Method for determining contents of seven active ingredients in ancient classical famous prescription Xiangsha Liujunzi decoction

Abstract

The invention belongs to the field of analysis of traditional Chinese medicine preparations, and particularly relates to a method for determining the contents of seven active ingredients in Xiangsha Liujunzi decoction of an ancient classical famous prescription. According to the method, the contents of liquiritin, apiose liquiritin, narirutin, hesperidin, nobiletin, costunolide and dehydrocostus lactone in the Xiangsha Liujunzi decoction are simultaneously separated and measured under the same experimental condition by utilizing a high performance liquid chromatography (HPLC). Experimental results show that the method for simultaneously determining the contents of a plurality of different index components in the preparation through HPLC is simple, rapid, accurate, reliable and good in repeatability, stability and the like, and can be used as a quality control method of the preparation.

Description

Technical Field

[0001] The invention belongs to the field of analysis of traditional Chinese medicine preparations, and particularly relates to a method for determining the contents of seven active ingredients in Xiangsha Liujunzi Decoction, an ancient classic prescription. Background Art

[0002] Xiangsha Liujunzi Decoction comes from Ke Yunbo's "Ancient and Modern Famous Doctors' Prescriptions", which can not only support the spleen and stomach qi, but also eliminate phlegm evil. "Ancient and Modern Famous Doctors' Prescriptions" records: It treats qi deficiency, swelling, phlegm and fluid accumulation, spleen and stomach disharmony, and various symptoms. Ginseng (1 qian), Atractylodes (2 qian), Poria (2 qian), Licorice (7 fen), Tangerine Peel (8 fen), Pinellia (1 qian), Amomum (8 fen), and ginger on the wood 2 qian, decocted in water and taken orally. Ke Yunbo said: The classics say: "The strong will recover when the qi moves, and the timid will become sick." People are born in the exchange of qi, and the vitality is always based on stomach qi. Food enters the yin, and the qi grows in the yang, circulating day and night, and circulating inside and outside. If the breath is not moving, there will be accumulation, or distension and loss of food, or phlegm and retention of fluid, so the muscles will be thin, wheezing, coughing, vomiting, and various symptoms will arise, and the spirit will be completely lost. The four gentlemen's qi points are generally kidney-benefiting.

[0003] (Qing Dynasty) Wu Qian compiled; Shan Tianji sorted. The fifth volume of the "Golden Mirror of the Medical Ancestors" records Xiangsha Liujunzi Decoction: ginseng (1 qian), white atractylodes (fried in soil, 2 qian), poria (1 qian), licorice (roasted, 5 fen), patchouli (or costus root, 1 qian), tangerine peel (1 qian), pinellia (processed, 1.5 qian), amomum (5 fen), the above eight ingredients, three slices of ginger, decocted in water and taken orally. Sijunzi Decoction with tangerine peel is called Yigongsan, which is suitable for patients with spleen deficiency and qi stagnation after ulceration. Sijunzi Decoction minus poria and adding dried ginger is called Lizhong Decoction, which is suitable for patients with spleen deficiency and cold stagnation after ulceration. Qi deficiency leads to yang deficiency, and yang deficiency produces cold, so warm and hot flavors are added to qi-tonifying medicines. Sijunzi Decoction with tangerine peel and pinellia is called Liujunzi Decoction, which is suitable for patients with qi deficiency and phlegm after ulceration. Liujunzi Decoction with Patchouli (or Costusroot) and Amomum villosum is called Xiangsha Liujunzi Decoction. It is suitable for those with stomach deficiency, phlegm and vomiting after ulcer.

[0004] Li Yuchuan et al. Study on the chemical components of the ethyl acetate fraction of Xiangsha Liujunzi Decoction decoction, Journal of Chinese Experimental Traditional Chinese Medicine, 2013, Vol. 19, No. 20, pp. 112-114. The traditional decoction method was used for extraction, and silica gel column chromatography and Sephadex LH-20 were used for separation repeatedly, and the structure was identified by spectral analysis. Results: 9 compounds were separated and identified. However, there are no patents or literature reports on the use of HPLC to simultaneously determine the content of multiple components in Xiangsha Liujunzi Decoction.

