Method for determining content of iridoid components

Abstract

The invention belongs to the field of quality analysis of traditional Chinese medicines, and particularly discloses a method for determining the content of iridoid components, which adopts HPLC (High Performance Liquid Chromatography) to simultaneously determine the contents of six iridoid components, namely morroniside, geniposide, loganin, picroside II, picroside I and harpagoside, in four medicinal materials, namely dogwood, cape jasmine, picrorhiza scrophularia and radix scrophulariae. According to the method, the content of iridoid components in multiple medicinal materials can be measured, the detection cost is reduced, the detection time is shortened, and the detection efficiency is improved. The detection method provided by the invention is stable and reliable, has the advantages of strong specificity, good reproducibility and the like, can ensure the safety and effectiveness of clinical medication of common medicinal materials such as dogwood, cape jasmine, rhizoma picrorhizae, radix scrophulariae and the like, and better meets the medical and market requirements.

Description

Technical Field

[0001] The invention relates to a method for determining the content of iridoid components, in particular to a method for determining the content of iridoid multi-components based on HPLC, and belongs to the field of traditional Chinese medicine preparation analysis. Background Art

[0002] Iridoids are secondary metabolites widely distributed in raw medicinal materials. From the perspective of chemical structure, the basic nucleus of iridoids is iridoid alcohol, which has structures such as cyclic ether and alcohol hydroxyl. It is more common in Gentianaceae, Lamiaceae, Rubiaceae, and Scrophulariaceae. It is a type of monoterpene compound with a hemiacetal and cyclopentane structure, which makes its properties unstable. Therefore, it mainly exists in plants in the form of glycosides. This type of compound is the main active ingredient of many traditional Chinese medicines and a key quality control ingredient in traditional Chinese medicines. With the in-depth research on it in recent years, it has been found that it has multiple pharmacological effects.

[0003] The chemical components of Gardenia mainly include iridoids, triterpenes, organic acids and volatile oils, among which iridoids are the main active ingredients of Gardenia. Many studies have shown that the iridoid components in Gardenia have a good therapeutic effect on Alzheimer's disease. Gardenia glycosides in Gardenia have immunomodulatory and anti-inflammatory effects. In addition, they also have a good therapeutic effect on organ ischemia-reperfusion injury. Morronoside and loganin in Cornus officinalis of Cornaceae can lower blood sugar, have obvious effects on heart function and vascular damage repair, and are often used to treat cardiovascular diseases caused by diabetes. Picroside I and Picroside II in Picrocoside of Scrophulariaceae can stabilize liver cell membranes, eliminate free radicals, protect liver damage caused by acute and subacute hepatitis, and have the effect of protecting the liver and promoting choleresis. Habao glycosides in Scrophulariaceae Scrophularia can improve the cardiac function of mice with acute myocardial infarction and have a protective effect on myocardial cells.

[0004] The invention discloses a method for simultaneously determining the contents of 6 components in Cornus officinalis, Gardenia jasminoides, Picrorhizon and Scrophularia ningpoensis, namely morroniside, geniposide, loganin, picroside II, picroside I and harpaoside, by HPLC. The method can realize accurate quantitative evaluation and control of 6 iridoid ether terpenoid components in the 4 medicinal materials, ensure the stability of product quality and the safety and effectiveness of clinical medication, and reduce the amount of solvent used, so as to be more environmentally friendly, save analysis costs, and reduce costs and increase efficiency. Summary of the invention

[0005] The technical problem to be solved by the present invention is to further optimize the detection method of Chinese medicinal materials, simplify the operation method and improve the detection efficiency. Through a large number of experiments, the inventor successfully combined the HPLC content determination of 6 components in Cornus officinalis, Gardenia jasminoides, Picrorhizon and Scrophularia ningpoensis, including morroniside, gardenia glycoside, loganin, picroside II, picroside I and harpaoside, to provide a more comprehensive and accurate new detection method.

