Rapid identification method for lavender germplasm and application of isovitexin in identification of lavandula angustifolia

Abstract

The invention relates to the technical field of medicinal material identification, in particular to a rapid lavender germplasm identification method and application of isovitexin in lavender edulis identification, the rapid lavender germplasm identification method comprises the following steps: step 1, extracting a sample to be detected to obtain an extracting solution of the sample to be detected; 2, detecting the content of isovitexin in the to-be-detected sample extracting solution; step 3, determining whether the to-be-detected sample contains lavandula angustifolia or not according to the content of isovitexin in the to-be-detected sample extracting solution. The systematic metabonomics research finds and confirms that isovitexin serves as a characteristic marker for lavender germplasm identification, lavandula dentata and lavandula angustifolia can be rapidly identified, and the method is easy and convenient to operate, low in cost, good in stability and reliability and convenient to apply and popularize.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal material identification, and is a method for rapid identification of lavender germplasm and the application of isovitexin in the identification of Lavandula dentata. Background Art

[0002] As a precious plant of the Lamiaceae family, lavender has attracted much attention due to its unique medicinal value. There are 39 known species of lavender in the world, and the most famous variety is Lavandula angustifolia (LA).

[0003] Lavandula angustifolia is a herbal medicine approved by the Herbal Medicines Committee of the European Medicines Agency. The flowers of Lavandula angustifolia are used to prepare herbal teas and oral preparations, mainly for relieving mental stress and improving sleep quality, and it is an approved medicinal lavender variety in Europe; in China, Lavandula angustifolia is also specified in modern preparations such as "Compound Gaoziban Tablets" and "Aiweixin Oral Liquid". However, the increasing number of imported Lavandula dentata (LD) has led to frequent species substitution or mixing in commercial products, especially in herbal teas and food supplements, which has raised concerns about the quality and efficacy of products.

[0004] At present, there are many studies and reports on the identification of the two. Chen Yan et al. (Comparative study on the flowers of Lavandula angustifolia Mill. and Lavandula dentata L. [J]. Northwest Pharmaceutical Journal, 2017, 32(05): 541-544.) proposed to identify the two by means of morphological identification, microscopic identification and determination of the content of active ingredients. The results showed that the two lavender species could be identified from the differences in calyx, the presence or absence of peduncles and bracts; from the color of the powder and the presence or absence of stone cells, calcium oxalate prisms and seed coat cells; and the content determination could be carried out from the content of volatile oil and the presence or absence of linalool. The research results could be used as the basis for the identification of the flowers of Lavandula angustifolia Mill. and Lavandula dentata L. However, this method was applicable only to the samples ground into powder and could not be applied. Li Chenyang et al. (Determination of the contents of four components such as luteoloside in Lavandula dentata L. and study on its fingerprint. Chinese Journal of Pharmaceutical Analysis, 2019, 39(12): 2234-2240) established the HPLC fingerprint of 11 batches of Lavandula dentata L. medicinal materials, obtained 11 common peaks, and determined 4 chemical components therefrom, namely luteoloside, luteolin-7-O-β-D-glucuronide, rosmarinic acid and luteolin. By comparing with the fingerprint of Lavandula angustifolia Mill., it was pointed out that there were significant differences in the number and area of chromatographic peaks between Lavandula dentata L. and Lavandula angustifolia Mill., indicating that there might be significant differences in their components. However, these components did not form a method that could be used for the identification of the two. Shi Yuge et al. (Analysis of volatile components of lavender and its quality control [D]) and Wang Qing et al. (Comparative analysis of infrared spectra of Lavandula angustifolia Mill. and Lavandula dentata L. [J]. The Journal of Light Scattering, 2013, 25(4)) took lavender as the research object, adopted two extraction techniques of steam distillation and headspace solid-phase microextraction, and analyzed the volatile chemical components of Lavandula angustifolia Mill., Lavandula dentata L. and different parts of Lavandula angustifolia Mill. by gas chromatography and gas chromatography-mass spectrometry. The principal component analysis and cluster analysis methods were used to identify and analyze the two lavender varieties. The experimental results showed that the two lavender species could be well identified and analyzed by combining the aroma components with the two statistical methods. Based on gas chromatography, the fingerprints of Lavandula angustifolia Mill. and Lavandula dentata L. were constructed, which could be used for the variety identification of Lavandula angustifolia Mill. and Lavandula dentata L. The infrared spectroscopy technology combined with cluster analysis and principal component analysis could also be well used for the identification of the varieties of Lavandula angustifolia Mill. and Lavandula dentata L. However, the mixture of the two could not be identified.

