Detection method for multi-components by single marker (QAM) of various active components in lonicerae flos

Abstract

The invention belongs to the technical field of traditional Chinese medicine component detection and analysis, and particularly relates to a method for detecting various active components in lonicerae flos by quantitative analysis of multi-components by single marker. According to the QAMS detection method for the various active ingredients in the lonicera confusa, based on a simple, convenient and rapid high performance liquid chromatography technology, QAMS of the active ingredients such as flavonoids, flavonoid glycosides, iridoid glycosides and organic acids in the lonicera confusa can be achieved, and qualitative and quantitative detection of the various active ingredients is included. The detection target objects comprise 14 active components, namely neochlorogenic acid, cumaric acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, swertiamarin, secologanin, rutin, quercitrin, galuteolin, ferulic acid, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C. The invention further provides a method for detecting the content of the isochlorogenic acid. Tests prove that the method disclosed by the invention meets methodological requirements, has the characteristics of simplicity and convenience in operation, rapidness in detection, high sensitivity, accuracy in determination and strong applicability, is beneficial to accurately evaluating the quality of the lonicerae flos and provides a reliable basis for safe application of the lonicerae flos.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine component detection and analysis, and particularly relates to a one-test-multiple-evaluation detection method for multiple active components in Lonicerae Japonicae Flos. BACKGROUND

[0002] Both Lonicerae Japonicae Flos and Lonicerae Japonicae Flos are the dried flower buds or early opened flowers of Caprifoliaceae plants, but their growth distribution, appearance characteristics and pharmacological effects are different. Lonicerae Japonicae Flos has strong adaptability, strong stress resistance, and low environmental requirements, and has good planting benefits. At the same time, the market price is usually cheaper than that of Lonicerae Japonicae Flos, which has more economic advantages for large-scale application.

[0003] In recent years, Lonicerae Japonicae Flos has attracted much attention due to its rich chemical components, significant medicinal value and important economic value. However, due to the wide planting area of Lonicerae Japonicae Flos, the quality of Lonicerae Japonicae Flos on the market is uneven, the content of effective components is greatly different, and the use effect cannot be guaranteed, which affects the rational use of high-quality resources and restricts the development of traditional Chinese medicine industry. Since the pharmacological effect of Lonicerae Japonicae Flos is mainly related to the organic acid and flavonoid components contained therein, it is necessary to establish a sensitive, high-resolution, fast and simple and practical multi-index component synchronous quantitative method to realize the effective analysis of multiple active components contained in Lonicerae Japonicae Flos.

[0004] High performance liquid chromatography (HPLC) has become an important technical means for researchers to analyze the chemical components of traditional Chinese medicine due to its strong separation ability, good reproducibility and fast analysis speed. The existing technology 1 (Determination of chlorogenic acid and four kinds of flavonoids in mulberry leaf and honeysuckle by HPLC. Strait Pharmaceutical, 2023, 35(09): 25-32.) determines the content of chlorogenic acid and four kinds of flavonoids in honeysuckle by HPLC method, and the linearity of the five compounds is good in the range of 1-100 μg / mL. The existing technology 2 (Establishment of HPLC method for determination of 7 kinds of organic acids in grape and grape wine) discloses a method for determining 7 kinds of organic acids in grape and grape wine by HPLC. The existing technology 3 (Simultaneous determination of syringid and 6 kinds of organic acids in honeysuckle from different producing areas by RP-HPLC) uses high performance liquid chromatography to separate and detect multiple organic acids in honeysuckle. Although the above studies use high performance liquid chromatography to analyze several active components such as flavonoids and organic acids in traditional Chinese medicine at the same time, the types of compounds separated and detected are still insufficient, which is difficult to fully reflect the complex chemical component characteristics of Lonicerae Japonicae Flos.

[0005] Therefore, how to establish a high performance liquid chromatography analysis method to realize the rapid separation and comprehensive detection of multiple active components in Lonicerae Japonicae Flos can provide theoretical basis and technical support for the quality evaluation and resource development of Lonicerae Japonicae Flos. SUMMARY

[0006] In order to overcome the deficiencies in the prior art, the purpose of the present application is to provide a quantitative analysis of multiple active ingredients in Lonicera japonica, which can realize the rapid separation and accurate detection of 14 kinds of active ingredients in Lonicera japonica, so as to provide theoretical basis and technical support for the quality evaluation and resource development of Lonicera japonica.

[0007] In order to achieve the above-mentioned purpose, the present application provides a quantitative analysis of multiple active ingredients in Lonicera japonica, comprising the following steps:

[0008] (1) Prepare mixed reference solution of different concentrations; the mixed reference solution includes rutin reference, quercitrin reference, osmanthus glycoside reference, secologanoside reference, swertiamarin reference, neochlorogenic acid reference, chlorogenic acid reference, caffeic acid reference, ferulic acid reference, coumaric acid reference, cryptochlorogenic acid reference, isochlorogenic acid A reference, isochlorogenic acid C reference, and isochlorogenic acid B reference;

[0009] Respectively, the high performance liquid chromatography analysis is carried out on the mixed reference solution of different concentrations; the swertiamarin reference in the mixed reference solution is used as an internal reference, and the relative correction factors of other references except swertiamarin are determined by combining the high performance liquid chromatography analysis results of the mixed reference solution;

[0010] Among them, the chromatographic conditions of high performance liquid chromatography analysis are: the mobile phase includes A phase and B phase; the A phase is 0.025% phosphoric acid solution, and the B phase is acetonitrile; the gradient elution program of the mobile phase is: 0min-5min, 12%-16%B; 5min-25min, 16%B-36%B;

[0011] (2) Prepare Lonicera japonica test solution; according to the chromatographic conditions in step (1), the high performance liquid chromatography analysis is carried out on the Lonicera japonica test solution, the chromatographic peaks of each active ingredient to be tested are located according to the relative retention time, and the content of swertiamarin in the Lonicera japonica test solution is calculated by external standard method; according to the determination results of the content of swertiamarin and the relative correction factors determined in step (1), the contents of multiple active ingredients in the test solution are calculated.

