Determination Method for Contents of Multiple Components in Wuwei Ganlu Medicated Bath Decoction Powder

Abstract

The present invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to a method for determining the contents of multiple components in Wuweiganlu medicinal bath powder. The method uses high performance liquid chromatography to simultaneously determine the contents of the main active components hyperoside, luteoloside, quercitrin, quercetin, and amentoflavone in Wuweiganlu medicinal bath powder. The present invention can effectively shorten the analysis time of the main active components in Wuweiganlu medicinal bath powder, has a good linear range, high accuracy, precision, and repeatability, the method is stable and feasible, and can be applied to the quality inspection of products.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine detection, and specifically relates to a method for determining the contents of multiple components in Wuweiganlu medicinal bath powder. Background Art

[0002] Wuweiganlu medicinal bath powder, whose prescription originates from the Four Medical Canons, is one of the few Tibetan medicinal bath prescriptions included in the Pharmacopoeia of the People's Republic of China (abbreviation: Chinese Pharmacopoeia) in 2020 edition. This prescription is a powder prepared by crushing five kinds of medicinal materials, namely Juniperus sabina, Rhododendron anthopogonoides, Ephedra sinica, Myricaria bracteata, and Artemisia sieversiana, into coarse powder, decocting with highland barley, and adding yeast for fermentation. It has the effects of relieving pain and activating collaterals, sweating and anti-inflammatory, and is a commonly used prescription in Tibetan medicine clinics for rheumatic arthritis, skin diseases, osteoarthropathy, gout, etc. Modern pharmacological research has confirmed that this medicinal bath has significant pharmacological effects in the prevention and treatment of acute and chronic gastroenteritis and radiation-induced intestinal injury, and has great clinical application value.

[0003] All five kinds of medicinal materials, namely Juniperus sabina, Rhododendron anthopogonoides, Ephedra sinica, Myricaria bracteata, and Artemisia sieversiana, in this prescription contain flavonoids. Network pharmacology analysis shows that the main active components of Tibetan medicine Wuweiganlu medicinal bath powder in the treatment of gouty arthritis are various flavonoid components such as luteoloside, quercetin, and luteolin. Juniperus sabina contains quercetin and taxusflavone, which have the effects of anti-inflammatory and antibacterial, and treating osteoarthritis; Myricaria bracteata and Artemisia sieversiana contain components such as quercetin and quercitrin, which have therapeutic effects on inflammation and cardiovascular diseases; Rhododendron anthopogonoides and Ephedra sinica contain components such as hyperoside, quercetin, and quercitrin, which have effects such as anti-inflammatory, treating osteoarticular diseases and cardiovascular diseases. Therefore, there are various components in Wuweiganlu medicinal bath powder that play anti-inflammatory, treating joint inflammation, and cardiovascular diseases. However, in the quality standard of Wuweiganlu medicinal bath powder included in the Chinese Pharmacopoeia, there is only a thin-layer identification of ephedrine hydrochloride, and there is no content determination item, which affects the quality stability and clinical efficacy of the product.

[0004] The present invention uses HPLC method to establish a method for determining the contents of flavonoid index components, namely hyperoside, luteoloside, quercitrin, quercetin, and taxusflavone, in Wuweiganlu medicinal bath powder, so as to provide a reference for the quality control of Tibetan medicine Wuweiganlu medicinal bath powder and the improvement and perfection of the quality standard included in the Chinese Pharmacopoeia. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for determining the contents of multiple components in Wuweiganlu medicinal bath powder.

[0006] To achieve the above purpose, the technical solution of the present invention is as follows:

[0007] The Wuweiganlu medicinal bath powder described in the present invention is a medicinal powder composed of five kinds of medicinal materials, namely Juniperus sabina, Rhododendron anthopogonoides, Ephedra sinica, Myricaria bracteata, and Artemisia sieversiana, in a weight ratio of 1:1:1:1:1.