[0005] In summary, there is a lack of simple and reliable identification methods for the identification of Lavandula angustifolia Mill. and Lavandula dentata L. in the prior art. Therefore, it is urgent to develop a rapid and accurate method for the identification of lavender germplasm. Summary of the Invention

[0006] The present invention provides a method for rapid identification of lavender germplasm and the application of isovitexin in the identification of Lavandula dentata, overcoming the deficiencies of the above-mentioned prior art and effectively solving the problems of difficult identification between Lavandula angustifolia and Lavandula dentata and complex steps of the identification method.

[0007] One of the technical solutions of the present invention is achieved by the following measures: A method for rapid identification of lavender germplasm is carried out according to the following steps: Step 1, extract the sample to be tested to obtain an extract of the sample to be tested; Step 2, detect the content of isovitexin in the extract of the sample to be tested; Step 3, determine whether the sample to be tested contains Lavandula dentata according to the content of isovitexin in the extract of the sample to be tested.

[0008] The following is a further optimization or / and improvement of the above-mentioned technical solution of the invention: In the above step 1, the sample to be tested is Lavandula dentata, Lavandula angustifolia or a mixed sample of Lavandula dentata and Lavandula angustifolia.

[0009] In the above step 1, the extraction of the sample to be tested is as follows: Using water as the extraction solvent for ultrasonic extraction. When extracting, 8 mL to 30 mL of water is added to each 1 g of the sample to be tested.

[0010] The temperature of the above ultrasonic extraction is 20°C to 60°C.

[0011] The time of the above ultrasonic extraction is 15 min to 60 min.

[0012] In the above step 2, the content of isovitexin in the extract of the sample to be tested is detected by liquid chromatography.

[0013] The detection of the content of isovitexin in the extract of the sample to be tested by the above liquid chromatography method includes: Preparing a series of standard solutions of isovitexin; Using liquid chromatography to separate and detect the series of standard solutions of isovitexin and the extract of the sample to be tested; Obtaining the standard curve of isovitexin according to the detection results of liquid chromatography, and combining the standard curve of isovitexin to obtain the content of isovitexin in the extract of the sample to be tested.

[0014] The above liquid chromatography method uses a C18 reversed-phase chromatographic column for separation. The chromatographic column is ACQUITY UPLC HSS T3 Column, 1.8 μm, 2.1 mm × 100 mm; the detector is an ultraviolet detector or a diode array detector, the detection wavelength is 338 nm, and the detection duration is 50 min.

[0015] The above liquid chromatography method uses a mobile phase of 0.1% formic acid aqueous solution and acetonitrile, adopts a gradient elution method, and the elution gradient is as follows: from 0 to 30 min, 85% acetonitrile; from 30 min to 40 min, from 85% to 60% acetonitrile; from 40 min to 42 min, from 60% to 85% acetonitrile; from 42 min to 50 min, 85% acetonitrile; the flow rate of the mobile phase is 0.3 mL / min, the column temperature is 35 °C, and the injection volume is 3.0 μL.

[0016] In the above step three, determining whether the test sample contains Lavandula dentata according to the content of isovitexin in the test sample extract includes: If isovitexin is detected in the test sample extract, it is determined that the test sample is Lavandula dentata or a mixed sample of Lavandula dentata and Lavandula angustifolia; If isovitexin is not detected in the test sample extract, it is determined that the test sample is Lavandula angustifolia.

[0017] The second technical solution of the present invention is achieved by the following measures: the application of isovitexin in the identification of Lavandula dentata.