[0006] The purpose of the present invention is to provide a method for determining the content of iridoid components. Morroniside and loganin are components in Cornus officinalis, Gardenia jasminoides is a component in Gardenia jasminoides, Picroside II and Picroside I are components in Picroside chinensis, and Harpaoside is a component in Scrophularia ningpoensis. The present invention adopts HPLC to simultaneously determine the contents of morroniside, Gardenia jasminoides, Loganin, Picroside II, Picroside I, and Harpaoside in Cornus officinalis, Gardenia jasminoides, Picroside chinensis, and Scrophularia ningpoensis, thereby achieving simultaneous evaluation and control of the quality of multiple medicinal materials, thereby providing an accurate basis for authenticity identification and intrinsic quality detection of the four medicinal materials.

[0007] A method for determining the content of iridoid components, comprising the following steps:

[0008] A. Preparation of test solution: Accurately weigh the powders of Cornus officinalis, Gardenia jasminoides, Rhizoma Picrorhizoma, and Scrophularia ningpoensis (passed through a No. 3 sieve), place them in a stoppered conical flask, accurately add methanol, sonicate, shake well, filter, and take the filtrate as the test solution;

[0009] B. Preparation of reference solution: Accurately weigh appropriate amount of morroniside, gardeniaside, loganin, picroside II, picroside I, and harpaoside reference substances, place in a volumetric flask, add methanol to dissolve, make up to volume, shake well, and obtain;

[0010] C.HPLC chromatographic conditions: chromatographic column is C18 chromatographic column, mobile phase A is trifluoroacetic acid solution, mobile phase B is trifluoroacetic acid-acetonitrile solution, gradient elution, detection wavelength is 240-260nm; flow rate: 0.5-1mL / min; column temperature: 25-30℃;

[0011] D. Determination method: Accurately aspirate the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine according to the chromatographic conditions described in step C.

[0012] Preferably, in step A, methanol is added and ultrasonicated for 30 minutes.

[0013] Preferably, the chromatographic column in step C is an Agilent ZORBAXSB-C18 chromatographic column with a specification of 4.6×250 mm and a particle size of 5 μm.

[0014] Preferably, the mobile phase A in step C is 0.1% trifluoroacetic acid solution, and the mobile phase B is 0.1% trifluoroacetic acid-acetonitrile.

[0015] Preferably, the gradient elution conditions in step C are:

[0016] Time, min Mobile phase, A% Mobile phase, B% 0 95 5 5 95 5 15 90 10 20 89 11 30 83 17 55 80 20 70 75 25 75 72 28 80 50 50 85 50 50 90 95 5 100 95 5 .

[0017] Preferably, in step C, the detection wavelength is 250 nm, the flow rate is 1 mL / min, and the column temperature is 25°C.

[0018] Compared with the prior art, the present invention has achieved the following beneficial technical effects:

[0019] 1) The present invention uses Gardenia jasminoides as an internal reference to establish a method for simultaneously detecting six iridoid terpenoid components, thereby achieving one-test-multiple-evaluations, improving detection efficiency, saving reagents, consumables and labor, reducing costs, increasing efficiency, and being energy-saving and environmentally friendly.

[0020] 2) The present invention uses a method based on HPLC to simultaneously quantify the contents of six pharmacopoeia index components in Cornus officinalis, Gardenia jasminoides, Picrorhizon and Scrophularia ningpoensis, including morroniside, geniposide, loganin, picroside II, picroside I and harpaoside, so as to achieve simultaneous evaluation and control of the quality of multiple medicinal materials.

[0021] 3) By comparing the results of the one-measurement-multiple-evaluation method and the standard curve method, the content results obtained by the two methods are similar, indicating that the content calculation results obtained by the one-measurement-multiple-evaluation method are consistent with those obtained by the standard curve method. Under different experimental conditions, the correction factors between the components have good reproducibility (RSD < 1.0%), and the content of 6 components including morroniside, gardenoside, loganin, picroside II, picroside I and harpaoside in Cornus officinalis, Gardenia jasminoides, Picrorhizon and Scrophularia ningpoensis can be effectively determined. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Chromatogram of reference solution