[0008] The method for determining the contents of multiple components in the Wuweiganlu medicinal bath decoction powder according to the present invention uses high performance liquid chromatography (HPLC) to simultaneously determine the contents of the main active components hyperoside, luteoloside, quercitrin, quercetin, and taxusflavone in the Wuweiganlu medicinal bath decoction powder. The main active components determined by the present invention are all flavonoid components. Among them, juniper contains quercetin and taxusflavone, which have the effects of anti-inflammatory and antibacterial, and treating osteoarthritis; myricaria and artemisia sieversiana contain components such as quercetin and quercitrin, which have the effects of treating inflammation and cardiovascular diseases; rhododendron anthopogonoides and ephedra contain components such as hyperoside, quercetin, and quercitrin, which have the effects of anti-inflammatory, treating osteoarticular diseases and cardiovascular diseases, etc.

[0009] The chromatographic conditions of the HPLC method described in the present invention are as follows: an ultraviolet-visible absorption detector, the mobile phase is acetonitrile - 0.4% phosphoric acid solution; gradient elution; flow rate 1 mL / min; detection wavelength 360 nm; column temperature 30 °C.

[0010] In the mobile phase system of the chromatographic conditions described in the present invention, it is acetonitrile - 0.4% phosphoric acid water. Under this condition, the methanol solutions of hyperoside, luteoloside, quercitrin, quercetin, and taxusflavone can have good peak shapes, and the chromatographic peaks of the test samples have good resolution, which is beneficial to the accurate analysis of the components.

[0011] The chromatographic column used in the HPLC method described in the present invention is Ultimate XB-C 18 (250 mm×4.6mm, 5 μm). This chromatographic column is an octadecylsilane-bonded silica gel chromatographic column, which is a relatively commonly used chromatographic column for analyzing flavonoids and can achieve the separation of the components in the test solution, which is beneficial to the accurate analysis of the components.

[0012] The gradient elution program of the chromatographic conditions described in the present invention includes the following steps: a) At 0 min, the proportion of acetonitrile is 15%; b) At 5 min, the proportion of acetonitrile is 15%; c) At 15 min, the proportion of acetonitrile is 20%; d) At 20 min, the proportion of acetonitrile is 20%; e) At 35 min, the proportion of acetonitrile is 30%; f) At 50 min, the proportion of acetonitrile is 50%; g) From 52 to 60 min, the proportion of acetonitrile is 15%.

[0013] Among them, the steps from a) to c) are for flushing to achieve complete separation of hyperoside and luteoloside; step d) is for separating quercitrin; steps e) and f) are for separating quercetin and taxusflavone; step g) is for restoring the gradient ratio before the program.

[0014] The chromatographic conditions described in the present invention adopt gradient elution with acetonitrile - 0.4% phosphoric acid water. The components in the test solution are relatively complex. To analyze the target components therein, good resolution is required. Under this condition, it can effectively reduce the tailing of the solvent peak, improve the signal-to-noise ratio, sensitivity, and the theoretical plate number of the chromatographic peaks of the target components.

[0015] The preparation method of the test solution described in the present invention is as follows: Take Wuweiganlu Medicated Bath Powder, pulverize it into fine powder, accurately weigh 2.0 g of the fine powder, place it in a 100 mL volumetric stoppered conical flask, add 25 mL of 80% methanol, tightly stopper it, weigh it, ultrasonically treat it (power 250 W, frequency 40 kHz) for 1 h, let it cool and weigh it again, make up the lost weight with 80% methanol, shake well, filter it through a 0.45 μm microporous filter membrane to obtain the test solution.

