Characteristic marker combination for identifying honeysuckle variety tea and identification method thereof

Abstract

The invention relates to a characteristic marker combination for identifying honeysuckle tea varieties and an identification method thereof, and belongs to the technical field of variety tea identification. The characteristic marker combination comprises chlorogenic acid, swertioside, secoxyloganin, galuteolin, isochlorogenic acid A and isochlorogenic acid C. The invention further discloses a kit for detecting the secoxyloganin. The contents of the characteristic markers are simultaneously detected through a high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS) technology, and different honeysuckle tea varieties can be rapidly and accurately identified by combining multivariate statistical analysis (such as principal component analysis or clustering analysis). The identification method provided by the invention has the characteristics of strong specificity, high stability and high sensitivity, is suitable for large-scale sample analysis, and can be widely applied to quality control, authenticity identification and market supervision of honeysuckle tea varieties.

Description

Technical Field

[0001] This invention relates to the field of tea variety identification technology, specifically to a method for identifying honeysuckle tea varieties based on a combination of characteristic markers. Background Technology

[0002] Honeysuckle (Lonicerae Japonicae Flos, LJF) is the dried flower bud or newly opened flower of the honeysuckle plant (Lonicera japonica), a member of the Caprifoliaceae family. Known as a "plant antibiotic," it is widely used in food, beverages, and traditional Chinese medicine. According to data from the National Association of Economic Forests, global demand for honeysuckle exceeds 20 million kilograms, with a market value of up to US$860 million.

[0003] The main functional components of honeysuckle include phenolic acids such as chlorogenic acid, isochlorogenic acid A and C, flavonoids such as luteolin, and iridoid glycosides such as strychnine and strychnine. Studies have shown that chlorogenic acid possesses antibacterial and antiviral properties, and exhibits anti-inflammatory, antioxidant, lipid-lowering, and hypoglycemic activities. Often hailed as "plant gold," it is widely used in various fields of the food industry, including as a food additive, food storage, food ingredient modification, food packaging material production, functional food ingredients, and prebiotics. Luteolin is easily hydrolyzed into luteolin by intestinal microorganisms after oral administration. Luteolin is a bioactive flavonoid polyphenol that is increasingly recognized for its health-promoting properties, bioavailability, and low toxicity, making it a valuable ingredient in food applications. Luteolin can alleviate myocardial remodeling and contractile dysfunction. Strychnine (3 mg / kg) effectively reduced inflammation in a mouse model. Isochlorogenic acid A promotes megakaryocyte differentiation and platelet production, offering hope for the treatment of radiation-induced thrombocytopenia. Both chlorogenic acid and isochlorogenic acid A can counteract the cytotoxicity induced by mixed mycotoxins, with isochlorogenic acid A being particularly effective by maintaining mitochondrial homeostasis and enhancing innate immune function associated with the RIG-I-like receptor pathway. Furthermore, it may alleviate rheumatoid arthritis and osteoporosis by modulating the IL-17 / MAPK signaling pathway. Isochlorogenic acid C is a novel cholesterol reverse transport promoter that can lower blood lipid levels without inducing hepatic steatosis.

[0004] Currently, the most frequently tested substances are chlorogenic acid and luteolin, which are present in higher concentrations in honeysuckle varieties. However, the content of isochlorogenic acid A and C, which are present in lower concentrations, as well as strychnine and oxylogenin, is not readily available.

[0005] With increasing public focus on health and natural therapies, the market demand for high-quality medicinal and edible honeysuckle tea is growing. Honeysuckle is widely cultivated in China, with Henan, Shandong, and Hebei provinces considered the main producing areas. Currently, the main honeysuckle variety grown in Fengqiu, Henan Province, is the Da Mao Hua variety. Over the years, a series of new honeysuckle varieties have been bred from Da Mao Hua, characterized by high yield, large flower buds, strong resistance, and rich active ingredients. While these new varieties show potential in agronomic performance, their functional component characteristics still lack evaluation to ensure they meet the quality standards for functional teas.

[0006] Honeysuckle tea is a type of tea with significant medicinal and edible value. It has numerous varieties, each exhibiting significant differences in chemical composition and efficacy. Currently, traditional identification methods primarily rely on appearance and sensory evaluation, which have relatively low accuracy. Therefore, developing a combination of characteristic markers based on chemical composition and its identification method is of great importance for the rapid and accurate identification of honeysuckle tea varieties. Summary of the Invention

[0007] This invention provides a combination of characteristic markers for identifying honeysuckle tea varieties and a method for doing so. The combination of characteristic markers includes chlorogenic acid, strychnine, strychnine oxidase, luteolin, isochlorogenic acid A, and isochlorogenic acid C. By simultaneously detecting the content of these characteristic markers and combining the results with data analysis, accurate identification of different honeysuckle tea varieties can be achieved.