[0023] The symbols in the accompanying drawings are described as follows: 1-morroniside, 2-gardeniside, 3-loganoside, 4-picroside II, 5-picroside I, 6-harpaoside;

[0024] Figure 2 Chromatogram of the test solution of Cornus officinalis, Gardenia jasminoides, Picrorhizon and Scrophularia ningpoensis

[0025] The symbols in the accompanying drawings are described as follows: 1-morroniside, 2-gardeniside, 3-loganoside, 4-picroside II, 5-picroside I, 6-harpaoside;

[0026] Figure 3 Chromatogram of reference solution of comparative example 1

[0027] Figure 4 Chromatogram of reference solution of comparative example 2

[0028] The symbols in the accompanying drawings are described as follows: 1-morroniside, 2-gardenoside, 3-loganoside, 4-picroside II, 5-picroside I, and 6-harpaoside. DETAILED DESCRIPTION

[0029] In order to facilitate a full understanding of the implementation of the present invention, the present invention is further described below through typical implementation cases. At the same time, since the following examples are only preferred implementations of the present invention, their descriptions are relatively specific and detailed, so they cannot be understood as limiting the scope of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principles and concepts of the present invention, several modifications and improvements can also be made, which should also belong to the scope of protection of the present invention.

[0030] Example 1 Study on HPLC Determination Methodology of Six Iridoids in Cornus officinalis, Gardenia jasminoides, Picrorhizon and Scrophularia ningpoensis

[0031] 1. Instruments and reagents:

[0032] 1.1 Instruments: Waters ACQUITY Arc liquid chromatograph (2998PDA detector, USA), electronic analytical balance (ME1002, METTLER TOLEDO), ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0033] 1.2 Reagents: Gardeniaside (batch number 110749-201919, content 97.1%), Morroniside (batch number 111998-202104, content 96.8%), Loganin (batch number 111640-201808, content 99.0%), Picroside II (batch number 111596-201805, content 93.2%), Picroside I (batch number 111727-201702, content 95.6%), Harpaoside (batch number 111730-201709, content 95.9%), all from China Food and Drug Inspection Institutes. Acetonitrile (Thermo Fisher Scientific Inc., batch number: F21L9F201, chromatographic grade), methanol (Thermo Fisher Scientific Inc., batch number: 213382, chromatographic grade), isopropanol (Shanghai Wokai Biotechnology Co., Ltd., batch number: 20181210, analytical grade), trifluoroacetic acid (Nanjing Chemical Reagent Co., Ltd., batch number: 201210681F, analytical grade), methanol (Nanjing Chemical Reagent Co., Ltd., batch number: 220616035F, analytical grade).

[0034] 2Chromatographic conditions:

[0035] Chromatographic column: Agilent ZORBAXSB-C18 chromatographic column (4.6mm×250mm, 5μm); mobile phase A is 0.1% trifluoroacetic acid solution, mobile phase B is 0.1% trifluoroacetic acid-acetonitrile; detection wavelength: 250nm; flow rate: 1mL / min; column temperature: 25℃; injection volume: 5μL. Gradient elution is performed according to the following table:

[0036] Time, min Mobile phase, A% Mobile phase, B% 0 95 5 5 95 5 15 90 10 20 89 11 30 83 17 55 80 20 70 75 25 75 72 28 80 50 50 85 50 50 90 95 5 100 95 5

[0037] 3 Methods and Results

[0038] 3.1 Preparation of test solution

[0039] Accurately weigh 0.1, 0.1, 0.1 and 1 g of powder of Cornus officinalis, Gardenia jasminoides, Picrorhizon and Scrophularia ningpoensis (passed through No. 3 sieve), place in a stoppered conical flask, add methanol to make up to 50 mL, ultrasonicate for 30 min, and filter through a 0.22 μm microporous filter membrane to obtain the product.

[0040] 3.2 Preparation of reference solution

[0041] Accurately weigh appropriate amounts of morroniside, gardeniaside, loganin, picroside II, picroside I, and harpaoside reference substances, place them in volumetric flasks respectively, add methanol to dissolve and dilute to the scale to obtain controls with concentrations of 0.10626, 4.38892, 0.370062, 0.8563, 3.8011, and 0.4818 mg / mL, respectively.