[0012] As a preferred scheme of the present application, the multiple active ingredients in Lonicera japonica are neochlorogenic acid, coumaric acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, secologanoside, rutin, quercitrin, osmanthus glycoside, ferulic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C.

[0013] As a preferred scheme of the present application, in step (1), the chromatographic column used in the high performance liquid chromatography analysis is Kromasil 100-5-C18 chromatographic column.

[0014] As a preferred scheme of the present application, in step (1), the high performance liquid chromatography analysis employs a Shimadzu LC-16 type high performance liquid chromatograph.

[0015] As a preferred scheme of the present application, in step (1), the column temperature for the high performance liquid chromatography analysis is 40℃, the flow rate is 1 mL / min, the detection wavelength is 240 nm, and the injection volume is 10 μL.

[0016] As a preferred scheme of the present application, in step (1), the determination process of the relative correction factor is as follows:

[0017] After high performance liquid chromatography analysis of the mixed control solution of different concentrations, the peak area of each control in the mixed control solution is obtained, and the relative correction factor of each control except for the rheomaikang control is calculated according to the relative correction factor calculation formula, taking the rheomaikang control as the internal reference.

[0018] The relative correction factor calculation formula is as follows: In the formula, f si is the relative correction factor; C s represents the concentration of the internal reference; A s is the peak area of the internal reference measured at a concentration of C s ; C i is the concentration of a certain control to be measured except for the internal reference; and A i is the peak area of a certain control to be measured except for the internal reference at a concentration of C i .

[0019] As a preferred scheme of the present application, in step (2), the preparation process of the honeysuckle test sample solution comprises the following steps: crushing, sieving and drying honeysuckle raw materials to obtain honeysuckle powder; and then ultrasonically extracting the honeysuckle powder in ethanol, and then filtering and diluting to obtain the honeysuckle test sample solution.

[0020] As a preferred scheme of the present application, the sieving employs a screen mesh with a pore size of 300-400 μm, and the drying is performed at a temperature of 40-80℃ for 5-20 h.

[0021] As a preferred scheme of the present application, the honeysuckle powder and ethanol are used in a ratio of (0.3-1.0) g:(5-20) mL, and the ultrasonic extraction is performed at a power of 100-500 W for 2-10 min. Further preferably, the honeysuckle powder and ethanol are used in a ratio of 0.5 g:10 mL, and the ethanol has a concentration of 70% (v / v).

[0022] As a preferred scheme of the present application, in step (2), the external standard method is used to calculate the content of jinyinhua test product solution according to a pre-constructed jinyinhua standard curve; the jinyinhua standard curve is constructed by performing high performance liquid chromatography analysis on jinyinhua control product solutions with different concentrations, and then taking the concentration of the jinyinhua control product solution as the abscissa and the peak area obtained after high performance liquid chromatography analysis as the ordinate.

[0023] The technical scheme of the present application has the following advantages and beneficial effects:

[0024] The one-test-multiple-evaluation detection method for multiple active components in jinyinhua provided by the present application is based on simple, rapid and efficient high performance liquid chromatography, and can realize comprehensive separation and synchronous detection of active components such as flavones, flavonoid glycosides, iridoid glycosides and organic acids in jinyinhua. The detection target of the method of the present application is 14 kinds of active components, including neochlorogenic acid, coumaric acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, jinyinhua, secologanin, rutin, quercitrin, luteoloside, ferulic acid, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C. The detection object of the present application includes but is not limited to jinyinhua, and the present application can realize the detection of the detection object as long as it contains the above-mentioned 14 kinds of active components.

[0025] The one-test-multiple-evaluation detection method for multiple active components in jinyinhua established by the present application uses a chromatographic column with octadecylsilane bonded silica gel as the filler, comprehensively controls the elution conditions and chromatography process, and uses the relative correction factor quantitative method, which is beneficial to realize comprehensive separation and synchronous detection of active components in jinyinhua.

[0026] Experiments prove that the method of the present application meets the methodological requirements, and has the characteristics of simple operation, rapid detection, high sensitivity, accurate measurement and strong applicability, and can be used as a means for rapid separation and accurate detection of more target components in traditional Chinese medicines, and is also beneficial to accurately evaluate the quality of jinyinhua, provide reliable basis for its safe application, and promote the rational development and utilization of this valuable traditional Chinese medicine resource, which has important practical significance for promoting the standardized development of jinyinhua industry. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The liquid chromatogram of the detection method of the present application Example 1 and Comparative Examples 1-4 is used to detect the mixed control solution;

[0028] Figure 2 The liquid chromatogram obtained by performing specificity test in the present application is shown in the following table:

[0029] Figure 3 The liquid chromatogram of the detection method of the present application Example 1 is used to detect jinyinhua samples from different producing areas. DETAILED DESCRIPTION

