A method for simultaneously determining five active ingredients in ormosia plants

Abstract

The application provides a method for simultaneously determining five active ingredients in plants of the genus Ormosia, and relates to the field of component identification technology.The method provided by the application comprises the following steps: respectively gradient diluting genistein solution, rutin solution, resveratrol solution, luteolin solution and kaempferol solution, establishing a standard curve of concentration and peak area after liquid chromatography analysis; gradient diluting a mixed solution containing genistein, rutin, resveratrol, luteolin and kaempferol, establishing a corresponding relationship between active ingredients and retention time after liquid chromatography analysis; obtaining the peak area of active ingredients in the test solution from the plants of the genus Ormosia based on the corresponding relationship after liquid chromatography analysis of the test solution, and calculating the content based on the peak area and the standard curve. The application can quickly explore the content difference of active ingredients in the plants of the genus Ormosia by establishing a liquid chromatography analysis method, and can provide theoretical guidance for the collection of the plants of the genus Ormosia and the control of planting environment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of component identification, and particularly relates to a method for simultaneously determining five active components in Ormosia plants. BACKGROUND

[0002] Ormosia belongs to Fabaceae, and is an important representative genus of woody plants. The "red beans" in "red beans grow in the southern country, and a few branches come out in spring" refer to the seeds of Ormosia plants. Ormosia plants have excellent wood properties and are widely used, and have outstanding development and utilization value. The whole plant of Ormosia fordii is used as "palm wood" in traditional Chinese medicine, and animal experiments show that the leaf extract of O. fordii has the effect of treating depression; the leaf color of O. minor changes with rainfall, and it is called "weather tree"; O. fruticosa has an elegant posture and is an excellent garden greening tree species. With the breakthroughs in seedling breeding and cultivation technology, the problem of large-scale planting has been gradually solved. Therefore, how to further scientifically develop and utilize the active components of Ormosia plants and fully tap their economic, medicinal and ecological values has become the current research focus.

[0003] Modern research has found that Ormosia plants contain a large amount of active components, and more than 50 active components (including flavonoid components and polyphenol components) have been isolated and identified. Among them, genistein is an isoflavone compound widely existing in Fabaceae plants, and has estrogen-like effects; rutin is one of the flavonoids widely used, and can enhance blood vessel elasticity and has anti-inflammatory effects; resveratrol has a significant effect on anti-aging and cardiovascular protection; luteolin is a potent anti-inflammatory flavonoid component, and also performs outstandingly in anti-allergy and neuroprotection; kaempferol is a broad-spectrum antioxidant, and has potential for metabolic syndrome and cancer prevention.

[0004] The active component contents of different parts of Ormosia plants are significantly different, and different conditions such as growth environment and planting technology will also cause certain differences in the active component contents in Ormosia plants. Therefore, it is crucial to establish a rapid and accurate active component detection method for guiding the rational use of Ormosia plants (such as optimal medicinal parts and high active component content variety breeding). This not only helps to improve the economic value of Ormosia plants, but also promotes their sustainable development and resource protection, and lays a foundation for subsequent deep processing and industrial application. SUMMARY

[0005] The purpose of the present application is to provide a method for simultaneously determining five active components in Ormosia plants, which can quickly explore the differences in active component contents in different parts of the same Ormosia plant and in the same part of different Ormosia plants by establishing a liquid chromatography analysis method, and can provide theoretical guidance for Ormosia plant provenance collection and planting environment control.

[0006] The application provides a method for simultaneously determining five active ingredients in an Ormosia plant.

[0007] The genistin solution, the rutin solution, the resveratrol solution, the luteolin solution and the kaempferol solution are gradiently diluted respectively, liquid chromatography analysis is performed, and a standard curve of concentration and peak area is established;

[0008] The mixed solution containing the genistin, the rutin, the resveratrol, the luteolin and the kaempferol is gradiently diluted, liquid chromatography analysis is performed, and a corresponding relationship between the active ingredients and the retention time is established;

[0009] After liquid chromatography analysis is performed on the test solution from the Ormosia plant, the peak areas of the genistin, the rutin, the resveratrol, the luteolin and the kaempferol in the test solution are obtained based on the corresponding relationship, and the contents are calculated based on the peak areas and the standard curve;