[0042] 3.3 Methodological investigation

[0043] 3.3.1 Investigation of linear relationship

[0044] Accurately pipette 2, 4, 5, 6, 8, 10 μL of the mixed reference solution, and inject and measure according to the chromatographic conditions under item "2". Draw the standard curve with the peak area as the ordinate and the injection volume as the abscissa, and obtain the linear regression equation and linear range of morroniside, gardenia jasminoides, loganin, picroside II, picroside I, and harpaoside. The results are shown in Table 1.

[0045] Table 1 Results of linear relationship investigation of 6 iridoid components

[0046] Element Regression equation <![CDATA[R 2 ]]> Linear range / (μg) Morroniside Y=1328301X-6908 0.9998 0.0308～0.1541 Gardenia glycoside Y=1177439X-26614 0.9998 0.1536～0.7681 Loganin Y=1077813X-3995 0.9998 0.0241～0.1203 Picroside II Y=915581X-45609 0.9998 0.3425～1.7126 Picroside I Y=695475X-12460 0.9999 0.1140～0.5702 Harpagoside Y=838164X-7755 0.9998 0.0626～0.3132

[0047] The experimental results showed that the six components, morroniside, gardeniaside, loganin, picroside II, picroside I and harpaoside, showed a good linear relationship within their respective mass ranges.

[0048] 3.3.2 Stability test

[0049] The same test solution was placed at room temperature and sampled at 0, 2, 4, 8, 12 and 24 h, respectively, and the peak areas were determined. The RSDs of the peak areas of morroniside, gardeniaside, loganin, picroside II, picroside I and harpaoside were 0.32%, 0.50%, 0.39%, 0.60%, 0.48% and 0.41%, respectively, indicating that the test results of the test solution were stable within 24 h, as shown in Table 2.

[0050] Table 2 Stability test of 6 iridoid components

[0051] Time (h) Morroniside Gardenia glycoside Loganin Picroside II Picroside I Harpagoside 0 92297 554176 54458 734263 217950 137453 2 92119 554768 54552 733111 218586 137688 8 92027 557619 54354 738772 218798 138924 12 92057 558843 54758 740411 219030 138547 18 92647 557372 54743 740648 219554 138583 24 92672 551424 54920 745121 220991 138099 average 92303 555700 54631 738721 219152 138216 RSD(%) 0.32 0.50 0.39 0.60 0.48 0.41

[0052] 3.3.3 Repeatability test

[0053] The same sample powder was accurately weighed 6 times, and the sample solution was prepared according to the sample solution preparation method under "3.1". The samples were injected according to the chromatographic method under "2", and the peak areas were recorded. The component contents were calculated. The results are shown in Table 3. The RSD (%) of the average content of each component for 6 times was less than 2%, indicating that the repeatability of this method was good.

[0054] Table 3 Repeatability test of 6 iridoid components

[0055]

[0056]

[0057] 3.3.4 Sample recovery test

[0058] Accurately weigh 0.05, 0.05, 0.05, and 0.5 g of Gardenia, Cornus officinalis, Picrorhizon, and Scrophularia ningpoensis powder (passed through No. 3 sieve), for a total of 6 portions, and add a certain amount of reference solution to each portion. Prepare samples according to the test solution treatment method under "3.1". Inject and determine according to the chromatographic conditions under 2.1, record the peak areas, and calculate the average recoveries (RSD) of morroniside, gardeniaside, loganin, picroside II, picroside I, and harpaoside. See Table 4.

[0059] Table 4 Recovery test of 6 iridoid components

[0060]

[0061]

[0062] 3.4 Establishment of the One Test, Multiple Evaluations Method

[0063] 3.4.1 Calculation of f

[0064] Accurately measure the mixed reference solution under "3.2" and inject 2, 4, 5, 6, 8, and 10 μL respectively according to the chromatographic conditions under "2". Use gardenia glycoside as the internal reference and calculate the f of morroniside, loganin, picroside II, picroside I, and harpaoside according to the following formulas. k / s , the results are shown in Table 5.