[0005] The present invention discloses a method for simultaneously determining the contents of seven components in Xiangsha Liujunzi Decoction by using HPLC, which can achieve accurate quantification of the seven active ingredients in the preparation and comprehensive evaluation and control of the preparation quality, ensure the stability of product quality and the safety and effectiveness of clinical medication, and provide important data reference for improving the quality standard of Xiangsha Liujunzi Decoction. The HPLC wavelength conversion wavelength-based determination method established by the present invention is simple, rapid, accurate, reliable, and has good repeatability and stability, and can be used as a quality control method for the preparation. Summary of the invention

[0006] In view of the defects in the prior art, the present invention aims to provide an HPLC wavelength switching method for simultaneously determining the contents of liquiritin, apigenin, naringin, hesperidin, nobiletin, costunolide and dehydrocostuslactone in Xiangsha Liujunzi Decoction. The method is simple, stable and reproducible, and can be used for quality control of the preparation.

[0007] The invention aims to provide a method for determining the multi-component content of Xiangsha Liujunzi Decoction, a classic ancient prescription. The Xiangsha Liujunzi Decoction is prepared from eight Chinese medicinal herbs, including Atractylodes macrocephala, Pinellia tuber, Licorice root, Tangerine peel, Ginseng, Poria cocos, Costusroot and Amomum villosum, through soaking, decocting and filtering.

[0008] The present invention adopts HPLC wavelength switching method to simultaneously determine the contents of liquiritin, apigenin, naringin, hesperidin, nobiletin, costusolide and dehydrocostusolide in the preparation, so as to achieve comprehensive evaluation and control of the quality of Xiangsha Liujunzi Decoction, thereby providing an accurate basis for authenticity identification and intrinsic quality detection of Xiangsha Liujunzi Decoction.

[0009] The specific steps include: 1) Preparation of standard solution Take appropriate amount of licorice glycoside, apigenin, naringenin, hesperidin, nobiletin, costusinol, and dehydrocostusinol reference substances, accurately weigh them, and prepare each single standard stock solution with methanol. Accurately pipette 1 ml of each single standard stock solution and dilute to 10 ml to obtain a mixed standard solution.

[0010] 2) Preparation of test solution Preparation of Xiangsha Liujunzi Decoction According to the prescription of Xiangsha Liujunzi Decoction, weigh the raw herbs (fried with bran) Atractylodes macrocephala, (ginger) Pinellia, (raw) Licorice, Tangerine peel, Ginseng, Poria, Aucklandia, Amomum villosum, soak all the raw herbs for 30 minutes before decoction, and Aucklandia villosum and Aucklandia lappa should also be soaked for 30 minutes before decoction. When the first decoction is about to be decocted to the predetermined amount, add them and decoct for 5-10 minutes. Decoction twice, the first decoction is 4 times the amount of water, boiled over high heat and then turned to low heat, decoct for 20-30 minutes; the second decoction is 3 times the amount of water, boiled over high heat and then turned to low heat, decoct for 10-20 minutes, filter the filtrates of both times with four layers of gauze, and combine the two filtrates to obtain the decoction.

[0011] Preparation of freeze-dried powder of Xiangsha Liujun Decoction The decoction obtained above is centrifuged, filtered to remove the drug residue, microfiltered, and then fixed to volume. The decoction is concentrated under reduced pressure in a rotary evaporator, fixed to volume, and the solution is divided into evaporating dishes and freeze-dried to prepare freeze-dried powder.

[0012] Weigh an appropriate amount of freeze-dried powder, re-dissolve, make up to volume, shake well, filter through a microporous filter membrane, and take the filtrate to obtain the product.

[0013] 3) HPLC chromatographic conditions: the chromatographic column is a C18 chromatographic column, the mobile phase A is acetonitrile, the mobile phase B is an aqueous phosphoric acid solution, the gradient elution, and the detection wavelength is 225-330 nm.

[0014] 4) Determination method: Accurately pipette the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine according to the chromatographic conditions described in step 3).

[0015] Preferably, in the step 2) of preparing the freeze-dried powder, the solvent for volume adjustment and reconstitution is a 50-70% by mass methanol aqueous solution, more preferably a 60% by mass methanol aqueous solution.

[0016] Preferably, the filter membrane in step 2) is a 0.22 μm microporous filter membrane.

[0017] Preferably, the chromatographic column in step 3) is an XB-C18 chromatographic column with specifications of 4.6×250 mm and 5 μm.

[0018] Preferably, the mobile phase B in step 3) is 0.05-0.2% phosphoric acid aqueous solution, more preferably 0.1% phosphoric acid aqueous solution.

[0019] Preferably, in step 3) gradient elution, the initial ratio is 18:22; the gradient elution process is as follows: Preferably, the flow rate in the chromatography condition of step 3) is 0.8 to 1.2 mL / min, and more preferably 1.0 mL / min.

