Method for simultaneously detecting contents of five coumarin components in heracleum moellendorffii hance

Abstract

The invention discloses a method for simultaneously detecting the content of five coumarin components in heracleum moellendorffii hance, and belongs to the technical field of natural compound detection and analysis. The detection method comprises the following detection steps: taking the saxifolin, the 7-hydroxycoumarin, the bergapten, the pimpinolide and the imperatorin as reference substances, and detecting the contents of the saxifolin, the 7-hydroxycoumarin, the bergapten, the pimpinolide and the imperatorin in the heracleum moellendorffii hance extract through HPLC (High Performance Liquid Chromatography). According to the present invention, the HPLC detection conditions are improved so as to qualitatively and quantitatively detect the pimpinellin, the 7-hydroxycoumarin, the bergamolactone, the pimpinellin and the imperatorin in the heracleum moellendorffii hemsl coumarin compound through the one-time HPLC detection.

Description

Technical Field

[0001] The invention belongs to the technical field of natural compound detection and analysis, and particularly relates to a method for simultaneously detecting the contents of five coumarin components in celery. Background Art

[0002] Heracleum moellendorffii Hance (HM), scientifically known as Northeastern Bull-wind-blooming Herb, also known as Short-haired Angelica, is a perennial herb belonging to the genus Angelicae in the Apiaceae family. Heracleum moellendorffii Hance is a perennial herbaceous plant of the Apiaceae family, growing approximately 0.7 to 1.5 meters tall. Its roots are relatively thick and grow deep into the soil. Its stem is erect, cylindrical, hollow, and grooved on the surface. It is covered with coarse hairs and has a slight, distinctive aroma. It has a thick, sheath-like base, and its stem-borne leaves have petioles covered with coarse hairs. Early petioles have three or five leaflets, and the leaves are either trifoliate or pinnately compound, with petioles. Under natural growth, Heracleum moellendorffii seedlings emerge from the soil in mid-to-early May. One-year-old Heracleum moellendorffii has oval leaves, and begins to bolt, flower, and set fruit in its second year. Heracleum flowers bloom from July to August, and fruit from August to September. The flowers are white, compound, umbellate, and approximately 20 cm in diameter. The fruit is a double-hanging fruit, broadly oval or nearly round in shape, 7-8mm long and approximately 7mm wide. The plant adapts to mild, cool, and humid environments, preferring loam and sandy loam rich in humus. It is highly cold-tolerant and can survive freezing temperatures of -4°C for several hours without freezing. The underground rhizomes of mature plants can withstand temperatures of -40°C without freezing to death. Temperature fluctuations affect the photosynthetic accumulation of the plant, a key factor affecting its growth. Research has determined that the optimal daily temperature for the plant is between 20 and 25°C.

[0003] As a wild vegetable commonly consumed in Northeast China for both medicinal and edible purposes, celery is widely loved for its unique taste and rich nutritional value. The main chemical components of celery include coumarins, polyphenols, flavonoids, polysaccharides and other active ingredients.

[0004] Coumarins are a class of lactone compounds that are widely distributed in nature. Their diverse chemical structures give them a wealth of biological activities. Consequently, they have found widespread application in a variety of fields, including pharmaceuticals, daily chemicals, and agrochemicals. In practice, coumarins are used as antifungals, natural antioxidants, and anticoagulants. Furthermore, as important secondary metabolites in plants, coumarins are crucial to plant protection mechanisms, helping them resist infection. Furthermore, in plant physiology, coumarins can act as antioxidants, enzyme inhibitors, and precursors of toxic substances. More notably, some coumarins affect plant growth, for example by interfering with the activity of growth hormones and regulators, and as important factors influencing plant respiration and photosynthesis.