[0030] In order to make the objectives, technical solutions and advantages of the present application clearer, the following will clearly and completely explain the present application with specific examples. Obviously, the described examples are part of the examples of the present application, rather than all the examples. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0031] In the following implementation, the dried honeysuckle flowers are purchased from a medicinal material company in Chongqing, and the production places are Xishan in Chongqing, Suiyang in Guizhou and Longhui in Hunan. Before determining the multiple active ingredients in the honeysuckle flowers, the present application pre-processes the honeysuckle flower raw materials. The pre-processing is a process of obtaining honeysuckle flower powder with uniform size by crushing, sieving and drying the raw materials. It can be understood that the pre-processing is a general pre-treatment step for detecting active substances in traditional Chinese medicinal materials, and the pre-processing is used to ensure the smooth extraction of subsequent active substances and the consistency of detection results. Therefore, the technicians can routinely select the pre-processing process and parameters, and the present application does not make additional limitations.

[0032] Specifically, in the examples and comparative examples of the present application, the pre-processing process of the dried honeysuckle flower raw materials is as follows: taking the dried honeysuckle flower sample, mechanically crushing for 10 min by using a crusher, then passing through a No. 3 sieve (sieve hole inner diameter 355±13 μm), and then putting the obtained honeysuckle flower powder into a drying machine to dry for 10 h, with the drying machine temperature set to 60℃. After drying, the honeysuckle flower powder is obtained for testing.

[0033] In the following implementation, the types and sources of the standard substances, i.e. the reference substances, of the multiple active ingredients are as follows: neochlorogenic acid reference substance (DSTDX001505), coumaric acid reference substance (DSTDX010301), chlorogenic acid reference substance (DSTDL002103), cryptochlorogenic acid reference substance (DSTDY003503), caffeic acid reference substance (DSTDK001301), swertiamarin reference substance (DSTDZ001401), secologanoside reference substance (DSTDD011101), rutin reference substance (DSTDL001702), quercitrin reference substance (DSTDY000603), jafferrin reference substance (DSTDM001602), ferulic acid reference substance (DSTDF008102), isochlorogenic acid A reference substance (DSTDY003703), isochlorogenic acid B reference substance (DSTDY003603), isochlorogenic acid C reference substance (DSTDY003804) purchased from Chengdu Lemaitian Pharmaceutical Technology Co., Ltd. The other raw materials and reagents not mentioned are also materials that can be obtained through commercial channels.

[0034] In the implementation of the present application, the names and structural formulas of the multiple active ingredients in the honeysuckle flowers are specifically as follows:

[0035]

[0036]

[0037] Example 1

[0038] This embodiment provides a method for detecting multiple active ingredients in honeysuckle using a single assay and multiple evaluation methods, including the following steps:

[0039] (1) Preparation of test solution and reference standard: Accurately weigh 0.500 g of honeysuckle powder and place it in a 100 mL centrifuge tube. Accurately add 10 mL of 70% (v / v) ethanol and sonicate for 4 min (ultrasonic power 250 W, frequency 50 kHz, temperature 60 °C). After sonication, shake well and filter with filter paper. Take 6 mL of the filtrate and filter it through a 0.45 μm filter head. After diluting five times, the test solution is obtained for subsequent liquid chromatography analysis.

[0040] Preparation of reference solution: Accurately weigh 10 mg each of rutin, quercetin, luteolin, strychnos nux-vomica, strychnos nux-vomica, neochlorogenic acid, chlorogenic acid, caffeic acid, ferulic acid, coumaric acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid C, and isochlorogenic acid B into a 100 mL volumetric flask, dissolve in methanol and dilute to volume to prepare a reference stock solution (100 μg / mL), and store at -4℃ for later use.

[0041] Accurately pipette the prepared reference stock solution and dilute it with methanol to 1.00 μg / mL, 10.00 μg / mL, 20.00 μg / mL, 40.00 μg / mL, 60.00 μg / mL, 80.00 μg / mL, and 100.00 μg / mL. Filter the solution through a 0.45 μm filter to obtain reference solutions of different concentrations for subsequent liquid chromatography analysis.

[0042] (2) Take 10 μL of reference solution of different concentrations and inject it into the high performance liquid chromatograph for high performance liquid chromatography analysis. After the analysis, the peak area of ​​each reference in the mixed reference solution is obtained. Using the dangyao glycoside reference as an internal reference, the relative correction factor of other references besides dangyao glycoside is calculated according to the relative correction factor calculation formula.

[0043] The formula for calculating the relative correction factor is as follows: In the formula, f si C is the relative correction factor. s Represents the concentration of the internal reference substance; A s The internal reference at a concentration of C s Peak area measured at time; C i A represents the concentration of a reference component other than the internal control;i the peak area of the test component in a certain control at a concentration of C i

[0044] Another 10 μL of the test sample solution of step (1) is injected into the high performance liquid chromatograph, and the chromatographic peaks of each test active ingredient are located according to the relative retention time, and the content of rhubarb glycoside in the test sample solution of honeysuckle is calculated by the external standard method; according to the determination result of the content of rhubarb glycoside and the aforementioned relative correction factor, the contents of multiple active ingredients in the test sample solution are calculated, thereby realizing the separation and content detection of 14 active ingredients in honeysuckle.

