A method for detecting the content of anti-inflammatory active ingredients in rhubarb and peony preparations

Abstract

The present invention provides a method for detecting the content of anti-inflammatory active ingredients in a rhubarb and peony preparation. The method utilizes specific solvents and chromatographic conditions, and employs multi-wavelength switching technology to simultaneously and quantitatively detect nine anti-inflammatory active ingredients in rhubarb and peony granules. The method is simple to operate, has good stability, high accuracy, short detection time, high efficiency, and low detection cost. The method calculates the content of the main active ingredients in the rhubarb and peony preparation, including gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and physcion, by calculating the relative correction factor f. This method provides a more comprehensive method for quality control of rhubarb and peony preparations.

Description

Technical Field

[0001] The invention belongs to the technical field of component detection of traditional Chinese medicine preparations, and particularly relates to a method for detecting the content of anti-inflammatory active components in a rhubarb and peony preparation. Background Art

[0002] The formula for Dahuang Mudan Granules is derived from Dahuang Mudan Decoction, first recorded in the "Golden Chamber" chapter on "Sores, Carbuncles, and Intestinal Carbuncles: Pulse, Symptoms, and Treatment." It was formulated by Zhang Zhongjing of the Eastern Han Dynasty for the early days of intestinal carbuncles. The formula, composed of rhubarb, peony bark, peach kernel, winter melon kernel, and Glauber's salt, is effective in purging heat, dissolving stagnation, and reducing swelling. Rhubarb, which clears heat and dissipates blood stasis, promotes bowel movements and detoxifies; moutan bark cools blood, clears heat, and promotes blood circulation and dissipates blood stasis. Together, these two ingredients work to clear damp-heat and stasis in the intestines and serve as the main ingredient. Peach kernel, with its blood-dissolving properties, aids the main ingredient in clearing stasis, while Glauber's salt softens and dissipates stagnation, assisting rhubarb in clearing excess heat and promoting rapid defecation. Winter melon kernel clears dampness, removes turbidity from the intestines, and drains pus and resolves carbuncles. Dahuang Mudan Decoction is a representative traditional Chinese medicine formula for treating appendicitis.

[0003] Dahuang Mudan Decoction (DRT) is a classic prescription widely used in clinical practice. It is not only used to treat appendicitis but also has proven efficacy in treating a variety of inflammatory-related diseases, including pancreatitis, colitis, pelvic inflammatory disease, hepatitis, cholecystitis, and suppurative diseases of the abdominal viscera. Research on DRT has primarily focused on its clinical efficacy, while quantitative determination of its active ingredients is remarkably lacking. Furthermore, its clinical use presents numerous inconveniences. Currently, granules are commonly used, but differences in the production process can significantly affect the content of its anti-inflammatory active ingredients, thereby impacting its clinical efficacy. For example, paeonol, an active ingredient in peony bark, has a boiling point of only 154°C, resulting in significant losses with prolonged decoction. Therefore, controlling the content of its active anti-inflammatory ingredients in DRT granules is crucial.

[0004] Aloe-emodin, rhein, rhein, rhein methyl ether and rhein phenol are the representative active ingredients of the main drug rhubarb; gallic acid, paeoniflorin and paeonol are the representative active ingredients of the main drug moutan bark; amygdalin is the representative active ingredient of the auxiliary drug peach kernel. Modern pharmacological studies have confirmed that these nine active ingredients all have significant anti-inflammatory activity, corresponding to the functions and indications of Dahuang Mudan Decoction and its preparations. Liu Li et al., Simultaneous determination of rhein, paeonol and paeoniflorin in Dahuang Mudan Decoction by HPLC [J], Chinese Journal of Pharmacovigilance, 2010, 7(3):139-141, discloses a method for determining the active ingredients in Dahuang Mudan Decoction, but it can only determine three of them. Since the number of active ingredients determined is small, it is impossible to fully control the quality of Dahuang Mudan Decoction and its preparations. Therefore, it is necessary to closely combine the functions and indications of Dahuang Mudan Decoction and its preparations to develop a method that can quantitatively determine more anti-inflammatory active ingredients to achieve more comprehensive quality control of Dahuang Mudan Decoction and its preparations. Summary of the Invention

[0005] To solve the above problems, the present invention establishes a method for detecting the content of anti-inflammatory active ingredients in a rhubarb and peony preparation, which comprises the following steps:

[0006] 1) Preparation of reference solution: Take the anti-inflammatory active ingredient reference substance and add an organic solvent to prepare a reference solution;

[0007] 2) Preparation of test solution: Take the Rhubarb and Peony preparation and extract it with methanol solution to obtain the test solution;