[0005] Studies have found that jasmine contains a variety of coumarin compounds, including four common furan coumarins: bergamotolactone, imperatorin, anise lactone, and isopsoralen; saxifragelin; and simple coumarins such as 7-hydroxycoumarin. However, how to simultaneously detect multiple coumarin compounds in jasmine using simple methods remains to be studied. Summary of the Invention

[0006] The present invention aims to provide a method for simultaneously detecting the contents of five coumarins in celery. By improving various detection factors in the HPLC detection method, the purpose of simultaneously qualitatively and quantitatively analyzing the coumarin compounds in celery can be achieved.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A method for simultaneously detecting the contents of five coumarins in celery comprises the following steps:

[0009] The contents of saxifragine, 7-hydroxycoumarin, bergamot lactone, anise lactone and imperatorin in the extract of Rhizoma Cibotii were determined by HPLC using saxifragine, 7-hydroxycoumarin, bergamot lactone, anise lactone and imperatorin as reference substances.

[0010] HPLC detection conditions include:

[0011] Chromatographic column: C-18 column;

[0012] Mobile phase: phase A: acetonitrile, phase B: 0.1 wt.% formic acid aqueous solution;

[0013] Flow rate: 1 mL / min;

[0014] Detection wavelength: 300nm;

[0015] Elution procedure: 0-10 min: phase A 20%, phase B 80%, 10-25 min: phase A 35%, phase B 65%, after 25 min: phase A 60%, phase B 40%.

[0016] The beneficial technical effects of the present invention are as follows:

[0017] The invention improves the HPLC detection conditions so that saxifragein, 7-hydroxycoumarin, bergamotolactone, anise lactone and imperatorin in the coumarin compounds of celery can be qualitatively and quantitatively detected through one HPLC detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1These are the HPLC chromatograms of the celery extract and the reference solution in Example 1, wherein A is the HPLC chromatogram of the reference solution and B is the HPLC chromatogram of the celery extract. DETAILED DESCRIPTION

[0019] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention.

[0020] In addition, for numerical ranges in the present invention, it is understood that each intervening value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or stated range, and any other stated value or intervening value in the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0022] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0023] Example 1

[0024] HPLC detection of five coumarin compounds in celery:

[0025] Preparation of reference solution: Accurately weigh 10 mg of bergamot lactone, imperatorin, anise lactone, 7-hydroxycoumarin, and saxifragein with a purity greater than 98%, respectively, and dissolve them in mass spectrometry grade methanol to prepare a 2 mg / mL solution. Subsequently, dilute it into standard solutions with different sample concentrations, filter through a 0.45 μm microporous filter membrane, and set aside.

[0026] Preparation of sample solution: Place the old mountain celery in an oven, dry it to constant weight, and grind it into powder with a grinder. Weigh 1 g of the experimental material, add 10 mL of methanol, ultrasonicate for 50 minutes (temperature 40°C, 100 Hz), collect the supernatant after centrifugation for 10 minutes, repeat three times, concentrate the collected supernatant in a vacuum centrifugal concentrator until there is no liquid, then dissolve it with 2 mL of methanol, filter it with a microporous filter membrane (0.45 μm) and take the clear filtrate for use.

[0027] HPLC detection conditions:

[0028] The mobile phase is one of the key factors affecting the separation effect of different bioactive components. The present invention tested the separation effect of methanol-water mobile phase system and acetonitrile-water mobile phase system on the coumarin components in old celery. A certain proportion of formic acid and phosphoric acid were also added to the aqueous phase to explore the selection of the best acid solution to improve the peak shape. It was finally found that acetonitrile-0.1wt.% formic acid aqueous solution had a good separation effect on citronellol, imperatorin, anise lactone, 7-hydroxycoumarin and saxifrage in old celery. Therefore, the chromatographic conditions selected in this example were: chromatographic column: Agilent Extend-C18 chromatographic column (4.6mm×250mm, 5μm), mobile phase A was acetonitrile, B was 0.1% formic acid aqueous solution, and gradient elution was performed. The gradient elution program is shown in Table 1; detection wavelength: 300nm; flow rate: 1mL / min; column temperature: 30℃; injection volume: 20μL.

[0029] Table 1 Elution program

[0030] Time (min) Mobile phase A (%) Mobile phase B (%) 0 20 80 10 35 65 25 60 40 28 60 40

[0031] Test results are shown in Figure 1 , where A is the HPLC chromatogram of the reference solution, and B is the HPLC chromatogram of the celery extract. Figure 1 In the figure, 1 is the chromatographic peak of saxifragein, 2 is the chromatographic peak of 7-hydroxycoumarin, 3 is the chromatographic peak of bergamot lactone, 4 is the chromatographic peak of anise lactone, and 5 is the chromatographic peak of imperatorin.

