A method for detecting a bacopa monnieri extract and applications thereof

Abstract

The application discloses a method for detecting an extract of Bacopa monnieri and application thereof, and the method comprises the following steps: using high performance liquid chromatography combined with a diode array detector system to analyze the extract of Bacopa monnieri, so as to identify the components in the extract of Bacopa monnieri; wherein a chromatographic column is a reversed-phase chromatographic column comprising a filler with bonded phase being octadecylsilane and carrier being silica gel; a mobile phase comprises mobile phase A and mobile phase B, wherein the mobile phase A is acetonitrile, and the mobile phase B is composed of a buffer saline solution and methanol. The method of the application guarantees that the separation degree of various components is high, the sensitivity is high, the accuracy is high, the concentration of each component can be more accurately determined, and Bacopaside I can be accurately separated.

Description

Technical Field

[0001] This invention relates to the field of analytical testing, specifically to a method for detecting purslane extract and its application. Background Technology

[0002] False purslane (Bacopa Monnieri) is an annual creeping plant of the Scrophulariaceae family with strong medicinal value. It contains abundant alkaloids and saponins, with the most important active ingredients being bacopasides (or bacopasaponins), which are classified into several subtypes. Currently, the main method for detecting and analyzing the active ingredients in false purslane is high-performance liquid chromatography (HPLC) as described in the United States Pharmacopeia. However, this method cannot completely separate various bacopasides, is easily interfered with by other compounds, and cannot accurately detect bacopaside I.

[0003] Therefore, it is still necessary to improve the detection and analysis methods for active ingredients in false purslane in order to more effectively control product quality. Summary of the Invention

[0004] This invention provides a method for detecting purslane extract and its application. The method uses a specific chromatographic column and mobile phase to effectively separate ten components: bacopaside A3, bacopaside II, bacopaside VI, J. bacopasaponin C, bacopasaponin C, bacopaside N1, bacopaside IV, bacopaside V, bacopaside I, and Apigenin. This method exhibits high resolution, sensitivity, and accuracy, and accurately separates bacopaside I. It can more accurately determine the concentration of each component, particularly accurately determining the content of bacopaside I, bacopaside A3, bacopaside II, J. bacipasagin C, and bacopasagin C, which have high medicinal value.

[0005] This invention provides a method for detecting Portulaca oleracea extract, the method comprising: analyzing the Portulaca oleracea extract using a high-performance liquid chromatography combined with a diode array detector system to identify the components in the Portulaca oleracea extract; wherein the chromatographic column is a reversed-phase chromatographic column containing a bonded phase of octadecylsilane and a support of silica gel; the mobile phase comprises mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile and mobile phase B is composed of a buffer salt aqueous solution and methanol.

[0006] Optionally, the buffer salt is a carbonate, preferably ammonium carbonate; in the aqueous buffer salt solution, the concentration of the buffer salt is 0.01-0.05%, preferably 0.01-0.025%, more preferably 0.025%, based on the weight of the aqueous buffer salt solution.

[0007] Optionally, in mobile phase B, the concentration of methanol is 30% by volume, based on the total volume of the buffer salt solution and methanol.

[0008] Optionally, the chromatographic column has a length of 200mm-300mm, an inner diameter of 2mm-6mm, and an average particle size of 1.7μm-5μm for the packing material.

[0009] Optionally, the chromatographic column is an ODS-HL column purchased from Shimadzu Corporation of Japan, wherein the average particle size of the packing material is 3 μm, the inner diameter is 4.6 mm, and the length is 250 mm.

[0010] Optionally, gradient elution is employed in the high-performance liquid chromatography; wherein the gradient elution time program is as follows:

[0011]

[0012] During the elution process, the sum of the volume percentages of mobile phase A and mobile phase B is 100%, based on the volume of the mobile phase.

[0013] Optionally, the components of the purslane extract include bacopaside A3, bacopaside II, bacopaside VI, J. bacopasaponin C, bacopasaponin C, bacopaside N1, bacopaside IV, bacopaside V, bacopaside I, and Apigenin.

[0014] Optionally, the resolution of bacopaside I, bacopaside A3, bacopaside II, J. bacipasagin C and bacopasagin C is not less than 1.5.

[0015] The present invention also provides a method for determining the concentration of components in a purslane extract, comprising determining the concentration of components in the purslane extract using the method described above.

[0016] Optionally, the method includes (1) detecting a standard solution containing each component using the method described above to obtain a standard curve of each component with peak area as the ordinate and mass concentration as the abscissa; (2) detecting the purslane extract using the method described above to obtain the peak area of ​​each component in the chromatogram of the purslane extract; and (3) calculating the concentration of each component in the purslane extract based on the peak area of ​​each component in the chromatogram of the obtained purslane extract and the standard curve. Attached Figure Description

[0017] For illustrative and non-limiting purposes, the invention will now be described with reference to embodiments thereof and the accompanying drawings, wherein:

[0018] Figure 1 The chromatogram of purslane extract 2 in acetonitrile + water as the mobile phase is shown.

[0019] Figure 2 The chromatogram of purslane extract 2 in tetrahydrofuran + water as the mobile phase is shown.

[0020] Figure 3 The chromatogram of Portulaca oleracea extract 2 in 10% tetrahydrofuran / acetonitrile + water as the mobile phase.

[0021] Figure 4 The chromatogram of purslane extract 2 in acetonitrile + methanol + water as the mobile phase is shown.

[0022] Figure 5 The chromatogram of purslane extract 2 on an ODS-35um 4.6*250mm column is shown.

[0023] Figure 6 The chromatogram of purslane extract 2 on a Hydro-RP 4um 4.6*250mm column is shown.

[0024] Figure 7 The chromatogram of purslane extract 2 on a Kinetex F55um 4.6*250mm column is shown.

[0025] Figure 8 The chromatogram of purslane extract 2 on an ODS-HL 3um 4.6*250mm column is shown.

[0026] Figure 9 The chromatogram of purslane extract 2 on an ODS-P 5.0um 250*4.6mm column is shown.

[0027] Figure 10 The chromatogram of purslane extract 2 on a Waters C18-5um 4.6*250 column is shown.