[0045] The chromatographic conditions of high performance liquid chromatography are as follows: Shimadzu LC-16 type high performance liquid chromatograph, Kromasil 100-5-C18 chromatographic column (4.6 mm x 250 mm, 5 μm, Nouryon), column temperature for chromatographic detection is 40°C, flow rate is 1 mL / min, detection wavelength is 240 nm, injection volume is 10 μL, and the mobile phase is composed of phase A and phase B. Phase A is 0.025% (v / v) phosphoric acid solution, phase B is chromatographic grade acetonitrile, and the gradient elution program is shown in Table 1:

[0046] Table 1. Gradient elution program for high performance liquid chromatography analysis

[0047] Time (min) Mobile phase A (%) Mobile phase B (%) 0～5 88→84 12→16 5～25 84→64 16％→36

[0048] Comparative Example 1

[0049] This comparative example provides a one-test-multiple-evaluation detection method for multiple active ingredients in honeysuckle, and the determination steps are basically the same as those of Example 1, and the difference between the two is that the gradient elution program for high performance liquid chromatography analysis used in this comparative example is as follows: 0 min-25 min, 10%-40% B, and the other parameters are the same as those of Example 1.

[0050] Comparative Example 2

[0051] This comparative example provides a one-test-multiple-evaluation detection method for multiple active ingredients in honeysuckle, and the determination steps are basically the same as those of Example 1, and the difference between the two is that the gradient elution program for high performance liquid chromatography analysis used in this comparative example is as follows: 0 min-25 min, 10%-35% B, and the other parameters are the same as those of Example 1.

[0052] Comparative Example 3

[0053] This comparative example provides a one-test-multiple-evaluation detection method for multiple active ingredients in honeysuckle, and the determination steps are basically the same as those of Example 1, and the difference between the two is that the gradient elution program for high performance liquid chromatography analysis used in this comparative example is as follows: 0 min-26 min, 9%-35% B, and the other parameters are the same as those of Example 1. ​

[0054] Comparative Example 4

[0055] The comparative example provides a quantitative analysis method for multiple active ingredients in Lonicera japonica. The determination steps are basically the same as those of Example 1, and the difference between the two is that the gradient elution program of high performance liquid chromatography analysis used in the comparative example is: 0 min-28 min, 7%-35% B, and the other parameters are the same as those of Example 1.

[0056] Test Example

[0057] The preparation method of the control stock solution used in the following test example is as follows: precisely weigh 10 mg of neochlorogenic acid, coumaric acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, swertiamarin, secologanoside, rutin, quercitrin, luteolin, ferulic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C control samples into a 100 mL volumetric flask, dissolve and dilute with methanol, and prepare a control stock solution (100 μg / mL). Store in a-4°C refrigerator for future use.

[0058] I. Elution condition optimization

[0059] Precisely take an appropriate amount of the above prepared control stock solution, dilute with methanol, and filter through a 0.45 μm filter head to obtain a mixed control sample solution (40 μg / mL). Determine the mixed control sample solution using the liquid chromatography conditions described in Example 1 and Comparative Examples 1-4, respectively, to obtain the liquid chromatograms as shown in Figure 1 . Among them, Figure 1 A-E in are the liquid chromatograms of Comparative Examples 1-4 and Example 1, respectively. Figure 1 The numbering of each peak in means: 1. neochlorogenic acid; 2. coumaric acid; 3. chlorogenic acid; 4. cryptochlorogenic acid; 5. caffeic acid; 6. swertiamarin; 7. secologanoside; 8. rutin; 9. quercitrin; 10. luteolin; 11. ferulic acid; 12. isochlorogenic acid A; 13. isochlorogenic acid B; 14. isochlorogenic acid C, and the same below.

[0060] The elution gradient condition is one of the key steps for effective separation of multiple components in high performance liquid chromatography analysis. From Figure 1It can be seen that when the target is separated by the determination method of Comparative Example 1, the chromatographic peaks of peak 1 and peak 2 are not separated on the same chromatographic peak after gradient elution, and the separation degree of peak 9 and peak 10 is poor (Figure A). When the target is separated by the determination method of Comparative Example 2, peak 1 and peak 2 are still not separated, and the separation degree of peak 9 and peak 10 is improved compared with Comparative Example 1 (Figure B). When the target is separated by the determination method of Comparative Example 3, peak 1 and peak 2 are separated after gradient elution, but the separation degree is poor, and the separation degree of peak 9 and peak 10 is obvious (Figure C). When the target is separated by the determination method of Comparative Example 4, the separation degree of peak 1 and peak 2 is obvious, and the separation degree of peak 9 and peak 10 is improved, but there is no peak at 0-8 min, and the whole chromatographic condition takes a long time (Figure D). When the target is separated by the determination method of Example 1, peak 1 and peak 2 are well separated, and peak 9, peak 10 and peak 11 are also well separated. In addition, Example 1 also shortens the overall analysis time compared with Comparative Examples 1-4 while achieving the separation of 14 peaks, and the analysis time is shortened to about 20 min. Therefore, the optimal elution gradient condition finally established is: 0 min-5 min, 12% B-16% B; 5 min-25 min, 16% B-36% B. This condition can realize the separation of 14 peaks while shortening the overall analysis time and improving the practicability and detection efficiency of the method.

[0061] Further separation degree analysis of the elution results shows that the separation degree between the target compound quercitrin and jacobinin (R=2.016), and the separation degree between jacobinin and ferulic acid (R=1.781) have all reached the baseline separation requirement (R≥1.5). This result shows that the chromatographic condition of the present application exhibits excellent performance in separation efficiency.