[0018] The present invention provides a method for rapid identification of lavender germplasm and the application of isovitexin in the identification of Lavandula dentata. Through systematic metabolomics research, isovitexin is discovered and confirmed as a characteristic marker for lavender germplasm identification, which can rapidly identify Lavandula dentata and Lavandula angustifolia. The method is simple to operate, low in cost, excellent in stability and reliability, and is convenient for popularization and application. Description of the Drawings

[0019] Att Figure 1 It is the total ion current chromatogram of metabolite analysis of Lavandula angustifolia and Lavandula dentata based on UHPLC-ESI-QE-Orbitrap-MS technology in Example 12 of the present invention.

[0020] Att Figure 2 It is the molecular network analysis diagram of metabolites of Lavandula angustifolia and Lavandula dentata in Example 12 of the present invention.

[0021] Att Figure 3 It is the schematic diagram of the content determination results of isovitexin in Lavandula angustifolia (LA) and Lavandula dentata (LD) in Example 12 of the present invention.

[0022] Att Figure 4 It is the liquid chromatogram of Lavandula dentata sample and Lavandula angustifolia sample in Example 13 of the present invention.

[0023] Att Figure 5 It is the relationship diagram between the mixing ratio of Lavandula dentata and the peak area of isovitexin in Example 14 of the present invention. Detailed Embodiments

[0024] The present invention is not limited by the following embodiments, and specific implementation manners can be determined according to the technical solution of the present invention and actual situations. Various chemical reagents and chemical supplies mentioned in the present invention are well-known and commonly used chemical reagents and chemical supplies in the prior art unless otherwise specified; percentages in the present invention are mass percentages unless otherwise specified; solutions in the present invention are aqueous solutions with water as the solvent unless otherwise specified. For example, a hydrochloric acid solution is an aqueous solution of hydrochloric acid; normal temperature and room temperature in the present invention generally refer to a temperature range of 15°C to 25°C, and are generally defined as 25°C.

[0025] The present invention will be further described below in conjunction with embodiments: Example 1: The method for rapid identification of lavender germplasm is carried out according to the following steps: Step 1, extract the sample to be tested to obtain an extract of the sample to be tested; Step 2, detect the content of isovitexin in the extract of the sample to be tested; Step 3, determine whether the sample to be tested contains Lavandula dentata according to the content of isovitexin in the extract of the sample to be tested.

[0026] Example 2: As an optimization of the above embodiment, in Step 1, the sample to be tested is Lavandula dentata, Lavandula angustifolia or a mixed sample of Lavandula dentata and Lavandula angustifolia.

[0027] Example 3: As an optimization of the above embodiment, in Step 1, the extraction of the sample to be tested is: ultrasonic extraction with water as the extraction solvent. When extracting, 8 mL to 30 mL of water is added to each 1 g of the sample to be tested.

[0028] Example 4: As an optimization of the above embodiment, the ultrasonic extraction temperature is 20°C to 60°C.

[0029] Example 5: As an optimization of the above embodiment, the ultrasonic extraction time is 15 min to 60 min.

[0030] Example 6: As an optimization of the above embodiment, in Step 2, high performance liquid chromatography is used to detect the content of isovitexin in the extract of the sample to be tested.

[0031] Example 7: As an optimization of the above embodiment, using high performance liquid chromatography to detect the content of isovitexin in the extract of the sample to be tested includes: Preparing a series of standard solutions of isovitexin; Using high performance liquid chromatography to separate and detect the series of standard solutions of isovitexin and the extract of the sample to be tested; Obtaining a standard curve of isovitexin according to the detection results of high performance liquid chromatography, and combining the standard curve of isovitexin to obtain the content of isovitexin in the extract of the sample to be tested.

[0032] Example 8: As an optimization of the above example, C18 reversed-phase chromatography column was used for separation in liquid chromatography. The chromatography column was ACQUITY UPLC HSS T3 Column, 1.8μm, 2.1mm×100mm; the detector was an ultraviolet detector or a diode array detector, the detection wavelength was 338nm, and the detection duration was 50min.

