A method for determining the content of sesquiterpenoid compounds in Hibiscus scabra

Abstract

The invention discloses a method for determining the content of sesquiterpenoid compounds in hibiscus chrysanthemum, comprising the following steps: step (1): preparation of a test solution; step (2): preparation of a reference solution; step (3): accurately aspirating 20 μl of a reference solution and a test solution, respectively injecting them into a liquid chromatograph to obtain a chromatogram; step (4): measuring the peak area according to the chromatogram, performing regression analysis on the reference solution, preparing a standard curve, and calculating the content of each sesquiterpenoid compound in the test solution according to the standard curve. The invention provides a method for determining the content of sesquiterpenoid compounds in hibiscus chrysanthemum, and the method is applied to determine the contents of three sesquiterpenoid components, namely, artemisinin, magnolia lactone and tansylide, in hibiscus chrysanthemum. The determination method has the characteristics of good linear relationship, high precision and sensitivity, good reproducibility and high recovery rate, and can efficiently, simply and quickly determine the content of sesquiterpenoid compounds in hibiscus chrysanthemum.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for determining the content of sesquiterpene compounds in Hibiscus scabra. Background Art

[0002] Due to the complexity of the chemical composition of plants in nature, a single indicator component cannot accurately reflect the effective ingredients of herbal medicines; and the content determination of a single component is formulated separately. During the implementation of the determination method, the consumption of reference materials is large, the detection cycle is long, time-consuming and labor-intensive, and the efficiency is low.

[0003] Chrysanthemum plants are rich in sesquiterpenoid compounds. Sesquiterpenoids have variable structures and diverse biological activities and have always been a hot topic in natural medicinal chemistry research. Hibiscus chrysanthemi is a representative plant of the Asteraceae family. Its main chemical components are terpenoids and flavonoids. The yield of sesquiterpenoid chemical components in separation is relatively high. At present, there is no method for determining the content of sesquiterpenoid compounds in Hibiscus chrysanthemi in the prior art. Therefore, it is necessary to establish a method for determining the content of sesquiterpenoid compounds in Hibiscus chrysanthemi to control the quality of medicinal materials or preparations containing the compounds. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a method for determining the content of sesquiterpenoid compounds in Hibiscus chrysanthemum, which can efficiently, simply and quickly determine the content of sesquiterpenoid compounds in Hibiscus chrysanthemum.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0006] Provided is a method for determining the content of sesquiterpene compounds in Hibiscus scabra, comprising the following steps:

[0007] Step (1): accurately weigh the coarse powder of the whole herb of Hibiscus scabra, pass it through a 20-mesh sieve, place it in a round-bottom flask, add chloroform, reflux extraction, filter it while hot with quantitative filter paper, combine the filtrate, recover the solvent, dissolve the residue with methanol, make up to volume in a volumetric flask, take 1 mL of it and filter it through a 0.45 μm microporous filter membrane, and take the filtrate as the test solution;

[0008] Step (2): Accurately weigh artemisinin, magnolia lactone, and tansylate, add methanol to make up to volume, and prepare individual standard stock solutions; accurately take each stock solution to prepare a mixed standard stock solution, and the concentrations of the above three compounds in the mixed standard stock solution are 400, 980, and 1560 μg mL, respectively. -1 ; Accurately pipette the mixed standard stock solution to prepare a series of reference solution concentrations;

[0009] Step (3): Accurately pipette 20 μl of the reference substance and the test substance solution, respectively, and inject them into the liquid chromatograph to obtain a chromatogram;

[0010] Step (4): Measure the peak area according to the chromatogram, perform regression analysis on the reference solution, prepare a standard curve, and calculate the content of each sesquiterpene compound in the test solution according to the standard curve.

[0011] Furthermore, in step (1), the mass volume ratio of the Hibiscus scabra whole herb coarse powder to chloroform is 1:20, and reflux extraction is performed for 1 hour, for a total of two times.

[0012] Furthermore, in step (3), the liquid chromatograph uses methanol-water (45:55, v / v) as the mobile phase; the constant flow rate is 1.0 mL·min -1 .