[0008] 3) Detection: HPLC chromatography was used for detection under the following chromatographic conditions: chromatographic column: phenyl column; mobile phase: mobile phase comprises phase A and phase B, phase A is acetonitrile, and phase B is 0.2% phosphoric acid aqueous solution;

[0009] The gradient elution program was as follows: -15 to 0 min, 5% A; 0 to 5 min, 5% A; 5 to 20 min, 5% A→20% A; 20 to 30 min, 20% A→40% A; 30 to 60 min, 40% A→70% A; 60 to 70 min, 70% A→5% A;

[0010] Detection wavelength: -15~10min, 270nm; 10.01~19min, 210nm; 19.01~27min, 230nm; 27.01~37min, 245nm; 37.01~70min, 254nm.

[0011] Furthermore, in step 1), the anti-inflammatory active ingredient reference substance is a reference substance of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol and / or physophanol methyl ether; the organic solvent is chloroform-methanol, acetone-methanol or methanol; and each 1 mL of the reference substance solution contains 20 to 2000 μg of the anti-inflammatory active ingredient.

[0012] Furthermore, the reference substance is chrysophanol or physophanol methyl ether, which is dissolved in 80% chloroform-methanol; the reference substance is rhein, which is dissolved in 80% acetone-methanol; the reference substance is emodin, gallic acid, amygdalin, paeoniflorin, paeonol or aloe-emodin, which is dissolved in methanol; the dissolved solution is then diluted with methanol.

[0013] Furthermore, in step 2), the rhubarb and peony preparation is ultrasonically extracted with 1 to 50 times the amount of methanol solution for 20 to 60 minutes; the rhubarb and peony preparation includes rhubarb and peony soup and rhubarb and peony granules, preferably rhubarb and peony granules.

[0014] Furthermore, in step 3), the phenyl chromatographic column is Agilent Zorbax SB-Phenyl, 4.6 mm×250 mm, 5 μm.

[0015] Furthermore, in step 3), the column temperature of the chromatographic conditions is 25-35°C, preferably 30°C; the flow rate is 0.8 mL / min-1.2 mL / min, preferably 1.00 mL / min, and the injection volume is 1-15 μL, preferably 5 μL.

[0016] Furthermore, in step 3), the detection wavelength is 270 nm, 210 nm, 230 nm, and 254 nm, which are the maximum absorption wavelengths of the peaks of the ultraviolet absorption spectra selected for gallic acid, amygdalin, paeoniflorin, aloe-emodin, rhein, emodin, chrysophanol, and physophanol methyl ether, respectively; and 245 nm, which is the minimum absorption wavelength of the peak valley of the ultraviolet absorption spectrum selected for paeonol.

[0017] Furthermore, it uses a standard curve method or a one-measurement-multiple-evaluation method to calculate the content; the one-measurement-multiple-evaluation method uses any one of the active ingredients as an internal reference, first detects the content of the internal reference, and uses a relative correction factor to calculate the content of the remaining active ingredients in the Rhubarb Mudan Granules.

[0018] Furthermore, the relative correction factor is obtained according to the following method:

[0019] Take the anti-inflammatory active ingredient reference substance, test it according to the above method, and calculate it according to the following formula:

[0020]

[0021] f i A is the correction factor of the object to be measured i; i is the peak area of ​​analyte i; C i is the concentration of the analyte i; f s is the correction factor of the internal reference s, A s is the peak area of ​​the internal reference s, C s is the concentration of the internal reference substance s.

[0022] Furthermore,

[0023] Taking gallic acid as the internal reference, the relative correction factors of amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, physophanol methyl ether and gallic acid were 0.6358, 0.5778, 0.5811, 1.7238, 0.9204, 0.8307, 2.3956, and 1.4878, respectively;

[0024] With amygdalin as the internal reference, the relative correction factors of gallic acid, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, physophanol methyl ether and amygdalin were 1.5728, 0.9085, 0.9136, 2.7145, 1.4472, 1.3073, 3.7688, and 2.3401, respectively;

[0025] With paeoniflorin as the internal reference, the relative correction factors of gallic acid, amygdalin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and physophanol methyl ether to paeoniflorin were 1.7343, 1.1025, 1.0068, 2.9940, 1.5949, 1.4403, 4.1526, and 2.5776, respectively;

[0026] With paeonol as the internal reference, the relative correction factors of gallic acid, amygdalin, paeoniflorin, aloe-emodin, rhein, emodin, chrysophanol, physophanol methyl ether and paeonol were 1.7265, 1.0981, 0.9896, 2.9905, 1.5928, 1.4302, 4.1237, and 2.5581, respectively;