[0010] When the liquid chromatography analysis is performed, the chromatography conditions are as follows: a chromatography column, a stationary phase is octadecyl bonded silica; a flow rate is 1 mL / min; a column temperature is 30-40 DEG C; a mobile phase A is acetonitrile; a mobile phase B is 0.1% formic acid aqueous solution; a sample injection amount is 5-20 mu L; a detection wavelength is 203 nm; gradient elution is as follows: 0 min-20 min, 5%-20% mobile phase B; 20 min-50 min, 20%-30% mobile phase B; 60 min-80 min, 30%-64% mobile phase B.

[0011] Optionally, the solvent in the mixed solution comprises acetonitrile; and / or, the mixed solution contains 260-280 mu g / mL of the genistin, 160-180 mu g / mL of the rutin, 18-22 mu g / mL of the resveratrol, 48-54 mu g / mL of the luteolin and 18-22 mu g / mL of the kaempferol.

[0012] Optionally, the Ormosia plant comprises Dalbergia hupeana, Ormosia fordii or Ormosia monophylla.

[0013] Optionally, the test solution comprises a root extract, a leaf extract, a young stem extract or a branch extract of the Ormosia plant.

[0014] Optionally, the test solution comprises an ethanol extract of the Ormosia plant.

[0015] Optionally, methanol is used to gradiently dilute the mixed solution.

[0016] Optionally, the extraction method of the test solution from the source of the Ormosia plant comprises: drying and grinding the plant tissue from the source of the Ormosia plant to obtain a tissue powder, then adding the tissue powder into 60% ethanol with a solid-liquid ratio of 1:40, extracting at 80°C for 1.5 hours, cooling, and filtering through a 0.45 μm microporous filter to obtain the test solution.

[0017] Optionally, when establishing the standard curve of the concentration and the peak area, the concentration of the active ingredient is used as the abscissa, and the peak area is used as the ordinate to establish the standard curve.

[0018] Optionally, the linear regression equation of the concentration and the peak area of genistein is Y=6903.85X-13883.1, R 2 =0.9993, and the linear range is 1.68 μg / mL-280 μg / mL.

[0019] Optionally, the linear regression equation of the concentration and the peak area of rutin is Y=16262.8X-23130.7, R 2 =0.9994, and the linear range is 1.08 μg / mL-180 μg / mL.

[0020] Optionally, the linear regression equation of the concentration and the peak area of resveratrol is Y=113850X-20268.9, R 2 =0.9994, and the linear range is 0.132 μg / mL-22 μg / mL.

[0021] Optionally, the linear regression equation of the concentration and the peak area of luteolin is Y=48847.8X-26223.4, R 2 =0.9994, and the linear range is 0.324 μg / mL-54 μg / mL.

[0022] Optionally, the linear regression equation of the concentration and the peak area of kaempferol is Y=33127X-6863.8, R 2 =0.9995, and the linear range is 0.132 μg / mL-22 μg / mL.

[0023] Optionally, the retention time of genistein is 28.95 min-30.60 min.

[0024] Optionally, the retention time of rutin is 33.80 min-35.55 min.

[0025] Optionally, the retention time of resveratrol is 35.75 min-36.85 min.

[0026] Optionally, the retention time of luteolin is 47.55 min-47.85 min.

[0027] Optionally, the retention time of kaempferol is 49.45-51.55 min. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 A chromatogram of liquid chromatography analysis of a genistin standard solution in some embodiments of the present application;

[0029] Figure 2 A chromatogram of liquid chromatography analysis of a rutin standard solution in some embodiments of the present application;

[0030] Figure 3 A chromatogram of liquid chromatography analysis of a rutin standard solution in some embodiments of the present application;

[0031] Figure 4 A chromatogram of liquid chromatography analysis of a rutin standard solution in some embodiments of the present application;

[0032] Figure 5 A chromatogram of liquid chromatography analysis of a rutin standard solution in some embodiments of the present application;

[0033] Figure 6 A standard curve of concentration of active ingredients versus peak area established in Preparation Example 1 of the present application;

[0034] Figure 7 A chromatogram of a mixed solution at one concentration during gradient dilution in Preparation Example 2 of the present application;

[0035] Figure 8 A liquid chromatogram when simultaneously determining five active ingredients in Caesalpinia japonica in Example 1 of the present application. DETAILED DESCRIPTION

[0036] In order to make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application. Unless otherwise defined, the technical terms or scientific terms used herein should have the usual meanings understood by a person of ordinary skill in the art.