[0062] II. Optimization of detection parameters

[0063] In order to minimize the influence of the composition of the mobile phase or the gradient program on the chromatographic peak shape, the chromatographic conditions are further explored in the present application. Under the optimal elution program of Example 1, Box-Behnken experimental design is adopted, the separation degree R1 (Y1) between quercitrin and jacobinin, and the separation degree R2 (Y1) between jacobinin and ferulic acid which have relatively poor separation degree are selected as response values, and three factors of injection volume (x1), column temperature (x2) and flow rate (x3) are optimized, and the factor levels and Box-Behnken design scheme and results are shown in Table 2. The variance analysis results of Box-Behnken are shown in Table 3. The design model of Box-Behnken and the statistical parameter results are shown in Table 4. *p<0.05 represents statistically significant difference.

[0064] Table 2, Box-Behnken factor levels and design experiment results

[0065]

[0066] Table 3, Box-Behnken design variance analysis results

[0067]

[0068]

[0069] Table 4, Box-Behnken design response model and statistical parameters

[0070]

[0071] Multivariate regression and variance analysis were performed on the data in Table 2, and the results are shown in Table 3. It was found that the two quadratic polynomial models p < 0.0001, the model fitting effect was extremely significant, the lack of fit term p > 0.05, not significant, indicating that other factors have little interference on the experimental results, the model has good fitting effect. By comparing the p value, the influence effect of the three factors on R1 (Y1) is flow rate (x3) > column temperature (x2) > injection volume (x1), and the influence effect on R2 (Y2) is column temperature (x2) > flow rate (x3) > injection volume (x1). In order to simplify the equation solution, the corresponding equation simplification was carried out under the premise of ensuring the fitting degree, and after rejecting the items with no significant correlation at the p < 0.1 level, the fitting equation of the response value and the influencing factor is shown in Table 4.

[0072] Further, response surface and contour maps were established to intuitively analyze the influence of each factor and its interaction on the response value. The results show that the flow rate has the most significant influence on the separation degree R1, and there is an interaction between the injection volume and the flow rate, and between the column temperature and the flow rate. The column temperature has the most significant influence on the separation degree R2, and there is a significant interaction between the column temperature and the flow rate. Then, the target of the separation degrees R1 and R2 is set as "maximum", and the weight of the two is equal, and the optimal level combination of each factor is obtained as follows: injection volume 9.31 μL, column temperature 41.69 ℃, and flow rate 1.10 mL / min. Under this condition, the response value R1 predicted by the model is 2.96, and the response value R2 predicted by the model is 2.01.

[0073] According to the optimal experimental conditions predicted by the model, HPLC determination was carried out under the same conditions, and the deviation between the measured value and the predicted value of the two response values was investigated. The results show that the deviation is not more than 5%, indicating that the model has good predictability. Considering that the method parameters should be as simple as possible to ensure the practicability of the method, therefore, the final chromatographic conditions are set as follows: injection volume 10 μL, column temperature 40 ℃, and flow rate 1 mL / min.

[0074] Since the polarities of quercitrin, luteoloside and ferulic acid in the test compound are very similar, and the isochlorogenic acid A, B and C are each other isomers, these factors significantly increase the technical difficulty of chromatographic separation. However, from the separation degree data, the separation effect between all compounds is more than 99.9% (R≥1.5), which proves that the method of the present application can realize accurate content determination of the test components.

[0075] III. Methodology verification

[0076] The present test example carries out a comprehensive investigation on the scientificity, reliability and applicability of the analysis method established by the present application through rigorous methodology verification.

[0077] 3.1, Specificity test

[0078] In order to verify the selectivity of the detection method established by the present application, the blank methanol, mixed control solution (40 μg / mL) and test sample solution (Chongqing Xiushan mountain silver flower sample prepared according to the method of Example 1) were respectively injected and analyzed according to the liquid chromatography conditions described in Example 1, and the chromatographic flow curve was compared to investigate the specificity of the method. The results are shown in Figure 2 .

[0079] Figure 2 It is shown that the blank methanol solution (A) has no interfering peak, the 14 kinds of control mixed solution (B) has good separation of each target peak, and the chromatogram of the mountain silver flower test sample solution (C) does not appear endogenous interfering peak, and there is no cross interference phenomenon near the target analyte. This indicates that the method has excellent specificity and can accurately distinguish target components from other interfering substances.

[0080] 3.2, Limit of quantification (LOQ)

[0081] The control stock solution was diluted step by step, and the limit of quantification of each component was determined by signal-to-noise ratio (S / N)≥10. The results are shown in Table 5. The results of Table 5 show that the LOQ of 14 target compounds is 0.04-0.40 μg / mL, indicating that the method has high sensitivity and can meet the quantitative needs of trace components.

[0082] 3.3, Precision test

[0083] The precision pipette the prepared reference stock solution, and dilute it with methanol to 40 μg / mL to obtain a mixed reference solution as the sample to be determined. Determine 6 times at different times within a day according to the liquid chromatography conditions described in Example 1, record the peak area, calculate the RSD%, and investigate the within-day precision of the method. Determine the mixed reference solution with the concentration of 40 μg / mL on the 1st day, the 2nd day, and the 3rd day according to the liquid chromatography conditions described in Example 1, record the peak area, calculate the RSD%, and investigate the within-day precision of the method. The results are shown in Table 5. The results in Table 5 show that the RSD values of the within-day precision (6 repeated determinations) and the inter-day precision (3 repeated determinations) are 0.21%-3.53% and 0.04%-3.59% respectively, indicating that the method has good repeatability, and the analysis results are stable and reliable.

