A HPLC analysis method for index components in the stems of Glechoma longituba

Abstract

The invention relates to an HPLC analysis method for index components aristolochic acid AⅡ, goniopedaline, piperonolactone B, aristolochic acid BⅡ and 5,6,7,8-tetramethoxyflavonoids in the stem of Cucurbita oleracea. The method of the invention can efficiently separate five index components and effectively determine the content of multiple index components in the stem of Cucurbita oleracea, with good separation effect, accurate and reliable method, and provides an effective analysis scheme for the research, development, utilization and quality control of Cucurbita oleracea medicinal materials.

Description

Technical Field

[0001] The invention belongs to the field of analysis and detection, and particularly relates to a HPLC analysis method for index components in the stem of Cucurbita ovata. Background Art

[0002] Fissistigma Griff, also known as Zuanshanfeng and Tiezuan, is one of the "Eighteen Zuan" of Yao medicine. The main components of this genus of plants are alkaloids, flavonoids, glycosides, organic acids, volatile oils, etc. It has the effects of promoting blood circulation and removing blood stasis, dispelling wind and dampness, anti-tumor, analgesia and anti-inflammatory, and is mainly used to treat arthritis, rheumatic bone pain, gout, etc. It is used both in folk and clinical settings, with significant therapeutic effects and high medicinal value.

[0003] Fissistigma retusum is one of the representative plants of this genus, with abundant resources and clear folk medicinal effects. However, there is currently no quality control method for Fissistigma retusum, and in-depth and standardized research cannot be carried out. Therefore, developing a method that can simultaneously detect the content of multiple index components of Fissistigma retusum and perform multi-index component control has far-reaching significance for the research, development, utilization and quality control of Fissistigma retusum medicinal materials. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides an HPLC analysis method for index components in the stems of Glechoma longituba, which solves the problem that there is no content determination method and quality control of Glechoma longituba. The method is feasible, accurate and has good separation effect.

[0005] The technical solution of the present invention to solve the technical problem is as follows:

[0006] A HPLC analysis method for index components in the stem of Cucurbita ovata, the chromatographic conditions are: C18 chromatographic column; mobile phase: phase A is 0.1% phosphoric acid aqueous solution, mobile phase B is acetonitrile, and the following gradient elution is adopted:

[0007] At 0-10 min, 80%-72% mobile phase A, 20%-28% mobile phase B;

[0008] At 10-30 min, 72%-68% mobile phase A, 28%-32% mobile phase B;

[0009] At 30-50 min, 68%-62% mobile phase A, 32%-38% mobile phase B;

[0010] At 50-70 min, 62%-46% mobile phase A, 38%-54% mobile phase B;

[0011] The index components are aristolochic acid lactone AⅡ, goniopedaline, piperonolactone B, aristolochic acid lactone BⅡ and 5,6,7,8-tetramethoxyflavone.

[0012] The method of the present invention, the mobile phase flow rate: 1mL min -1 The column temperature was 25°C.

[0013] Dual wavelengths were used for detection, with the detection wavelengths being 223nm and 254nm.

[0014] In a specific example of the present invention, the chromatographic column is ACE EXCEL 5C18 (article number EXL-121-2546U), 250×4.6 mm.

[0015] The method of the present invention specifically comprises the following steps:

[0016] Step a, preparation of the test solution: crush the dried stems of the genus Achyranthes, pass them through a No. 4 sieve, accurately weigh 5 g, place them in a 25 mL volumetric flask, add methanol to make up the volume, weigh them; ultrasonicate for 30 min, cool, weigh, make up the weight loss with methanol, shake well, filter, remove the filtrate, and obtain the test solution;

[0017] Step b, preparation of reference solution: accurately weigh appropriate amounts of aristolochic acid lactam AⅡ, goniopedaline, piperonyl lactam B, aristolochic acid lactam BⅡ and 5,6,7,8-tetramethoxyflavone reference substances, dissolve them in methanol and prepare. Measure appropriate amounts of mother liquor respectively, dilute with methanol, and prepare a mixed reference solution containing aristolochic acid lactam AⅡ, goniopedaline, piperonyl lactam B, aristolochic acid lactam BⅡ and 5,6,7,8-tetramethoxyflavone.