[0028] Figure 11 The chromatogram of Portulaca oleracea extract 2 was obtained using an ODS-HL 3um 4.6*250mm (UP) column at 30℃ with acetonitrile + methanol + water as the mobile phase.

[0029] Figure 12 The chromatogram of Portulaca oleracea extract 2 was obtained using an ODS-HL 3um 4.6*250mm (UP) column at 40℃ with acetonitrile, methanol and water as the mobile phase.

[0030] Figure 13 The chromatogram of the pseudopurslane extract 2 was obtained by gradient elution at 45°C using an ODS-HL 3um 4.6*250mm (UP) column with acetonitrile, methanol and water as the mobile phase.

[0031] Figure 14 The chromatogram of purslane extract 2 was obtained using acetonitrile + 30% methanol / water / 0.05% formic acid as the mobile phase and an ODS-HL3um 4.6*250mm column.

[0032] Figure 15 The chromatogram of purslane extract 2 on an ODS-HL 3um 4.6*250mm column with acetonitrile + 30% methanol / water / 0.05% ammonium acetate as the mobile phase is obtained.

[0033] Figure 16 The chromatogram of Portulaca oleracea extract 2 was obtained using acetonitrile + 30% methanol / water / 0.05% sodium ethylenediaminetetraacetate as the mobile phase and an ODS-HL 3um 4.6*250mm column.

[0034] Figure 17 The chromatogram of purslane extract 2 on an ODS-HL 3um 4.6*250mm column with acetonitrile + 30% methanol / water / 0.05% ammonium oxalate as the mobile phase is shown.

[0035] Figure 18 The chromatogram of purslane extract 2 on an ODS-HL 3um 4.6*250mm column with acetonitrile + 30% methanol / water / 0.05% ammonium carbonate as the mobile phase is obtained.

[0036] Figure 19 The chromatogram of purslane extract 1 on an ODS-HL 3um 4.6*250mm column with acetonitrile + 30% methanol / water / 0.05% ammonium carbonate as the mobile phase is obtained.

[0037] Figure 20The chromatogram of Portulaca oleracea extract 1 on an ODS-HL 3um 4.6*250mm column with acetonitrile + 30% methanol / water / 0.01% ammonium carbonate as the mobile phase is obtained.

[0038] Figure 21 The chromatogram of purslane extract 2 on an ODS-HL 3um 4.6*250mm column with acetonitrile + 30% methanol / water / 0.025% ammonium carbonate as the mobile phase is obtained.

[0039] Figure 22 The chromatogram of purslane extract 1 on an ODS-HL 3um 4.6*250mm column with acetonitrile + 30% methanol / water / 0.025% ammonium carbonate as the mobile phase is obtained.

[0040] Figure 23 The chromatogram of the mixed standard solution was obtained using acetonitrile + 30% methanol / water / 0.025% ammonium carbonate as the mobile phase and an ODS-HL3um 4.6*250mm column.

[0041] Figure 24 This is a purity graph for Apigenin.

[0042] Figure 25 This is a purity diagram of Bacopaside I.

[0043] Figure 26 This is a purity diagram of bacopaside VI.

[0044] Figure 27 This is a purity diagram of bacopaside A3.

[0045] Figure 28 This is a purity chart for bacopaside II.

[0046] Figure 29 The purity diagram of J. bacopasaponin C.

[0047] Figure 30 This is a purity graph of bacopasaponin C.

[0048] Figure 31 This is a purity diagram of bacopaside N1.

[0049] Figure 32 This is a purity graph of bacopaside IV.

[0050] Figure 33 This is a purity graph for bacopaside V.

[0051] Figure 34This is the hydrogen NMR spectrum of bacopaside VI.

[0052] in

[0053] Figures 1-23 The numbers in the middle correspond to the following components:

[0054] 1. bacopaside A3; 2. bacopaside II; 3. J. bacopasaponin C; 4. bacopasaponin C; 5. bacopaside VI; 6. bacopaside I; 7. apigenin; 8. bacopaside N1; 9. bacopaside IV; 10. bacopaside V. Detailed Implementation

[0055] To facilitate understanding of the present invention, a more complete description will be given below with reference to embodiments. Preferred embodiments of the invention are shown in the embodiments, and these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein.

[0056] Currently, most methods for detecting Portulaca oleracea extract adopt the methods described in the United States Pharmacopeia (USP). These methods involve using a liquid chromatography system and a UV detector at a wavelength of 205 nm, with a flow rate of 1.5 mL / min, an injection volume of 20 μL, and a column temperature of 27 ± 1 °C. Mobile phase A consists of dissolving 0.14 g of anhydrous potassium dihydrogen phosphate in 900 mL of water, adding 0.5 mL of phosphoric acid, diluting with water to 1000 mL, mixing, filtering, and degassing. Mobile phase B uses filtered and degassed acetonitrile.

[0057] The gradient elution time program for the mobile phase is shown in Table 1 below.

[0058] Table 1. Gradient elution time schedule for the mobile phase in the USP method.

[0059] Time (min) Mobile phase A (%) Mobile phase B (%) 0 70 30 25 60 40 26 70 30 30 70 30

[0060] The USP method primarily separates five substances: bacopaside I, bacopaside A3, bacopaside II, J. bacopasaponin C (a jujubogenin isomer of bacopasaponin C), and bacopasaponin C. It does not provide further research on other components, and the separation degree of each component is not high, resulting in low accuracy. This invention provides a method for detecting *Portulaca oleracea* extract. By selecting appropriate chromatographic conditions, it can separate up to 10 components with high separation degree, high sensitivity, good repeatability, and high accuracy, while also accurately separating Bacopaside I. Specifically, the inventors discovered that selecting a suitable chromatographic column can better separate isomers and homologues in *Portulaca oleracea* extract. Elution was performed using an acetonitrile / methanol-water solution with added buffer salt as the mobile phase. Methanol is a protic solvent with high selectivity for separating acids, bases, or highly electronegative compounds. Acetonitrile is a polar molecule with unbonded electrons in its carbon-nitrogen triple bond, which can bind to compounds containing empty orbitals, exhibiting a different compound selectivity than methanol. A buffer salt, such as ammonium carbonate, was added to the mobile phase. These components of the mobile phase improved the retention time of each component and optimized the elution of Bacopaside I.