[0033] Example 9: As an optimization of the above example, the mobile phase of liquid chromatography was 0.1% formic acid aqueous solution and acetonitrile, and gradient elution was used. The elution gradient was as follows: from 0 to 30min, 85% acetonitrile; from 30min to 40min, 85% to 60% acetonitrile; from 40min to 42min, 60% to 85% acetonitrile; from 42min to 50min, 85% acetonitrile; the flow rate of the mobile phase was 0.3mL / min, the column temperature was 35°C, and the injection volume was 3.0μL.

[0034] Example 10: As an optimization of the above example, in step three, to determine whether the test sample contains Lavandula dentata according to the content of isovitexin in the extract of the test sample, it includes: If isovitexin is detected in the extract of the test sample, it is determined that the test sample is Lavandula dentata or a mixed sample of Lavandula dentata and Lavandula angustifolia; If isovitexin is not detected in the extract of the test sample, it is determined that the test sample is Lavandula angustifolia.

[0035] Example 11: Application of isovitexin in the identification of Lavandula dentata Lavandula angustifolia and Lavandula dentata are two medicinal plants with different medicinal values but similar morphologies. The present invention compared the prototype components compounds that enter the blood and brain of Lavandula angustifolia and Lavandula dentata. By high-resolution mass spectrometry analysis of the migratory chemical components in the brain tissues of model mice after administration of Lavandula angustifolia and Lavandula dentata, the experimental results showed that a total of 34 prototype compounds were detected in the brain tissues of the mice after administration. Among them, a total of 23 compounds were detected in the Lavandula angustifolia group, mainly cinnamic acid and its derivatives; a total of 21 compounds were detected in the Lavandula dentata group, mainly glycoside compounds of luteolin and apigenin. This means that they have different biological activities, so an accurate identification method needs to be proposed for their identification. Through a large number of experiments and previous research, the present invention selected the unique isovitexin in Lavandula dentata as a marker for differentiating the two, and can quickly and accurately identify Lavandula angustifolia and Lavandula dentata as well as the mixed samples of Lavandula dentata and Lavandula angustifolia.

[0036] Example 12: Discovery and confirmation of the marker First, based on the UHPLC-Q Exactive Orbitrap-MS technology, metabolomic analyses of lavender plants (Lavandula angustifolia and Lavandula dentata) were performed in positive and negative ion modes, respectively. Figure 1 Representative total ion chromatograms (TICs) of Lavandula angustifolia, Lavandula dentata, and their mixed samples are shown. Among them, Figures A and B are the total ion chromatograms (TIC) of Lavandula angustifolia in positive and negative ion modes, respectively; Figures C and D are the total ion chromatograms (TIC) of Lavandula dentata in positive and negative ion modes, respectively; Figures E and F are the total ion chromatograms (TIC) of the mixed sample of Lavandula angustifolia and Lavandula dentata in positive and negative ion modes, respectively. A total of 1610 characteristic signals with response values greater than 10 6 were obtained in the negative ion mode, and 1210 characteristic peaks with MS2 information were obtained therefrom.

[0037] Secondly, a molecular network analysis method based on GNPS was used to characterize the chemical components of extracts from Lavandula angustifolia and Lavandula dentata. Through the processing of MS / MS data, network analysis identified 8 major molecular families and 20 independent nodes, as shown in Figure 2 . Among them, Figure A is a visualization of the feature-based molecular network (FBMN), showing the annotated and unannotated compounds. The red and green nodes represent the compounds (96 species) annotated by the FBMN molecular network, and 54 of the green nodes were further classified by MOLNETENHANCER. Figure B shows the distribution of the six major types of compounds identified.

[0038] Based on the above research, by integrating OPLS-DA (orthogonal partial least squares discriminant analysis) and volcano plot analysis, a total of 15 significantly different metabolites were identified. Among them, as shown in Figure 3 , isovitexin was not detected in Lavandula angustifolia and was only present in Lavandula dentata. Therefore, it was confirmed that isovitexin can be used as a reliable germplasm identification marker for the identification of Lavandula angustifolia and Lavandula dentata.

[0039] Example 13: The specific implementation process of this rapid lavender germplasm identification method is as follows: Weigh 1.0000 g of each of the 6 groups of samples to be tested, add 10 mL of water, and perform ultrasonic extraction at 25 °C for 40 min. Then filter to obtain the extract of the samples to be tested.