[0008] The feature marker combination includes the following components: chlorogenic acid, strychnine, strychnine, luteolin, isochlorogenic acid A, and isochlorogenic acid C.

[0009] Identification methods:

[0010] Sample preparation: Fresh honeysuckle flowers were dried in an oven at 55℃ for 24 hours to obtain honeysuckle tea. The honeysuckle tea sample was pulverized and dissolved in 70% ethanol at a solid-liquid ratio of 1:16 (m / v), and the pH was adjusted to 4 with dilute sulfuric acid solution. The honeysuckle solution was ultrasonically treated (250W, 40kHz) for 30 minutes, and the extract was centrifuged at 4000rpm for 30 minutes, and the supernatant was collected. Then, the precipitate was redissolved in 70% ethanol, and the extraction process was repeated once. The supernatants from the two extractions were combined and concentrated by rotary evaporation to obtain the honeysuckle tea extract.

[0011] Detection method: High performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS) is used to simultaneously detect the content of characteristic marker combinations in the sample.

[0012] Data analysis: The test results are compared with a database of characteristic marker contents of known varieties, and the variety to which the sample belongs is determined through multivariate statistical analysis (such as principal component analysis or cluster analysis).

[0013] advantage:

[0014] The combination of characteristic markers provided by this invention has the characteristics of high specificity and high stability, and can accurately reflect the chemical characteristics of different honeysuckle tea varieties.

[0015] High-performance liquid chromatography or liquid chromatography-mass spectrometry (LC-MS) is used, which provides high detection sensitivity and good repeatability, making it suitable for large-scale sample analysis.

[0016] Technical effects:

[0017] This method is simple and quick to operate, and can be widely used for quality control and authenticity identification of honeysuckle tea varieties. Attached Figure Description

[0018] Figure 1 Camellia bud morphology of 10 representative honeysuckle varieties. (A) Da Mao Hua (B) Yang Jiao No. 1 (C) Jiu Feng No. 1 (D) Ju Hua No. 1 (E) Bei Hua No. 1 (F) Feng Hua No. 1 (G) Bainong Jin No. 1 (H) Bainong Jin No. 2 (I) Yu Jin No. 1 (J) Yu Jin No. 2

[0019] Figure 2 HPLC chromatograms of the base peak intensities of 10 representative honeysuckle tea varieties. The R curve represents 14 common peaks (blue vertical dashed line), and the numbers 1 to 6 represent the peak positions of chlorogenic acid, strychnine, loganin, apigenin, isochlorogenic acid A, and isochlorogenic acid C, respectively.

[0020] Figure 3 Principal component analysis chart of 10 representative honeysuckle tea varieties.

[0021] Figure 4 Comparison of the main components in tea extracts from 10 representative honeysuckle varieties. (A) Chlorogenic acid. (B) Isochlorogenic acid. (C) Strychnos nuciferoside. (D) Luteolin. (E) Isochlorogenic acid A. (F) Isochlorogenic acid C. (G) Phenolic acids. (AF) Molecular formulas are shown at the top of each figure. Duncan's multiple range test (DMRT) was used to determine the statistical significance of differences between groups. Different lowercase letters indicate significant differences between each other (P<0.05). In intergroup comparisons, the same group may have insignificant or significant differences with one or more other groups, and crossover exists. Detailed Implementation

[0022] It should be understood that the following detailed description is merely illustrative and intended to further explain the content of this application, rather than limiting the scope of the invention. Unless otherwise expressly stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application pertains.

[0023] The embodiments of the present invention will be described in detail below with reference to specific examples. These examples are merely illustrative and are intended to further clarify the technical solutions and applications of the present invention, so that those skilled in the art can better understand and implement the present invention. It should be noted that the following examples are not intended to limit the present invention, and any equivalent substitutions or modifications made based on the spirit of the present invention are within the scope of protection of the present invention.

[0024] Example 1: Preparation of Honeysuckle Variety Tea Extract

[0025] (1) Experimental materials:

[0026] The raw materials used in this embodiment for preparing honeysuckle tea were collected in May 2023 from the germplasm resource nursery of Xinfeng Agricultural Planting Professional Cooperative in Fengqiu County, Xinxiang City, Henan Province, China. Fresh flower buds of 10 honeysuckle varieties were collected and identified as honeysuckle buds of the Caprifoliaceae family. The fresh flower buds of the 10 honeysuckle varieties were dried in an oven at 55℃ for 24 hours to obtain 10 varieties of honeysuckle tea.

