Preparation method and quality detection method of anti-pharyngitis traditional Chinese medicine compound extract

Abstract

The invention provides a quality detection method of an anti-pharyngitis traditional Chinese medicine compound extract, which adopts high performance liquid chromatography for qualitative and quantitative analysis of iridin and arctiin, and adopts ultraviolet-visible spectrophotometry for quantitative analysis of total flavonoids. Furthermore, comprehensive evaluation is carried out by taking the contents of iridin, arctiin and total flavonoids as well as the yield of the extract as indexes, the extraction time and the solvent amount which have great influence on the water extraction effect are investigated, and the extraction process of the anti-pharyngitis traditional Chinese medicine compound extract is optimized; the high performance liquid chromatography and the ultraviolet-visible spectrophotometric method are good in linear relation, high in precision, good in repeatability and accurate in determination result, and qualitative and quantitative analysis are combined to lay a foundation for establishing the quality standard of the anti-pharyngitis traditional Chinese medicine compound extract.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, in particular to a preparation method and a quality detection method of an anti-pharyngitis traditional Chinese medicine compound extract. Background Art

[0002] Acute pharyngitis is a common and frequently occurring disease in otolaryngology clinics, accounting for 7% to 17% of pharyngeal diseases. Clinically, acute pharyngeal inflammation is characterized by sore throat, redness and swelling of the pharynx, granular protrusions on the pharyngeal floor, subtle swelling of the posterior nucleus, and systemic wind-heat symptoms. If acute pharyngitis is not treated promptly and effectively, it may lead to chronic pharyngitis, chronic tracheitis and bronchitis, nephritis, heart disease, and other conditions. Clinically, acute pharyngitis is often treated with antibiotics or hormones. However, due to issues such as antibiotic resistance and side effects of hormone therapy, current clinical treatments are insufficient. Research is urgently needed on therapeutic drugs with improved clinical efficacy and minimal side effects.

[0003] Traditional Chinese medicine, especially Chinese herbal compound, has unique advantages in the prevention and treatment of acute pharyngitis. Chinese herbal compound has become an important preventive and therapeutic drug for acute pharyngitis because it can regulate the pathogenesis of acute pharyngitis through the interaction of multiple pathways, multiple targets and multiple components. Jindengshangen Decoction: Jindengshangen Decoction: Jindengshangen Decoction, Belamcanda Root, Sautéed Burdock Fructus, Sophora flavescens, Licorice, Platycodon grandiflorum, has the effects of dispelling wind and resolving phlegm, clearing away heat and detoxifying, and relieving swelling and sore throat. As a classic Chinese herbal compound for anti-pharyngitis, it has high development and application value, but its quality control method is currently lacking. In order to ensure the safety, stability, effectiveness and controllability of compound drug extracts, it is necessary to find a simple, accurate and reliable method for quality testing. Summary of the Invention

[0004] In view of the above technical problems, the purpose of the present invention is to provide a preparation method and quality detection method of an anti-pharyngitis Chinese medicine compound extract, so as to perform quality detection on the anti-pharyngitis Chinese medicine compound extract in a simple, accurate and reliable way, and then optimize the extraction process.

[0005] To achieve the above-mentioned and other related purposes, the present invention provides a quality detection method for an anti-pharyngitis Chinese herbal compound extract, wherein the anti-pharyngitis Chinese herbal compound comprises the following components: Chinese lantern, Belamcanda chinensis, stir-fried burdock fruit, Sophora flavescens root, Licorice root, and Platycodon grandiflorum; the quality detection method is as follows:

[0006] Step 1: Use high performance liquid chromatography to perform qualitative and quantitative analysis, specifically including the following steps:

[0007] 11) Preparation of test solution: Weigh the components of the anti-pharyngitis Chinese herbal compound, mix them, and prepare a Chinese herbal compound extract by water extraction. Dissolve the Chinese herbal compound extract in a solvent, centrifuge, and dilute the supernatant to obtain a test solution.