[0061] In an embodiment of the present invention, a method for detecting Portulaca oleracea extract includes: analyzing the Portulaca oleracea extract using a high-performance liquid chromatography (HPLC) combined with a diode array detector (PDA) system to identify the components in the Portulaca oleracea extract; wherein the chromatographic column is a reversed-phase chromatographic column packed with a packing material in which the bonded phase is octadecylsilane and the support is silica gel; the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile and mobile phase B is composed of a buffer salt aqueous solution and methanol.

[0062] High-performance liquid chromatography (HPLC) is a commonly used analytical instrument in the field of science. It typically consists of a high-pressure pump, injector, column, detector, and integrator or data processing system. HPLC is a chromatographic method that uses a high-pressure pump to inject a specified mobile phase into a packed column to separate and determine the sample. The injected sample is carried into the column by the mobile phase, where the components are separated and detected by the detector. The integrator or data processing system records and processes the chromatographic signals to obtain a liquid chromatogram.

[0063] A photodiode array detector (PDA), also known as a photodiode array detector, is a detector formed by closely arranging a series of photodiodes on a crystalline silicon substrate. Each diode acts as an exit slit of a monochromator; the more diodes, the higher the resolution. Currently, PDAs are used in photodiode array detectors.

[0064] The chromatographic column is a reversed-phase column containing an octadecylsilane (C18) bonded phase and a silica gel support, such as spherical silica gel. Optionally, the column may employ end-capping technology, with a carbon content of 5-30%, for example 23%, and a pH range of 1.5-9.0. The column length is 200-300 mm, for example 250 mm; the inner diameter is 2 mm-6 mm, for example 4.6 mm; the average particle size of the packing material is 1.7 μm-5 μm, for example 3 μm; and the specific surface area is 400-500 m². 2 / g, for example 450m 2 / g; pore size is The chromatographic column can be any of the following models purchased from Shimadzu Corporation of Japan: ODS-HL column (average particle size of packing material 3 μm, inner diameter 4.6 mm, length 250 mm), Shim-pack Velox column (average particle size of packing material 2.7 μm, inner diameter 4.6 mm, length 150 mm), or ODS-P column (average particle size of packing material 5.0 μm, inner diameter 4.6 mm, length 250 mm); preferably, the ODS-HL column (average particle size of packing material 3 μm, inner diameter 4.6 mm, length 250 mm). The average particle size of the packing material and the column length can be adjusted as needed. Generally, after changing the average particle size of the packing material and the column length, the ratio of the average particle size of the packing material to the column length (N value) should be within the range of -25% to +50% of the original value.

[0065] The buffer salt may be a carbonate, preferably ammonium carbonate. In the aqueous buffer salt solution, the concentration of the buffer salt is 0.01-0.05%, preferably 0.01-0.03%, 0.02-0.03%, or 0.01-0.025%, more preferably 0.025%, based on the weight of the aqueous buffer salt solution. In mobile phase B, the concentration of methanol is 30% by volume, based on the total volume of the aqueous buffer salt solution and methanol.

[0066] In the mobile phase, mobile phases A and B can be degassed before entering the chromatographic column.

[0067] Gradient elution is employed in the high-performance liquid chromatography (HPLC); wherein the gradient elution time program is 0 min → 45 min: 12% mobile phase A → 50% mobile phase A; 45 min → 46 min: 50% mobile phase A → 12% mobile phase A; 46 min → 50 min: 12% mobile phase A → 12% mobile phase A, based on the volume of the mobile phase; or it can be represented by the following table:

[0068]

[0069] During elution, the sum of the volume percentages of mobile phase A and mobile phase B in the mobile phase is 100%, based on the volume of the mobile phase. Those skilled in the art can appropriately adjust the proportions of the mobile phase components to ensure that the system suitability meets the requirements, and that the final elution intensity of the mobile phase is not weaker than the elution intensity of the original gradient.

[0070] Those skilled in the art may adjust the gradient elution program as needed, but the following conditions must be met:

[0071]

[0072] in,

[0073] t G1 : represents the gradient elution time of the original method;

[0074] t G2 The adjusted gradient elution time;

[0075] F1 represents the flow rate in the original method;

[0076] F2 represents the flow rate in the adjusted method;

[0077] dc1 is the inner diameter of the chromatographic column in the original method;

[0078] dc2 is the inner diameter of the chromatographic column in the adjusted method;

[0079] L1 is the column length in the original method; and

[0080] L2 is the column length in the adjusted method;

[0081] Therefore, maintaining the same elution volume factor for columns of different specifications ensures the gradient.

[0082] The changes are the same, and the differences in the size of different instrument systems need to be taken into account.

[0083] In high-performance liquid chromatography (HPLC), the column temperature is 20-60℃, preferably 30-50℃, more preferably 40-50℃; the injection volume is 10-50 μL, preferably 20-40 μL, more preferably 30 μL; the flow rate is 0.1-2.5 ml / min, preferably 0.1-1.5 ml / min, more preferably 0.5-1.0 ml / min, preferably 0.7 ml / min; and the detection wavelength is 205 nm. Unless otherwise specified, the column temperature can be adjusted within ±5℃ of the above values; other parameters can be routinely adjusted according to the Chinese Pharmacopoeia (2020 edition).

[0084] The components in the purslane extract include bacopaside A3 (CAS: 157408-08-7), bacopaside II (CAS: 382146-66-9), and bacopaside VI (its 1H NMR spectrum can be found here). Figure 34 ), J. bacopasaponin C (also known as Bacopaside X, CAS: 94443-88-6), bacopasaponin C (CAS: 178064-13-6), bacopaside N1 (CAS: 871706-74-0), bacopaside IV (CAS: 155545-03-2), bacopaside V (CAS: 620592-16-7), bacopaside I (CAS: 382148-47-2), and Apigenin (CAS: 520-36-5).