[0020] Preferably, the wavelength switching procedure in step 3) is: Preferably, the column temperature of the chromatography condition in step 3) is 23-27°C, more preferably 25°C.

[0021] Compared with the prior art, the present invention has the following advantages: 1. The present invention establishes a quality detection method for Xiangsha Liujunzi Decoction based on the HPLC wavelength switching method, which realizes one test and multiple evaluations, improves the detection efficiency, saves reagents, consumables and labor, reduces costs, increases efficiency, and is energy-saving and environmentally friendly.

[0022] 2. The present invention simultaneously and quantitatively determines the contents of seven active ingredients in Xiangsha Liujunzi Decoction under the same experimental conditions. The determination method of the present invention can accurately and comprehensively evaluate the quality of Xiangsha Liujunzi Decoction. It is simple and rapid, and has good stability and repeatability, which is conducive to improving the quality standard of Xiangsha Liujunzi Decoction. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the HPLC chromatogram of the mixed reference solution.

[0024] Figure 2 It is the HPLC chromatogram of the test solution. DETAILED DESCRIPTION

[0025] The beneficial effects of the present invention are further described below by way of examples. It should be correctly understood that the examples of the present invention are provided only for the purpose of illustrating the present invention, rather than limiting the present invention. Therefore, simple improvements to the present invention under the premise of the method of the present invention all fall within the scope of protection claimed in the present invention.

[0026] The materials used in the experiment can be purchased or prepared according to existing public technologies; In the following embodiments, various processes and methods not described in detail are conventional methods well known in the art.

[0027] 1. Apparatus and Materials 1.1 Instrument Agilent-1260 high performance liquid chromatograph (Agilent) with UV detector; XS105DU electronic balance, ME 204E electronic balance (Mettler-Toledo); KQ-300DE digitally controlled ultrasonic instrument (Kunshan Ultrasonic Instrument Co., Ltd.), freeze dryer, water purifier.

[0028] 1.2 Materials Eight kinds of crude drugs, including ginseng, Poria, licorice, Atractylodes macrocephala, dried orange peel, Pinellia tuber, costusroot and Amomum villosum, were purchased from the Zhejiang University of Chinese Medicine's Decoction Piece Company. Reference substances, including liquiritin, apigenin, naringenin, hesperidin, nobiletin, costusolide and dehydrocostusolide, were purchased from Chengdu Munster Technology Co., Ltd.; methanol and acetonitrile (chromatographic grade, Thermo Fisher); purified water was used for decoction, ultrapure water was used for mobile phase preparation, and other reagents such as phosphoric acid were of analytical grade.

[0029] 2. Methods and Results 2.1 Chromatographic conditions An XB-C18 chromatographic column (4.6 mm×250 mm, 5 μm) was used, with acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, with an initial ratio of 18:22; the elution gradient was (0-8 min, 18% A; 8-20 min, 18%-23% A; 20-35 min, 23%-40% A; 35-50 min, 40%-60% A; 50-65 min, 60%-75% A; 65-70 min, 75%-12% A), with a flow rate of 1 ml / min, a column temperature of 25°C, and an injection volume of 10 μl.

[0030] Detection wavelength: 237nm (0-20min to detect licorice glycosides, liquiritin and naringenin); 283nm (20-33min to detect hesperidin); 330nm (33-50min to detect nobiletin); 225nm (50-68min, to detect costunolide and dehydrocostuslactone) 68-70min (237nm).

[0031] 2.2 Solution preparation 2.2.1 Preparation of standard solution Take appropriate amount of licorice glycoside, apigenin, naringenin, hesperidin, nobiletin, costusinol, and dehydrocostusinol reference substances, accurately weigh them, and prepare each single standard stock solution with methanol. Accurately pipette 1 ml of each single standard stock solution and dilute to 10 ml to obtain a mixed standard solution.

[0032] 2.2.2 Preparation of test solution 2.2.2.1 Preparation of Xiangsha Liujunzi Decoction According to the prescription of Xiangsha Liujunzi Decoction, weigh 12 g of raw herbs (fried with bran) Atractylodes macrocephala, 9 g of (ginger) Pinellia, 6 g of (raw) Licorice, 9 g of dried orange peel, 9 g of ginseng, 15 g of Poria, 6 g of costus root, and 3 g of Amomum villosum. Soak all the raw herbs for 30 min before decoction. Amomum villosum and costus root should also be soaked for 30 min before decoction. When the first decoction is about to be decocted to the predetermined amount, add them and decoct for 5 min. Decoction twice, the first decoction is 4 times the amount of water, boiled over high heat and then turned to low heat, decoct for 25 min; the second decoction is 3 times the amount of water, boiled over high heat and then turned to low heat, decoct for 15 min, filter the filtrates of both times with four layers of gauze, and combine the two filtrates to obtain the decoction.