[0040] Prepare a series of standard solutions of isovitexin with concentrations of 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 50 μg / mL (the solvent is methanol).

[0041] The liquid chromatography analysis conditions are as follows: The chromatographic column is: ACQUITY UPLC HSS T3 Column, 1.8μm, 2.1mm×100mm.

[0042] The mobile phase is: A: 0.1% formic acid aqueous solution; B: acetonitrile; The elution gradient is: 0 to 30 min, 85% acetonitrile; 30 min to 40 min, 85% to 60% acetonitrile; 40 min to 42 min, 60% to 85% acetonitrile; 42 min to 50 min, 85% acetonitrile.

[0043] The flow rate of the mobile phase is 0.3 mL / min, the column temperature is 35°C, and the injection volume is 3.0 μL.

[0044] The detector is a diode array detector, and the detection wavelength is 338 nm.

[0045] Under the above chromatographic conditions, a series of standard solutions of isovitexin were separated and detected, and the standard curve of isovitexin was obtained as: y = 22010x - 2290.2; R 2 = 0.9999.

[0046] Under the above chromatographic conditions, 6 groups of test sample extracts were separated and detected. The detection results are shown in Table 1, and the chromatograms of some samples are shown in Figure 4 (The upper figure is for Sample No. 1, and the lower figure is for Sample No. 6).

[0047] Example 14: Detection of a mixed sample of Lavandula angustifolia and Lavandula dentata Lavandula dentata and Lavandula angustifolia were mixed in the ratios of 9:1, 8:2, 7:3, 6:4, and 5:5, and the mixed samples were detected according to the analysis method of Example 13. The detection results are shown in Table 2.

[0048] A graph was plotted of the mixing ratio of Lavandula dentata and the peak area of isovitexin. As Figure 5 shown, the results indicate that there is a significant negative correlation between the mixing ratio (the proportion of Lavandula dentata ranging from 90% to 50%) and the peak area, suggesting that the content of isovitexin decreases as the proportion of Lavandula dentata decreases; isovitexin, as a characteristic component of Lavandula dentata, its detection can effectively distinguish the two lavender varieties. In addition, based on the linear relationship between the proportion of Lavandula dentata and the peak area, the identification method of the present invention can also be applied to the evaluation of the mixing ratio in a mixed sample of Lavandula dentata and Lavandula angustifolia.

[0049] Example 15: Detection of samples after different temperature treatments Samples of *Lavandula dentata* and *Lavandula angustifolia* were respectively left standing in an oven at 25 °C, 60 °C, 100 °C, and 120 °C for 40 min, and after cooling, were ultrasonically extracted at 25 °C for 40 min. The extract was detected according to the analysis method of Example 13, and the detection results are shown in Table 3.

[0050] In Table 3, isovitexin could be detected in the samples of *Lavandula dentata* after treatment at different temperatures, while isovitexin could not be detected in the samples of *Lavandula angustifolia* after treatment at different temperatures; the treatment temperature was not related to the peak area of isovitexin in *Lavandula dentata*. This indicates that under different temperatures (25 °C, 60 °C, 100 °C, 120 °C), the peak area of isovitexin in *Lavandula dentata* fluctuates less and has high overall stability; isovitexin was not detected in *Lavandula angustifolia* under any temperature treatment, further verifying that it does not contain this component and there is no situation where other internal components are converted into isovitexin; it shows that temperature does not affect the differentiation between *Lavandula dentata* and *Lavandula angustifolia*, and the detection result of isovitexin is only related to the species.

[0051] Example 16: The methodology of the rapid identification method for lavender germplasm of the present invention was verified. The detection linear range of isovitexin was 0.2 μg / mL to 100 μg / mL, and the detection limit was 0.15 μg / mL; the detection precision RSD < 5.38%, the recovery rate was between 93.5% and 102.4%, and in terms of sample stability, the RSD of the peak area within 24 h < 3%. The method has good accuracy and stability.

[0052] The samples of *Lavandula angustifolia* used in Examples 12 to 16 above were collected from Yining City, Xinjiang Uygur Autonomous Region, China, and the samples of *Lavandula dentata* were from the Urumqi Chinese medicinal materials market, and the record shows that its origin is Pakistan. All samples were identified by experts from the Xinjiang Uygur Autonomous Region Academy of Analysis and Testing.

