A method for quantitative detection of active ingredients in Ophiopogon japonicus and its application

Abstract

This disclosure relates to a method for quantitatively detecting active ingredients in Ophiopogon japonicus and its application. The method includes: S1, placing the Ophiopogon japonicus to be tested in an extract, shaking, sonicating, centrifuging, and then mixing with an internal standard to obtain a test solution; S2, using liquid chromatography-mass spectrometry to detect the content of active ingredients in the test solution; the active ingredients include flavonoids and saponins. This method can simultaneously and rapidly quantitatively analyze multiple active substances in Ophiopogon japonicus, including steroidal saponins with different physicochemical properties, high levels of isoflavones, and ketones, which is of great significance for evaluating the quality level of Ophiopogon japonicus.

Description

Technical Field

[0001] This disclosure relates to the field of traditional Chinese medicine analysis technology, specifically to a method for quantitative detection of active ingredients in Ophiopogon japonicus and its application. Background Technology

[0002] The content of active substances saponins and flavonoids in Ophiopogon japonicus is one of the important indicators for evaluating its quality. The 2020 edition of the Chinese Pharmacopoeia only lists one evaluation indicator for Ophiopogon japonicus: total saponin content greater than 0.12%. However, this standard alone is insufficient for comprehensive quality evaluation and control of Ophiopogon japonicus. Currently, there is a lack of comprehensive evaluation methods for Ophiopogon japonicus that combine monomeric compounds with effective fraction groups and are based on multiple indicator components. Traditional methods for detecting flavonoid saponins include colorimetry, spectrophotometry, thin-layer chromatography, and ultraviolet liquid chromatography. Although these methods can meet the detection requirements of some common saponin and flavonoid compounds, they cannot meet the requirements for accurate qualitative and quantitative analysis of multiple saponin and flavonoid substances, as well as the speed and accuracy of detecting large batches of samples.

[0003] Therefore, there is an urgent need to provide a simple, easy-to-operate, fast, and highly accurate method for the quantitative detection of active ingredients in Ophiopogon japonicus. Summary of the Invention

[0004] The purpose of this disclosure is to provide a simple, easy-to-operate, fast, and highly accurate method for the quantitative detection of active ingredients in Ophiopogon japonicus.

[0005] On the one hand, this disclosure provides a method for quantitatively detecting active ingredients in Ophiopogon japonicus, the method comprising:

[0006] S1. Place the Ophiopogon japonicus to be tested in the extract and shake, sonicate, and centrifuge it. Then mix it with the internal standard to obtain the test solution.

[0007] S2. The content of the active ingredient in the test solution is determined using liquid chromatography-mass spectrometry.

[0008] The active ingredients include flavonoids and saponins;

[0009] The flavonoids include at least one of the following: Ophiopogon flavanone A, Ophiopogon methyl flavanone B, methyl Ophiopogon hyperflavonone A, Ophiopogon methyl flavanone A, 8-aldehyde isoOphiopogon flavanone B, 6-aldehyde isoOphiopogon flavanone A, Ophiopogon flavanone C, 6-aldehyde isoOphiopogon dihydro isoflavonone A, Ophiopogon flavanone E, Ophiopogon flavanone D, and Ophiopogon flavanone F;

[0010] The saponin compounds include at least one of Rusque saponin, Ophiopogonin B, Ophiopogonin Ra, Ophiopogonin A, Ophiopogonin B monosulfate, Ophiopogonin D', Ophiopogonin D, Ophiopogonin C, and deacetylated Ophiopogonin A.

[0011] Optionally, the extract includes at least one of methanol, ethanol, acetonitrile, and acetone; preferably methanol.

[0012] Optionally, the Ophiopogon japonicus to be tested is prepared by the following steps: the dried Ophiopogon japonicus tuber is crushed and passed through a 40-60 mesh sieve.

[0013] Optionally, the weight ratio of the Ophiopogon japonicus to be tested to the extract is 1:(20-50).

[0014] Optionally, the conditions for oscillation processing include: vortex oscillation for 30-90 seconds;

[0015] The conditions for ultrasonic treatment include: ultrasonic power of 250-500W, ultrasonic frequency of 30-60kHz, ultrasonic time of 30-60min, and ultrasonic temperature of 20-30℃.

[0016] The conditions for centrifugation include: a centrifugation rate of 6000-10000 r / min and a centrifugation time of 3-5 min.

[0017] Optionally, step S1 further includes: passing the supernatant obtained by centrifugation through a 0.22 μm filter membrane and mixing it with an internal standard to obtain the test solution;

[0018] The internal standard is a standard solution containing flavonoids and saponins at a concentration of 10-100 μg / mL;

[0019] The weight ratio of the tested Ophiopogon japonicus to the internal standard is (10) 4 -10 5 ): 1.