[0018] Step c, inject the test solution and the reference solution into the high performance liquid chromatograph according to the above chromatographic conditions for chromatographic analysis. The concentrations of the mother solutions of the reference substances of aristolochic acid lactam AⅡ, goniopedaline, piperonolactam B, aristolochic acid lactam BⅡ and 5,6,7,8-tetramethoxyflavone are 1.54 mg / mL, 0.69 mg / mL, 0.64 mg / mL, 0.23 mg / mL and 6.29 mg / mL respectively. The concentration of the mixed reference solution is 375 μg / mL of aristolochic acid lactam AⅡ, 300 μg / mL of goniopedaline, 540 μg / mL of piperonolactam B, 320 μg / mL of aristolochic acid lactam BⅡ and 1541.2 μg / mL of 5,6,7,8-tetramethoxyflavone.

[0019] Advantages of the present invention:

[0020] The present invention uses modern chromatography technology to study traditional Chinese medicine for multi-index components in the stem of Achyranthes bidentata, which has far-reaching scientific research significance; a high performance liquid chromatography (HPLC) method is used to provide a multi-index component analysis method in the stem of Achyranthes bidentata. This method fills the gap that there is no HPLC method used in the current domestic research on the multi-component detection of Achyranthes bidentata medicinal materials; the detection of multi-index components in the stem of Achyranthes bidentata is accurate and reliable, with good separation effect, which provides an important basis for the in-depth study and quality control of Achyranthes bidentata, and has great application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 HPLC comparison chromatograms of the test sample solution under different chromatographic column conditions in Example 1 of the present invention.

[0022] Figure 2 HPLC comparison chromatograms of the test sample solutions under different mobile phase conditions in Example 2 of the present invention.

[0023] Figure 3 The HPLC comparison chromatograms of the test sample solution under different elution conditions in Example 3 of the present invention are shown.

[0024] Figure 4 This is a chromatogram of a mixed reference substance for the method of the present invention (peak 1: aristolochic acid lactam AⅡ, peak 2: goniopedaline, peak 3: piperonolactam B, peak 4: aristolochic acid lactam BⅡ, peak 5: 5,6,7,8-tetramethoxyflavone).

[0025] Figure 5 The present invention provides a chromatogram of five index components detected in the stem of Cucurbita ovata using the method described in the present invention (peak 1: aristolochic acid amine AⅡ, peak 2: goniopedaline, peak 3: piperonolactam B, peak 4: aristolochic acid amine BⅡ, peak 5: 5,6,7,8-tetramethoxyflavone). DETAILED DESCRIPTION

[0026] The present invention is further described in detail below with reference to the accompanying drawings and in conjunction with the embodiments. However, the present invention is not limited to the examples given. The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention is further described in detail below with reference to specific examples and with reference to data.

[0027] Instruments and reagents:

[0028] Lc-20AT high performance liquid chromatograph (Shimadzu Corporation, Japan); EXL-121-2546U ACE EXCEL 5C18, 250*4.6mm, V18-2503, A231359 (ACE Co., Ltd.); Shiding SHB-Ⅲ circulating water multi-purpose vacuum pump (Zhengzhou Great Wall Science and Technology Industry and Trade Co., Ltd.)

[0029] Aristolochic acid lactone AⅡ, goniopedaline, piperonolactone B, aristolochic acid lactone BⅡ and 5,6,7,8-tetramethoxyflavone (the reference substances were prepared according to the literature (Liu Huizhen. Study on the chemical composition and anti-gout effect of Aristolochic acid glutinosa [D]. Jiangxi University of Traditional Chinese Medicine, 2023. DOI: 10.27180 / d.cnki.gjxzc.2023.000149).