[0008] 12) Preparation of Reference Solution: Weigh and dissolve the standard solutions of irisin and arctiin, respectively, then mix and dilute the two solutions to prepare the first reference solution;

[0009] 13) Preparation of negative control solution: Prepare a Chinese herbal compound extract not containing Belamcanda chinensis by the water extraction method in step 11), add a solvent to dissolve, centrifuge, and dilute the supernatant to obtain a Belamcanda chinensis negative control solution; prepare a Chinese herbal compound extract not containing stir-fried Arctium lappa by the water extraction method in step 11), add a solvent to dissolve, centrifuge, and dilute the supernatant to obtain a stir-fried Arctium lappa negative control solution;

[0010] 14) Detection: The test solution, the first reference solution, and the negative control solution were assayed by high performance liquid chromatography (HPLC). The retention times were compared for qualitative analysis, and the irisin and arctiin in the test solution were quantified using a standard curve method.

[0011] Step 2: Quantitative analysis is performed using UV-visible spectrophotometry, specifically comprising the following steps:

[0012] 21) Preparation of test solution: Prepare a Chinese herbal compound extract using the water extraction method in step 11); weigh the Chinese herbal compound extract, dissolve it in a solvent, sonicate, centrifuge, and dilute the supernatant to obtain a test solution;

[0013] 22) Preparation of reference solution: Weigh rutin standard and prepare a second reference solution;

[0014] 23) The test solution and the second reference solution were measured by UV-visible spectrophotometry, and the standard curve method was used for quantitative determination to determine the content of total flavonoids in the test solution.

[0015] The present invention also provides a method for preparing an anti-pharyngitis Chinese medicine compound extract, which is characterized in that the Chinese medicine compound extract is prepared by a double water extraction method, and the content and transfer rate of irisin, arctiin and total flavonoids in the obtained Chinese medicine compound extract are detected by the quality detection method of the anti-pharyngitis Chinese medicine compound extract according to any one of claims 1 to 9, and the double water extraction method is finally determined to specifically include: soaking in 8 to 9 times the amount of water for 30 to 40 minutes and then decocting for 1 to 2 hours in the first water extraction; and decocting in 5 to 7 times the amount of water for the second water extraction for 1 to 2 hours.

[0016] As described above, the quality detection method and extraction method of the anti-pharyngitis Chinese medicine compound extract of the present invention have the following beneficial effects:

[0017] The method for detecting the quality of the Chinese herbal compound extract of the present invention can accurately determine the contents of irisin, arctiin and total flavonoids contained in the Chinese herbal compound extract to control the quality of the Chinese herbal compound extract. The method is simple, accurate and rapid and has good application prospects.

[0018] The high-performance liquid chromatography method adopted in the present invention can simultaneously determine the contents of irisin and arctiin at one wavelength, and can quantitatively determine the content of total flavonoids by using ultraviolet-visible spectrophotometry with rutin as a standard. The determination method is simple, highly sensitive, and has good reproducibility, and can be used for quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The chromatogram showing the specificity of the HPLC determination of Jindengshangen Decoction in Example 1 of the present invention is shown; wherein A is the mixed reference solution; B is the test solution; C is the Belamcanda chinensis negative reference solution; and D is the stir-fried Burdock Fructus negative reference solution. DETAILED DESCRIPTION

[0020] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0021] Please refer to the accompanying drawings. It should be noted that the illustrations provided in this embodiment are merely schematic illustrations of the basic concept of the present invention. Therefore, the drawings only show components relevant to the present invention and are not drawn according to the number, shape, and size of components in actual implementation. In actual implementation, the type, quantity, and proportion of each component may be varied arbitrarily, and the component layout may also be more complex.

[0022] The first aspect of the present invention provides a quality detection method for an anti-pharyngitis Chinese medicine compound extract, wherein the anti-pharyngitis Chinese medicine compound comprises the following components: Chinese lantern, Belamcanda chinensis, stir-fried burdock fruit, Sophora flavescens root, Licorice root, and Platycodon grandiflorum; the quality detection method is as follows:

[0023] Step 1: Use high performance liquid chromatography to perform qualitative and quantitative analysis, specifically including the following steps:

[0024] 11) Preparation of test solution: Weigh the components of the anti-pharyngitis Chinese herbal compound, mix them, and prepare a Chinese herbal compound extract using a water extraction method; dissolve the Chinese herbal compound extract in a solvent, centrifuge, and dilute the supernatant to obtain a test solution;