[0037] The present application provides a method for simultaneously determining five active ingredients in a plant of the genus Ormosia, comprising the following steps:

[0038] S1, respectively gradient dilute genistin solution, rutin solution, resveratrol solution, luteolin solution, and kaempferol solution, and establish a standard curve of concentration versus peak area after liquid chromatography analysis;

[0039] S2. gradient dilution of a mixed solution containing genistein, rutin, resveratrol, luteolin, and kaempferol, and performing liquid chromatography analysis to establish a correspondence between the active ingredients and the retention time;

[0040] S3. After liquid chromatography analysis of the test solution derived from the genus Rhododendron, the peak areas of genistin, rutin, resveratrol, luteolin, and kaempferol in the test solution were obtained based on the corresponding relationships, and the contents were calculated based on the peak areas and the standard curve.

[0041] Specifically, when performing steps S1 to S3 for liquid chromatography analysis, the chromatographic conditions used are the same, which are: chromatographic column: the stationary phase is octadecyl bonded silica gel; flow rate: 1 mL / min; column temperature: 30°C-40°C; mobile phase A: acetonitrile; mobile phase B: 0.1% formic acid aqueous solution; injection volume: 5 μL-20 μL; detection wavelength: 203 nm; gradient elution: 0 min-20 min, 5%-20% mobile phase B; 20 min-50 min, 20%-30% mobile phase B; 60 min-80 min, 30%-64% mobile phase B.

[0042] In fact, during the execution of step S1, standard genistin solution, rutin solution, resveratrol solution, luteolin solution, and kaempferol solution can be pre-prepared. Specifically, acetonitrile can be used as a solvent to prepare the standard solution.

[0043] In some embodiments, when performing the gradient dilution of the standard solution in step S1, methanol can be used as the solvent for the gradient dilution. In addition, to improve the accuracy of the standard curve, 4-8 gradients can be set during the gradient dilution process for liquid chromatography analysis, and a standard curve of concentration and peak area can be established using the concentration of the active ingredient as the horizontal axis (X) and the peak area as the vertical axis (Y).

[0044] In fact, when performing liquid chromatography analysis in step S1, the retention time of each active ingredient under the current chromatographic conditions can also be obtained. In some embodiments, when performing liquid chromatography analysis in step S1, the chromatograms of a certain concentration standard solution are as follows: Figures 1 to 5 As shown, from Figure 1 It can be seen from the figure that the retention time of genistin is 29.020min. Figure 2 It can be seen that the retention time of rutin is 34.833min. Figure 3 It can be seen that the retention time of resveratrol is 35.764min. Figure 4 It can be seen that the retention time of luteolin is 47.467min. Figure 5 It can be seen that the retention time of kaempferol is 50.304min.

[0045] In some embodiments, 260 μg / mL-280 μg / mL of genistin, 160 μg / mL-180 μg / mL of rutin, 18 μg / mL-22 μg / mL of resveratrol, 48 μg / mL-54 μg / mL of luteolin, and 18 μg / mL-22 μg / mL of kaempferol are dissolved in the mixed solution. In addition, the solvent in the mixed solution can be acetonitrile. In fact, by carrying out gradient dilution to the mixed solution in step S2, it is possible to obtain chromatograms of mixed solutions of different concentrations under current chromatographic conditions, and according to obtaining a spectrogram when carrying out chromatographic analysis of the standard solution, it is possible to identify the corresponding relationship between the active ingredient and the retention time in the mixed solution.

[0046] In practice, the Ormosia plant used in step S3 includes Ormosia henryi, Ormosia xylocarpa, or Ormosia microphylla. Furthermore, the test solution used can be a root extract, leaf extract, young stem extract, or branch extract of the Ormosia plant. Specifically, the test solution includes an ethanol extract of the Ormosia plant.