[0016] The Wuweiganlu Medicated Bath Powder described in the present invention is a powder prepared by traditional crude drug processing. Under the premise of determining the extraction solvent and extraction method, the present invention studied the effects of extraction time and solid-to-liquid ratio on 5 target components in the test solution through single-factor experiments. The results showed that for ultrasonic treatment for 30, 60, and 90 min, the content of the target components in the test solution was less after 30 min. There was no significant difference in the content of the target components in the test solution after 60 min and 90 min of ultrasonic treatment. Therefore, 60 min of ultrasonic treatment was selected; under the same conditions, the amount of the extraction solvent used was positively correlated with the content of 5 target components in the test solution within a certain range. When the solid-to-liquid ratio was 1:12.5, 1:15, and 1:20, the change in the content of the target components in the test solution was not significant. Therefore, a solid-to-liquid ratio of 1:12.5 was selected.

[0017] The high performance liquid chromatography method described in the present invention also includes the preparation method of the reference solution. The method is as follows: Accurately weigh hyperoside, luteoloside, quercitrin, quercetin, and taxusflavone respectively, dissolve them with methanol and make up the volume, shake well to prepare the reference stock solution. Take the reference stock solutions of hyperoside, luteoloside, quercitrin, quercetin, and taxusflavone according to the volume ratio of 2:2:2.5:1:1 and place them in the same volumetric flask, dilute and make up the volume with methanol, shake well to prepare the mixed reference solution.

[0018] The pure methanol solution described in the present invention is methanol with a purity exceeding 99.9%.

[0019] The treatment method of the test sample in the present invention is ultrasonic treatment for 60 min, and the Wuweiganlu medicinal bath decoction powder and 80% methanol water are in a weight-to-volume ratio of 1:12.5. By comparing the effects of steam distillation extraction and ultrasonic extraction on 5 index components in the test sample solution, the inventor of the present invention found that the effects of steam distillation extraction and ultrasonic extraction on the component content are not significant, but distillation extraction will increase the content of other components with larger polarity, affecting the detection of content. Therefore, the present invention selects the method of ultrasonic treatment with organic solvents.

[0020] The high performance liquid chromatography method described in the present invention also includes a preparation method for a blank solution: the method for the blank solution is: without adding the Wuweiganlu medicinal bath decoction powder, and the remaining operations are the same as those for the preparation method of the test sample solution, to obtain the blank solution.

[0021] Advantages of the present invention:

[0022] (1) The present invention establishes a high performance liquid chromatography method for simultaneously quantitatively determining the index components hyperoside, luteoloside, quercitrin, quercetin, and amentoflavone in the Wuweiganlu medicinal bath decoction powder and conducts method validation. The results show that this method is scientific and feasible;

[0023] (2) Compared with single analysis, the present invention can effectively reduce the injection times and analysis time of the instrument, has a short detection time, accurate detection results, and is simple and easy to implement;

[0024] (3) The present invention has a wide linear range, good repeatability, a stable and feasible sample treatment method, and the obtained test sample solution has good stability at room temperature;

[0025] (4) The method of the present invention is stable and accurate, can be used for the quality evaluation of the Wuweiganlu medicinal bath decoction powder, and can provide a reference for improving the quality standard of the Wuweiganlu medicinal bath decoction powder. Description of the drawings

[0026] Figure 1 HPLC chart of the blank solution;

[0027] Figure 2 HPLC chart of the mixed control solution;

[0028] Figure 3 HPLC chart of the test sample solution;

[0029] Figure 4 Linear relationship chart of the hyperoside reference substance;

[0030] Figure 5 Linear relationship chart of the luteoloside reference substance;

[0031] Figure 6 Linear relationship chart of the quercitrin reference substance;

[0032] Figure 7 It is the linear relationship diagram of quercetin reference substance;

[0033] Figure 8 It is the linear relationship diagram of amentoflavone reference substance. Specific implementation manners

[0034] The following experimental examples are used to further illustrate but not limit the present invention.

[0035] In the following specific examples, the Wumei Ganlu medicated bath decoction powder used was purchased from Tibet Shenhou Pharmaceutical Co., Ltd., with batch numbers 20231001, 20231002, 20231101, 20231202, 20240201, and 20240202.

