A method for determining the contents of seven active ingredients in the ancient classic prescription Xiangsha Liujunzi Decoction

Abstract

The present invention belongs to the field of analysis of traditional Chinese medicine preparations, and specifically relates to a method for determining the contents of seven active ingredients in the classic ancient prescription Xiangsha Liujunzi Decoction. This method utilizes high-performance liquid chromatography (HPLC) under the same experimental conditions to simultaneously separate and determine the contents of liquiritin, apigenin, naringin, hesperidin, nobiletin, costunolide, and dehydrocostuslactone in Xiangsha Liujunzi Decoction. Experimental results demonstrate that the HPLC method established herein for simultaneously determining the contents of multiple different index components in a preparation is simple, rapid, accurate, reliable, and exhibits good repeatability and stability, making it suitable for use as a quality control method for the preparation.

Description

Technical Field

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

[0002] Xiangsha Liujunzi Decoction, from Ke Yunbo's "Ancient and Modern Famous Doctors' Prescriptions," can both strengthen the spleen and stomach qi and dispel phlegm. "Ancient and Modern Famous Doctors' Prescriptions" states: "It treats qi deficiency, swelling, phlegm and fluid accumulation, spleen-stomach disharmony, and various symptoms." Ingredients: ginseng (1 qian), white atractylodes (2 qian), poria (2 qian), licorice (7 fen), dried tangerine peel (8 fen), pinellia (1 qian), amomum (8 fen), and ginger (2 qian). Boil in water and drink. Ke Yunbo commented: "The classics state: 'Strong men recover when their qi flows, while weak men become ill when their qi is retained.'" Humans are born from qi, and vital energy is ultimately rooted in stomach qi. Food enters the yin, nourishes the yang, and circulates day and night, circulating internally and externally. If the breath is disrupted, accumulation can occur, leading to distension and loss of appetite, or the generation of phlegm and fluid retention, resulting in muscle wasting, wheezing, coughing, vomiting, and other symptoms, all of which can lead to a loss of spiritual function. The Four Gentlemen Qi are generally beneficial to the kidneys.

[0003] Compiled by Wu Qian (Qing Dynasty); edited by Shan Tianji. Volume 5 of the Golden Mirror of the Medical Ancestral Collection describes Xiangsha Liujunzi Decoction: ginseng (1 qian), white atractylodes (roasted in earth, 2 qian), poria (1 qian), licorice (roasted, 5 fen), patchouli (or costus root, 1 qian), tangerine peel (1 qian), pinellia (processed, 1.5 qian), and amomum villosum (5 fen). These eight ingredients, along with three slices of ginger, are decocted in water and taken orally. Sijunzi Decoction with tangerine peel is called Yigongsan and is suitable for those with spleen deficiency and qi stagnation after ulcers. Sijunzi Decoction with the addition of poria and dried ginger is called Lizhong Decoction and is suitable for those with spleen deficiency and cold stagnation after ulcers. Qi deficiency leads to yang deficiency, and yang deficiency generates cold, so warming and heating properties are added to qi-tonifying herbs. Sijunzi Decoction with tangerine peel and pinellia is called Liujunzi Decoction and is suitable for those with qi deficiency and phlegm after ulcers. Liujunzi Decoction with Patchouli (or Costus Root) and Amomum villosum is called Xiangsha Liujunzi Decoction. It is suitable for those with stomach deficiency, phlegm and fluid vomiting after ulcer.

[0004] Li Yuchuan et al. (Study on the Chemical Constituents of the Ethyl Acetate Fraction of the Decoction of Xiangsha Liujunzi Decoction), Journal of Experimental Traditional Chinese Medicine, Vol. 19, No. 20, 2013, pp. 112-114. The decoction was extracted using a traditional decoction method, separated by repeated silica gel column chromatography and Sephadex LH-20 chromatography, and structurally identified by spectral analysis. Results: Nine compounds were isolated and identified. However, there are no patents or published reports on the simultaneous determination of multiple components in Xiangsha Liujunzi Decoction using HPLC.