[0025] 12) Preparation of Reference Solution: Weigh and dissolve the standard solutions of irisin and arctiin, respectively, then mix and dilute the two solutions to prepare the first reference solution;

[0026] 13) Preparation of negative control solution: Prepare a Chinese herbal compound extract not containing Belamcanda chinensis by the water extraction method in step 11), add a solvent to dissolve, centrifuge, collect the supernatant, and dilute to obtain a Belamcanda chinensis negative control solution; prepare a Chinese herbal compound extract not containing stir-fried Arctium lappa by the water extraction method in step 11), add a solvent to dissolve, centrifuge, collect the supernatant, and dilute to obtain a stir-fried Arctium lappa negative control solution;

[0027] 14) Detection: The test solution, the first reference solution, and the negative control solution were assayed by high performance liquid chromatography (HPLC). The retention times were compared for qualitative analysis, and the irisin and arctiin in the test solution were quantified using a standard curve method.

[0028] Step 2: Quantitative analysis is performed using UV-visible spectrophotometry, specifically comprising the following steps:

[0029] 21) Preparation of test solution: Prepare a Chinese herbal compound extract using the water extraction method in step 11); weigh the Chinese herbal compound extract, dissolve it in a solvent, sonicate, centrifuge, and dilute the supernatant to obtain a test solution;

[0030] 22) Preparation of reference solution: Weigh rutin standard and prepare a second reference solution;

[0031] 23) The test solution and the second reference solution were measured by UV-visible spectrophotometry, and the standard curve method was used for quantitative determination to determine the content of total flavonoids in the test solution.

[0032] The CAS number of the irisin is 491-74-7, the CAS number of the arctiin is 20362-31-6, and the CAS number of the rutin is 153-18-4.

[0033] Fragrant Lantern is the dried persistent calyx or the persistent calyx with fruit of Physalis alkekengi L. var. franchetii (Mast.) Makino, a plant of the Solanaceae family. It is bitter and cold in nature and enters the Lung meridian. It has the effects of clearing heat and detoxifying, relieving sore throat and resolving phlegm, and promoting diuresis and relieving stranguria. It is primarily used to treat sore throat, hoarseness, cough due to phlegm and heat, dysuria, and painful stranguria caused by heat.

[0034] Stir-fried burdock seeds are prepared by stir-frying the dried, mature fruits of Arctium lappa L., a plant of the Asteraceae family. They are pungent, bitter, and cold in nature, and enter the lung and stomach meridians. They have the effects of dispersing wind-heat, clearing the lungs and relieving rashes, and detoxifying and reducing swelling. They are primarily used to treat wind-heat colds, coughs with excessive sputum, measles, rubella, sore throats, mumps, erysipelas, and carbuncles and sores.

[0035] Belamcanda chinensis (L.) DC. is the dried rhizome of the Iridaceae plant. It is bitter and cold in nature. It enters the Lung meridian. It clears heat and detoxifies, eliminates phlegm, and relieves sore throats. It is used to treat heat, phlegm, and fire stagnation, sore throat, excessive phlegm, and cough and asthma.

[0036] Sophora tonkinensis (Sophora tonkinensis Gagnep) is a plant of the genus Sophora in the Fabaceae family. It is bitter and cold in nature and enters the lung and stomach meridians. It clears heat and detoxifies, relieves sore throats, and reduces swelling. It is primarily used to treat sore throats and swollen gums.

[0037] Platycodon grandiflorum (Jacq.) A.DC., a plant of the Campanulaceae family, is a dried root. It is bitter, pungent, and neutral in nature. It enters the Lung meridian, clearing the lungs, dispelling phlegm, and draining pus.

[0038] Licorice is the dried root and rhizome of Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., or Glycyrrhiza glabra L. (all belonging to the Leguminosae family). It is neutral in nature and sweet in taste. It enters the Heart, Stomach, Spleen, and Lung meridians. It tonifies the spleen and replenishes Qi, relieves cough and expectoration, relieves pain, and harmonizes the medicinal properties.