[0030] Preparation of test solution: Crush the dried stems of Glechoma longituba, pass through a No. 4 sieve, accurately weigh 5 g, place in a 25 mL volumetric flask, add methanol to make up the volume, weigh and sonicate for 30 min, cool, weigh, make up the lost weight with methanol, shake well, filter, remove the filtrate, and obtain the test solution;

[0031] Preparation of reference solution: Accurately weigh appropriate amounts of aristolochic acid lactam AⅡ, goniopedaline, piperonyl lactam B, aristolochic acid lactam BⅡ and 5,6,7,8-tetramethoxyflavone reference substances, dissolve them in methanol and prepare reference substance mother solutions with concentrations of 1.54 mg / mL, 0.69 mg / mL, 0.64 mg / mL, 0.23 mg / mL and 6.29 mg / mL, respectively. Take appropriate amounts of the mother solutions, dilute with methanol, and prepare mixed reference solution containing 375 μg / mL of aristolochic acid lactam AⅡ, 300 μg / mL of goniopedaline, 540 μg / mL of piperonyl lactam B, 320 μg / mL of aristolochic acid lactam BⅡ and 1541.2 μg / mL of 5,6,7,8-tetramethoxyflavone.

[0032] Example 1 Screening of Chromatographic Conditions

[0033] 1. Screening of chromatographic columns

[0034] 1.1 Chromatographic conditions

[0035] The detection wavelengths were 223 nm and 254 nm, the column temperature was 25 °C, and the flow rate was 1 mL min -1 , mobile phase A is 0.1% phosphoric acid aqueous solution, mobile phase B is acetonitrile, gradient elution,

[0036] The elution method is as follows:

[0037] 0~10min, 80%~70%A, 20%~30%B;

[0038] 10~30min, 70%~66%A, 30%~34%B;

[0039] 30~50min, 66%~56%A, 34%~44%B;

[0040] 50~70min, 56%~46%A, 44%~54%B.

[0041] The chromatographic columns examined are as follows:

[0042] Column-1: YMC-Pack ODS-A analytical column, 250×4.6mm, 5μm

[0043] Chromatographic column-2: ACE EXCEL 5C18 (Cat. No. EXL-121-2546U), 250×4.6mm

[0044] 1.2 Experimental steps: Inject the test solution and reference solution into the HPLC according to the above chromatographic conditions. The chromatograms of the test solution under different chromatographic column conditions are shown in Figure 1 The results showed that when using column-1, the minimum separation between piperonyl lactam B and adjacent peaks was less than 1, and the minimum separation between aristolochic acid lactam AⅡ and adjacent peaks was greater than 1 and less than 1.5; when using column-2, the minimum separation between piperonyl lactam B and adjacent peaks was greater than 1.5, and the minimum separation between aristolochic acid lactam AⅡ and adjacent peaks was close to 1.5. The A and B phases and elution methods were subsequently adjusted to optimize the conditions.

[0045] Table 1 HPLC results of chromatographic columns-1 and-2

[0046]

[0047] 2. Mobile phase screening

[0048] 2.1 Chromatographic conditions

[0049] The chromatographic column is ACE EXCEL 5C18 (article number EXL-121-2546U), 250×4.6mm

[0050] The detection wavelengths were 223 nm and 254 nm, the column temperature was 25 °C, and the flow rate was 1 mL min -1 , gradient elution,

[0051] The elution method is as follows

[0052] 0~10min, 80%~70%A, 20%~30%B;

[0053] 10~30min, 70%~66%A, 30%~34%B;

[0054] 30~50min, 66%~56%A, 34%~44%B;

[0055] 50~70min, 56%~46%A, 44%~54%B.

[0056] The mobile phases investigated were as follows:

[0057] Mobile phase-1: Mobile phase A is water, mobile phase B is methanol;

[0058] Mobile phase-2: mobile phase A is water, mobile phase B is acetonitrile;

[0059] Mobile phase-3: Mobile phase A was 0.1% phosphoric acid water, and mobile phase B was acetonitrile.

[0060] 2.2 Experimental steps: Inject the test solution and reference solution into the HPLC according to the above chromatographic conditions. The chromatograms of the test solution under different mobile phase conditions are shown in Figure 2 Using mobile phase-3, the minimum separation between aristolochic acid lactam AⅡ and adjacent peaks was closer to 1.5, and the elution mode was further adjusted to optimize the conditions.