[0027] With aloe-emodin as the internal reference, the relative correction factors of gallic acid, amygdalin, paeoniflorin, paeonol, rhein, emodin, chrysophanol, physophanol methyl ether and aloe-emodin were 0.5803, 0.3690, 0.3354, 0.3372, 0.5340, 0.4822, 1.3910, and 0.8636, respectively;

[0028] With rhein as the internal reference, the relative correction factors of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, emodin, chrysophanol, and physophanol methyl ether to rhein were 1.0879, 0.6918, 0.6282, 0.6319, 1.8776, 0.9045, 2.6070, and 1.6194, respectively;

[0029] With emodin as the internal reference, the relative correction factors of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, chrysophanol, and physophanol methyl ether to emodin were 1.2059, 0.7666, 0.6960, 0.7003, 2.0801, 1.1098, 2.8879, and 1.7921, respectively;

[0030] With chrysophanol as the internal reference, the relative correction factors of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, physophanol methyl ether and chrysophanol were 0.4176, 0.2655, 0.2411, 0.2425, 0.7209, 0.3843, 0.3470, and 0.6211, respectively;

[0031] With physophanol as the internal reference, the relative correction factors of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol and physophanol were 0.6734, 0.4280, 0.3885, 0.3909, 1.1620, 0.6196, 0.5589 and 1.6122, respectively.

[0032] The present invention discloses a method for detecting the content of active ingredients in a rhubarb and peony preparation. The method can simultaneously and quantitatively detect nine anti-inflammatory active ingredients in rhubarb and peony granules by using a specific solvent and chromatographic conditions and a multi-wavelength switching technology. The method is simple to operate, has good stability, high accuracy, short detection time, high efficiency, and low detection cost. The method calculates the content of the main anti-inflammatory active ingredients in the rhubarb and peony preparation, namely gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and physcion, by calculating the content of the main anti-inflammatory active ingredients in the rhubarb and peony preparation, thereby providing a more comprehensive method for quality control of the rhubarb and peony preparation.

[0033] The experiments show that the one-measurement-multiple-evaluation method of the present invention and the standard curve method mutually verify each other, have no obvious difference, and the results are reliable.

[0034] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0035] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 HPLC chromatogram of mixed reference substance;

[0037] Figure 2 HPLC chromatogram of the test solution (1-gallic acid; 2-amygdalin; 3-peoniflorin; 4-paeonol; 5-aloe-emodin; 6-rhein; 7-emodin; 8-chrysophanol; 9-emodin methyl ether);

[0038] Figure 3 HPLC chromatogram of negative control solution lacking rhubarb (1-gallic acid; 2-amygdalin; 3-peoniflorin; 4-paeonol);

[0039] Figure 4 HPLC chromatogram of negative control solution lacking Paeonia suffruticosa suffruticosa (2-amygdalin; 5-aloe-emodin; 6-rhein; 7-emodin; 8-chrysophanol; 9-emodin methyl ether);

[0040] Figure 5 HPLC chromatogram of peach kernel negative control solution (1-gallic acid; 3-paeoniflorin; 4-paeonol; 5-aloe-emodin; 6-rhein; 7-emodin; 8-chrysophanol; 9-chrysophanol methyl ether).

[0041] Figure 6 The names and structures of the nine anti-inflammatory active ingredients in Dahuang Mudan Granules;

[0042] Figure 7 UV absorption spectra of 9 anti-inflammatory active ingredients in Dahuang Mudan Granules; DETAILED DESCRIPTION

[0043] Example 1 Detection method of the present invention

[0044] 1. Detection method

[0045] (1) Preparation of mixed reference substances

[0046] Gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and physophanol methyl ether reference substances were accurately weighed into respective volumetric flasks. Chrysophanol and physophanol methyl ether were dissolved and brought to volume in 80% chloroform-methanol, rhein was dissolved and brought to volume in 80% acetone-methanol, and the other components were dissolved and brought to volume in methanol to prepare stock solutions of 2173.8 μg, 3562.0 μg, 1710.8 μg, 6068.2 μg, 838.0 μg, 2800.0 μg, 1018.4 μg, 800.2 μg, and 402.4 μg of each reference substance per mL, and stored at 4°C for later use.

[0047] Accurately pipette each reference substance stock solution into the same volumetric flask, add methanol to make up to volume, and prepare a mixed reference substance solution containing 217.38μg, 356.20μg, 171.08μg, 606.82μg, 83.8μg, 280.00μg, 101.84μg, 80.00μg, and 40.24μg per 1mL, and further dilute with methanol to form a series of reference substance solutions.