[0047] Preparation Example 1

[0048] This preparation example 1 provides a method for establishing a standard curve of the concentration and peak area of ​​an active ingredient, comprising the following steps:

[0049] S0, respectively, dissolving genistin (CAS: 529-59-9), rutin (CAS: 153-18-4), resveratrol (CAS: 501-36-0), luteolin (CAS: 491-70-3), and kaempferol (CAS: 520-18-3) in acetonitrile by stirring to prepare 280 μg / mL genistin solution, 180 μg / mL rutin solution, 22 μg / mL resveratrol solution, 54 μg / mL luteolin solution, and 22 μg / mL kaempferol solution;

[0050] S1, the solution prepared in S0 was diluted with acetonitrile to obtain 168 μg / mL genistin solution, 108 μg / mL rutin solution, 13.2 μg / mL resveratrol solution, 32.4 μg / mL luteolin solution, and 13.2 μg / mL kaempferol solution, which were then graded diluted with methanol and analyzed by liquid chromatography on an LC-20AT high performance liquid chromatograph (Shimadazu, Japan) (chromatographic column: InertSustain C18 (250 mm × 4.6 mm, 5μm); flow rate: 1mL / min; column temperature: 35℃; mobile phase A: acetonitrile; mobile phase B: 0.1% formic acid aqueous solution; injection volume: 20μL; detection wavelength: 203nm; gradient elution: 0min-20min, 5%-20% mobile phase B; 20min-50min, 20%-30% mobile phase B; 60min-80min, 30%-64% mobile phase B). The standard curve was established using the concentration of the active ingredient as the abscissa and the peak area as the ordinate, respectively. Figure 6 As shown in AE.

[0051] from Figure 6 As can be seen from A in the figure, the concentration of genistin has a good linear relationship with the peak area when it is between 1.68μg / mL and 280μg / mL. The equation of the standard curve is: Y=6903.85X-13883.1, R 2 =0.9993; from Figure 6 As can be seen from B, the concentration of rutin has a good linear relationship with the peak area when it is between 1.08μg / mL and 180μg / mL. The equation of the standard curve is: Y=16262.8X-23130.7, R 2 =0.9994; from Figure 6 As can be seen from C in the figure, the concentration of resveratrol has a good linear relationship with the peak area when it is between 0.132μg / mL and 22μg / mL. The equation of the standard curve is: Y=113850X-20268.9, R 2 =0.9994; from Figure 6 As can be seen from D in the figure, luteolin has a good linear relationship with the peak area when the concentration is between 0.324μg / mL and 54μg / mL. The equation of the standard curve is: Y=48847.8X-26223.4, R 2 =0.9994; from Figure 6 As can be seen from E, the concentration of kaempferol has a good linear relationship with the peak area when it is between 0.132μg / mL and 22μg / mL. The equation of the standard curve is: Y=33127X-6863.8, R 2 =0.9995.

[0052] Preparation Example 2

[0053] The present preparation example 2 provides a method for establishing the correspondence between active ingredients and retention time, comprising the following steps:

[0054] S2, configure a mixed solution containing 280 pg / mL genistein, 180 pg / mL rutin, 22 pg / mL resveratrol, 54 pg / mL luteolin, and 22 pg / mL kaempferol, gradient dilute the mixed solution with methanol, and perform liquid chromatography analysis (chromatographic conditions are the same as those in preparation example 1) in an LC-20AT high-performance liquid chromatograph (Japan, Shimadazu) to obtain the correspondence between active ingredients and retention time of the mixed solution at different concentrations.

[0055] The liquid chromatography characterization of the mixed solution at one of the concentrations during gradient dilution in preparation example 2 is shown in Figure 7 As can be seen from Figure 7 , the five active ingredients in the mixed solution can be well phase-separated during liquid chromatography analysis, and the retention time of each of the five active ingredients is relatively stable during the entire gradient dilution and liquid chromatography analysis process as shown in Table 1.