[0085] In the chromatogram of this invention, Apigenin, bacopaside I, bacopaside VI, bacopaside A3, bacopaside II, J. bacopasaponin C, bacopasaponin C, bacopaside N1, bacopaside IV, and bacopaside V elute in sequence. For example, in a preferred embodiment, the retention time of bacopaside A3 is 35.83 min, bacopaside II is 36.42 min, bacopaside VI is 25.63 min, J. bacopasaponin C is 38.01 min, bacopasaponin C is 39.10 min, bacopaside N1 is 40.51 min, bacopaside IV is 42.08 min, bacopaside V is 42.78 min, bacopaside I is 22.88 min, and Apigenin is 11.03 min. In this invention, the retention times of each component can be appropriately shifted according to the operating conditions, but the elution order of the chromatographic peaks remains unchanged.

[0086] In the chromatograms of this invention, the resolution (R) between the chromatographic peaks of each component and adjacent chromatographic peaks is generally not less than 1.5; in particular, the resolution of bacopaside I, bacopaside A3, bacopaside II, J. bacipasagin C, and bacopasagin C, which have high medicinal value, is not less than 1.5, and may even be greater than 1.5. Resolution (R) is used to evaluate the degree of separation between the analyte and the separated substances, and is a key indicator for measuring the separation efficiency of a chromatographic system.

[0087] In an embodiment of the present invention, a method for determining the concentration of components in a purslane extract includes determining the concentration of components in the purslane extract using the method described above.

[0088] Optionally, the method includes

[0089] (1) Using the method described above, the standard solution containing each component was tested to obtain a standard curve of each component with peak area as the ordinate and mass concentration as the abscissa.

[0090] (2) The method described above was used to detect the purslane extract to obtain the peak area of ​​each component in the chromatogram of the purslane extract.

[0091] (3) The concentration of each component in the purslane extract was calculated based on the peak area of ​​each component in the chromatogram of the obtained purslane extract and the standard curve.

[0092] The correlation coefficients of the linear equations of the standard curves obtained in (1) are all greater than 0.999, indicating that the standard curves obtained by the method of the present invention have good linearity and high sensitivity.

[0093] The order of the above steps can be adjusted by those skilled in the art as needed. For example, steps (1) and (2) can be performed simultaneously or sequentially, or step (2) can be performed before step (1).

[0094] The detection method of this invention can be used to simultaneously identify and distinguish multiple components in Portulaca oleracea extract, and then quantitatively analyze the important components therein, greatly improving the detection efficiency of components, especially active components, in Portulaca oleracea extract. It can separate and detect up to 10 components, with the resolution of each component not less than 1.5 and the purity angles all less than the purity threshold. Therefore, this detection method can completely separate the components and accurately separate Bacopaside I. Moreover, the spiked recovery rate of each component is above 85%, so the detection method has high accuracy and can more accurately determine the concentration of each component. The relative standard error (RSD) of the concentration of each component determined by this detection method is less than 5%, so the detection method has high precision and good repeatability.

[0095] The present invention will be described below through specific embodiments. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific operating techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. In the present invention, the operating environment unless otherwise specified shall be understood as being at normal pressure and temperature. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0096] Example

[0097] instrument:

[0098] Waters 2698 high performance liquid chromatograph combined with 2998PDA diode array detector;

[0099] Ultrasonic cleaner with heating function (KUDOS);

[0100] Mettler balance (model: XSE204).

[0101] raw material:

[0102] Purslane Extract 1: Purslane extract from the United States Pharmacopeia (USP), product code 1048244;

[0103] Purslane extract 2: purchased from Natural Remedies, India, product code NRBME40E;

[0104] Methanol and acetonitrile were of chromatographic grade and purchased from Merck, Germany.

[0105] Ammonium acetate, ammonium carbonate, ammonium oxalate, formic acid, and acetic acid were all of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.

[0106] Bacopaside I standard, purchased from Chromadex, with a purity greater than 95%;

[0107] Bacopaside II, Bacopaside A3, Bacopaside IV, Bacopaside V, J. Bacopasaponin C, Bacopasaponin C, Bacopaside VI, Bacopaside N1, and Apigenin were all purchased from Dest Company, and their purity was greater than 95%.

[0108] Preparation of standard solutions

[0109] According to the required concentration, add the required weight of standard to a 5ml volumetric flask, dilute with methanol and bring to volume, and store at 4-8℃ for later use.

[0110] Preparation of mobile phase B

[0111] Weigh 0.25 g of ammonium carbonate standard, dissolve it in water, and dilute to 1000 ml to obtain an ammonium carbonate aqueous solution. Mix 700 ml of this ammonium carbonate aqueous solution with 300 ml of methanol, degas by sonication, and filter to obtain mobile phase B, which is ready for use.

[0112] Preparation of purslane extract samples

[0113] Weigh approximately 0.2 g of Portulaca oleracea extract and add it to a 50 mL volumetric flask. Then add 40 mL of methanol solution to the volumetric flask and shake well. Place the volumetric flask in a 60 °C water bath and sonicate at 53 Hz for 20 min. Remove it, cool it to room temperature, and dilute it to the mark with methanol solution. Shake well and filter through a 0.45 μm filter to obtain the Portulaca oleracea extract sample solution.

[0114] Example 1 Selection of mobile phase

[0115] Mobile phase 1

[0116] The *Portulaca oleracea* extract sample solution 2, prepared according to the "Preparation of *Portulaca oleracea* Extract Samples" method, was analyzed using a Shim-pack Velox 2.7µm 150mm*4.6mm column. The mobile phase was acetonitrile (A): water, flow rate was 0.7mL / min, column temperature was 35℃, and detection wavelength was 205nm. Elution was performed using a full gradient of A: 5%→95%, B: 95%→5% (0→100min) for 100 minutes. The resulting chromatogram is shown below. Figure 1 The separation of components in the purslane extract was poor.

[0117] Mobile phase 2

[0118] Using sample solution of *Portulaca oleracea* extract 2, a Shim-pack Velox 2.7µm 150mm*4.6mm column was used. Mobile phase A: tetrahydrofuran, B: water, flow rate 0.7mL / min, column temperature 35℃, detection wavelength 205nm, and elution was performed using a full gradient elution of A: 5%→95%, B: 95%→5% (0→100min) for 100 minutes. The resulting chromatogram is shown below. Figure 2 The separation of components in the purslane extract was poor.