[0084] 3.4, Sample loading recovery test

[0085] Precisely weigh 3 portions of the honeysuckle powder from Chongqing Xiushan with the determined content, 0.500 g each, and place them in 100 mL centrifuge tubes. Add 10 mL of a mixed solution of 70% ethanol containing 14 standard substances (neochlorogenic acid 0.301 mg, coumaric acid 0.300 mg, chlorogenic acid 3.000 mg, cryptochlorogenic acid 1.010 mg, caffeic acid 0.100 mg, swertiamin 0.501 mg, secologanin 3.000 mg, rutin 0.301 mg, quercitrin 0.100 mg, galuteolin 0.200 mg, ferulic acid 0.300 mg, isochlorogenic acid A 0.601 mg, isochlorogenic acid B 2.998 mg, and isochlorogenic acid C 1.001 mg) to each portion. Perform chromatographic analysis according to the determination method described in Example 1. By comparing the measured value with the added amount, calculate the sample loading recovery rate and RSD% of each target component to verify the accuracy of the method. The results are shown in Table 5. The results in Table 5 show that the recovery rates of the 14 components are between 97.03%-102.26%, and the RSD is ≤2.82%, indicating that the method has high accuracy, the matrix effect is controllable, and is suitable for the analysis of complex samples.

[0086] 3.5, Stability test

[0087] The precision pipette the prepared reference stock solution, and dilute it with methanol to 40 μg / mL to obtain a mixed reference solution as the sample to be determined. Place the sample to be determined in a room temperature 25°C, 50% relative humidity environment, and determine it at 0, 3, 6, 9, 12, and 24 h according to the liquid chromatography conditions described in Example 1, record the peak area, calculate the RSD%, and investigate the stability. The results are shown in Table 5. The results in Table 5 show that the mixed reference solution is placed at room temperature (25°C) for 24 hours, and the RSD of the peak area of each component is 0.36%-2.95% (Table 5), proving that the sample solution has good stability within the detection period and meets the actual analysis requirements.

[0088] Table 5, Quantitative limit, accuracy, precision and stability results (n = 6)

[0089]

[0090]

[0091] 3.6, Linearity test

[0092] An appropriate amount of the above prepared control stock solution was precisely pipetted, diluted with methanol to 1.00 μg / mL, 10.00 μg / mL, 20.00 μg / mL, 40.00 μg / mL, 60.00 μg / mL, 80.00 μg / mL, 100.00 μg / mL, filtered through a 0.45 μm filter, and determined according to the liquid chromatography conditions described in Example 1. The standard curve was plotted with the peak area (y) versus the concentration (x), and the linearity of the method was evaluated by calculating the regression equation and R 2 values. The results are shown in Table 6.

[0093] Table 6, Standard curve and linear range results of the target compound

[0094]

[0095]

[0096] The results in Table 6 show that, within the concentration range of 1.00-100.00 μg / mL, the peak areas of the 14 analytes have a good linear relationship with the concentration; the regression equation correlation coefficients (R 2 ) are all greater than 0.9990, indicating that the method has a wide linear range and is suitable for accurate quantification of samples of different concentrations.

[0097] Four, Calculation of correction factors and robustness test

[0098] 4.1, Calculation of relative correction factors

[0099] Under the chromatographic conditions defined in the present application, the mixed control solution in the "3.6 Linearity Test" was precisely pipetted, and the peak areas of neochlorogenic acid, coumaric acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, secologanoside, rutin, quercitrin, galuteolin, ferulic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C in Lonicera japonica were determined with jatrorrhizine as the internal standard, and the relative correction factors were calculated according to the formula: The relative correction factors of the control substances other than jatrorrhizine were calculated. Among them, A s represents the peak area of the internal reference control substance, C s represents the concentration of the internal reference control substance, A i represents the peak area of the component to be tested, and Ci Concentration of the representative internal standard control si Correction factor of the representative internal standard to the analyte. The calculation results of the relative correction factors are shown in Table 7.

[0100] Table 7, Calculation results of relative correction factors of 14 analytes

[0101]

[0102]

[0103] 4.2, Durability test of relative correction factors

[0104] 4.2.1, Effect of flow rate on relative correction factors

[0105] The effects of different flow rates (0.8 mL / min, 1.0 mL / min, 1.2 mL / min) on relative correction factors were investigated respectively, and the experimental results are shown in Table 8.

[0106] Table 8, Effect of flow rate on relative correction factors

[0107]

[0108] From Table 8, it can be seen that the relative correction factors of astringin to neochlorogenic acid, coumaric acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, secologanoside, rutin, quercitrin, lavandulifolin, ferulic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C are stable under different flow rates.

[0109] 4.2.2, Effect of column temperature on relative correction factors

[0110] The effects of different column temperatures (30℃, 40℃, 50℃) on relative correction factors were investigated respectively, and the experimental results are shown in Table 9.

[0111] Table 9, Effect of column temperature on relative correction factors

[0112]

[0113] From Table 9, it can be seen that the relative correction factors of astringin to neochlorogenic acid, coumaric acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, secologanoside, rutin, quercitrin, lavandulifolin, ferulic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C are stable under different column temperatures.