[0036] Hyperoside reference substance (purity 98.3%), luteoloside reference substance (purity 98.7%), quercitrin reference substance (purity 98.2%), amentoflavone reference substance (purity 99.0%) were purchased from Chengdu Lemeitian Medical Technology Co., Ltd.; quercetin reference substance (purity 99.8%) was purchased from the National Institutes for Food and Drug Control; methanol and acetonitrile were of chromatographic grade, and other reagents were of analytical grade, all purchased from Sinopharm Chemical Reagent Co., Ltd. Example

[0037] Simultaneously determine the contents of hyperoside, luteoloside, quercitrin, quercetin, and amentoflavone in the Wumei Ganlu medicated bath decoction powder by high performance liquid chromatography, including the following steps:

[0038] (1) Preparation of test solution

[0039] Take the Wumei Ganlu medicated bath decoction powder, pulverize it into fine powder, accurately weigh 2.0 g of the fine powder, place it in a 100 mL volumetric stoppered conical flask, add 25 mL of 80% methanol, tightly stopper, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 1 h, let it cool and weigh again, make up the lost weight with 80% methanol, shake well, filter with a 0.45 μm microporous filter membrane to obtain the test solution.

[0040] (2) Preparation of mixed reference substance solution

[0041] Accurately weigh 9.98 mg, 9.99 mg, 8.14 mg, 9.98 mg, and 10.00 mg of hyperoside, luteoloside, quercitrin, quercetin, and amentoflavone reference substances respectively, place them in 10 mL volumetric flasks, dissolve with methanol and make up the volume, shake well to prepare hyperoside, luteoloside, quercitrin, quercetin, and amentoflavone with mass concentrations of 981.00 μg·mL -1 、986.00 μg·mL -1, 799.30 μg·mL -1 , 996.00 μg·mL -1 and 990.00 μg·mL -1 of the reference substance stock solutions. Accurately pipette 4 mL, 4 mL, 5 mL, 2 mL, and 2 mL of the hyperoside, luteoloside, quercitrin, quercetin, and amentoflavone reference substance stock solutions respectively into the same 20 mL volumetric flask, dilute to the mark with methanol, shake well, and prepare a mixed reference substance solution containing hyperoside, luteoloside, quercitrin, quercetin, and amentoflavone with mass concentrations of 196.21 μg·mL -1 , 197.20 μg·mL -1 , 199.84 μg·mL -1 , 99.60 μg·mL -1 and 99.00 μg·mL -1 respectively.

[0042] (3) Preparation of the blank solution

[0043] Without adding Wuweiganlu Yaoyu Decoction Powder, prepare the blank solution according to the preparation method of the test solution in step (1) with the remaining operations being the same.

[0044] (4) Chromatographic conditions

[0045] The chromatographic column is Ultimate XB-C 18 (250 mm × 4.6 mm, 5 μm); the mobile phase is acetonitrile - 0.4% phosphoric acid solution. At 0 min, the proportion of acetonitrile is 15%; at 5 min, the proportion of acetonitrile is 15%; at 15 min, the proportion of acetonitrile is 20%; at 20 min, the proportion of acetonitrile is 20%; at 35 min, the proportion of acetonitrile is 30%; at 50 min, the proportion of acetonitrile is 50%; from 52 to 60 min, the proportion of acetonitrile is 15%. The detection wavelength is 360 nm; the column temperature is 30 °C; the injection volume is 10 μL.

[0046] (6) Methodology investigation

[0047] System suitability test investigation: Take the blank solution, the mixed reference substance solution, and the test solution, inject and determine according to the chromatographic conditions, record the chromatograms, and the results are shown in Figure 1 , Figure 2 , Figure 3No interfering peaks appeared in the chromatogram of the blank solution at the retention times of hyperoside, luteoloside, quercitrin, quercetin, and amentoflavone. In the chromatogram of the test solution, the resolution between hyperoside, luteoloside, quercitrin, quercetin, and amentoflavone and the adjacent chromatographic peaks was greater than 1.5, the tailing factor was between 0.95 and 1.05, the theoretical plate number was greater than 5,000, and the retention times of the chromatographic peaks of each component were consistent with those in the reference solution. The chromatographic system suitability test met the requirements for content determination.