[0020] Optionally, in step S2, when using liquid chromatography-mass spectrometry for detection, the mass spectrometry analysis conditions include: mass spectrometry precursor ion of 328.852-914.487 m / z, mass spectrometry daughter ion of 76.942-736.856 m / z, cone voltage of 2-30 V, and collision energy of 10-66 V.

[0021] Optionally, the limit of detection for quantitative detection is 0.0008-0.3036 mg / kg, the limit of quantification is 0.002-0.2089 mg / kg, and the coefficient of determination is 0.9907-0.9998.

[0022] On the other hand, this disclosure also provides the application of the above method in the quantitative detection of active ingredients in Ophiopogon japonicus.

[0023] Through the above technical solution, this disclosure provides a method for quantitatively detecting active ingredients in Ophiopogon japonicus and its application. This method can simultaneously and rapidly quantitatively analyze multiple active substances in Ophiopogon japonicus, including steroidal saponins with different physicochemical properties, high levels of isoflavones and other ketones, which is of great significance for evaluating the quality level of Ophiopogon japonicus.

[0024] Other features and advantages of this disclosure will be described in detail in the following detailed description section. Attached Figure Description

[0025] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings:

[0026] Figure 1 The graph shows the recovery rate of 20 saponin flavonoid compounds in Ophiopogon japonicus using the method provided in this disclosure. Detailed Implementation

[0027] The specific embodiments of this disclosure will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit this disclosure.

[0028] On the one hand, this disclosure provides a method for quantitatively detecting active ingredients in Ophiopogon japonicus, the method comprising:

[0029] S1. Place the Ophiopogon japonicus to be tested in the extract and shake, sonicate, and centrifuge it. Then mix it with the internal standard to obtain the test solution.

[0030] S2. The content of the active ingredient in the test solution is determined using liquid chromatography-mass spectrometry.

[0031] The active ingredients include flavonoids and saponins;

[0032] The flavonoids include at least one of the following: Ophiopogon flavanone A, Ophiopogon methyl flavanone B, methyl Ophiopogon hyperflavonone A, Ophiopogon methyl flavanone A, 8-aldehyde isoOphiopogon flavanone B, 6-aldehyde isoOphiopogon flavanone A, Ophiopogon flavanone C, 6-aldehyde isoOphiopogon dihydro isoflavonone A, Ophiopogon flavanone E, Ophiopogon flavanone D, and Ophiopogon flavanone F;

[0033] The saponin compounds include at least one of Rusque saponin, Ophiopogonin B, Ophiopogonin Ra, Ophiopogonin A, Ophiopogonin B monosulfate, Ophiopogonin D', Ophiopogonin D, Ophiopogonin C, and deacetylated Ophiopogonin A.

[0034] According to this disclosure, the extract comprises at least one of methanol, ethanol, acetonitrile, and acetone; preferably methanol.

[0035] According to this disclosure, the Ophiopogon japonicus to be tested is prepared by the following steps: the dried Ophiopogon japonicus tuber is crushed and passed through a 40-60 mesh sieve.

[0036] According to this disclosure, the weight ratio of the Ophiopogon japonicus to be tested to the extract is 1:(20-50).

[0037] According to this disclosure, the conditions for oscillation processing include: vortex oscillation for 30-90 seconds;

[0038] The conditions for ultrasonic treatment include: ultrasonic power of 250-500W, ultrasonic frequency of 30-60kHz, ultrasonic time of 30-60min, and ultrasonic temperature of 20-30℃.

[0039] The conditions for centrifugation include: a centrifugation rate of 6000-10000 r / min and a centrifugation time of 3-5 min.

[0040] Step S1 further includes: passing the supernatant obtained by centrifugation through a 0.22 μm filter membrane and mixing it with an internal standard to obtain the test solution;

[0041] The internal standard is a standard solution containing flavonoids and saponins with a concentration of 10-100 μg / mL;

[0042] The weight ratio of the tested Ophiopogon japonicus to the internal standard is (10) 4 -10 5 ): 1.

[0043] According to this disclosure, in step S2, when using liquid chromatography-mass spectrometry for detection, the mass spectrometry analysis conditions include: the mass spectrometry precursor ion is 328.852-914.487 m / z, the mass spectrometry daughter ion is 76.942-736.856 m / z, the cone voltage is 2-30 V, and the collision energy is 10-66 V.

[0044] According to this disclosure, the limit of detection for quantitative detection is 0.0008-0.3036 mg / kg, the limit of quantitation is 0.002-0.2089 mg / kg, and the coefficient of determination is 0.9907-0.9998.

[0045] On the other hand, this disclosure provides the application of the above method in the quantitative detection of active ingredients in Ophiopogon japonicus.

[0046] The present disclosure will be further illustrated by the following examples, but the present disclosure is not limited thereto.

