, neohesperidin, chrysophanol and aurantosid, and atractylodes lactone I.

[0037] (3) System suitability test Examine the effects of column temperature 25℃, 30℃, 35℃ and flow rate 0.9, 1.0, 1.1 mL / min on f, respectively. The results are shown in Table 5, RSD<2.0%, indicating that different column temperature and flow rate have no significant effect on f, and the system suitability is good.

[0038] Table 5: Effect of flow rate and column temperature on correction factor f

[0039] 6. Content calculation

[0040] Cs is the internal standard concentration (mg / ml) As is the internal standard peak area A k is the peak area of the component to be tested in the test sample V k is the volume of the test sample (ml) W k is the sample weight of the test sample (g) f k / s is the relative correction factor of the component to be tested in the test sample.

[0041] Example 2: Methodology investigation and validation 1. Linearity 0.96 mg of caffeic acid, 2.01 mg of chicoric acid, 1.89 mg of naringin, 7.24 mg of naringin, 1.95 mg of hesperidin, 8.82 mg of neohesperidin, 0.76 mg of chrysophanol, 0.54 mg of aurantiamarin, 0.26 mg of atractylodin were precisely weighed into 50 ml volumetric flasks, dissolved with methanol and diluted to the mark, and shaken well. 0.5, 1.0, 2.0, 3.0, 4.0, and 10 ml of the above control solution were precisely pipetted into 10 ml volumetric flasks, diluted to the mark with water, shaken well, and determined according to the above chromatographic conditions. Linear regression was performed with the concentration (X) of each component against the peak area (Y) of each component. The results are shown in Table 6.

[0042] Table 6: Linear relationship of 9 components

[0043] 2. Stability The test sample solution (batch number: 250801) was taken in an appropriate amount and determined at 0, 4, 8, 12 h, and 24 h according to the above chromatographic conditions. The RSDs of the chromatographic peak areas of the 9 components were 0.92%, 1.08%, 1.42%, 0.94%, 1.85%, 0.39%, 1.63%, 1.47%, and 0.36%, respectively, indicating that the test sample solution was stable within 48 h.

[0044] 3. Precision The precision of the instrument was good, as indicated by the RSDs of the chromatographic peak areas of the 9 components, which were 0.35%, 0.69%, 1.33%, 0.78%, 0.78%, 1.03%, 1.44%, 0.90% and 0.79% respectively, as measured by continuously injecting the reference solution 6 times under the chromatographic conditions.

[0045] 4. Repeatability Six samples of the same batch (batch number: 250801) were prepared into 6 test sample solutions according to the test sample solution preparation method, and determined under the chromatographic conditions. The contents and RSDs of the 9 components were as shown in the following table, which were 1.44%, 1.68%, 0.72%, 0.29%, 0.57%, 0.45%, 1.09%, 1.14% and 1.29% respectively, indicating that the repeatability of the method was good.

[0046] 5. Recovery rate A total of 6 samples of 1 g each of the known content of Zhizhu Tongbian Granules (batch number: 250801) were precisely weighed, and placed in stoppered conical flasks. The same amount of reference substance mixed solution as the test sample (1.91 mg of caffeic acid, 4.00 mg of chicoric acid, 3.98 mg of naringinioside, 36.35 mg of naringin, 4.00 mg of hesperidin, 35.89 mg of neohesperidin, 1.35 mg of emodin, 0.87 mg of aurantotic acid and 0.45 mg of atractylodin lactone I were precisely weighed, dissolved in methanol and made up to 500 ml) was precisely added to each flask. The weight was determined, and the samples were ultrasonically extracted for 40 minutes (power: 250 W, frequency: 40 kHz). After being shaken and filtered, 4 ml of pectinase was added to the filter residue in a flash extractor for flash extraction. Then, the supernatant was collected by centrifugation at 5000 r / min for 15 min, combined with the methanol extract, passed through a silica gel column, eluted with cyclohexane-ethyl acetate (1:1), evaporated, dissolved in an appropriate amount of methanol and made up to 50 ml, to obtain the test sample extract.

[0047] The recovery rate test results are shown in Table 7.

[0048] Table 7: Recovery rate test results

[0049] 6. Sample content determination Take 10 batches of Zhishutongbian granules, prepare test solution, respectively, precision control product solution and test solution 10 μl injection liquid chromatograph, determination and calculation of content. The results are shown in Table 5 below.

[0050] Table 8: Content of 9 components in 10 batches of samples (mg / g)

[0051] Example 3: comparative test Detection was carried out according to the method of CN 115754061 A.