[0048] Investigation of linear relationship: Accurately pipette 0.5, 0.5, 1, 2.5, and 2.5 mL of the mixed reference solution into 20, 10, 10, 10, and 5 mL volumetric flasks respectively, add methanol to dilute to the mark, shake well, and prepare mixed reference solutions with different concentrations. Inject and determine according to the chromatographic conditions. Using the mass concentration of the reference solution as the abscissa ( X ), and the peak area as the ordinate ( Y ), perform linear regression. The results showed that hyperoside had a good linear relationship with the integrated peak area value within the range of 4.91 - 98.10 μg / mL, and the equation was Y = 23.31 X + 0.0441 ( r = 1.000 0). Luteoloside had a good linear relationship with the integrated peak area value within the range of 4.93 - 98.60 μg / mL, and the equation was Y = 18.48 X + 30.1030 ( r = 0.999 6). Quercitrin had a good linear relationship with the integrated peak area value within the range of 4.99 - 99.92 μg / mL, and the equation was Y = 18.89 X + 0.3095 ( r = 1.000 0). Quercetin had a good linear relationship with the integrated peak area value within the range of 2.50 - 49.80 μg / mL, and the equation was Y = 40.52 X +1.9915 ( r = 0.999 9). Amentoflavone had a good linear relationship with the integrated peak area value within the range of 2.48 - 49.50 μg / mL, and the equation was Y = 26.634 X + 17.5190 ( r = 0.999 9). The results were as shown in Figure 4 and Figure 5 and Figure 6 and Figure 7 and Figure 8 .

[0049] Precision test: Take the test solution, inject samples according to the chromatographic conditions, and continuously measure 6 times. The RSD (n = 6) of the peak areas of hyperoside, luteoloside, quercitrin, quercetin, and amentoflavone are 0.26%, 0.40%, 0.31%, 0.23%, and 0.18% respectively, indicating good instrument precision.

[0050] Repeatability test: Take the powder of the same batch of Wuweiganlu Medicated Bath Decoction Powder (batch number 20231001), and prepare 6 portions of test solutions in parallel according to the preparation method of the test solution. Inject samples and measure according to the chromatographic conditions. The average contents of hyperoside, luteoloside, quercitrin, quercetin, and amentoflavone are 0.2351, 0.2675, 0.3974, 0.1171, and 0.0791 mg·g -1 respectively, and the RSD (n = 6) are 0.49%, 0.42%, 0.12%, 0.51%, and 0.42% respectively, indicating good repeatability of this method.

[0051] Stability test: Prepare 1 portion of the test solution again, inject samples at 0, 2, 4, 8, 12, and 24 h at room temperature according to the chromatographic conditions. The RSD (n = 6) of the peak areas of hyperoside, luteoloside, quercitrin, quercetin, and amentoflavone are 0.73%, 0.23%, 0.56%, 0.27%, and 0.52%. It shows that the test solution is stable within 24 h.

[0052] Spiked recovery test: Accurately weigh 9 portions of the Wuweiganlu Medicated Bath Decoction Powder with known content (batch number 20231001), 2 g for each portion, divide them into 3 groups of high, medium, and low doses, accurately add high, medium, and low dose mixed reference substance solutions respectively, and then prepare the test solution according to the method under "2.1.1". Measure and calculate that the average recoveries of hyperoside, luteoloside, quercitrin, quercetin, and amentoflavone are 98.0%, 96.3%, 100.2%, 100.4%, 99.6%, and the RSD (n = 6) are 0.52%, 1.79%, 0.54%, 0.70%, and 0.80% respectively.