[0048] (2) Preparation of test solution

[0049] Accurately weigh 5.0 g of Dahuang Mudan granules into a stoppered conical flask, accurately add 50 mL of 50% methanol-water solution, weigh, ultrasonicate for 30 min, cool, weigh again, make up the weight loss with methanol solution, shake well, let stand, filter through a 0.45 μm microporous membrane, and take the filtrate to obtain the test solution.

[0050] (3) HPLC chromatography detection

[0051] The chromatographic column was an Agilent Zorbax SB-Phenyl (4.6 mm × 250 mm, 5 μm); mobile phase A was acetonitrile, and mobile phase B was 0.2% aqueous phosphoric acid; the flow rate was 1.0 mL / min; the injection volume was 5 μL, and the column temperature was 30°C; the gradient elution program was:

[0052]

[0053] Detection wavelength switching procedure

[0054]

[0055] (4) The contents of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and physophanol methyl ether in Dahuang Mudan granules were calculated using the standard curve method or the one-measurement-multiple-evaluation method;

[0056] The standard curve method uses the concentration of each component in a series of concentration reference solutions as the horizontal axis and the peak area of ​​each component as the vertical axis to draw a standard curve and obtain a linear regression equation. The content of each component in Dahuang Mudan Granules is calculated based on the linear regression equation.

[0057] The one-test-multiple-evaluation method uses any one of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and physophanol methyl ether as an internal reference. The content of the internal reference is first detected, and the content of the remaining active ingredients in Dahuang Mudan Granules is calculated using relative correction factors.

[0058] Taking gallic acid as the internal reference, the relative correction factors of amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, physophanol methyl ether and gallic acid were 0.6358, 0.5778, 0.5811, 1.7238, 0.9204, 0.8307, 2.3956, and 1.4878, respectively;

[0059] With amygdalin as the internal reference, the relative correction factors of gallic acid, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, physophanol methyl ether and amygdalin were 1.5728, 0.9085, 0.9136, 2.7145, 1.4472, 1.3073, 3.7688, and 2.3401, respectively;

[0060] With paeoniflorin as the internal reference, the relative correction factors of gallic acid, amygdalin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and physophanol methyl ether to paeoniflorin were 1.7343, 1.1025, 1.0068, 2.9940, 1.5949, 1.4403, 4.1526, and 2.5776, respectively;

[0061] With paeonol as the internal reference, the relative correction factors of gallic acid, amygdalin, paeoniflorin, aloe-emodin, rhein, emodin, chrysophanol, physophanol methyl ether and paeonol were 1.7265, 1.0981, 0.9896, 2.9905, 1.5928, 1.4302, 4.1237, and 2.5581, respectively;

[0062] With aloe-emodin as the internal reference, the relative correction factors of gallic acid, amygdalin, paeoniflorin, paeonol, rhein, emodin, chrysophanol, physophanol methyl ether and aloe-emodin were 0.5803, 0.3690, 0.3354, 0.3372, 0.5340, 0.4822, 1.3910, and 0.8636, respectively;

[0063] With rhein as the internal reference, the relative correction factors of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, emodin, chrysophanol, and physophanol methyl ether to rhein were 1.0879, 0.6918, 0.6282, 0.6319, 1.8776, 0.9045, 2.6070, and 1.6194, respectively;

[0064] With emodin as the internal reference, the relative correction factors of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, chrysophanol, and physophanol methyl ether to emodin were 1.2059, 0.7666, 0.6960, 0.7003, 2.0801, 1.1098, 2.8879, and 1.7921, respectively;

[0065] With chrysophanol as the internal reference, the relative correction factors of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, physophanol methyl ether and chrysophanol were 0.4176, 0.2655, 0.2411, 0.2425, 0.7209, 0.3843, 0.3470, and 0.6211, respectively;

[0066] With physophanol as the internal reference, the relative correction factors of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol and physophanol were 0.6734, 0.4280, 0.3885, 0.3909, 1.1620, 0.6196, 0.5589 and 1.6122, respectively.

[0067] The following test examples further illustrate the beneficial effects of the present invention.

[0068] Experimental Example 1 Study on the anti-inflammatory active ingredients in Dahuang Mudan granules

[0069] 1. Instruments and reagents

[0070] 1.1 Instrument

[0071] Ultimate 3000 high-performance liquid chromatograph (Thermo Fisher Scientific, USA), Agilent 1260 Infinity high-performance liquid chromatograph (Agilent Technologies, USA), AUW220D analytical balance (Shimadzu Corporation, Japan); FA2004 analytical balance (Shanghai Lichen Technology Instrument Co., Ltd.); G-100S ultrasonic cleaner (Shenzhen Geneng Cleaning Equipment Co., Ltd.); LCK2000 decoction machine (Tianjin Sanyan Precision Machinery Co., Ltd.).