[0032] Among them, the standard curve was drawn with the concentration of the reference substance (mg / mL) as the horizontal axis and the peak area as the vertical axis, and the linear regression equations of bergamot lactone, imperatorin, anise lactone, 7-hydroxycoumarin and saxifragein were obtained and shown in Table 2.

[0033] Table 2 Regression equations of each reference substance

[0034]

[0035]

[0036] Table 2 shows that the R2 of the five target compounds is greater than 0.998, indicating that the regression equations for the five target analytes have a good linear relationship, indicating that the analysis method for bergamot lactone, imperatorin, anise lactone, 7-hydroxycoumarin and saxifragenol established in this experiment has high accuracy and sensitivity.

[0037] Example 2

[0038] Validation of analytical methods:

[0039] Precision: 20 μL of the reference solution was taken and injected continuously 6 times according to the chromatographic conditions in Example 1 to determine the peak areas and elution times of bergamotolactone, imperatorin, anise lactone, 7-hydroxycoumarin, and saxifragenol. The precision results were expressed as relative standard deviations (RSDs) calculated using SPSS 15.0. The measurement results are shown in Table 3.

[0040] Table 3 Precision test results

[0041]

[0042]

[0043] Note: RT a :retention time, peak time; PA b :peakarea, peak surface; same below.

[0044] Stability: Six identical sample solutions were prepared according to the sample solution preparation method in Example 1. The peak areas and elution times of bergamotolactone, imperatorin, anise lactone, 7-hydroxycoumarin, and saxifragenol in the sample solutions were determined at 0, 2, 4, 8, 12, and 24 h after preparation according to the chromatographic conditions in Example 1. The RSDs were calculated using SPSS 15.0. The results are shown in Table 4.

[0045] Table 4 Stability test results

[0046]

[0047] Repeatability: Six identical sample solutions were prepared according to the sample solution preparation method in Example 1. The peak areas and elution times of bergamotolactone, imperatorin, aniseed lactone, 7-hydroxycoumarin, and saxifragenol were determined according to the chromatographic conditions in Example 1, and the RSDs were calculated. The results are shown in Table 5.

[0048] Table 5 Repeatability test results

[0049]

[0050]

[0051] Recovery: Accurately weigh 6 portions of celery powder with known index component content, prepare a sample solution according to the sample solution preparation method in Example 1, accurately add an appropriate amount of the mixed standard solution, and measure the spiked recoveries of the five coumarin compounds according to the chromatographic conditions in Example 1, and calculate the RSDs. The results are shown in Table 6. The recovery calculation formula is as follows:

[0052]

[0053] In the above formula: C1 is the content of the sample compound after spiked, mg / g; C2 is the content of the sample compound, mg / g; C3 is the spiked amount, mg / g.

[0054] Table 6 Recovery rate experimental results

[0055]

[0056] The results in Tables 3 to 6 show that the detection method provided by the present invention has high accuracy and good stability, and can be used for the simultaneous qualitative and quantitative detection of five coumarin components in celery.

[0057] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for simultaneously detecting the contents of five coumarin components in old celery, characterized in that: The following steps are involved: The contents of saxifragin, 7-hydroxycoumarin, bergamot lactone, anise lactone and imperatorin in the extract of Rhizoma Cibotii were determined by HPLC, with saxifragin, 7-hydroxycoumarin, bergamot lactone, anise lactone and imperatorin as reference substances. HPLC detection conditions include: Chromatographic column: C-18 column; Mobile phase: A phase acetonitrile, B phase 0.1wt.% formic acid aqueous solution; Flow rate: 1 mL / min; Detection wavelength: 300nm; Elution procedure: 0-10 min: phase A 20%, phase B 80%, 10-25 min: phase A 35%, phase B 65%, after 25 min: phase A 60%, phase B 40%.

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