[0056] Table 1 Correspondence table between active ingredients and retention time

[0057]

[0058] Example 1

[0059] The present example 1 provides a method for simultaneously determining five active ingredients in Bursera simaruba, comprising:

[0060] S3, after collecting the root system (81.47 g, from the Jiangxi Academy of Forestry greenhouse base, five years old, plant height 1.2 m-1.3 m, 3 plants, collection time May 8, 2022, DPG-1) of Bursera simaruba, drying and grinding to obtain tissue powder, adding 60% ethanol by volume at a solid-liquid ratio of 1:40, mixing uniformly, and then using a heating reflux device to extract in a 80°C water bath environment for 1.5 h, filtering while hot, and cooling the extraction solution to room temperature, then constant volume and filtering through a 0.45 pm microporous filter membrane to obtain a test solution; perform liquid chromatography analysis (chromatographic conditions are the same as those in preparation example 1) on the test solution in an LC-20AT high-performance liquid chromatograph (Japan, Shimadazu) as shown in Figure 8 , and according to the correspondence table shown in Table 1, obtain the peak area of genistein, rutin, resveratrol, luteolin, and kaempferol in the test solution, respectively, and calculate the content of the active ingredients according to the standard curve shown in Figure 6 as shown in Table 2.

[0061] The test solution prepared in Example 1 is placed at room temperature for 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, and then liquid chromatography analysis is performed under the same chromatographic conditions, and the RSDs of the peak areas of genistein, rutin, resveratrol, luteolin and kaempferol at different storage times are 0.16%, 0.45%, 0.26%, 0.33% and 0.25% respectively, which indicates that the test solution has good stability at room temperature within 48 h.

[0062] The test solution prepared in Example 1 is continuously injected for liquid chromatography analysis under the same chromatographic conditions, and the RSDs of the peak areas of genistein, rutin, resveratrol, luteolin and kaempferol in different batches are 0.15%, 0.42%, 0.36%, 0.38% and 0.32% respectively, which indicates that the determination method provided by the present application has good repeatability.

[0063] Examples 2-3

[0064] Examples 2-3 respectively provide a method for simultaneously determining five active ingredients in Terminalia ferdinandiana, which is different from Example 1 in that Example 2 collects the young stem segments (206.22 g, from the greenhouse base of Jiangxi Forestry Academy, five years old, plant height 1.2 m-1.3 m, 3 plants, collection time May 8, 2022, DPJ-1) of Terminalia ferdinandiana, and Example 3 collects the leaves (88.19 g, from the greenhouse base of Jiangxi Forestry Academy, five years old, plant height 1.2 m-1.3 m, 3 plants, collection time May 8, 2022, DPY-1) of Terminalia ferdinandiana.

[0065] Table 2 Content of active ingredients in Examples 1-3

[0066]

[0067] Examples 4-9

[0068] Examples 4-9 respectively provide a method for simultaneously determining five active ingredients in Terminalia ferdinandiana, which is different from Example 1 as shown in Table 3, and the content of the active ingredients after liquid chromatography analysis is shown in Table 4.

[0069] Table 3 Differences between Examples 4-9 and Example 1

[0070]

[0071] Table 4 Content of active ingredients in Examples 4-9

[0072]

[0073] Examples 10-18

[0074] Examples 10 to 18 provide a method for simultaneously determining five active ingredients in Pterocarpus tataricus. The differences from Example 1 are shown in Table 5 below. The contents of the active ingredients calculated after liquid chromatography analysis are shown in Table 6 below.

[0075] Table 5 Differences between Examples 10 to 18 and Example 1

[0076]

[0077] Table 6 Content of active ingredients in Examples 10 to 18

[0078]

[0079] Example 19 to Example 20

[0080] Examples 19 and 20 provide a method for simultaneously determining five active ingredients in Azadirachta indica. The differences from Example 1 are shown in Table 7 below. The contents of the active ingredients calculated after liquid chromatography analysis are shown in Table 8 below.

[0081] Table 7 Differences between Examples 19 to 20 and Example 1

[0082]

[0083] Table 8 Content of active ingredients in Examples 19 to 20

[0084]

[0085] Example 21

[0086] This Example 21 provides a method for simultaneously determining five active ingredients in small-leaf red beans. The differences from Example 1 are shown in Table 9 below. The content of the active ingredients calculated after liquid chromatography analysis is shown in Table 10 below.