[0039] In some embodiments of the present invention, the water extraction method in step 11) specifically comprises the following steps:

[0040] The Chinese medicine compound components of Jin Deng Lan, Belamcanda chinensis, fried Burdock Fruit, Sophora flavescens, Licorice and Platycodon grandiflorum were weighed according to the mass ratio of 8-10:5-7:8-10:5-7:2-4:3.5-5.5, and the mass ratio of each component was preferably 9:6:9:6:3:4.5. Water of 7-9 times, 7-8 times or 8-9 times the total mass of the Chinese medicine compound components was added, and the mixture was soaked at room temperature for 20-40 min, 20-25 min and 25 ~30min, 30~35min or 35~40min, decocted for 1~2h, 1~1.5h or 1.5~2h, filtered with gauze, added 5~7 times, 5~6 times or 6~7 times the amount of water (i.e. 5~7 times the total mass of the components of the traditional Chinese medicine compound), decocted for 1~2h, 1~1.5h or 1.5~2h, filtered, combined the filtrate, concentrated under reduced pressure, and dried to constant weight to obtain the traditional Chinese medicine compound extract.

[0041] In some embodiments of the present invention, the solvent in step 11) is 40-60% methanol. For example, it is 40-45% methanol, 45-50% methanol, 50-55% methanol, or 55-60% methanol. Preferably, it is 50% methanol, wherein the 50% methanol is a 50% by volume methanol-water solution.

[0042] In some embodiments of the present invention, the centrifugal speed in step 11) is 12,000 to 16,000 r / min, for example, 12,000 to 13,000 r / min, 13,000 to 14,000 r / min, 14,000 to 15,000 r / min, or 15,000 to 16,000 r / min. Preferably, the centrifugal speed is 14,000 r / min.

[0043] In some embodiments of the present invention, the centrifugation time in step 11) is 4 to 7 minutes, for example, 4 to 5 minutes, 5 to 6 minutes, or 6 to 7 minutes. Preferably, the centrifugation time is 5 minutes.

[0044] In some embodiments of the present invention, the dilution factor in step 11) is 8 to 13 times, for example, 8 to 9 times, 9 to 10 times, 10 to 11 times, 11 to 12 times, or 12 to 13 times. Preferably, the dilution factor is 10 times.

[0045] In some embodiments of the present invention, the first reference solution in step 12) is a mixed reference solution of irisin and arctiin.

[0046] Preferably, the solvent of the mixed reference solution is 40-60% methanol, for example, 40-45% methanol, 45-50% methanol, 50-55% methanol, or 55-60% methanol. Preferably, it is 50% methanol, wherein the 50% methanol is a 50% by volume methanol-water solution.

[0047] Preferably, the concentration of the mixed reference solution is 0.2-0.4 mg of irisin and 2.3-2.7 mg of arctiin per 1 mL of solution. For example, 0.2-0.25 mg, 0.25-0.3 mg, 0.3-0.35 mg, or 0.35-0.4 mg of irisin per 1 mL of solution; for example, 2.3-2.4 mg, 2.4-2.5 mg, 2.5-2.6 mg, or 2.6-2.7 mg of arctiin per 1 mL of solution; preferably, 0.3 mg of irisin and 2.5 mg of arctiin per 1 mL of solution.

[0048] In some embodiments of the present invention, the HPLC method in step 14) is performed using a Discovery C 18, 25cm×4.6mm, 5μm; injection volume 4-6μL, preferably 5μL; column temperature 25-40℃, 25-30℃, 30-35℃ or 35-40℃, preferably 40℃; flow rate 0.8-1.3mL / min, 0.8-0.9mL / min, 0.9-1.0mL / min, 1.0-1.1mL / min, 1.1-1.2mL / min or 1.2-1.3mL / min, preferably 1.0mL / min; acetonitrile as mobile phase A, and 1-3mmol / L ammonium acetate / 0.05-0.2% acetic acid aqueous solution as mobile phase B, Mobile phase B can be 1-2 mmol / L ammonium acetate / 0.05-0.2% acetic acid aqueous solution, 2-3 mmol / L ammonium acetate / 0.05-0.2% acetic acid aqueous solution, 1-3 mmol / L ammonium acetate / 0.05-0.1% acetic acid aqueous solution, 1-3 mmol / L ammonium acetate / 0.1-0.15% acetic acid aqueous solution or 1-3 mmol / L ammonium acetate / 0.15-0.2% acetic acid aqueous solution; in a preferred embodiment of the present invention, mobile phase B is 2 mmol / L ammonium acetate / 0.1% acetic acid aqueous solution, wherein 0.1% acetic acid is 0.1% acetic acid aqueous solution by volume.