[0119] Mobile phase 3

[0120] Using sample solution of *Portulaca oleracea* extract 2, a Shim-pack Velox 2.7µm 150mm*4.6mm column was used. Mobile phase A: 10% tetrahydrofuran / acetonitrile, B: water, flow rate 0.7mL / min, column temperature 35℃, detection wavelength 205nm, with a full gradient elution of A: 5%→95%, B: 95%→5% (0→100min) for 100 minutes. The resulting chromatogram is shown below. Figure 3 The separation of components in the purslane extract was poor.

[0121] Mobile phase 4

[0122] Using the sample solution of *Portulaca oleracea* extract 2, a Shim-pack Velox 2.7µm 150mm*4.6mm column was employed. The mobile phases were A: acetonitrile, B: methanol, and C: water. The flow rate was 0.7 mL / min, the column temperature was 35℃, and the detection wavelength was 205 nm. Elution was performed using a full gradient of A: 5%→60%, B: 0%→40%, C: 95%→5% (0→60 min) for 60 minutes. The resulting chromatogram is shown below. Figure 4 Among them, the components in the purslane extract showed good separation.

[0123] Based on the above experimental results, the separation degree of each component is better when acetonitrile, methanol and water are used as the mobile phase. Therefore, this mixed mobile phase of acetonitrile, methanol and water is selected as the basis for the research of this invention.

[0124] Example 2: Selection of Chromatographic Column

[0125] Column 1

[0126] Sample solution of *Portulaca oleracea* extract was used. An ODS-35um 4.6*250mm column was employed with the mobile phases A: acetonitrile, B: methanol, and C: water. The flow rate was 0.7 mL / min, the column temperature was 35℃, and the detection wavelength was 205 nm. Elution was performed using a full gradient of A: 5%→60%, B: 0%→40%, C: 95%→5% (0→60 min) for 60 minutes. The resulting chromatogram is shown below. Figure 5 The separation of components in the purslane extract was poor.

[0127] Column 2

[0128] Sample solution of *Portulaca oleracea* extract was used. A Hydro-RP 4µm 4.6*250mm column was employed with mobile phases A: acetonitrile, B: methanol, and C: water. The flow rate was 0.7 mL / min, column temperature was 35℃, and detection wavelength was 205 nm. Elution was performed using a full gradient of A: 5%→60%, B: 0%→40%, C: 95%→5% (0→60 min) for 60 min. The resulting chromatogram is shown below. Figure 6 The separation of components in the purslane extract was poor.

[0129] Column 3

[0130] Sample solution of *Portulaca oleracea* extract was used. A Kinetex F5 5µm 4.6*250mm column was employed with mobile phases A: acetonitrile, B: methanol, and C: water. The flow rate was 0.7 mL / min, column temperature was 35℃, and detection wavelength was 205 nm. Elution was performed using a full gradient of A: 5%→60%, B: 0%→40%, C: 95%→5% (0→60 min) for 60 min. The resulting chromatogram is shown below. Figure 7 The separation of components in the purslane extract was poor.

[0131] Column 4

[0132] Sample solution of *Portulaca oleracea* extract was used. An ODS-HL 3µm 4.6*250mm (UP) column was employed with mobile phases A: acetonitrile, B: methanol, and C: water. The flow rate was 0.7 mL / min, column temperature was 35℃, and detection wavelength was 205 nm. Elution was performed using a full gradient of A: 5%→60%, B: 0%→40%, C: 95%→5% (0→60 min) for 60 min. The resulting chromatogram is shown below. Figure 8 Among them, the components in the purslane extract showed good separation.

[0133] Column 5

[0134] Sample solution of *Portulaca oleracea* extract was used. An ODS-P 5.0µm 250*4.6mm column was employed with the mobile phases A: acetonitrile, B: methanol, and C: water. The flow rate was 0.7 mL / min, the column temperature was 35℃, and the detection wavelength was 205 nm. Elution was performed using a full gradient of A: 5%→60%, B: 0%→40%, C: 95%→5% (0→60 min) for 60 minutes. The resulting chromatogram is shown below. Figure 9 Among them, the components in the purslane extract showed good separation.

[0135] Column 6

[0136] Sample solution of *Portulaca oleracea* extract was used. A Waters C18-5µm 4.6*250 column was employed with mobile phases A: acetonitrile, B: methanol, and C: water. The flow rate was 0.7 mL / min, column temperature was 35℃, and detection wavelength was 205 nm. Elution was performed using a full gradient of A: 5% → 60%, B: 0% → 40%, C: 95% → 5% (0 → 60 min) for 60 min. The resulting chromatogram is shown below. Figure 10 The separation of components in the purslane extract was poor.

[0137] As can be seen from the experimental results of Example 2, the ODS-HL 3um 4.6*250mm (UP) column, the Shim-packvelox 2.7um 150mm*4.6mm column, and the ODS-P 5.0um 250*4.6mm column all achieved high resolution.

[0138] Example 3: Selection of Column Temperature

[0139] Temperature 1

[0140] Repeat the operation steps of column 4 in Example 2, except that the column temperature is 30°C. The resulting chromatogram is shown below. Figure 11 .

[0141] Temperature 2

[0142] Repeat the operation steps of column 4 in Example 2, except that the column temperature is 40°C. The resulting chromatogram is shown below. Figure 12 .

[0143] Temperature 3

[0144] Repeat the operation steps of column 4 in Example 2, except that the column temperature is 45°C. See the chromatogram obtained. Figure 13 .

[0145] Based on the above experimental results, column temperature has no significant effect on the separation degree of each component. To protect the HPLC pressure, 45℃ was chosen as the column temperature.