[0033] 2.2.2.2 Preparation of freeze-dried powder of Xiangsha Liujun Decoction Take the decoction obtained under "2.2.2.1", centrifuge at 5500 rpm for 20 min, remove the residue by suction filtration, microfilter, and then adjust the volume to 250 ml. Concentrate under reduced pressure in a rotary evaporator at 55℃ and 50 rpm, adjust the volume to 100 ml, divide into evaporating dishes, and freeze-dry to prepare freeze-dried powder.

[0034] Weigh an appropriate amount of freeze-dried powder, dissolve it in 60% methanol, make up to 10 ml, shake well, filter through a 0.22μm microporous filter membrane, and take the filtrate to obtain the product.

[0035] 2.2.3 Preparation of negative test solution Prepare decoctions lacking dried tangerine peel, licorice and costus root according to the proportion of each drug in the prescription of Xiangsha Liujunzi Decoction, and prepare negative test solutions lacking dried tangerine peel, licorice and costus root according to the method under "2.2.2".

[0036] 2.2.4 System suitability and specificity tests The reference solution, the test solution and the negative test solution lacking dried orange peel, licorice and costus root were determined and analyzed according to the chromatographic conditions under "2.1". The 7 components, including liquiritin, apigenin, naringenin, hesperidin, nobiletin, costunolide and dehydrocostusolide, were well separated from the adjacent peaks, and the separation degree was greater than 1.5; the chromatographic peaks of other components in the negative sample did not interfere with the 7 components, indicating that the analytical method has good specificity. See the chromatogram for details. Figure 1 (reference substance), Figure 2 (Test sample).

[0037] 2.2.5 Investigation of linear relationship Accurately pipette 1.2, 1.0, 0.8, 0.6, 0.4, 0.2 and 0.1 ml of each single standard stock solution, dilute to 10 ml, and obtain a 120%-10% mixed standard solution. Perform the determination according to the chromatographic conditions under "2.1" and draw a standard curve, where the abscissa is the concentration of the reference substance (X, μg / ml) and the ordinate is the peak area (Y). The linear range, linear regression equation and correlation coefficient of the 7 components are shown in Table 1. The results show that the 7 components have a good linear relationship within their respective concentration ranges.

[0038] Table 1 Linear range, linear regression equation and correlation coefficient of 7 components 2.2.6 Limit of detection and limit of quantification The method used in the experiment for the detection limit and quantification limit is based on the standard deviation of the response value and the slope of the standard curve. The calculation formula is LOD=3.3δ / S. Where LOD: detection limit; δ: deviation of the response value; S: slope of the standard curve. δ can be measured by the following methods: ① standard deviation of the blank value; ② residual standard deviation of the standard curve or standard deviation of the intercept.

[0039] The method used to determine the standard deviation δ of the response value in this experiment was the residual standard deviation of the standard curve. The detection limits and quantification limits of the seven components are shown in Table 2. The results show that the seven components are within the range of the detection limit and quantification limit, indicating that they can be accurately quantified.

[0040] Table 2 Detection limits and quantification limits of 7 components 2.2.7 Stability test The same sample was placed at room temperature for 0, 3.5, 7, 10.5, 14, 24, and 48 hours, and then determined according to the chromatographic conditions under "2.1". The results showed that the contents of liquiritin, apigenin, naringenin, hesperidin, nobiletin, costunolide, and dehydrocostuslactone were 1.96%, 2.37%, 3.26%, 1.50%, 2.88%, 2.43%, and 1.67%, respectively, indicating that each component can remain stable within 48 hours at room temperature. The RSD values ​​of the seven components are shown in Table 3.

[0041] Table 3 Test results of stability of test products 2.2.8 Repeatability test Six samples of Xiangsha Liujunzi Decoction test solution were prepared in parallel according to the method in "2.2.2" and determined according to the chromatographic conditions in "2.1". The results showed that the RSD values ​​of liquiritin, apigenin, naringenin, hesperidin, nobiletin, costus lanolin, and dehydrocostus lanolin were 1.72%, 2.50%, 2.15%, 0.65%, 1.10%, 1.50%, and 1.68%, respectively, which met the requirements of the pharmacopoeia within their respective concentration ranges, indicating that the methodology had good repeatability. The repeatability results are shown in Table 4.