[0047] Instrument conditions

[0048] The mass spectrometry conditions are shown in Table 1: capillary (kV): 2.20; ion source: ESI+; cone voltage: 12V; source temperature (°C): 150; desolvation temperature (°C): 500; cone gas flow rate (L / Hr): 150; desolvation gas flow rate (L / Hr): 1000.

[0049] Chromatographic conditions: Injection volume 5 μL; Flow rate: 0.3 mL / min; Mobile phase: A: 0.1% formic acid in water; B: acetonitrile; Column: CORTECS UPLC C18 1.6 μm 2.1*100 mm Column; Gradient elution program: 0-10 min, 40-44% (B); 10-16 min, 44-70% (B); 16-16.01 min, 70-40% (B); 16.01-20 min, 40% (B);

[0050] Preparation of standard solutions

[0051] Preparation of standard stock solutions: All purchased saponin flavonoid standards are solid powder standards. They are dissolved in chromatographic grade methanol solution to prepare standard stock solutions with a concentration range of 250-2000 μg / mL in 20mL brown glass bottles and stored in a -20℃ refrigerator for later use.

[0052] Preparation of mixed standard stock solution: Used as calibration, spiking and control reagent solutions, take the standard stock solutions of 20 target analytes and place them in 20 mL volumetric flasks, dilute with methanol and make up to the mark to obtain a mixed standard stock solution containing 20 Ophiopogon japonicus saponins and flavonoids at a concentration of 100 μg / mL. Store at -20℃ for later use.

[0053] Mixed standard working solution: Used for plotting solvent standard curves. Accurately pipette 100μL, 80μL, 60μL, 40μL, 20μL, 10μL, and 5μL of the mixed standard stock solution of 20 Ophiopogon japonicus saponins and flavonoids into dark brown glass bottles containing 9900μL, 9920μL, 9940μL, 9960μL, 9980μL, 9910μL, and 9995μL of 50% methanol solution, respectively. This prepares a gradient solution of 20 saponins and flavonoids with standard concentrations of 1000μg / L, 800μg / L, 600μg / L, 400μg / L, 200μg / L, 100μg / L, and 50μg / L. Prepare and use immediately.

[0054] Preparation of internal standard solutions: Dissolve 2 mg of each of the two internal standards in 20 mL of methanol solution to prepare internal standard solutions with a concentration of 100 μg / mL. Store in 25 mL brown glass bottles at -20℃ for later use.

[0055] Pretreatment method for Ophiopogon japonicus: Dry the Ophiopogon japonicus tuber (70℃, 10h), pulverize into fine powder, pass through a 50-mesh sieve, weigh 1.0g of sample powder, transfer to a 50mL polyethylene centrifuge tube, add 25mL of methanol-water solution (50:50, v / v), vortex for 60 seconds, extract by sonication (40KHz, 25℃) for 45min, centrifuge at 8000rpm for 5min, add 1mL of extract (or standard solution of calibration curve) to a 2mL centrifuge tube + 10μL of internal standard (100ppm), draw 1mL with a syringe, pass through a 0.22μm organic filter membrane, and inject into a 1.5mL dark brown vial.

[0056] Table 1 Mass Spectrometry Analysis Conditions

[0057]

[0058]

[0059] Method Validation

[0060] Standard curve establishment: Following the preparation method of the mixed standard working solution described above, a series of mixed standard solutions of 20 saponins and flavonoids with gradient concentrations were prepared. These solutions were then analyzed by HPLC-MS / MS, with each concentration measured three times and the average value taken. Standard curves were established using Masslynx 4.2 software, and the regression equations for each saponin and flavonoid were obtained, as shown in Table 2. It can be seen that the linear range is good, and the standard curves for each saponin and flavonoid exhibit a good linear relationship.

[0061] Table 2

[0062]

[0063]

[0064] Repeatability test

[0065] Accurately weigh 1g of Ophiopogon japonicus sample powder, divide it into 6 portions, and prepare Ophiopogon japonicus test solutions according to the above Ophiopogon japonicus treatment method. Analyze each test solution by HPLC-MS / MS, and take the average value. The specific results are shown in Table 3. The RSD range is 0.75-6.83%. It can be seen that this method has good reproducibility for the detection of 20 saponins and flavonoids in Ophiopogon japonicus tuber.

[0066] Table 3 Repeatability Tests

[0067]

[0068]

[0069] Stability test

[0070] Take a sample of Ophiopogon japonicus powder and prepare a test solution according to the above-mentioned Ophiopogon japonicus treatment method. After 0, 2, 6, 12 and 24 hours after the pretreatment extraction, the sample was detected by HPLC-MS / MS. Each test solution was detected 3 times and the average value was taken. The RSD value was calculated to be 0.20-6.08%, as shown in Table 4. It can be seen that the content of saponins and flavonoids in the Ophiopogon japonicus sample test solution has good stability after 24 hours.