[0146] Example 4 Selection of Buffer Salt

[0147] Buffer salt 1

[0148] Sample solution of *Portulaca oleracea* extract was used. An ODS-HL 3µm 4.6*250mm (UP) column was employed. Mobile phases A: acetonitrile, B: 30% methanol / water + 0.05% formic acid. The flow rate was 0.7 mL / min, column temperature was 45℃, and detection wavelength was 205 nm. Elution was performed using a full gradient elution for 80 minutes, A: 0%→100%, B: 100%→0% (0→80 min). The resulting chromatogram is shown below. Figure 14 The sample contained a large number of separated components with high separation, and the Bacopaside I peak had appeared, but the peak shape was poor and the baseline was drifted.

[0149] Buffer salt 2

[0150] Sample solution of *Portulaca oleracea* extract was used. An ODS-HL 3µm 4.6*250mm (UP) column was employed. Mobile phase A: acetonitrile, B: 30% methanol / water + 0.05% ammonium acetate, flow rate 0.7 mL / min, column temperature 45℃, detection wavelength 205 nm. Elution was performed using a full gradient elution for 80 minutes, A: 0%→100%, B: 100%→0% (0→80 min). The resulting chromatogram is shown below. Figure 15 The sample contained a large number of separated components with high separation, and the Bacopaside I peak had appeared with a good peak shape, but the baseline drift was severe.

[0151] Buffer salt 3

[0152] Using sample solution of *Portulaca oleracea* extract, an ODS-HL 3µm 4.6*250mm (UP) column was employed. Mobile phase A: acetonitrile; Mobile phase B: 30% methanol / water + 0.05% sodium ethylenediaminetetraacetate; Flow rate: 0.7 mL / min; Column temperature: 45℃; Detection wavelength: 205 nm; Elution was performed using a full gradient elution for 80 minutes, A: 0%→100%, B: 100%→0% (0→80 min). The resulting chromatogram is shown below. Figure 16 The sample contained a large number of separated components with high separation, and the Bacopaside I peak had appeared, but the peak shape was poor.

[0153] Buffer salt 4

[0154] Sample solution of *Portulaca oleracea* extract was used. An ODS-HL 3µm 4.6*250mm (UP) column was employed. Mobile phase A: acetonitrile, B: 30% methanol / water + 0.05% ammonium oxalate, flow rate 0.7 mL / min, column temperature 45℃, detection wavelength 205 nm. Elution was performed using a full gradient elution for 80 minutes, A: 0%→100%, B: 100%→0% (0→80 min). The resulting chromatogram is shown below. Figure 17 The separation of the components was extremely poor, and the baseline drift was severe.

[0155] Buffer salt 5

[0156] Using sample solution of *Portulaca oleracea* extract, an ODS-HL 3µm 4.6*250mm (UP) column was used. Mobile phase A: acetonitrile, B: 30% methanol / water + 0.05% ammonium carbonate, flow rate 0.7 mL / min, column temperature 45℃, detection wavelength 205 nm. Elution was performed using a full gradient elution for 80 minutes, A: 0%→100%, B: 100%→0% (0→80 min). The resulting chromatogram is shown below. Figure 18 The sample contained a large number of separated components with high separation degree. The Bacopaside I peak had appeared with good peak shape and stable baseline.

[0157] The experimental results above show that ammonium carbonate buffer can further improve the separation of the components.

[0158] Example 5

[0159] Sample name: Sample solution using purslane extract 1

[0160] Instrumentation: A Waters 2698 high-performance liquid chromatograph combined with a 2998 PDA diode array detector was used.

[0161] Chromatographic conditions include:

[0162] Column: ODS-HL 3um 4.6*250mm

[0163] Column temperature: 45℃

[0164] Injection volume: 30 μL

[0165] Flow rate: 0.7 mL / min

[0166] Detection wavelength: 205nm

[0167] Mobile phase A is acetonitrile.

[0168] Mobile phase B consists of 0.025% ammonium carbonate aqueous solution and methanol, wherein the volume ratio of the ammonium carbonate aqueous solution to methanol is 70% by volume and 30% by volume, respectively;

[0169] Elution program: Gradient elution, the specific elution program is shown in Table 2 below, where the volume percentages of mobile phase A and mobile phase B are used.

[0170] Table 2 shows the elution procedure of the mobile phase in Example 5.

[0171]

[0172]

[0173] The chromatogram obtained by this method is as follows: Figure 22 The retention times and separation degrees of each component are shown in Table 3 below.

[0174] Table 3 shows the retention times and resolutions of each component in the chromatogram of Example 5.

[0175]

[0176] The resolution of each component in this detection method is not less than 1.5. Therefore, this detection method can completely separate each component and accurately separate Bacopaside I.

[0177] Example 6

[0178] Repeat the steps described in Example 5, except that mobile phase B consists of 70% by volume of 0.05% ammonium carbonate aqueous solution and 30% by volume of methanol.

[0179] The chromatogram of the obtained purslane extract is shown below. Figure 19 As shown.

[0180] The retention times and resolutions of each component in the obtained chromatograms are shown in Table 4 below.

[0181] Table 4 shows the retention times and resolutions of each component in the chromatograms of Example 6.

[0182]

[0183] In this detection method, the resolution of Bacopaside I is 0.642, which is less than 1.5. The resolutions of bacopaside A3, bacopaside II, J. bacipasagin C, and bacopasagin C are not less than 1.5.

[0184] Example 7

[0185] Repeat the steps described in Example 5, except that mobile phase B consists of 70% by volume of 0.01% ammonium carbonate aqueous solution and 30% by volume of methanol.

[0186] The chromatogram of the obtained purslane extract is shown below. Figure 20 As shown.

[0187] The retention times and resolutions of each component in the obtained chromatograms are shown in Table 5 below.

[0188] Table 5 shows the retention times and resolution of each component in the chromatogram of Example 7.

[0189]

[0190] In this detection method, the resolution of apigenin was 1.126, which is less than 1.5, and the baseline fluctuation was large. However, the resolutions of the five components with high medicinal value, bacopaside I, bacopaside A3, bacopaside II, J, bacipasagin C, and bacopasagin C, were all greater than 1.5, indicating that they could be separated well.

[0191] Example 8

[0192] Repeat the steps of Example 5, except that the test sample is replaced with a sample solution of *Portulaca oleracea* extract 2. The resulting chromatogram is shown below. Figure 21 As shown.