[0027] Example 2: Appearance characteristics of 10 honeysuckle tea varieties

[0028] Description of the appearance characteristics of honeysuckle tea varieties: Da Mao Hua (traditional variety) has a dried flower bud weight of 15.91±0.057g, the buds are slightly curved, yellowish-green, hairy, and fragrant; Yang Jiao No. 1 (traditional variety) has a dried flower bud weight of 20.49±0.056g, the buds are green, curved like horns, thicker at the top and thinner at the bottom, with a large degree of curvature and dense fine hairs; Feng Hua No. 1 (Yu 2017002) has a dried flower bud weight of 25.74±0.024g, strong upright growth, short internodes, and dense epidermal hairs; Yu Jin No. 1 (Yu 2015001) has a dried flower bud weight of 24.62±0.046g, long flowering period, large flower buds, high yield, strong resistance, and dense and long epidermal hairs; Yu Jin No. 2 (Yu 2015002) Dried flower buds weighed 17.46±0.041g, with strong upright growth, large flowers, early germination, and a rich aroma; the flower buds were red. Bainongjin No. 1 (Yu 2021001) Dried flower buds weighed 29.7±0.025g, with upright growth and thick branches, large and thin, dark green leaves with clear veins and dense long hairs on the back; the flower buds were slender. Bainongjin No. 2 (Yu 2021002) Dried flower buds weighed 22.64±0.038g, with weak upright growth, few but strong taproots, thick stems, short internodes, ovate, dark green leaves with dense long hairs on the back; the flower buds were swollen at the top and slender at the bottom, forming a long, slightly curved rod. Jiufeng No. 1 (Lu S-SV-LJ-004-2005) has a dry flower bud weight of 20.97±0.039g, with stout and upright stems and branches, almost no long branches, many flowering branches, heart-shaped leaves with blunt and rounded tips, thick leaves, dark green color, and abundant hairs, and large flower buds; Beihua No. 1 (00116092015) has a dry flower bud weight of 16.07±0.046g, with large, yellowish-green, rod-shaped flower buds that are thicker at the top and thinner at the bottom, slightly curved, densely covered with downy hairs, long flower buds that are easy to pick, good resistance to adverse conditions and upright growth, and wide adaptability; Juhua No. 1 (Ji S-SV-LJ-039-2013) has a dry flower bud weight of 19.64±0.026g, strong upright growth, large, dark green leaves with long leaf hairs, stout stems and branches, short internodes, few bare branches, long flowering period, large and numerous flower buds, and strong salt and alkali tolerance, drought resistance, and disease resistance.

[0029] Example 3: Preparation and Analysis of Honeysuckle Variety Tea Extract

[0030] To further identify characteristic markers of honeysuckle tea varieties, chemical components in 10 representative honeysuckle tea varieties were detected using high-performance liquid chromatography (HPLC) and principal component analysis (PCA). The specific detection methods are as follows:

[0031] (1) Sample preparation

[0032] After pulverization, tea powder from 10 varieties was accurately weighed, including Da Mao Hua, Yang Jiao No. 1, Feng Hua No. 1, Ju Hua No. 1, Yu Jin No. 1, Yu Jin No. 2, Jiu Feng No. 1, Bai Jin No. 1, Bai Jin No. 2, and Bei Hua No. 1, with weights of 2.0001 g, 4.9994 g, 5.0003 g, 5.0007 g, 5.003 g, 2.4462 g, 4.2482 g, 2.0004 g, 5.0012 g, and 5.0003 g, respectively. These tea powders were dissolved in 70% ethanol solution at a material-to-liquid ratio of 1:16 (m / v), and the pH was adjusted to 4 with dilute sulfuric acid solution. The honeysuckle solution was ultrasonically treated (250 W, 40 kHz) for 30 minutes, and the extract was centrifuged at 4000 rpm for 30 minutes, collecting the supernatant. The precipitate was then redissolved in 70% ethanol, and the extraction process was repeated once. The supernatants from the two extractions were combined and concentrated by rotary evaporation to obtain the extract. An appropriate amount of the extract was then accurately weighed and dissolved in 75% methanol to a concentration of 5 mg / mL. The solution was then filtered through a 0.22 μm organic filter membrane for later use.