[0013] The beneficial effects of the present invention are:

[0014] The invention provides a method for determining the content of sesquiterpenoid compounds in hibiscus chrysanthemum. The method is applied to determine the contents of three sesquiterpenoid components, namely artemisinin, magnolia lactone and tansylide in hibiscus chrysanthemum. The determination method has the characteristics of good linear relationship, high precision and sensitivity, good reproducibility and high recovery rate, and can efficiently, simply and quickly determine the content of sesquiterpenoid compounds in hibiscus chrysanthemum. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 HPLC chromatograms of the artemisinin, magnolia lactone, and tansylate reference substances provided in Example 1;

[0016] Figure 2 HPLC chromatogram of the Hibiscus chrysanthemum test sample provided in Example 1;

[0017] Figure 3 The standard curve of artemisinin provided in Example 1;

[0018] Figure 4 The standard curve of magnolia lactone provided in Example 1;

[0019] Figure 5 This is the standard curve of tansylate provided in Example 1. DETAILED DESCRIPTION

[0020] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0021] Instrument: DIONEX high-performance liquid chromatography (HPLC) system (DIONEX Co., München, Germany): DIONEX P680 pump, UVD170U detector, Chromeleon version 6.50 data processing workstation. Dikma DiamonsilTM C18 chromatographic column (250 mm × 4.6 mm id, 5 μm), coupled with Dikma EasyGuard C18 guard column (20 mm × 4.6 mm id).

[0022] Example 1

[0023] Selection of extraction method for sesquiterpenoid compounds from Hibiscus chrysanthemum:

[0024] Sesquiterpenoids have good solubility in chloroform. In this embodiment, the chloroform extract was tested by TLC and found to be able to completely extract the sesquiterpenoids in the Hibiscus chrysanthemum medicinal material. This embodiment designed a four-factor three-level table (see Table 1-2), fixed the extraction solvent chloroform, and explored the difference in the total extraction amount of Hibiscus chrysanthemum sesquiterpenoids under different extraction methods, extraction time, extraction times and solvent times. The sum of the peak areas of Hibiscus chrysanthemum sesquiterpenoids was used as an indicator.

[0025] Table 1L9(34) orthogonal table

[0026]

[0027]

[0028] Table 2 Analysis of variance of orthogonal experiment

[0029]

[0030] According to the results of orthogonal experiments, the optimized extraction method was determined to be A1B2C2D2, that is, 20 times the amount of solvent chloroform, reflux extraction for 1 hour, for a total of two times.

[0031] Preparation of test samples:

[0032] Accurately weigh 5.000 g of coarse powder of the whole herb of Hibiscus scabra, pass it through a 20-mesh sieve, place it in a round-bottom flask, add 20 times the amount of chloroform as the solvent, reflux extraction for 1 hour, twice in total, filter it while hot with quantitative filter paper, combine the filtrate, recover the solvent, dissolve the residue with methanol, make up to the volume in a 50 mL volumetric flask, take 1 mL from it and filter it through a 0.45 μm microporous filter membrane, and take the filtrate as the test solution.

[0033] Establishment of HPLC conditions:

[0034] (I) Selection of solvent

[0035] This example investigates different solvent ratios, including methanol-water, acetonitrile-water, methanol-acetonitrile-water, methanol-acid water, and acetonitrile-acid water. Finally, the separation conditions of the mobile phase of methanol-water are the best. Methanol-water (45:55, v / v) is used as the mobile phase; the constant flow rate is 1.0 mL·min-1; and the detection wavelength of 210 nm is used as the detection wavelength for content determination.

[0036] (II) Methodological Validation

[0037] 1. Standard curve

[0038] Accurately weigh 4.0 mg of artemisinin, 5.2 mg of magnolia lactone, and 4.9 mg of tansylate, add methanol to a 1 mL volumetric flask, and prepare a concentration of 4.0 mg mL -1 , 5.2mg·mL -1 , 4.9mg·mL -1 Accurately pipette a certain amount of artemisinin, magnolia lactone and tansylate individual standard stock solutions into the same volumetric flask, and use methanol to make a mixed standard stock solution. The concentrations of the above three reference substances in the mixed standard stock solution are 1560, 400, and 980 μg mL, respectively. -1 The mixed standard stock solution was accurately pipetted to prepare a series of 6 reference solution concentrations. Among them, the concentrations of artemisinin were 400, 200, 100, 50, 25, and 12.5 mg·mL -1 ; The concentrations of magnolia lactone were 980, 490, 245, 122.5, 61.25, and 30.625 mg·mL -1 ; The concentrations of tansylate were 1560, 780, 390, 195, 97.5, and 48.7 mg·mL -1 . Accurately pipette 20 μl of the reference substance and the test solution of Example 2, and inject them into the HPLC chromatograph system respectively to obtain the chromatogram ( Figures 1-2 ). According to the chromatogram, the peak area is measured, and the concentration (x) is used as the horizontal axis and the peak area (y) is used as the vertical axis. The reference solution is subjected to regression analysis to prepare a standard curve ( Figures 3 to 5 ). The regression curve, correlation coefficient and linear range are shown in Table 3. The results show that each compound has a good linear relationship within the corresponding range.