[0193] Repeat the steps of Example 5, except that the test sample is replaced with a mixed standard solution of each component; wherein the mixed standard solution includes bacopaside A3, bacopaside II, bacopaside IV, bacopaside X, bacopasaponin C, bacopaside N1, bacopaside IV, bacopaside V, bacopaside I and Apigenin, and its preparation method is as follows:

[0194] (1) Weigh out the following at room temperature and normal pressure: Bacopaside I 33.42 mg, Bacopaside II 21.75 mg, Bacopaside A 315.60 mg, Bacopaside IV 4.87 mg, Bacopaside V 5.21 mg, Bacopaside VII 18.27 mg, Bacopasaponin C 17.28 mg, Bacopaside VI 4.03 mg, Bacopaside N1 4.86 mg, J. bacopasaponin C 5.61 mg, and Apigenin 7.04 mg.

[0195] (2) Add each of the weighed components to 10 mL of methanol, sonicate to dissolve, and dilute to the mark to obtain standard solutions of each component. Then, transfer 0.5 mL of the standard solutions of each component to prepare a mixed standard solution.

[0196] The chromatogram of the mixed standard solution of each component is as follows: Figure 23 As shown.

[0197] The purity of each component was analyzed using the following methods:

[0198] The purity charts of each component were obtained using Waters' built-in Empower3 software, as shown below:

[0199] See the purity chart for Apigenin. Figure 24 ;

[0200] See the purity chart for Bacopaside I. Figure 25 ;

[0201] See the purity chart for bacopaside VI. Figure 26 ;

[0202] See the purity chart for bacopaside A3. Figure 27 ;

[0203] See the purity chart for bacopaside II. Figure 28 ;

[0204] See the purity diagram of J. bacopasaponin C. Figure 29 ;

[0205] See the purity chart for bacopasaponin C. Figure 30 ;

[0206] See the purity chart for bacopaside N1. Figure 31 ;

[0207] See the purity chart for bacopaside IV. Figure 32 ;

[0208] See the purity chart for bacopaside V. Figure 33 .

[0209] As shown in the purity diagram above, the relevant purity angles and purity thresholds of each component are as shown in Table 6.

[0210] Table 6. Purity angles and purity thresholds of each component.

[0211]

[0212] Conclusion: This detection method can be used to simultaneously determine and identify multiple components, greatly improving the detection efficiency of components, especially active ingredients, in *Portulaca oleracea* extract. Furthermore, the resolution of each component is not less than 1.5, and the purity angle is less than the purity threshold. Therefore, this detection method can completely separate the components and accurately separate Bacopaside I.

[0213] Methodological evaluation

[0214] Example 9: Investigation of linear range, limit of detection, and limit of quantitation

[0215] (1) Prepare five sets of mixed standard solutions, which includes the following steps:

[0216] Weigh 16.44 mg of bacopaside I, 13.20 mg of bacopaside A, 18.24 mg of bacopaside II, 15.24 mg of J. bacopasaponin C, and 14.44 mg of bacopasaponin C into 25 mL volumetric flasks, add methanol and sonicate to dissolve, then dilute to the mark and store at 2-8 °C to obtain the initial mixed standard solution.

[0217] Then, 1 mL of the above initial mixed standard solution was transferred and diluted with methanol to 10 mL to obtain the first mixed standard solution, wherein the first mixed standard solution contains 0.0655 mg / mL of bacopaside I, 0.0520 mg / mL of bacopaside A3, 0.0717 mg / mL of bacopaside II, 0.0609 mg / mL of J. bacopasaponin C and 0.0576 mg / mL of bacopasaponin C;

[0218] Take 1 mL of the above initial mixed standard solution and dilute it with methanol to 8 mL to obtain a second mixed standard solution, wherein the second mixed standard solution contains 0.0819 mg / mL of bacopaside I, 0.0650 mg / mL of bacopaside A3, 0.0897 mg / mL of bacopaside II, 0.0761 mg / mL of J. bacopasaponin C and 0.0720 mg / mL of bacopasaponin C;

[0219] Take 1.5 mL of the above initial mixed standard solution and dilute it to 10 mL with methanol to obtain the third mixed standard solution, wherein the third mixed standard solution contains 0.0982 mg / mL of bacopaside I, 0.0781 mg / mL of bacopaside A3, 0.1077 mg / mL of bacopaside II, 0.0914 mg / mL of J. bacopasaponin C and 0.0865 mg / mL of bacopasaponin C;

[0220] Take 2 mL of the above initial mixed standard solution and dilute it with methanol to 8 mL to obtain a fourth mixed standard solution, wherein the fourth mixed standard solution contains 0.1638 mg / mL bacopaside I, 0.1300 mg / mL bacopaside A3, 0.1793 mg / mL bacopaside II, 0.1523 mg / mL J bacopasaponin C and 0.1441 mg / mL bacopasaponin C;

[0221] Take 2 mL of the above initial mixed standard solution and dilute it with methanol to 4 mL to obtain the fifth mixed standard solution, which contains 0.3275 mg / mL bacopaside I, 0.2600 mg / mL bacopaside A3, 0.3586 mg / mL bacopaside II, 0.3045 mg / mL J. bacopasaponin C and 0.2881 mg / mL bacopasaponin C.

[0222] (2) Using the operating conditions described in Example 5, the above five groups of mixed standard solutions were tested. Based on the obtained chromatograms, a standard curve was plotted with peak area (Y) as the ordinate and mass concentration (X) as the abscissa. The linear equation, linear correlation coefficient, limit of detection, and limit of quantitation are shown in Table 7. The limit of detection for each analyte was calculated using S / N = 3, and the limit of quantitation for each analyte was calculated using a signal-to-noise ratio (S / N = 10). The results showed that the linear relationship of each component within its linear range was good, and the correlation coefficient R... 2 All values ​​were greater than 0.999, indicating that the method has high sensitivity and can be used for quantitative analysis.

[0223] Table 7 shows the linear equations, linear correlation coefficients, limits of detection, and limits of quantitation for each component.

[0224]

[0225] Example 10: Precision Examination

[0226] (1) Weigh 0.216g, 0.2172g, 0.2148g, 0.1797g, 0.231g and 0.1839g of purslane extract 2 respectively, and prepare 6 groups of purslane extract 2 solutions according to the steps in "Products of purslane extract samples" described above.