[0033] (2) HPLC analysis method

[0034] Tea extracts from 10 representative honeysuckle varieties were analyzed using an Agilent 1260 HPLC system, with three replicates for each sample.

[0035] The analytical conditions were as follows: Column: Agilent Microsorb-MV 100-5C18 column (4.6 × 250 mm, 5 μm); Column temperature: 25 °C; Solvent system included acetonitrile (A) and 0.1% phosphoric acid solution (B). The gradient program was as follows: 0–8 min, 14–19% A; 8–14 min, 19% A; 14–34 min, 19–31% A; 34–35 min, 31–90% A; 35–40 min, 90% A. Other chromatographic parameters were as follows: Injection volume: 25 μL; Flow rate: 0.7 mL / min; Detection wavelength: 240 nm.

[0036] (3) Data Analysis

[0037] HPLC results were recorded and analyzed using an Agilent ChemStation B.04.03 workstation. HPLC data were processed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" (2012A version). The contents of characteristic markers were calculated using the respective standard curves and are expressed as mean ± standard deviation (SD). One-way ANOVA was performed using SPSS 25.0 software, followed by Duncan's multiple range test (DMRT) to assess statistical significance. For the classification of LJF extracts, principal component analysis (PCA) and trend observation were performed using SIMCA (version 14.1).

[0038] Experimental results

[0039] The test results showed that the contents of six characteristic markers (chlorogenic acid, strychnine, strychnine, luteolin, isochlorogenic acid A, and isochlorogenic acid C) in the extracts of 10 representative honeysuckle tea varieties were significantly different. This combination can characterize the quality of honeysuckle tea varieties.

[0040] Large-flowered chrysanthemum (%): Chlorogenic acid content 2.50±0.41, strychnine content 1.17±0.06, isochlorogenic acid A content 1.52±0.06, strychnine content 0.19±0.02, luteolin content 0.10±0.01, isochlorogenic acid C content 0.29±0.02

[0041] Yangjiao No. 1 (%): Chlorogenic acid content is 3.48±0.02, strychnine content is 1.01±0.01, isochlorogenic acid A content is 1.63±0.02, dangyao glycoside content is 0.19±0, luteolin content is 0.13±0.02, and isochlorogenic acid C content is 0.53±0.01.

[0042] Fenghua No. 1 (%): Chlorogenic acid content is 3.32±0.36, strychnine content is 0.75±0.06, isochlorogenic acid A content is 1.86±0.08, thymol content is 0.20±0.01, luteolin content is 0.10±0.01, and isochlorogenic acid C content is 0.46±0.01.

[0043] Bainongjin No. 1 (%): Chlorogenic acid content is 1.40±0.11, strychnine content is 0.74±0, isochlorogenic acid A content is 0.82±0.03, thymol content is 0.36±0.02, luteolin content is 0.11±0.01, and isochlorogenic acid C content is 0.56±0.

[0044] Bainongjin No. 2 (%): Chlorogenic acid content is 4.20±0.14, Strychnos nux-ointense glycoside content is 1.09±0.08, Isochlorogenic acid A content is 1.40±0.02, Dangyao glycoside content is 0.19±0.01, Luteinoside content is 0.11±0.01, and Isochlorogenic acid C content is 0.90±0.02.

[0045] Yujin No. 1 (%): Chlorogenic acid content is 2.46±0.18, strychnoside content is 1.37±0.05, isochlorogenic acid A content is 1.68±0.11, thymol content is 0.36±0.02, luteolin content is 0.10±0.03, and isochlorogenic acid C content is 0.69±0.04.

[0046] Yujin No. 2 (%): Chlorogenic acid content is 2.97±0.39, strychnine content is 4.11±0.08, isochlorogenic acid A content is 0.69±0.04, dangyao glycoside content is 0.02±0, luteolin content is 0.17±0.01, and isochlorogenic acid C content is 0.23±0.01.

[0047] Jiufeng No. 1 (%): Chlorogenic acid content is 2.40±0.44, strychnine content is 0.75±0.18, isochlorogenic acid A content is 0.96±0.24, dangyao glycoside content is 0.07±0.02, luteolin content is 0.09±0.01, and isochlorogenic acid C content is 0.14±0.04.

[0048] Beihua No. 1 (%): Chlorogenic acid content is 4.61±0.10, strychnine content is 0.97±0.09, isochlorogenic acid A content is 1.67±0.10, dangyao glycoside content is 0.22±0.01, luteolin content is 0.14±0.02, and isochlorogenic acid C content is 0.67±0.06.