[0071] Table 4

[0072]

[0073]

[0074] Add recycling test

[0075] The recovery experiment was conducted at three spiking levels: high (15 mg / kg), medium (10 mg / kg), and low (5 mg / kg). A blank control group was also included. Six portions of dried Ophiopogon japonicus root powder from the same batch were precisely weighed, each 1.0 g (accurate to 0.1 g). A mixed solution of twenty active substances was added to each sample, with a volume of 100 μL. The specific amounts added are shown in Table 5. After processing according to the Ophiopogon japonicus pretreatment method, the samples were analyzed by HPLC-MS / MS. Each sample was analyzed three times, and the average value was taken. The average recovery rate was calculated using the following formula: Recovery rate (%) = ((detected amount - original amount) / spiked amount) * 100%.

[0076] Table 5

[0077] Group Added concentration (mg / L) Theoretical detection concentration (μg / L) Volume added (μL) high concentration 150 150 100 medium concentration 100 600 100 low concentration 50 200 100

[0078] Table 6

[0079]

[0080]

[0081] The recoveries of 20 saponins and flavonoids at three spiking levels (low, medium, and high) are shown in Table 6. Figure 1 As shown, the average recovery rates of the 20 saponins and flavonoids ranged from 70% to 130%, with RSDs ranging from 0.11% to 16.46%, all less than 20%, indicating good recovery rates.

[0082] It is evident that the method disclosed herein can quantitatively detect various saponins and flavonoids in Ophiopogon japonicus, providing a simple, feasible, accurate, and rapid detection method for controlling the quality of Ophiopogon japonicus.

[0083] The preferred embodiments of this disclosure have been described in detail above with reference to the accompanying drawings. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0084] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0085] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. A method for quantitatively detecting active ingredients in Ophiopogon japonicus, characterized in that, The method includes: S1. Place the Ophiopogon japonicus to be tested in the extract and perform shaking, sonication, and centrifugation, then mix with the internal standard to obtain the test solution. The shaking conditions include: vortexing for 30-90 seconds; the sonication conditions include: sonication power of 250-500W, sonication frequency of 30-60kHz, sonication time of 30-60min, and sonication temperature of 20-30℃; the centrifugation conditions include: centrifugation speed of 6000-10000r / min and centrifugation time of 3-5min. S2. The content of the active ingredient in the test solution is determined using liquid chromatography-mass spectrometry. The active ingredients include flavonoids and saponins; The flavonoids are Ophiopogon flavanone A, Ophiopogon methyl flavanone B, methyl Ophiopogon hyperflavonone A, Ophiopogon methyl flavanone A, 8-aldehyde isoOphiopogon flavanone B, 6-aldehyde isoOphiopogon flavanone A, Ophiopogon flavanone C, 6-aldehyde isoOphiopogon dihydro isoflavonone A, Ophiopogon flavanone E, Ophiopogon flavanone D and Ophiopogon flavanone F; The saponin compounds are Rusque saponin, Ophiopogonin B, Ophiopogonin Ra, Ophiopogonin A, Ophiopogonin B monosulfate, Ophiopogonin D', Ophiopogonin D, Ophiopogonin C and deacetylated Ophiopogonin A; The extract is methanol; The weight ratio of the tested Ophiopogon japonicus to the extract is 1:(20-50). Chromatographic conditions: Mobile phase: A: 0.1% formic acid in water; B: acetonitrile; Column: CORTECS UPLC C18 1.6μm 2.1*100mm Column; Gradient elution program: 0-10 min, 40-44% B; 10-16 min, 44-70% B; 16-16.01 min, 70-40% B; 16.01-20 min, 40% B.

2. The method according to claim 1, wherein, The tested Ophiopogon japonicus was prepared by the following steps: the dried Ophiopogon japonicus tubers were crushed and passed through a 40-60 mesh sieve.

3. The method according to claim 1, wherein, Step S1 further includes: passing the supernatant obtained by centrifugation through a 0.22 μm filter membrane and mixing it with an internal standard to obtain the test solution.

4. The method according to claim 1, wherein, In step S2, when using liquid chromatography-mass spectrometry (LC-MS) for detection, the mass spectrometry analysis conditions include: precursor ion 328.852-914.487 m / z, daughter ion 76.9429-736.8560 m / z, cone voltage 2-30 V, and collision energy 10-66 V.

5. The method according to claim 1, wherein, The detection limit for the quantitative assay is 0.0008-0.3036 mg / kg, the quantitation limit is 0.002-0.2089 mg / kg, and the coefficient of determination is 0.9907-0.9998.

6. The application of the method according to any one of claims 1-5 in the quantitative detection of active ingredients in Ophiopogon japonicus.

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