[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 components in the preparation and comprehensive evaluation and control of the preparation quality, thereby ensuring the stability of product quality and the safety and effectiveness of clinical medication, and providing 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] To address the shortcomings of the prior art, the present invention provides an HPLC wavelength-switching method for simultaneously determining the contents of liquiritin, apigenin, naringin, hesperidin, nobiletin, costunolide, and dehydrocostuslactone in Xiangsha Liujunzi Decoction. This method is simple, stable, and reproducible, and can be used for quality control of this 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 ternata, Licorice root, Tangerine peel, Ginseng, Poria cocos, Costus root and Amomum villosum, through soaking, decocting and filtering.

[0008] The present invention uses an HPLC wavelength switching method to simultaneously determine the contents of liquiritin, apigenin, naringin, hesperidin, nobiletin, costunolide, and dehydrocostuslactone in the preparation. This method enables comprehensive quality evaluation and control of Xiangsha Liujunzi Decoction, providing an accurate basis for authenticity identification and intrinsic quality testing of Xiangsha Liujunzi Decoction.

[0009] The specific steps include:

[0010] 1) Preparation of standard solution

[0011] Take appropriate amount of liquiritin, apigenin, naringenin, hesperidin, nobiletin, costunolide and dehydrocostuslactone reference substances, accurately weigh them, and prepare the single standard stock solution with methanol. Accurately pipette 1 ml of each single standard stock solution and dilute to 10 ml to obtain the mixed standard solution.

[0012] 2) Preparation of test solution

[0013] Preparation of Xiangsha Liujunzi Decoction

[0014] According to the recipe for Xiangsha Liujunzi Decoction, weigh the raw herbs (fried with bran) Atractylodes macrocephala, (ginger) Pinellia ternata, (raw) Licorice root, dried tangerine peel, ginseng, Poria cocos, costus root, and Amomum villosum. Soak all the herbs for 30 minutes before decoction. Amomum villosum and costus root should also be soaked for 30 minutes before decoction. When the first decoction is almost reduced to the desired amount, add them and decoct for 5-10 minutes. Decoction twice: first, with 4 times the amount of water, boil over high heat, then reduce to simmer, and simmer for 20-30 minutes; second, with 3 times the amount of water, boil over high heat, then reduce to simmer, and simmer for 10-20 minutes. Filter both filtrates through four layers of gauze and combine the two filtrates.

[0015] Preparation of freeze-dried powder of Xiangsha Liujun Decoction

[0016] The decoction obtained above was centrifuged, filtered to remove the medicinal residue, microfiltered, and then fixed to volume. The solution was concentrated under reduced pressure in a rotary evaporator, fixed to volume, and the solution was divided into evaporating dishes and freeze-dried to prepare freeze-dried powder.

[0017] Weigh an appropriate amount of freeze-dried powder, re-dissolve, adjust to volume, shake well, filter through a microporous filter membrane, and take the filtrate to obtain the product.

[0018] 3) HPLC conditions: C18 column, acetonitrile as mobile phase A, phosphoric acid as mobile phase B, gradient elution, detection wavelength 225-330 nm.

[0019] 4) Determination method: Accurately aspirate the reference solution and the test solution, inject them into the liquid chromatograph, and determine them according to the chromatographic conditions described in step 3).

[0020] Preferably, in the step 2) of preparing the lyophilized 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.

[0021] Preferably, the filter membrane in step 2) is a 0.22 μm microporous filter membrane.

[0022] Preferably, the chromatographic column in step 3) is an XB-C18 chromatographic column with a specification of 4.6×250 mm and 5 μm.

[0023] Preferably, the mobile phase B in step 3) is a 0.05-0.2% phosphoric acid aqueous solution, more preferably a 0.1% phosphoric acid aqueous solution.