[0052] Preparation of test solution: take the product under the item of weight difference, mix well, take an appropriate amount, grind finely, take about 1.5 g, accurately weigh, put in a 50 ml conical flask with a stopper, accurately add 25 ml of chloroform, tightly stop, ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, add 25 ml of 50% methanol to the filter residue, ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, combine the two parts of the filtrate, rotary evaporation to evaporate the organic solvent, then pass through a macroporous adsorption resin column (resin type LSA-5B type), first elute with water to remove impurities, the solution is clear, then elute with ethyl acetate 5 times the column volume, collect the ethyl acetate eluate, concentrate and evaporate to dryness, then dissolve in 50% methanol to 50 ml, and you get it.

[0053] Preparation of mixed control solution: as in Example 1.

[0054] Chromatographic conditions: precision control product solution and control mixed solution, on high performance liquid chromatograph for detection, chromatographic column: NanoChrom ChromCore-C18, 4.6x250mm, 5μm; mobile phase A: 0.1% phosphoric acid aqueous solution; mobile phase B: acetonitrile (containing 1% isopropanol); injection volume 10 μl; gradient elution; detection wavelength 245 nm; flow rate 1 ml / min; column temperature 30℃.

[0055] Gradient elution program:

[0056] Complete 1 sample analysis program, record time 0-75 min, test solution and control mixed solution chromatogram respectively as shown in Figure 3 and Figure 4 .

[0057] According to the peak time and single reference standard positioning, peaks 1-8 in the chromatogram of the mixed reference standard solution were identified as coffee acid, chicoric acid, naringin, hesperidin, neohesperidin, atractylodes lactone I, aurantosid, and chrysophanol, respectively. Naringoside in the mixed reference standard solution could not be detected by the method of CN115754061 A. At the same time, according to the chromatogram of the mixed reference standard solution, peaks 1-6 in the test sample solution were identified as coffee acid, chicoric acid, naringin, atractylodes lactone I, aurantosid, and chrysophanol, respectively, while naringoside, hesperidin, and neohesperidin could not be detected in the chromatogram of the test sample solution.

[0058] The peak time, separation degree, and peak area of each component in the test sample solution are shown in Table 9.

[0059] Table 9: Separation degree and peak area of the test sample solution

[0060] The results show that naringoside, hesperidin, and neohesperidin could not be detected by the method of CN115754061 A, and the peak separation degree of naringin was low and the peak area was small. Therefore, the method could not simultaneously detect the nine components in Zhizhushu Tongbian Granules.

Claims

1. A method for determining the content of multiple indicators in Zhishu Tongbian granules, wherein the granules are made from five Chinese medicinal herbs: Atractylodes macrocephala, Taraxacum mongolicum, Citrus aurantium, Cassia tora, and Prunus armeniaca, characterized in that... The determination method includes the following steps: (1) Extract the sample to be tested with methanol, filter it, collect the extract, add methanol and pectinase to the filter residue, perform flash extraction, centrifuge, collect the supernatant and combine it with the methanol extract, pass the combined solution through a silica gel column, elute with a cyclohexane-ethyl acetate mixture, evaporate the eluent to dryness, dissolve it in methanol and make up to volume to obtain the test solution. (2) The test solution is detected by high performance liquid chromatography (HPLC), wherein the HPLC contains an ultraviolet detector and the detection wavelength of the ultraviolet detector is set to 270-290 nm; the HPLC column is packed with octadecylsilane-bonded silica gel; the mobile phase A is phosphate buffer solution with pH 2.0-2.5 and the mobile phase B is acetonitrile; gradient elution is performed, the column temperature is 25-35℃ and the flow rate is 0.9-1.1 mL / min; (3) Using the one-test-multiple-evaluation method, caffeic acid was used as an internal reference, and the contents of chicoric acid, naringin, naringin, hesperidin, neohesperidin, rhein, cassia seed extract and atractylodes lactone I were calculated by relative correction factors.

2. The method according to claim 1, characterized in that: The concentration of the pectinase is 1-3 mg / ml.

3. The method according to claim 1, characterized in that: The volume ratio of the cyclohexane-ethyl acetate mixed solution is 1:

1.

4. The method according to claim 1, characterized in that: The chromatographic column is a NanoChromChromCore-C18, 4.6×250mm, 5μm.

5. The method according to claim 1, characterized in that: The phosphate buffer solution is potassium dihydrogen phosphate buffer solution with a pH of 2.

1.

6. The method according to claim 1, characterized in that, The gradient elution procedure is as follows: 。 7. The method according to claim 1, characterized in that: The medicinal material source of the Citrus aurantium is sour orange ( Citrus aurantium L. ).

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