[0072] 1.2 Drug testing

[0073] Dahuang Mudan Granules (Nanchong Traditional Chinese Medicine Hospital Traditional Chinese Medicine Preparation Center, batch number: 20220601, 20220602, 20220603, 20220701, 20220702, 20220703); Gallic acid reference substance (Gallic acid, batch number: 110831-201906, purity: 91.5%), amygdalin reference substance (Amygdalin, batch number: 110820-202109, purity: 93.1%), paeoniflorin reference substance (Paeoniflorin, batch number: 110736-202145, purity: 94.6%), paeonol reference substance (Paeonol, batch number: 110708-201908, purity: 99.8%), aloe-emodin reference substance (Aloe-emodin, batch number: 110795-202011, purity: 97.5%) , rhein reference substance (Rhein, batch number: 110757-201607, purity: 99.3%), emodin reference substance (Emodin, batch number: 110758-201913, purity: 96.0%), chrysophanol reference substance (Chrysophanol, batch number: 110796-201922, purity: 99.4%), and physcion methyl ether reference substance (Physcion, batch number: 110758-202218, purity: 98.9%) were all purchased from China Food and Drug Inspection Institutes; acetonitrile was chromatographic grade, water was Yibao purified water, and the other reagents were of analytical grade.

[0074] 2 Methods and Results

[0075] 2.1 Preparation of Rhubarb and Peony Granules: According to the prescription ratio of the classical prescription Rhubarb and Peony Decoction, Chinese medicinal pieces (rhubarb 12g, peony bark 3g, peach kernel 9g, wax gourd kernel 30g, and Glauber's salt 9g) were obtained. The peony bark was cold-soaked and extracted with 6 times the amount of 75% ethanol for 12h, stirred frequently, filtered, and the filtrate was concentrated under reduced pressure at 45°C to a clear paste for later use. The residue was added with 10 times the amount of water of rhubarb, peach kernel, and wax gourd kernel, decocted and extracted twice, each time for 30min, Glauber's salt was added to dissolve, and the mixture was concentrated under reduced pressure at 70°C to a thick paste. The thick paste was mixed with 1.5 times the amount of dextrin and the clear paste, granulated, dried at low temperature, and packaged.

[0076] 2.2 Preparation of mixed reference solution: Accurately weigh appropriate amounts of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and physcion methyl ether reference substances into respective volumetric flasks. Dissolve chrysophanol and physcion methyl ether in 80% chloroform-methanol and adjust to volume. Dissolve rhein in 80% acetone-methanol and adjust to volume. Dissolve the other ingredients in methanol and adjust to volume. Prepare a solution containing 2173.8 μg, 3562.0 μg, 1710.8 μg, 6068.2 μg, 83 8.0μg, 2800.0μg, 1018.4μg, 800.2μg, and 402.4μg of each reference substance stock solution were stored at 4°C for use; an appropriate amount of each reference substance stock solution was accurately pipetted into the same volumetric flask, and methanol was added to make up the volume to prepare a mixed reference substance solution containing 217.38μg, 356.20μg, 171.08μg, 606.82μg, 83.8μg, 280.00μg, 101.84μg, 80.00μg, and 40.24μg per 1mL.

[0077] 2.3 Preparation of test solution: Accurately weigh 5.0 g of Dahuang Mudan granules into a stoppered conical flask, accurately add 50 mL of 50% methanol-water solution, weigh, ultrasonicate for 30 min, cool, weigh again, make up the weight loss with methanol solution, shake well, let stand, filter through a 0.45 μm microporous membrane, and take the filtrate to obtain the test solution.

[0078] 2.4 Preparation of negative control solution: Weigh the prescription doses of rhubarb and peony root without rhubarb, peony root bark and peach kernel respectively, and prepare the negative control solutions without rhubarb, peony root bark and peach kernel respectively according to the preparation method of rhubarb and peony granules and the preparation method of test solution.

[0079] 2.5 Chromatographic conditions: The chromatographic column was an Agilent Zorbax SB-Phenyl (4.6 mm × 250 mm, 5 μm); the mobile phase A was acetonitrile, and the mobile phase B was 0.2% aqueous phosphoric acid; the flow rate was 1.0 mL / min; the injection volume was 5 μL, and the column temperature was 30°C.

[0080] The gradient elution program is shown in Table 1.

[0081] Table 1: HPLC conditional gradient elution program for Dahuang Mudan granules

[0082]

[0083] The multi-wavelength switching procedure is shown in Table 2.