[0065]

[0066] A in the formula s is the peak area of ​​the internal reference, C s is the internal reference concentration, A k is the peak area of ​​a component to be measured, Ck is the concentration of a component to be tested.

[0067] Table 5 Correction factors (f k / s )

[0068]

[0069] 3.4.2 Calculation of relative retention value

[0070] The relative retention time is used for positioning, that is, the retention time of the chromatographic peak of each component to be tested and the chromatographic peak of gardenia glycoside is used for determination. The relative retention time calculation formula is: RRT = T k / T s (T s is the retention time of the reference, T k is the retention time of other components to be measured), and the results are shown in Table 6.

[0071] Table 6 Relative retention value (RRT, n = 6)

[0072] sample Morroniside Gardenia glycoside Loganin Picroside II Picroside I Harpagoside 1 0.75 1 1.04 1.44 1.95 2.74 2 0.75 1 1.04 1.44 1.95 2.73 3 0.75 1 1.04 1.44 1.96 2.73 4 0.75 1 1.04 1.44 1.96 2.73 5 0.75 1 1.04 1.44 1.96 2.73 6 0.75 1 1.04 1.44 1.96 2.74 average 0.75 1 1.04 1.44 1.96 2.73 RSD(%) 0 0 0 0 0.26 0.19

[0073] 4 Comparison of results between one-test-multiple-evaluation method and external standard method

[0074] 4.1 Comparison of the results of one-test-multiple-evaluations and the results of content detection by external standard method

[0075] The contents of six iridoid components, including morroniside, geniposide, loganin, picroside II, picroside I and harpaoside, in 10 batches of Cornus officinalis, Gardenia jasminoides, Picrorhizon and Scrophularia ningpoensis from the same manufacturer were determined by external standard method and one-measurement-multiple-evaluation method, respectively. See Table 7. The results showed that there was no significant difference in the contents of iridoid components measured by the two methods.

[0076] Table 7 One-test-multiple-evaluation results and external standard method content detection results

[0077]

[0078]

[0079] In summary, this study used high performance liquid chromatography (HPLC) to establish a method for the simultaneous determination of 6 iridoid components, including morroniside, gardenoside, loganin, picroside II, picroside I, and harpaoside, in Gardenia jasminoides, Cornus officinalis, Picrorhizon, and Scrophularia ningpoensis. This method was established by investigating the extraction method, chromatographic conditions, elution gradient, etc., and further, using Gardenia jasminoides as the internal reference, the relative correction factors of morroniside, loganin, picroside II, picroside I, and harpaoside were determined by the multi-point calibration method, and the content determination method of these five components was established by the one-standard multiple-measurement method; the relative correction factors (f k / s ) were 0.8948, 1.0968, 1.2849, 1.7008, and 1.4051, and the relative retention values ​​(RRT) were 0.75, 1.04, 1.44, 1.96, and 2.73, respectively; 10 batches of Gardenia, Cornus officinalis, Coptis chinensis, and Scrophularia ningpoensis were determined by QAMS method, and the contents of the six components obtained by QAMS method were compared with the content determination values ​​of the external standard method (ESM), and the RSDs (%) were all less than 1.0%. The calculated retention values ​​obtained by QAMS method were compared with the actual retention values ​​of the ESM method, and the RSDs (%) were all less than 1.0%, indicating that this method is accurate and low-cost, which can ensure the safety and effectiveness of the clinical use of Gardenia, Cornus officinalis, Coptis chinensis, and Scrophularia ningpoensis, and better meet medical and market needs.