[0024] Preferably, in step 3) gradient elution, the initial ratio is 18:22; the gradient elution process is as follows:

[0025]

[0026] Preferably, the flow rate in the chromatography condition of step 3) is 0.8 to 1.2 mL / min, more preferably 1.0 mL / min.

[0027] Preferably, the wavelength switching procedure in step 3) is:

[0028]

[0029] Preferably, the column temperature of the chromatography condition in step 3) is 23-27°C, more preferably 25°C.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] 1. The present invention establishes a quality detection method for Xiangsha Liujunzi Decoction based on the HPLC wavelength switching method, which realizes multiple evaluations with one test, improves the detection efficiency, saves reagents, consumables and labor, reduces costs, increases efficiency, and is energy-saving and environmentally friendly.

[0032] 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, rapid, stable and repeatable, and is conducive to improving the quality standard of Xiangsha Liujunzi Decoction. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the HPLC chromatogram of the mixed reference solution.

[0034] Figure 2 is the HPLC chromatogram of the test solution. DETAILED DESCRIPTION

[0035] The beneficial effects of the present invention are further described below by way of examples. It should be understood that the examples are provided for illustrative purposes only and are not intended to limit the present invention. Therefore, simple modifications to the present invention based on the method of the present invention fall within the scope of protection claimed in the present invention.

[0036] The materials used in the experiment can be purchased or prepared according to existing public technology;

[0037] In the following embodiments, various processes and methods not described in detail are conventional methods well known in the art.

[0038] 1. Instruments and Materials

[0039] 1.1 Instrument

[0040] Agilent-1260 high-performance liquid chromatograph (Agilent) with UV detector; XS105DU electronic balance and ME 204E electronic balance (Mettler-Toledo); KQ-300DE digitally controlled ultrasonic analyzer (Kunshan Ultrasonic Instrument Co., Ltd.); freeze dryer and water purifier.

[0041] 1.2 Materials

[0042] Eight crude drugs (ginseng, Poria cocos, licorice, Atractylodes macrocephala, dried tangerine peel, Pinellia ternata, costus root, and Amomum villosum) were purchased from the Zhejiang University of Chinese Medicine's Medicinal Pieces Company. Reference substances (liquiritin, apigenin, naringenin, hesperidin, nobiletin, costunolide, and dehydrocostusolide) were purchased from Chengdu Munster Technology Co., Ltd. Methanol and acetonitrile (chromatographic grade, Thermo Fisher Scientific) were used. Purified water was used for decoction, ultrapure water was used for mobile phase preparation, and all other reagents, including phosphoric acid, were of analytical grade.

[0043] 2. Methods and Results

[0044] 2.1 Chromatographic conditions

[0045] An XB-C18 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 at 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.

[0046] Detection wavelength: 237nm (0-20min, detection of apigenin, liquiritin and naringenin); 283nm (20-33min, detection of hesperidin); 330nm (33-50min, detection of nobiletin); 225nm (50-68min, detection of costunolide and dehydrocostuslactone); 68-70min (237nm).

[0047] 2.2 Solution preparation

[0048] 2.2.1 Preparation of standard solution

[0049] Take appropriate amount of liquiritin, apigenin, naringenin, hesperidin, nobiletin, costunolide and dehydrocostuslactone reference substances, accurately weigh them, and prepare the single standard stock solution with methanol. Accurately pipette 1 ml of each single standard stock solution and dilute to 10 ml to obtain the mixed standard solution.