[0084] Table 2: Multi-wavelength switching program for HPLC conditions of Dahuang Mudan granules

[0085]

[0086]

[0087] 3. Methodological Investigation

[0088] 3.1 Specificity test: According to "2.5 Chromatographic Conditions", the mixed reference solution, each negative control solution and the test solution were tested. The chromatogram showed that the test solution chromatographic peak was clearly visible at the same retention time as the reference solution, while the negative control solution had no interfering chromatographic peak, indicating good specificity. Figures 1 to 5 .

[0089] 3.2 Linear Relationship: Take the mixed reference solution and dilute it with methanol to form eight concentration gradient solutions. The concentration gradients of each component are shown in Table 3. Test according to "2.5 Chromatographic Conditions" and perform linear regression analysis using the peak area integral value (Y) against the mass concentration (X, μg / mL). The results are shown in Table 4.

[0090] Table 3 Concentration gradient of each component in the mixed control solution

[0091]

[0092] Table 4 Results of linear relationship tests of various components

[0093]

[0094]

[0095] 3.3 Precision test

[0096] The same mixed reference solution was injected six times in succession. The peak area of ​​each reference substance was recorded, and the RSD values ​​of the peak areas for gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and physophanol methyl ether were calculated. The results indicate good instrument precision (see Table 5).

[0097] 3.4 Repeatability test

[0098] The same test solution was injected six times in succession. The peak area of ​​each target peak was recorded, and the RSD values ​​for gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and physophanol methyl ether were calculated. The results showed good reproducibility of the method (see Table 5).

[0099] 3.5 Stability test

[0100] The same test solution was tested at room temperature at 0, 2, 4, 6, 12, and 24 hours. The peak area of ​​each target peak at each time point was recorded, and the RSD values ​​of the peak areas of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and physophanol methyl ether were calculated. The results showed that the test solution was stable at room temperature for 24 hours, as shown in Table 5.

[0101] Table 5 Precision, repeatability and stability test results

[0102]

[0103] 4. Establishment of a one-test-multiple-evaluation approach

[0104] 4.1 Positioning of target chromatographic peaks: The premise of achieving multiple evaluations of multiple components is to accurately position the target chromatographic peaks. Generally, relative retention time is used to position the target peaks, that is, a certain component is used as an internal standard, and the relative retention time of each peak to be measured (R = t i / t s , R is the relative retention time, t s is the retention time of the internal reference peak, t i =Retention time of the peak to be measured). However, due to the complexity of the compound preparation and the diversity of ingredients, there is a large error in locating the target peak using only relative retention time. Each type of ingredient has its own unique ultraviolet absorption spectrum. The nine anti-inflammatory active ingredients in the present invention have different structures and involve various structural types of ingredients, and their ultraviolet absorption spectra are also different. Figures 6-7 Therefore, it is more accurate to locate the target peak by combining relative retention time with UV absorption spectrum.

[0105] The relative retention times of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and physophanol methyl ether are shown in Table 6.

[0106] 4.2 Establishment of relative correction factor: The correction factor is based on the proportional relationship between the amount of a substance and the instrument response value within a certain linear range. However, in actual operation, due to system errors, operational errors, etc., the calculated correction factor may have deviations. The relative correction factor can eliminate the system error to a certain extent, reduce the accidental error and show higher accuracy. i / s) is calculated as:

[0107]

[0108] f i A is the correction factor of the object to be measured i; i is the peak area of ​​analyte i; C i is the concentration of the analyte i; f s is the correction factor of the internal reference s, A s is the peak area of ​​the internal reference s, C s is the concentration of the internal reference substance s.

[0109] In order to ensure the accuracy, scientificity and rigor of the experiment, 9 components were measured under certain fluctuation conditions (different HPLC, chromatographic columns, injection volumes, flow rates, and column temperatures) to finally determine the relative factor values.

[0110] 4.2.1. Determination by Different HPLC Methods: Under the same chromatographic conditions, the components were detected using an Ultimate 3000 HPLC and an Agilent 1260 Infinity HPLC, respectively, with gallic acid as the internal reference. Relative correction factors were calculated. The results are shown in Table 7.

[0111] 4.2.2. Determination on different chromatographic columns: Under the same chromatographic conditions, Agilent Zorbax SB-Phenyl (4.6 mm × 250 mm, 5 μm), Agilent Zorbax SB-C18 (4.6 mm × 250 mm, 5 μm), and Agilent Eclipse XDB-C18 (4.6 mm × 250 mm, 5 μm) columns were used to detect the components using gallic acid as the internal reference. The relative correction factors were calculated. The results are shown in Table 7.