[0227] (2) Using the operating conditions of Example 5, the prepared purslane extract solutions 2 of 6 groups were tested, and the peak areas of each component in the obtained chromatograms were recorded. Based on the standard curve relationship obtained in Example 9, the contents of bacopaside I, bacopaside A3, bacopaside II, J. bacopasaponin C and bacopasaponin C in the purslane extract and their relative standard deviations (RSDs) were calculated, as shown in Table 8. The relative standard errors (RSDs) of the concentrations of each component determined by this detection method were all less than 5%. Therefore, this detection method has high precision and good repeatability.

[0228] Table 8. Content and RSD of each component in the extract of *Portulaca oleracea*

[0229]

[0230] The results above show that there was no significant difference in the saponin content of the six groups of Portulaca oleracea extract 2 determined by this method, with an RSD of 2.2-3.0%, indicating that the method is stable and reliable and meets the requirements of the test samples.

[0231] Example 11 Accuracy Examination

[0232] (1) Three groups of samples were weighed for the spiked recovery test. The first group consisted of two samples weighing 0.2108 g each, the second group consisted of two samples weighing 0.2149 g each, and the third group consisted of two samples weighing 0.2208 g each. One of the two samples was used as the sample matrix and the other as the spiked matrix. All samples were prepared into methanol solutions according to the steps in "Preparation of False Purslane Extract Samples" described above.

[0233] (2) 7.15 mg of bacopaside I, 5.16 mg of bacopaside A3, 7.28 mg of bacopaside II, 6.09 mg of J. bacopasaponin C, and 5.91 mg of bacopasaponin C were added to the spiked matrix samples of Group 1; 14.34 mg of bacopaside I, 10.35 mg of bacopaside A3, 14.55 mg of bacopaside II, 12.18 mg of J. bacopasaponin C, and 11.82 mg of bacopasaponin C were added to the spiked matrix samples of Group 2; and 21.52 mg of bacopaside I, 15.53 mg of bacopaside A3, 21.83 mg of bacopaside II, 18.27 mg of J. bacopasaponin C, and 17.73 mg of bacopasaponin C were added to the spiked matrix samples of Group 3. C; Obtain all spiked solutions.

[0234] (3) The sample matrix solution and the spiked solution were tested according to the operating conditions in Example 5.

[0235] (4) Based on the above chromatograms and the standard curve obtained in Example 9, calculate the content of each component in the matrix solution and the spiked solution, and then calculate the spiked recovery rate according to the following formula:

[0236]

[0237] The concentrations and recoveries of each component are shown in Table 9. The recoveries of all components are in the range of 89.79% to 113.13%, indicating that the detection method is highly accurate and can determine the concentration of each component more precisely.

[0238] Table 9 Spiked recoveries of each component

[0239]

[0240] As can be seen from the above embodiments and comparative examples, the detection method of the present invention can detect 10 components at once: bacopaside A3, bacopaside II, bacopaside VI, J. bacopasaponin C, bacopasaponin C, bacopaside N1, bacopaside IV, bacopaside V, bacopaside I, and Apigenin. The separation degree of each component is very high, and Bacopaside I can be accurately separated. At the same time, the detection method has high sensitivity, high precision, and high accuracy, and can accurately detect the concentration of each component.

[0241] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can occur depending on design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A method for detecting *Purslane spp.* extract, the method comprising: The extract of Portulaca oleracea was analyzed using a high-performance liquid chromatography (HPLC) system with a diode array detector to identify its components. The chromatographic column was a reversed-phase column containing an octadecylsilane bonded phase and a silica gel support. The mobile phase consisted of mobile phase A and mobile phase B, where mobile phase A was acetonitrile and mobile phase B was composed of a buffer salt solution and methanol.

2. The method according to claim 1, wherein, The buffer salt is a carbonate, preferably ammonium carbonate; in the aqueous buffer salt solution, the concentration of the buffer salt is 0.01-0.05%, preferably 0.01-0.025%, more preferably 0.025%, based on the weight of the aqueous buffer salt solution.

3. The method according to claim 1 or 2, wherein, In mobile phase B, the concentration of methanol is 30% by volume, based on the total volume of the buffer salt solution and methanol.

4. The method according to claim 1 or 2, wherein, The chromatographic column has a length of 200mm-300mm and an inner diameter of 2mm-6mm, with the packing material having an average particle size of 1.7μm-5μm.

5. The method according to claim 4, wherein the chromatographic column is an ODS-HL chromatographic column purchased from Shimadzu Corporation of Japan, wherein the average particle size of the packing material is 3 μm, the inner diameter is 4.6 mm, and the length is 250 mm.

6. The method according to claim 1 or 2, wherein, Gradient elution is used in the high-performance liquid chromatography; wherein the gradient elution time program is as follows: During the elution process, the sum of the volume percentages of mobile phase A and mobile phase B is 100%, based on the volume of the mobile phase.

7. The method according to claim 1 or 2, wherein, The components in the purslane extract include bacopaside A3, bacopaside II, bacopaside VI, J. bacopasaponin C, bacopasaponin C, bacopaside N1, bacopaside IV, bacopaside V, bacopaside I, and Apigenin.

8. The method according to claim 7, wherein, The separation ratio of bacopaside I, bacopaside A3, bacopaside II, J. bacipasagin C and bacopasagin C is not less than 1.

5.

9. A method for determining the concentration of a component in a Portulaca oleracea extract, comprising determining the concentration of a component in the Portulaca oleracea extract using the method of any one of claims 1-8.

10. The method according to claim 9, wherein, It includes (1) Using the method of any one of claims 1-8, a standard solution containing each component is tested to obtain a standard curve of each component with peak area as the ordinate and mass concentration as the abscissa. (2) The method of any one of claims 1-8 is used to detect the purslane extract to obtain the peak area of ​​each component in the chromatogram of the purslane extract; (3) The concentration of each component in the purslane extract was calculated based on the peak area of ​​each component in the chromatogram of the obtained purslane extract and the standard curve.

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