[0087] Table 9 Differences between Example 21 and Example 1

[0088]

[0089] Table 10 Content of active ingredients in Example 21

[0090]

[0091] While the embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations of these embodiments are possible. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as set forth in the claims. Furthermore, the invention described herein is susceptible to other embodiments and may be practiced or implemented in a variety of ways.

Claims

1. A method for simultaneously determining five active ingredients in plants of the genus Rhododendron, characterized in that: include: Genistin solution, rutin solution, resveratrol solution, luteolin solution, and kaempferol solution were gradiently diluted and analyzed by liquid chromatography. A standard curve was established using the concentration of the active ingredient as the abscissa and the peak area as the ordinate. The linear regression equation between the concentration of genistin and the peak area was: Y = 6903.85X - 13883.1, R 2 =0.9993, the linear range is 1.68μg / mL-280μg / mL; the linear regression equation of rutin concentration and peak area is: Y=16262.8X-23130.7, R 2 =0.9994, the linear range is 1.08μg / mL-180μg / mL; the linear regression equation between resveratrol concentration and peak area is: Y=113850X-20268.9, R 2 =0.9994, the linear range is 0.132μg / mL-22μg / mL; the linear regression equation between the concentration and peak area of ​​luteolin is: Y=48847.8X-26223.4, R 2 =0.9994, the linear range is 0.324μg / mL-54μg / mL; the linear regression equation of kaempferol concentration and peak area is: Y=33127X-6863.8, R 2 =0.9995, linear range: 0.132μg / mL-22μg / mL; A mixed solution containing genistin, rutin, resveratrol, luteolin, and kaempferol was diluted gradiently, and liquid chromatography analysis was performed to establish a correspondence between the active ingredients and the retention time. The retention time of genistin was 28.95 min-30.60 min, the retention time of rutin was 33.80 min-35.55 min, the retention time of resveratrol was 35.75 min-36.85 min, the retention time of luteolin was 47.55 min-47.85 min, and the retention time of kaempferol was 49.45 min-51.55 min. After liquid chromatography analysis of the test solution derived from the genus Echinops, the peak areas of genistin, rutin, resveratrol, luteolin, and kaempferol in the test solution were obtained based on the corresponding relationship, and the contents were calculated based on the peak areas and the standard curve; When conducting liquid chromatography analysis, the chromatographic conditions are as follows: chromatographic column: octadecyl bonded silica gel as the stationary phase; flow rate: 1 mL / min; column temperature: 30°C-40°C; mobile phase A: acetonitrile; mobile phase B: 0.1% formic acid aqueous solution; injection volume: 5 μL-20 μL; detection wavelength: 203 nm; gradient elution: 0 min-20 min, 5%-20% mobile phase B; 20 min-50 min, 20%-30% mobile phase B; 60 min-80 min, 30%-64% mobile phase B.

2. The method according to claim 1, characterized in that The solvent in the mixed solution includes acetonitrile; and / or the mixed solution dissolves 260 μg / mL-280 μg / mL of genistein, 160 μg / mL-180 μg / mL of rutin, 18 μg / mL-22 μg / mL of resveratrol, 48 μg / mL-54 μg / mL of luteolin, and 18 μg / mL-22 μg / mL of kaempferol.

3. The method according to claim 1, characterized in that The plant of the genus Rhododendron includes Aesculus tataricus, Aesculus occidentalis or Aesculus microphylla; and / or, the test solution includes a root extract, a leaf extract, a young stem extract or a branch extract of the plant of the genus Rhododendron; and / or, the test solution includes an ethanol extract of the plant of the genus Rhododendron; and / or, the mixed solution is gradiently diluted using methanol.

4. The method according to claim 1 or 3, characterized in that The extraction method of the test solution derived from the genus Echinops comprises: drying and grinding plant tissue derived from the genus Echinops to obtain tissue powder, adding the powder to 60% ethanol by volume at a material-liquid ratio of 1:40, extracting at 80° C. for 1.5 hours, cooling the powder, and filtering the powder through a 0.45 μm microporous filter membrane to obtain the test solution.

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