[0061] Table 2 HPLC results of mobile phase-1, mobile phase-2 and mobile phase-3

[0062]

[0063] 3. Elution gradient screening

[0064] 3.1 Chromatographic conditions

[0065] The chromatographic column is ACE EXCEL 5C18 (article number EXL-121-2546U), 250×4.6mm

[0066] The detection wavelengths were 223 nm and 254 nm, the column temperature was 25 °C, the flow rate was 1 mL min-1, the mobile phases were 0.1% phosphoric acid aqueous solution (A) and acetonitrile (B), and the gradient elution was performed.

[0067] The elution method was investigated as follows:

[0068] Elution method-1

[0069] 0~10min, 66%~56%A, 34%~44%B;

[0070] 10~20min, 56%~46%A, 44%~54%B.

[0071] Elution method-2

[0072] 0~10min, 74%~66%A, 26%~34%B;

[0073] 10~30min, 66%~56%A, 34%~44%B;

[0074] 30~40min, 56%~46%A, 44%~54%B.

[0075] Elution method-3

[0076] 0~10min, 80%~70%A, 20%~30%B;

[0077] 10~30min, 70%~66%A, 30%~34%B;

[0078] 30~50min, 66%~56%A, 34%~44%B;

[0079] 50~70min, 56%~46%A, 44%~54%B.

[0080] Elution method-4

[0081] 0~10min, 80%~72%A, 20%~28%B;

[0082] 10~30min, 72%~68%A, 28%~32%B;

[0083] 30~50min, 68%~62%A, 32%~38%B;

[0084] 50~70min, 62%~46%A, 38%~54%B.

[0085] 3.2 Experimental steps: Inject the test solution and reference solution into the HPLC according to the above chromatographic conditions. The chromatograms of the test solution under different elution conditions are shown in Figure 3 Using elution mode-4, the minimum separation between aristolochic acid lactam AⅡ and adjacent peaks was greater than 1.5.

[0086] 3.3 Conclusion

[0087] The present invention adopts the following HPLC chromatographic conditions for dual-wavelength detection: the chromatographic column is ACE EXCEL 5C18 (article number EXL-121-2546U), 250×4.6 mm, the column temperature is 25° C., the flow rate is 1 mL·min-1, the detection wavelengths are 223 nm and 254 nm, the mobile phase is 0.1% phosphoric acid water (A) and acetonitrile (B), and the gradient elution is performed. The gradient elution conditions are:

[0088] 0~10min, 80%~72%A, 20%~28%B;

[0089] 10~30min, 72%~68%A, 28%~32%B;

[0090] 30~50min, 68%~62%A, 32%~38%B;

[0091] 50~70min, 62%~46%A, 38%~54%B

[0092] The separation effect is best.

[0093] Table 3 HPLC results of elution method-1, elution method-2, elution method-3 and elution method-4

[0094]

[0095] Example 2 Methodological verification of the chromatographic method of the present invention

[0096] 1. Linear relationship test: Accurately measure an appropriate amount of mixed reference solution and dilute it step by step to obtain a series of mixed reference solutions. Detect according to the HPLC chromatographic conditions of the present invention, and use the peak area Y to the concentration X (μg·mL -1 ) were used for linear regression, and the results are shown in Table 4.

[0097] Table 4 Investigation of linear relationships among five index components

[0098]

[0099] 2. Precision test: According to the preparation method of the test solution, prepare 1 sample solution, inject it 6 times continuously according to the HPLC chromatographic conditions, record the peak area, and calculate the RSD value. As shown in Table 5, the results show that the method has good precision.

[0100] 3. Repeatability test: According to the preparation method of the test solution, 6 test solutions were prepared, and the samples were analyzed by HPLC chromatography, the peak area was recorded, and the RSD value was calculated. As shown in Table 5, the results show that the method has good repeatability.

[0101] 4. Stability test: According to the preparation method of the test solution, prepare 1 portion of the test solution, inject the sample at 0, 4, 8, 12, 16, and 20 hours according to the HPLC chromatographic conditions, record the peak area, and calculate the RSD value. As shown in Table 5, it shows that the test sample is stable within 20 hours at room temperature.