[0114] Five, Comparison of determination results between external standard method and quantitative analysis of multiple components by one marker method

[0115] To verify the practicality and convenience of the method of the present invention, the content of multiple active ingredients in honeysuckle from different origins was determined by high performance liquid chromatography (HPLC). Specifically, honeysuckle raw materials from Xiushan in Chongqing, Suiyang in Guizhou, and Longhui in Hunan were used. Three parallel test solutions were prepared according to the sample preparation method and chromatographic conditions described in Example 1, and analyzed by HPLC. A mixed solution of the aforementioned reference standard (40 μg / mL) was used as a control.

[0116] For quantitative testing, the contents of thymol, neochlorogenic acid, coumaric acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, strychnos nuciferoside, rutin, quercetin, luteolin, ferulic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C in the *Lonicera japonica* sample from Xiushan, Chongqing, were first determined using the external standard method (i.e., substituting the peak areas of each analyte into a pre-established standard curve to calculate the content of each analyte). Then, the one-test-multiple-evaluation method established in Example 1 of this invention was used to calculate the contents of thymol, neochlorogenic acid, coumaric acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, strychnos nuciferoside, rutin, quercetin, luteolin, ferulic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C in the same *Lonicera japonica* sample. The results of the two methods were then compared.

[0117] The comparison results of the single-method multi-evaluation method and the external standard method for honeysuckle samples from different origins are shown in Tables 10, 11, and 12. In addition, the comparison results of the liquid chromatograms of the honeysuckle test solutions from different origins are shown in [Table 10]. Figure 3 As shown. Figure 3 In the figures, A is the chromatogram of the mixed solution of reference standards; B is the chromatogram of honeysuckle sample from Xiushan, Chongqing; C is the chromatogram of honeysuckle sample from Suiyang, Guizhou; and D is the chromatogram of honeysuckle sample from Longhui, Hunan.

[0118] Table 10. Simultaneous determination of active ingredient content in honeysuckle (Xiushan, Chongqing) using the single-test-multiple-evaluation method and external standard method.

[0119]

[0120] Table 11. Simultaneous determination of active ingredient content in Lonicera japonica (Suiyang, Guizhou) using the single-test-multiple-evaluation method and external standard method.

[0121]

[0122]

[0123] Table 12. Simultaneous determination of active ingredient content in Lonicera japonica (Longhui, Hunan) using the single-test-multiple-evaluation method and external standard method.

[0124]

[0125] Depend on Figure 3From the results of Tables 10, 11 and 12, it can be seen that the contents of the active ingredients in honeysuckle measured by the two methods have no significant difference, and the relative deviation is less than 5%, which indicates that the one measurement multiple evaluation method established in the present application is reliable. Further comprehensive analysis and comparison of the results show that the present application successfully detects the main active ingredients in honeysuckle samples from Longhui in Hunan, Suiyang in Guizhou and Xiushan in Chongqing, and detects 14, 13 and 12 kinds of compounds, respectively. According to the results of the number and content of the detected compounds, it is fully embodied that the chemical components of honeysuckle from different producing areas have obvious differences. Among them, the content of chlorogenic acid (32.389±0.916 mg / g) in the sample from Longhui in Hunan is the highest, and the contents of neochlorogenic acid (4.971±0.122 mg / g) and isochlorogenic acid B (11.776±0.364 mg / g) are also significantly higher than those of the other two producing areas. The sample from Suiyang in Guizhou is characterized by high contents of disacetylmonospergualin (13.042±0.395 mg / g) and chlorogenic acid (19.636±0.234 mg / g), and the content of caffeic acid (1.440±0.040 mg / g) is significantly higher than that of other regions. The content of each component in the sample from Xiushan in Chongqing is relatively low, especially the contents of neochlorogenic acid (0.540±0.016 mg / g), chlorogenic acid (7.105±0.077 mg / g) and isochlorogenic acid B (5.498±0.152 mg / g) are only 1 / 10, 1 / 5 and 2 / 5 of those of the sample from Longhui in Hunan. This highlights the significant influence of producing area on the chemical component spectrum of traditional Chinese medicine, thereby providing a scientific basis for the producing area identification and clinical application of honeysuckle.

[0126] From the above, it can be seen that through systematic comparison and contrast of the current reports, it is found that the existing detection methods for the main components of honeysuckle still have significant limitations. At present, the related detection means can only determine the contents of 2-7 main components, and it is difficult to comprehensively and truly reflect the inherent quality characteristics of honeysuckle. Since the medicinal value of honeysuckle is reflected by the synergistic effect of multiple active ingredients, the incompleteness of the detected components is likely to lead to incorrect judgments in the activity research, which not only affects the scientific cognition of the pharmacological mechanism of honeysuckle, but also may mislead the quality evaluation and application promotion of health products based on honeysuckle.

[0127] The application first establishes a high performance liquid chromatography analysis method for simultaneously detecting 14 main effective components in Honeysuckle. The application can more systematically and intuitively improve the effect of multi-component separation, improve the overall detection efficiency, and has strong popularization and application potential by optimizing the chromatographic elution conditions, injection volume, column temperature and flow rate, and simultaneously using relative correction factors for quantification. Especially, the method established by the application is not only simple in operation and fast in analysis, but also has better specificity, linear relationship, precision, recovery rate and stability. At the same time, the verification test also confirms that the content determination result of the application is accurate, reliable, stable and good in reproducibility, and the detection result can more comprehensively reflect the complex chemical component characteristics of Honeysuckle, thereby effectively being applied to the quality control of Honeysuckle and its preparations, providing meaningful technical and data support for further standardizing the quality evaluation system, providing a reference for formulating a scientific and reasonable multi-component quality control standard for traditional Chinese medicines in the future, and being helpful for breaking through the limitations of single component evaluation in traditional Chinese medicine quality control. Therefore, the research of the application is not only helpful for elucidating the pharmacodynamic material basis of Honeysuckle, but also provides a scientific basis for future new drug research and development and functional product development, and is conducive to improving the quality of Honeysuckle from the source, developing its higher economic value in the fields of medicine, health food and functional beverage, etc.