[0050] 2.2.2 Preparation of test solution

[0051] 2.2.2.1 Preparation of Xiangsha Liujunzi Decoction

[0052] According to the recipe for Xiangsha Liujunzi Decoction, weigh 12 g of (fried) Atractylodes macrocephala (rice root), 9 g of (ginger) Pinellia ternata, 6 g of (raw) Licorice root, 9 g of dried tangerine peel, 9 g of Panax ginseng, 15 g of Poria cocos, 6 g of costus root, and 3 g of Amomum villosum. Soak all the herbs for 30 minutes before decoction. Amomum villosum and costus root should also be soaked for 30 minutes before decoction. When the first decoction is almost reduced to the desired amount, add them and decoct for 5 minutes. Decoction the decoction twice: first, with 4 times the amount of water, boil over high heat, then reduce to simmer, and simmer for 25 minutes; second, with 3 times the amount of water, boil over high heat, then reduce to simmer, and simmer for 15 minutes. Filter both filtrates through four layers of gauze and combine the two filtrates.

[0053] 2.2.2.2 Preparation of freeze-dried powder of Xiangsha Liujun Decoction

[0054] Take the decoction obtained under "2.2.2.1" and centrifuge at 5500 rpm for 20 min. Filter to remove the residue, microfilter, and dilute to 250 ml. Concentrate under reduced pressure on a rotary evaporator at 55°C and 50 rpm to a volume of 100 ml. Aliquot into evaporating dishes and freeze-dry to prepare lyophilized powder.

[0055] Weigh an appropriate amount of freeze-dried powder, dissolve it in 60% methanol, make up to 10 ml, shake well, filter through a 0.22 μm microporous membrane, and obtain the filtrate.

[0056] 2.2.3 Preparation of negative test solution

[0057] Prepare decoctions lacking dried tangerine peel, licorice and costus root according to the proportions 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".

[0058] 2.2.4 System adaptability and specificity test

[0059] The reference solution, test solution, and negative test solution lacking dried tangerine peel, licorice, and costus root were analyzed according to the chromatographic conditions in 2.1. The seven components, including liquiritin, apigenin, naringenin, hesperidin, nobiletin, costunolide, and dehydrocostusolide, were well separated from adjacent peaks, with resolutions greater than 1.5. The chromatographic peaks of other components in the negative sample did not interfere with the seven components, indicating good specificity of the analytical method. See the chromatogram for details. Figure 1 (reference substance), Figure 2 (Test sample).

[0060] 2.2.5 Investigation of linear relationships

[0061] Accurately pipette 1.2, 1.0, 0.8, 0.6, 0.4, 0.2, and 0.1 ml of each individual standard stock solution and dilute to 10 ml to obtain a 120%-10% mixed standard solution. Perform the assay according to the chromatographic conditions in "2.1" and plot a standard curve with the reference concentration (X, μg / ml) on the abscissa and the peak area (Y) on the ordinate. The linear ranges, linear regression equations, and correlation coefficients for the seven components are shown in Table 1. The results show good linearity for each of the seven components within their respective concentration ranges.

[0062] Table 1 Linear range, linear regression equation and correlation coefficient of 7 components

[0063]

[0064] 2.2.6 Limit of detection and limit of quantification

[0065] The limit of detection and limit of quantification (LOD) were determined based on the standard deviation of the response and the slope of the standard curve. The calculation formula is LOD = 3.3δ / S. Where LOD is the limit of detection; δ is the deviation of the response; and S is the slope of the standard curve. δ can be determined by: ① the standard deviation of the blank; ② the residual standard deviation or the standard deviation of the intercept of the standard curve.

[0066] The standard deviation δ of the response values ​​in this experiment was determined using the residual standard deviation of the standard curve. The limits of detection and quantification for the seven components are shown in Table 2. The results show that all seven components are within the limits of detection and quantification, indicating that they can be accurately quantified.

[0067] Table 2 Detection limits and quantification limits of 7 components

[0068]

[0069] 2.2.7 Stability test

[0070] The same sample was stored at room temperature for 0, 3.5, 7, 10.5, 14, 24, and 48 hours, then assayed according to the chromatographic conditions in "2.1." The results showed that the concentrations 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 remained stable at room temperature for 48 hours. The RSD values ​​for the seven components are shown in Table 3.