[0049] Giant Flower No. 1 (%): Chlorogenic acid content is 2.85±0.10, strychnine content is 1.25±0.02, isochlorogenic acid A content is 1.21±0.05, dangyao glycoside content is 0.18±0, luteolin content is 0.14±0.04, and isochlorogenic acid C content is 0.41±0.06.

[0050] It should be noted that the above embodiments are merely preferred embodiments of this application, and are intended to better illustrate the technical solution of this application, rather than to limit the scope of protection of this application. For those skilled in the art, various modifications, substitutions, or improvements can be made to the technical solution of this application without departing from the technical principles thereof, and all such equivalent technical solutions should be covered within the scope of protection of the claims of this application.

Claims

1. A combination of characteristic markers for identifying honeysuckle tea varieties, characterized in that, The combination of characteristic markers includes the following components: chlorogenic acid, strychnine, strychnine, luteolin, isochlorogenic acid A, and isochlorogenic acid C.

2. The combination of feature markers according to claim 1, characterized in that, The combination of characteristic markers was detected by high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS).

3. A method for identifying honeysuckle tea varieties, characterized in that, Includes the following steps: a. Sample preparation: Fresh honeysuckle flowers were dried in an oven at 55°C for 24 hours to obtain honeysuckle tea; the honeysuckle tea sample was pulverized and extracted with 70% ethanol solution at a material-to-liquid ratio of 1:16 (m / v), and the pH was adjusted to 4 with dilute sulfuric acid solution; the honeysuckle solution was ultrasonically treated (250 W, 40 kHz) for 30 minutes, and the extract was centrifuged at 4000 rpm for 30 minutes, and the supernatant was collected; the precipitate was redissolved in 70% ethanol and the extraction was repeated once; the supernatants from the two extractions were combined and concentrated by rotary evaporation to obtain honeysuckle tea extract; b. Detection method: High performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS) is used to simultaneously detect the content of characteristic marker combinations in the sample; c. Data analysis: Compare the test results with a database of characteristic marker contents of known varieties, and determine the variety to which the sample belongs through multivariate statistical analysis (such as principal component analysis or cluster analysis).

4. The identification method according to claim 3, characterized in that, The high-performance liquid chromatography (HPLC) analysis conditions are as follows: Column: Agilent Microsorb-MV 100-5 C18 column (4.6 × 250 mm, 5 μm); Column temperature: 25°C; Solvent system: acetonitrile (A) and 0.1% phosphoric acid solution (B); Gradient program: 0–8 min, 14–19% A; 8–14 min, 19% A; 14–34 min, 19–31% A; 34–35 min, 31–90% A; 35–40 min, 90% A; Injection volume: 25 μL; Flow rate: 0.7 mL / min; Detection wavelength: 240 nm.

5. The identification method according to claim 3, characterized in that, In the data analysis step, principal component analysis (PCA) and cluster analysis are used to perform multivariate statistical analysis on the detection results.

6. The identification method according to claim 3, characterized in that, The content of the combination of characteristic markers was calculated using a standard curve and expressed as mean ± standard deviation (SD).

7. The identification method according to claim 3, characterized in that, The method is suitable for large-scale sample analysis and can quickly and accurately identify honeysuckle tea varieties.

8. A quality control method for a honeysuckle tea variety, characterized in that, Includes the following steps: The honeysuckle tea sample was tested using the identification method described in claim 3; Based on the test results, determine whether the honeysuckle tea sample meets the quality standards for a specific variety.

9. The quality control method according to claim 8, characterized in that, The quality standard includes the content range of the characteristic marker combination.

10. A method for identifying the authenticity of honeysuckle tea varieties, characterized in that, Includes the following steps: The honeysuckle tea sample was tested using the identification method described in claim 3; Based on the test results, determine whether the honeysuckle tea sample is a specific variety.

11. The method for identifying authenticity according to claim 9, characterized in that, The method described is applicable to the identification of genuine and counterfeit honeysuckle tea products on the market.

12. A database of honeysuckle tea varieties, characterized in that, The content data of characteristic marker combinations from multiple honeysuckle tea varieties are used to compare with the test results of the sample to be tested.

13. The database according to claim 11, characterized in that, The database processes data through principal component analysis (PCA) and cluster analysis to facilitate rapid comparison and identification.

14. A reagent kit for identifying honeysuckle tea varieties, characterized in that, It includes reagents and standards for detecting the combination of characteristic markers described in claim 1.

15. The reagent kit according to claim 13, characterized in that, The kit is suitable for high performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS) techniques.

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