[0128] The above only describes the preferred embodiments of the application, and it should be noted that those skilled in the art can make some improvements and refinements without departing from the principles of the application, and these improvements and refinements should also be considered within the protection scope of the application.

Claims

1. A method for detecting multiple active ingredients in honeysuckle using a single assay and multiple evaluation methods, characterized in that, Includes the following steps: (1) Prepare mixed reference solutions of different concentrations; the mixed reference solutions include rutin reference, quercetin reference, luteolin reference, strychnos nux-vomica reference, strychnos nux-vomica reference, strychnos nux-vomica reference, neochlorogenic acid reference, chlorogenic acid reference, caffeic acid reference, ferulic acid reference, coumaric acid reference, cryptochlorogenic acid reference, isochlorogenic acid A reference, isochlorogenic acid C reference, and isochlorogenic acid B reference; High-performance liquid chromatography (HPLC) analysis was performed on mixed reference solutions of different concentrations. Using the dangyao glycoside reference standard in the mixed reference solution as an internal reference, the relative correction factors of other reference standards besides dangyao glycoside were determined based on the HPLC analysis results of the mixed reference solution. The chromatographic conditions for high performance liquid chromatography (HPLC) analysis were as follows: the mobile phase consisted of phase A and phase B; phase A was 0.025% phosphoric acid solution, and phase B was acetonitrile; the gradient elution program for the mobile phase was: 0 min to 5 min, 12% to 16% B; 5 min to 25 min, 16% B to 36% B. (2) Prepare the honeysuckle test solution; perform high performance liquid chromatography analysis on the honeysuckle test solution according to the chromatographic conditions in step (1), locate the chromatographic peaks of each active ingredient to be tested according to the relative retention time, and calculate the content of dangyao glycoside in the honeysuckle test solution by external standard method; calculate the content of multiple active ingredients in the test solution according to the determination results of dangyao glycoside content and the relative correction factor determined in step (1).

2. The method for detecting multiple active ingredients in honeysuckle according to claim 1, characterized in that, The active ingredients in honeysuckle include neochlorogenic acid, coumaric acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, strychnos nux-vomica glycoside, rutin, quercetin, luteolin, ferulic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C.

3. The method for detecting multiple active ingredients in honeysuckle according to claim 1, characterized in that, In step (1), the high performance liquid chromatography analysis uses a Kromasil 100-5-C18 column.

4. The method for detecting multiple active ingredients in honeysuckle according to claim 1, characterized in that, In step (1), the instrument used for the high performance liquid chromatography analysis is a Shimadzu LC-16 high performance liquid chromatograph.

5. The method for detecting multiple active ingredients in honeysuckle according to claim 1, characterized in that, In step (1), the high performance liquid chromatography analysis uses a column temperature of 40℃, a flow rate of 1mL / min, a detection wavelength of 240nm, and an injection volume of 10μL.

6. The method for detecting multiple active ingredients in honeysuckle according to claim 1, characterized in that, In step (1), the process of determining the relative correction factor is as follows: After high-performance liquid chromatography analysis of mixed reference solutions of different concentrations, the peak areas of each reference in the mixed reference solution were obtained. Using dangyao glycoside reference as an internal reference, the relative correction factors of other references besides dangyao glycoside were calculated according to the relative correction factor calculation formula. The formula for calculating the relative correction factor is as follows: In the formula, f si C is the relative correction factor. s Represents the concentration of the internal reference substance; A s The internal reference at a concentration of C s Peak area measured at time; C i A represents the concentration of a reference component other than the internal control; i For a reference standard analyte other than the internal reference, at a concentration of C i Peak area at that time.

7. The method for detecting multiple active ingredients in honeysuckle according to any one of claims 1 to 6, characterized in that, In step (2), the preparation process of the honeysuckle test solution includes the following steps: crushing, sieving and drying the honeysuckle raw material to obtain honeysuckle powder; then ultrasonically extracting the honeysuckle powder in ethanol, and then filtering and diluting to obtain the honeysuckle test solution.

8. The method for detecting multiple active ingredients in honeysuckle according to claim 7, characterized in that, The sieve used for sieving has a mesh size of 300–400 μm; the drying temperature is 40–80 °C and the drying time is 5–20 h.

9. The method for detecting multiple active ingredients in honeysuckle according to claim 7, characterized in that, The ratio of honeysuckle powder to ethanol is (0.3-1.0) g : (5-20) mL; the ultrasonic extraction power is 100-500 W and the time is 2-10 min.

10. The method for detecting multiple active ingredients in honeysuckle according to claim 1, characterized in that, In step (2), the external standard method is to calculate the content of dangyao glycoside in the honeysuckle test solution based on the pre-constructed dangyao glycoside standard curve; the dangyao glycoside standard curve is constructed by performing high performance liquid chromatography analysis on dangyao glycoside reference solutions of different concentrations, and then constructing the curve with the concentration of the dangyao glycoside reference solution as the abscissa and the peak area obtained after high performance liquid chromatography analysis as the ordinate.

Images