[0112] 4.2.3 Determination of different injection volumes: Under the same chromatographic conditions, the injection volumes were 1, 2, and 4 μL, respectively, and gallic acid was used as the internal reference to detect each component. The relative correction factors were calculated. The results are shown in Table 7.

[0113] 4.2.4 Determination at different flow rates: Under the same chromatographic conditions, the flow rates were selected as 0.9, 1.0, and 1.1 mL / min, respectively, with gallic acid as the internal reference for detection of each component. The relative correction factors were calculated. The results are shown in Table 7.

[0114] 4.2.5 Determination at different column temperatures: Under the same chromatographic conditions, column temperatures of 25, 30, and 35°C were selected, respectively, and gallic acid was used as the internal reference to detect each component. The relative correction factors were calculated. The results are shown in Table 7.

[0115] Through multiple investigations of different HPLC, different chromatographic columns, different injection volumes, different flow rates, and different column temperatures, the relative correction factors were corrected and the values ​​of the relative correction factors were finally determined, as shown in Table 8.

[0116] The same internal reference substances were used in the same experiment, sequentially using amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and physophanol methyl ether as internal reference substances, to obtain relative calibration factors for each. The relative calibration factors corresponding to each internal reference substance were combined to form a table of relative calibration factors for the nine anti-inflammatory active ingredients in Dahuang Mudan Granules, as shown in Table 9. Ultimately, qualitative and quantitative analysis of all nine ingredients can be performed using only a single reference substance.

[0117] 5. Verification of the One Test, Multiple Evaluations Method

[0118] 5.1 Six batches of Dahuang Mudan Granules were prepared according to the Dahuang Mudan Granules preparation method. Test sample solutions were prepared according to the test sample preparation method. The content of each component was calculated using the standard curve method and the one-sided multiple evaluation method, respectively. The difference in the detection results between the two methods was compared using relative error as an indicator. The results showed that the relative error of the detection results of the two methods was small, indicating that there was no significant difference in the detection results between the two methods. The established one-sided multiple evaluation method for the nine active ingredients in Dahuang Mudan Granules has good accuracy. The results are shown in Tables 10-18.

[0119] Table 6 Relative retention time test results of each component (n=6)

[0120]

[0121] Table 7 Results of relative correction factors of various components under different liquid phases, chromatographic columns, injection volumes, flow rates, and column temperatures with gallic acid as the internal reference

[0122]

[0123]

[0124] Table 8 Relative correction factors of various components using gallic acid as internal reference

[0125]

[0126] Table 9: Results of relative correction factors test for 9 anti-inflammatory active ingredients (n=3)

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138] From the comparison of the above content determination methods, it can be seen that: using any one of the active ingredients of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, or physcion as the internal reference, the relative correction factor of each component was determined to establish a one-measurement, multiple-evaluation detection method. The relative errors of the content determination results of the nine anti-inflammatory active ingredients in six batches of Dahuang Mudan Granules were compared with the detection results determined by the standard curve method, all less than 5%, indicating that there was no significant difference between the two detection methods. Therefore, the one-measurement, multiple-evaluation method of the present invention provides a scientific and reasonable relative correction factor and can be used as a quantitative determination method for the nine anti-inflammatory active ingredients of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, or physcion in Dahuang Mudan Granules.

Claims

1. A method for detecting the content of anti-inflammatory active ingredients in a rhubarb and peony preparation, characterized in that: It includes the following steps: 1) Preparation of reference solution: An anti-inflammatory active ingredient reference substance is prepared by adding an organic solvent to a reference solution; the anti-inflammatory active ingredient reference substances are gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and physophanol methyl ether reference substances; the reference substances chrysophanol and physophanol methyl ether are dissolved in 80% chloroform-methanol; the reference substance rhein is dissolved in 80% acetone-methanol; the reference substances emodin, gallic acid, amygdalin, paeoniflorin, paeonol, and aloe-emodin are dissolved in methanol; the dissolved solution is then diluted with methanol; 2) Preparation of test solution: extracting a rhubarb and peony preparation with methanol to obtain a test solution; the rhubarb and peony preparation is rhubarb and peony granules; 3) Detection: HPLC chromatography was used under the following conditions: chromatographic column: Agilent Zorbax SB-Phenyl, 4.6 mm × 250 mm, 5 μm; column temperature: 30°C; flow rate: 1.00 mL / min; injection volume: 5 μL; mobile phase: mobile phase A: acetonitrile; phase B: 0.2% aqueous phosphoric acid. The gradient elution program was as follows: -15 to 0 min, 5% A; 0 to 5 min, 5% A; 5 to 20 min, 5% A→20% A; 20 to 30 min, 20% A→40% A; 30 to 60 min, 40% A→70% A; 60 to 70 min, 70% A→5% A; Detection wavelength: -15 to 10 min, 270 nm; 10.01 to 19 min, 210 nm; 19.01 to 27 min, 230 nm; 27.01 to 37 min, 245 nm; 37.01 to 70 min, 254 nm.