[0080] Comparative Example 1 HPLC Determination of Six Iridoids in Cornus officinalis, Gardenia jasminoides, Picrorhizon and Scrophularia ningpoensis

[0081] 1. Instruments and reagents: Same as Example 1

[0082] 2Chromatographic conditions:

[0083] Chromatographic column: Agilent ZORBAXSB-C18 chromatographic column (4.6mm×250mm, 5μm); mobile phase A is 0.25% trifluoroacetic acid solution, mobile phase B is 0.25% trifluoroacetic acid-acetonitrile solution; detection wavelength: 240nm; flow rate: 0.5mL / min; column temperature: 30℃; injection volume: 5μL. Gradient elution is performed according to the following table:

[0084] Time, min Mobile phase, A% Mobile phase, B% 0 95 5 50 95 5 60 91 9 75 86 14 90 73 27

[0085] 3. Preparation of reference substance and test solution: Same as Example 1

[0086] 4 Experimental results

[0087] Take the above solutions respectively, perform HPLC analysis under the above chromatographic conditions, and record the chromatograms. Figure 3 Under this chromatographic condition, not all of the six components eluted, but only three components eluted, and their retention times were relatively late.

[0088] Comparative Example 2 HPLC Determination of Six Iridoids in Cornus officinalis, Gardenia jasminoides, Picrorhizon and Scrophularia ningpoensis

[0089] 1. Instruments and reagents: Same as Example 1

[0090] 2Chromatographic conditions:

[0091] Chromatographic column: Agilent ZORBAXSB-C18 chromatographic column (4.6mm×250mm, 5μm); mobile phase A is 0.05% trifluoroacetic acid solution, mobile phase B is 0.05% trifluoroacetic acid-acetonitrile solution; detection wavelength: 260nm; flow rate: 1mL / min; column temperature: 25℃; injection volume: 5μL. Gradient elution is performed according to the following table:

[0092] Time, min Mobile phase, A% Mobile phase, B% 0 90 10 20 70 30 30 30 70 40 30 70 50 90 10 53 90 10

[0093] 3. Preparation of reference substance and test solution: Same as Example 1

[0094] 4 Experimental results

[0095] Take the above solutions respectively, perform HPLC analysis under the above chromatographic conditions, and record the chromatograms. Figure 4 Under this chromatographic condition, the resolution was low and the two chromatographic peaks of gardenia jasminoides and loganin were inseparable.

Claims

1. A method for determining the content of iridoid components, characterized in that: The method adopts HPLC to simultaneously determine the contents of morroniside, gardeniaside, loganin, picroside II, picroside I and harpaoside in the medicinal materials of Cornus officinalis, Gardenia jasminoides, Picrorhizon and Scrophularia ningpoensis.

2. The content determination method according to claim 1, characterized in that: The following steps are involved: A. Preparation of test solution: Accurately weigh the powders of Cornus officinalis, Gardenia jasminoides, Rhizoma Picrorhizoma, and Scrophularia ningpoensis, place them in a stoppered conical flask, accurately add methanol, sonicate, shake well, filter, and take the filtrate as the test solution; B. Preparation of reference solution: Accurately weigh appropriate amount of morroniside, gardeniaside, loganin, picroside II, picroside I, and harpaoside reference substances, place in a volumetric flask, add methanol to dissolve, make up to volume, shake well, and obtain; C.HPLC chromatographic conditions: the chromatographic column is C18 chromatographic column, the mobile phase A is trifluoroacetic acid solution, the mobile phase B is trifluoroacetic acid-acetonitrile solution, gradient elution, the detection wavelength is 240-260nm; the flow rate is 0.5-1mL / min; Column temperature: 25-30°C; D. Determination method: Accurately aspirate the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine according to the chromatographic conditions described in step C.

3. The content determination method according to claim 2, characterized in that: Step A: Add methanol and sonicate for 30 min.

4. The content determination method according to claim 2, characterized in that: The chromatographic column in step C is an Agilent ZORBAXSB-C18 chromatographic column with a specification of 4.6×250 mm and a particle size of 5 μm.

5. The content determination method according to claim 2, characterized in that: In step C, the mobile phase A is 0.1% trifluoroacetic acid solution, and the mobile phase B is 0.1% trifluoroacetic acid-acetonitrile.

6. The content determination method according to claim 2, characterized in that: The gradient elution conditions in step C are: 。 7. The content determination method according to claim 2, characterized in that: Step C: detection wavelength is 250 nm, flow rate is 1 mL / min, column temperature is 25°C.

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