[0071] Table 3 Test results of stability of test products

[0072]

[0073] 2.2.8 Repeatability test

[0074] Six replicates of Xiangsha Liujunzi Decoction test solutions were prepared in parallel according to the method in "2.2.2" and assayed using the chromatographic conditions in "2.1." The results showed that the RSDs for liquiritin, apigenin, naringenin, hesperidin, nobiletin, costunolide, and dehydrocostuslactone were 1.72%, 2.50%, 2.15%, 0.65%, 1.10%, 1.50%, and 1.68%, respectively. These values ​​met the pharmacopoeial requirements within their respective concentration ranges, demonstrating good reproducibility of the methodology. The reproducibility results are shown in Table 4.

[0075] Table 4 Repeatability test results

[0076]

[0077] 2.2.9 Accuracy test

[0078] Take 6 portions of the test sample of known concentration, each about 0.2g, in a 10ml volumetric flask, add 60% methanol and ultrasonically dissolve, and dilute to the scale to obtain the test solution. Use a pipette to accurately transfer 1ml of the test solution to 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 methodological accuracy was good.

[0079] Table 5 Accuracy test

[0080]

[0081] 2.2.10 Durability test

[0082] The assay was tested to determine whether it could accurately quantify the sample under subtle changes in flow rate, temperature, and mobile phase ratio. The experiment varied the column temperature by ±2°C, the flow rate by ±0.2 mL / min, and the phosphoric acid content of the mobile phase. The results are shown in Tables 6-8. These results demonstrate that the assay can accurately quantify the sample even with subtle changes in flow rate, column temperature, and mobile phase ratio, demonstrating the robustness of the assay.

[0083] Table 6 Durability-Flow Rate Change

[0084]

[0085] Table 7 Durability - Column Temperature Change

[0086]

[0087] Table 8 Durability - Mobile Phase Change

[0088]

[0089] 2.2.11 Sample content determination

[0090] Replace the decoction and prepare the test solution according to "2.2.2". Detect according to the chromatographic conditions under "2.1". Calculate the content of each component by external standard method. The results are shown in Table 9.

[0091] Table 9 Determination of content of different batches of decoction

[0092]

[0093] The HPLC method established in the present invention can simultaneously and quantitatively determine the contents of seven active ingredients in Xiangsha Liujunzi Decoction, 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 uses an HPLC wavelength switching method to simultaneously determine the contents of liquiritin, liquiritin apigenin, naringin, hesperidin, nobiletin, costunolide, and dehydrocostuslactone in a preparation; the method comprises the following steps: 1) Preparation of standard solution Take appropriate amounts of liquiritin, apigenin, naringenin, hesperidin, nobiletin, costunolide, and dehydrocostuslactone reference substances, accurately weigh them, and prepare a 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 The Xiangsha Liujunzi decoction was centrifuged, filtered to remove the residue, microfiltered, and then fixed to volume; the solution was concentrated under reduced pressure in a rotary evaporator, fixed to volume, and divided into evaporating dishes, and freeze-dried to prepare freeze-dried powder; Weigh an appropriate amount of freeze-dried powder, re-dissolve, adjust to volume, shake well, filter through a microporous filter membrane, and take the filtrate to obtain the product; the solvent for adjusting to volume and re-dissolving is a 50-70% by mass methanol aqueous solution; 3) HPLC conditions: XB-C18 column, 4.6 × 250 mm, 5 μm; flow rate: 0.8–1.2 mL / min; column temperature: 23–27°C; mobile phase A: acetonitrile; mobile phase B: 0.05–0.2% aqueous phosphoric acid; gradient elution; detection wavelength: 225–330 nm; initial acetonitrile: aqueous phosphoric acid ratio: 18:22; gradient elution procedure: ； The wavelength switching procedure is: ； 4) Determination method: Accurately aspirate the reference solution and the test solution, inject them into the liquid chromatograph, and determine them according to the chromatographic conditions described in step 3).

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

Images