[0102] Table 5 Precision, repeatability and stability results

[0103]

[0104] 5. Sample recovery test: Accurately weigh 5 g of 5 parts of the powder of the concave leaf guarana, add each reference substance, place in a 25 mL volumetric flask, add methanol to the scale, weigh the mass, ultrasonically treat for 30 minutes, cool, weigh again, make up the weight loss with methanol, shake well, and obtain the sample solution. Inject 10 μL according to the HPLC chromatographic conditions, calculate the content of each component, the average recovery rate and the RSD value according to the external standard method, and the results are shown in Table 6.

[0105] Table 6 Results of sample recovery test for 5 index components (n=5)

[0106]

[0107]

[0108] Example 3 Detection of the contents of aristolochic acid AⅡ, goniopedaline, piperonolactam B, aristolochic acid BⅡ and 5,6,7,8-tetramethoxyflavone in the stems of Cucurbita ovata by the chromatographic method of the present invention

[0109] 1. Chromatographic conditions:

[0110] The chromatographic column is ACE EXCEL 5C18 (Cat. No. EXL-121-2546U), 250×4.6 mm, column temperature 25°C, flow rate 1 mL·min -1 , detection wavelengths were 223 nm and 254 nm, and the mobile phases were 0.1% phosphoric acid aqueous solution (A) and acetonitrile (B).

[0111] The gradient elution conditions were as follows:

[0112] 0~10min, 80%~72%A, 20%~28%B;

[0113] 10~30min, 72%~68%A, 28%~32%B;

[0114] 30~50min, 68%~62%A, 32%~38%B;

[0115] 50~70min, 62%~46%A, 38%~54%B.

[0116] 2. Inject the test solution and reference solution into the high performance liquid chromatograph for determination. The HPLC chromatogram is shown in Figure 4 , Figure 5 The peak area of ​​each index component was recorded and the content was calculated. The results are shown in Table 7.

[0117] Table 7 Determination results of the contents of 5 index components

[0118]

[0119] The results showed that the content of aristolochic acid lactone in the stem extract of Cucurbita ovata was 8.711 mg / g; the content of gohopedaline was 3.921 mg / g; the content of piperonolactone B was 3.612 mg / g; the content of aristolochic acid lactone BⅡ was 1.312 mg / g; and the content of 5,6,7,8-tetramethoxyflavone was 35.650 mg / g.

Claims

1. A HPLC analysis method for index components in the stem of Cucurbita ovata, characterized in that: Chromatographic conditions: ACEEXCEL 5 C18, 250×4.6 mm, chromatographic column; mobile phase: phase A is 0.1% phosphoric acid aqueous solution, phase B is acetonitrile, detection wavelengths are 223 nm and 254 nm, preparation of test solution: dried stems of Achyranthes bidentata, crushed and sieved, accurately weighed, placed in a volumetric flask, methanol was added to make up the volume, weighed, ultrasonicated, cooled, weighed, supplemented with methanol to make up the lost weight, shaken, filtered, and the filtrate was taken to obtain the test solution; preparation of reference solution: accurately weigh appropriate amounts of aristolochic acid lactam AⅡ, goniopedaline, piperonyl lactam B, aristolochic acid lactam BⅡ and 5,6,7,8-tetramethoxyflavone reference substances, respectively, dissolved in methanol to obtain the reference solution, and the following gradient elution was used: At 0-10 min, 80%-72% mobile phase A, 20%-28% mobile phase B; At 10-30 min, 72%-68% mobile phase A, 28%-32% mobile phase B; At 30-50 min, 68%-62% mobile phase A, 32%-38% mobile phase B; At 50-70 min, 62%-46% mobile phase A, 38%-54% mobile phase B; The index components are aristolochic acid lactone AⅡ, goniopedaline, piperonolactone B, aristolochic acid lactone BⅡ and 5,6,7,8-tetramethoxyflavone.

2. The method according to claim 1, characterized in that The mobile phase flow rate: 1 mL min -1 .

3. The method according to claim 1, characterized in that The column temperature was 25°C.

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