2. The method for detecting the content of the compound according to claim 1, wherein: In step 1), the reference solution contains 20-2000 μg of anti-inflammatory active ingredient per 1 mL.

3. The content detection method according to claim 1, wherein: In step 2), the Rhubarb and Peony preparation is added with 1 to 50 times the amount of methanol solution and ultrasonically extracted for 20 to 60 minutes.

4. The method for detecting the content of the compound according to claim 1, wherein: In step 3), the detection wavelengths are 270 nm, 210 nm, 230 nm, and 254 nm, which are the maximum absorption wavelengths of the peaks of the ultraviolet absorption spectra selected for gallic acid, amygdalin, paeoniflorin, aloe-emodin, rhein, emodin, chrysophanol, and physophanol methyl ether, respectively, and 245 nm, which is the minimum absorption wavelength of the peak valley of the ultraviolet absorption spectrum selected for paeonol.

5. The method for detecting the content of a compound according to any one of claims 1 to 4, wherein: The content is calculated by using a standard curve method or a one-measurement-multiple-evaluation method; the one-measurement-multiple-evaluation method uses any one of the active ingredients as an internal reference, first detects the content of the internal reference, and then uses a relative correction factor to calculate the content of the remaining active ingredients in the Rhubarb Mudan granules.

6. The content detection method according to claim 5, characterized in that: The relative correction factor is calculated according to the following formula: f i is the correction factor of the test object i; A i is the peak area of ​​analyte i; C i is the concentration of analyte i; f s is the correction factor of the internal reference s, A s is the peak area of ​​the internal reference s, C s is the concentration of the internal reference substance s.

7. The content detection method according to claim 5, characterized in that: Taking gallic acid as the internal reference, the relative correction factors of amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, physophanol methyl ether and gallic acid were 0.6358, 0.5778, 0.5811, 1.7238, 0.9204, 0.8307, 2.3956, and 1.4878, respectively; With amygdalin as the internal reference, the relative correction factors of gallic acid, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, physophanol methyl ether and amygdalin were 1.5728, 0.9085, 0.9136, 2.7145, 1.4472, 1.3073, 3.7688, and 2.3401, respectively; With paeoniflorin as the internal reference, the relative correction factors of gallic acid, amygdalin, paeonol, aloe-emodin, rhein, emodin, chrysophanol, and physophanol methyl ether to paeoniflorin were 1.7343, 1.1025, 1.0068, 2.9940, 1.5949, 1.4403, 4.1526, and 2.5776, respectively; With paeonol as the internal reference, the relative correction factors of gallic acid, amygdalin, paeoniflorin, aloe-emodin, rhein, emodin, chrysophanol, physophanol methyl ether and paeonol were 1.7265, 1.0981, 0.9896, 2.9905, 1.5928, 1.4302, 4.1237, and 2.5581, respectively; With aloe-emodin as the internal reference, the relative correction factors of gallic acid, amygdalin, paeoniflorin, paeonol, rhein, emodin, chrysophanol, physophanol methyl ether and aloe-emodin were 0.5803, 0.3690, 0.3354, 0.3372, 0.5340, 0.4822, 1.3910, and 0.8636, respectively; With rhein as the internal reference, the relative correction factors of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, emodin, chrysophanol, and physophanol methyl ether to rhein were 1.0879, 0.6918, 0.6282, 0.6319, 1.8776, 0.9045, 2.6070, and 1.6194, respectively; With emodin as the internal reference, the relative correction factors of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, chrysophanol, and physophanol methyl ether to emodin were 1.2059, 0.7666, 0.6960, 0.7003, 2.0801, 1.1098, 2.8879, and 1.7921, respectively; With chrysophanol as the internal reference, the relative correction factors of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, physophanol methyl ether and chrysophanol were 0.4176, 0.2655, 0.2411, 0.2425, 0.7209, 0.3843, 0.3470, and 0.6211, respectively; With physophanol as the internal reference, the relative correction factors of gallic acid, amygdalin, paeoniflorin, paeonol, aloe-emodin, rhein, emodin, chrysophanol and physophanol were 0.6734, 0.4280, 0.3885, 0.3909, 1.1620, 0.6196, 0.5589 and 1.6122, respectively.

Images