[0042] Table 4 Repeatability test results 2.2.9 Accuracy test Take 6 portions of the test sample of known concentration, about 0.2g each, in a 10ml volumetric flask, add 60% methanol ultrasonically to dissolve, and dilute to the mark to obtain the test solution. Use a pipette to accurately transfer 1ml of the test solution into a centrifuge tube, add an equal volume of a mixed standard solution of equal concentration with a pipette, mix evenly, and obtain the sample recovery test solution, inject the liquid phase, determine the content, and calculate the sample recovery rate. The accuracy results are shown in Table 5. The average sample recovery rates of the seven index components were 99.86%, 101.92%, 98.29%, 96.47%, 95.00%, 95.61%, and 100.34%, respectively. Within their respective content ranges, the RSD values ​​of the sample recovery rates met the requirements, indicating that the accuracy of the methodology is good.

[0043] Table 5 Accuracy test 2.2.10 Durability test The test method was investigated to determine whether it can accurately quantify when there are slight changes in flow rate, temperature and mobile phase ratio. The experiment changed the column temperature by ±2°C, the flow rate by ±0.2mL / min, and the phosphoric acid content of the mobile phase. The experimental results are shown in Tables 6-8. The experimental results show that the methodology can still accurately quantify when there are slight changes in flow rate, column temperature and mobile phase, indicating that the methodology has good durability.

[0044] Table 6 Durability - Flow Rate Change Table 7 Durability - Column Temperature Change Table 8 Durability - Mobile Phase Change 2.2.11 Sample content determination Instead of decoction, prepare the test solution according to "2.2.2", detect according to the chromatographic conditions under "2.1", and calculate the content of each component by external standard method. The results are shown in Table 9.

[0045] Table 9 Determination of content of different batches of decoction The HPLC method established in the present invention can quantitatively determine the contents of seven effective ingredients in Xiangsha Liujunzi Decoction at the same time, has the characteristics of simple operation, good precision and high repeatability, and can comprehensively and more effectively control the quality of the medicine.

Claims

1. A method for determining the contents of seven active ingredients in the ancient classic prescription Xiangsha Liujunzi Decoction, characterized in that: The method adopts HPLC wavelength switching method to simultaneously determine the contents of liquiritin, apigenin, naringin, hesperidin, nobiletin, costusolide and dehydrocostusolide in the preparation; the method comprises the following steps: 1) Preparation of standard solution Take appropriate amount of licorice glycoside, apigenin, naringenin, hesperidin, nobiletin, costusinol, and dehydrocostusinol reference substances, weigh them accurately, and prepare each single standard stock solution with methanol. Accurately pipette 1 ml of each single standard stock solution and dilute to 10 ml to obtain a mixed standard solution; 2) Preparation of test solution Preparation of freeze-dried powder of Xiangsha Liujun Decoction Take Xiangsha Liujunzi decoction, centrifuge, filter to remove the residue, microfilter, and then fix the volume; reduce the pressure and concentrate in a rotary evaporator, fix the volume, divide the solution into evaporating dishes, and freeze-dry to prepare freeze-dried powder; Weigh an appropriate amount of freeze-dried powder, re-dissolve, adjust volume, shake well, filter through a microporous filter membrane, and take the filtrate to obtain; 3) HPLC chromatographic conditions: the chromatographic column is a C18 column, the flow rate is 0.8-1.2 mL / min, the column temperature is 23-27°C, the mobile phase A is acetonitrile, the mobile phase B is phosphoric acid aqueous solution, the gradient elution, the detection wavelength is 225-330 nm; the initial ratio of acetonitrile: phosphoric acid aqueous solution is 18:22; the gradient elution process is as follows: ； The wavelength switching procedure is: ； 4) Determination method: Accurately pipette the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine according to the chromatographic conditions described in step 3).

2. The method for determining the content according to claim 1, characterized in that: In the step 2) of preparing the freeze-dried powder, the solvent for volume adjustment and re-dissolution is a methanol aqueous solution with a mass fraction of 50-70%.

3. The method for determining the content according to claim 1, characterized in that: The filter membrane in step 2) is a 0.22 μm microporous filter membrane.

4. The method for determining the content according to claim 1, characterized in that: The chromatographic column in step 3) is an XB-C18 chromatographic column with specifications of 4.6×250 mm and 5 μm.

5. The method for determining the content according to claim 1, characterized in that: In the step 3), the mobile phase B is a 0.05-0.2% phosphoric acid aqueous solution.

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