[0039] Table 3 Linear relationship between artemisinin, magnolia lactone and tansylate

[0040]

[0041] 2. Precision

[0042] Take three concentrations of reference substances, high, medium and low, and analyze them according to the HPLC determination method. Inject the sample three times in a day, measure the peak area of ​​each reference substance, and calculate the intra-day precision. Inject the sample once a day for three consecutive days, measure the peak area of ​​each reference substance, and calculate the inter-day precision. The results are shown in Table 4.

[0043] Table 4 Precision of artemisinin, magnolia lactone and tansylate (n=3)

[0044]

[0045] 3. Repeatability

[0046] According to the method for preparing test samples, prepare a test sample, inject it continuously for 6 times, determine the peak area of ​​each reference substance by HPLC analysis method, and calculate RSD (%), see Table 5.

[0047] Table 5 Repeatability of artemisinin, magnolia lactone and tansylate

[0048]

[0049] 4. Sample recovery rate

[0050] 5.000±0.005g of Hibiscus chinensis crude powder was accurately weighed, three portions were used as blanks, and the other 9 portions were divided into three groups (n=3), high, medium and low, and three different concentrations of reference substances were added respectively. The test sample solution was prepared according to the test sample preparation method, and analyzed according to the HPLC analysis method, the peak area of ​​each reference substance was determined, and the recovery rate of high, medium and low concentrations was calculated. The results are shown in Table 6.

[0051] Table 6 Sample recovery

[0052]

[0053] Example 2

[0054] Determination of Sesquiterpenoids in Hibiscus scabra

[0055] Take 5.0056g, 5.0013g and 4.9942g of crude powder of Hibiscus chinensis, prepare the test solution according to the test solution preparation method, analyze according to the HPLC analysis method established in Example 1, and calculate the content of each component according to the standard curve. The content of artemisinin in Hibiscus chinensis is 0.40mg·g- 1 The content of magnolia lactone in crude drug is 3.21 mg·g- 1 The content of tansylate in crude drug is 7.48 mg g -1 Herbal medicine.

[0056] The invention provides a method for determining the content of sesquiterpenoid compounds in hibiscus chrysanthemum. The method is applied to determine the contents of three sesquiterpenoid components, namely artemisinin, magnolia lactone and tansylide in hibiscus chrysanthemum. The determination method has the characteristics of good linear relationship, high precision and sensitivity, good reproducibility and high recovery rate, and can efficiently, simply and quickly determine the content of sesquiterpenoid compounds in hibiscus chrysanthemum.

[0057] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0058] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for determining the content of sesquiterpenoid compounds in Hibiscus scabra, characterized in that: The method simultaneously determines the contents of three sesquiterpenoid compounds, namely, artemisinin, magnolia lactone and tansylide, in hibiscus chrysanthemum, including the following steps: Step (1): accurately weigh the coarse powder of the whole herb of Hibiscus scabra, pass it through a 20-mesh sieve, place it in a round-bottom flask, add chloroform, reflux extraction, filter it while hot with quantitative filter paper, combine the filtrate, recover the solvent, dissolve the residue with methanol, make up to volume in a volumetric flask, take 1 mL of it and filter it through a 0.45 μm microporous filter membrane, and take the filtrate as the test solution; Step (2): Accurately weigh artemisinin, magnolia lactone, and tansylate, add methanol to make up to volume, and prepare individual standard stock solutions; accurately take each stock solution to prepare a mixed standard stock solution. The concentrations of the above three compounds in the mixed standard stock solution are 400, 980, and 1560 μg mL, respectively. -1 ; Accurately pipette the mixed standard stock solution to prepare a series of reference solution concentrations; Step (3): Accurately pipette 20 μl of the reference substance and the test substance solution, respectively, and inject them into the liquid chromatograph to obtain a chromatogram; Step (4): Measure the peak area according to the chromatogram, perform regression analysis on the reference solution, prepare a standard curve, and calculate the content of each sesquiterpene compound in the test solution according to the standard curve; The mass volume ratio of Hibiscus chinensis whole herb crude powder to chloroform was 1:20, and reflux extraction was performed for 1 h, twice in total. The liquid chromatography conditions included: a 250 mm × 4.6 mm id, 5 μm Dikma DiamonsilTM C18 column coupled with a 20 mm × 4.6 mm id Dikma EasyGuard C18 guard column; a methanol-water mobile phase with a volume ratio of 45:55; a constant flow rate of 1.0 mL·min -1 .

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