[0053] In summary, the present invention provides a rapid identification method for lavender germplasm and the application of isovitexin in the identification of *Lavandula dentata*. Through systematic metabolomics research, isovitexin was discovered and confirmed as a characteristic marker for lavender germplasm identification, and a simple and reliable detection method was established. The method is easy to operate, low in cost, good in stability and reliability, and has a wide range of applications, providing good technical support for the supervision and quality control of lavender germplasm resources.

[0054] The above technical features respectively constitute the embodiments of the present invention, which have strong adaptability and implementation effects. Non-essential technical features can be added or subtracted according to actual needs to meet the requirements of different situations.

Claims

1. A method for rapid identification of lavender germplasm, characterized in that It is carried out according to the following steps: Step 1: Extract the sample to be tested to obtain an extract of the sample to be tested; Step 2: Detect the content of isovitexin in the extract of the sample to be tested; Step 3: Determine whether the sample to be tested contains Lavandula dentata according to the content of isovitexin in the extract of the sample to be tested.

2. The rapid identification method of lavender germplasm according to claim 1, characterized in that In Step 1, the sample to be tested is Lavandula dentata, Lavandula angustifolia or a mixed sample of Lavandula dentata and Lavandula angustifolia.

3. The rapid identification method of lavender germplasm according to claim 1 or 2, characterized in that In Step 1, the extraction of the sample to be tested is as follows: ultrasonic extraction is carried out with water as the extraction solvent. When extracting, 8 mL to 30 mL of water is added to every 1 g of the sample to be tested.

4. The rapid identification method of lavender germplasm according to claim 3, characterized in that The ultrasonic extraction temperature is 20 °C to 60 °C.

5. The rapid identification method of lavender germplasm according to claim 3 or 4, characterized in that The ultrasonic extraction time is 15 min to 60 min.

6. The rapid identification method of lavender germplasm according to any one of claims 1 to 5, characterized in that In Step 2, the content of isovitexin in the extract of the sample to be tested is detected by liquid chromatography.

7. The rapid identification method of lavender germplasm according to claim 6, characterized in that Detecting the content of isovitexin in the extract of the sample to be tested by liquid chromatography includes: Preparing a series of standard solutions of isovitexin; Separating and detecting the series of standard solutions of isovitexin and the extract of the sample to be tested by liquid chromatography; Obtaining a standard curve of isovitexin according to the detection results of liquid chromatography, and combining the standard curve of isovitexin to obtain the content of isovitexin in the extract of the sample to be tested.

8. The rapid identification method of lavender germplasm according to claim 7, characterized in that The liquid chromatography method uses a C18 reversed-phase chromatographic column for separation. The chromatographic column is ACQUITY UPLC HSS T3 Column, 1.8 μm, 2.1 mm × 100 mm; the detector is an ultraviolet detector or a diode array detector, the detection wavelength is 338 nm, and the detection duration is 50 min; or / and, the liquid chromatography method uses a mobile phase of 0.1% formic acid aqueous solution and acetonitrile, and gradient elution is adopted. The elution gradient is: 0 to 30 min, 85% acetonitrile; 30 min to 40 min, 85% to 60% acetonitrile; 40 min to 42 min, 60% to 85% acetonitrile; 42 min to 50 min, 85% acetonitrile; the flow rate of the mobile phase is 0.3 mL / min, the column temperature is 35 °C, and the injection volume is 3.0 μL.

9. The rapid identification method of lavender germplasm according to any one of claims 1 to 8, characterized in that Step 3: Determine whether the sample to be tested contains Lavandula dentata according to the content of isovitexin in the extract of the sample to be tested, including: If isovitexin is detected in the extract of the sample to be tested, it is determined that the sample to be tested is Lavandula dentata or a mixed sample of Lavandula dentata and Lavandula angustifolia; If isovitexin is not detected in the extract of the sample to be tested, it is determined that the sample to be tested is Lavandula angustifolia.

10. Application of isovitexin in the identification of Lavandula dentata.

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