[0053] (7) Determination of multi-components in Wuweiganlu Medicated Bath Decoction Powder

[0054] Take six batches of Wuweiganlu Medicated Bath Decoction Powder (batch numbers: 202231001, 20231002, 20231101, 20231102, 20240201, 20240202), prepare test solutions respectively, inject samples according to the chromatographic conditions, and the results are shown in Table 1.

[0055] Table 1 Determination results of 5 components in Wuweiganlu Medicated Bath Decoction Powder (n = 2)

[0056]

[0057] The present invention established a method for the simultaneous quantitative determination of hyperoside, luteoloside, quercitrin, quercetin, and amentoflavone, which are the index components in Wuwei Ganlu medicated bath powder, by high performance liquid chromatography (HPLC), and the method was verified by methodology. The results showed that the method is scientific and feasible. Compared with individual analysis, this method can effectively reduce the injection times and analysis time of the instrument, shorten the experimental time, and reduce the experimental cost. At the same time, the method of the present invention has a wide linear range, good repeatability, a stable and feasible sample treatment method, and the obtained test solution has good stability at room temperature. The method of the present invention is stable and accurate, can be used for the quality evaluation of Wuwei Ganlu medicated bath powder, and can provide a reference for the improvement of the quality standard of this product.

[0058] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than a limitation on the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for determining the contents of multiple components in Wuweiganlu medicinal bath decoction powder, characterized in that, Determine the contents of hyperoside, luteoloside, quercitrin, quercetin, and taxusflavone in Wuweiganlu Medicated Bath Powder simultaneously by high performance liquid chromatography (HPLC); the chromatographic conditions of the HPLC are as follows: an octadecylsilyl bonded silica gel chromatographic column with a specification of 250 mm × 4.6 mm, 5 μm; an ultraviolet-visible absorption detector, the mobile phase is acetonitrile - 0.4% phosphoric acid solution; gradient elution; the flow rate is 1 mL / min; the detection wavelength is 360 nm; the column temperature is 30°C; the gradient elution program of the chromatographic conditions includes the following steps: a) At 0 min, the proportion of acetonitrile is 15%; b) At 5 min, the proportion of acetonitrile is 15%; c) At 15 min, the proportion of acetonitrile is 20%; d) At 20 min, the proportion of acetonitrile is 20%; e) At 35 min, the proportion of acetonitrile is 30%; f) At 50 min, the proportion of acetonitrile is 50%; g) At 52 - 60 min, the proportion of acetonitrile is 15%; The preparation method of the test solution in the HPLC is as follows: Take Wuweiganlu Medicated Bath Powder, pulverize it into fine powder, add 80% methanol, and filter through a microporous membrane to obtain the test solution.

2. The method for determining the contents of multiple components in Wuwei Ganlu Medicinal Bath Decoction according to claim 1, wherein: The preparation method of the reference solution in the HPLC is as follows: Weigh hyperoside, luteoloside, quercitrin, quercetin, and taxusflavone precisely respectively, dissolve them in methanol and make up the volume to a fixed volume, shake well to prepare the reference stock solution; Take the reference stock solutions of hyperoside, luteoloside, quercitrin, quercetin, and taxusflavone according to the volume ratio of 2:2:2.5:1:1 and place them in the same volumetric flask, dilute with methanol to a fixed volume, shake well to prepare the mixed reference solution.

3. The method for determining the contents of multiple components in Wuwei Ganlu Medicinal Bath Decoction according to claim 1, wherein: The injection volume in the HPLC is 10 μL.

4. The method for determining the contents of multiple components in the Wuweiganlu medicinal bath decoction powder according to claim 1, wherein The Wuweiganlu Medicated Bath Powder is a pharmaceutical composition composed of five Chinese herbal medicines, namely Juniperus Sabina L., Rhododendron anthopogonoides Maxim., Ephedra sinica Stapf, Myricaria bracteata Royle, and Artemisia sieversiana Ehrhart ex Willd, in a weight ratio of 1:1:1:1:1.

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