[0049] The gradient elution program is as follows:

[0050] 0-14 min, 80% → 80% mobile phase B, 20% → 20% mobile phase A;

[0051] 14-18 min, 80% → 75% mobile phase B, 20% → 25% mobile phase A;

[0052] 18-23 min, 75% → 70% mobile phase B, 25% → 30% mobile phase A;

[0053] 23-33 min, 70% → 70% mobile phase B, 30% → 30% mobile phase A;

[0054] 33-33.1 min, 70% → 10% mobile phase B, 30% → 90% mobile phase A;

[0055] 33.1-38 min, 10% → 10% mobile phase B, 90% → 90% mobile phase A;

[0056] 38-38.1 min, 10% → 80% mobile phase B, 90% → 20% mobile phase A;

[0057] 38.1~48min, 80%→80% mobile phase B, 20%→20% mobile phase A.

[0058] In some embodiments of the present invention, the quantification of irisin and arctiin by the standard curve method in step 14) comprises the following steps:

[0059] A) preparing a series of first reference solutions of different concentrations of irisin and arctiin standards;

[0060] B) subjecting a series of first reference substance solutions of different concentrations obtained in step A) to high performance liquid chromatography to obtain a linear relationship between the chromatographic peak area of ​​at least one component of irisin and arctiin and the concentration of the corresponding component, plotting a corresponding standard working curve, and calculating the regression equation of the standard working curve of at least one component of irisin and arctiin;

[0061] C) subjecting the test solution to high performance liquid chromatography (HPLC) and substituting the obtained chromatographic peak area of ​​at least one of irisin and arctiin into the regression equation of the standard working curve of the corresponding component in step B) to calculate the content of the corresponding component in the test solution.

[0062] In some embodiments of the present invention, the solvent in step 21) is 50-70% ethanol. For example, it is 50-55% ethanol, 55-60% ethanol, 60-65% ethanol, or 65-70% ethanol. Preferably, it is 60% ethanol, which is a 60% by volume ethanol-water solution.

[0063] In some embodiments of the present invention, the ultrasonication time in step 21) is 20 to 40 minutes, for example, 20 to 25 minutes, 25 to 30 minutes, 30 to 35 minutes, or 35 to 40 minutes. Preferably, the ultrasonication time is 30 minutes.

[0064] In some embodiments of the present invention, the centrifugal speed in step 21) is 8000-12000 r / min, for example, 8000-9000 r / min, 9000-10000 r / min, 10000-11000 r / min, or 11000-12000 r / min. Preferably, the centrifugal speed is 10000 r / min.

[0065] In some embodiments of the present invention, the centrifugation time in step 21) is 8 to 12 minutes, for example, 8 to 9 minutes, 9 to 10 minutes, 10 to 11 minutes, or 11 to 12 minutes. Preferably, the centrifugation time is 10 minutes.

[0066] In some embodiments of the present invention, the dilution factor in step 21) is 9 to 13 times, for example, 9 to 10 times, 10 to 11 times, 11 to 12 times, 12 to 12.5 times, or 12.5 to 13 times. Preferably, the dilution factor is 12.5 times.

[0067] The preparation method of the test solution in step 21) specifically includes: preparing a Chinese herbal compound extract by the water extraction method in step 11), weighing 4-6 g of the Chinese herbal compound extract, placing it in a conical flask, adding 15-30 mL of 50-70% ethanol solution, ultrasonically extracting for 20-40 min, centrifuging at 8000-12000 r / min, taking the supernatant into a volumetric flask, adding 50-70% ethanol solution to the scale, and obtaining a test solution.

[0068] In some embodiments of the present invention, the solvent of the second reference substance solution in step 22) is 50-70% ethanol. For example, it is 50-55% ethanol, 55-60% ethanol, 60-65% ethanol, or 65-70% ethanol. Preferably, it is 60% ethanol, wherein the 60% ethanol is a 60% by volume ethanol-water solution.

[0069] In some embodiments of the present invention, the concentration of the second reference substance solution in step 22) is 0.8-1.2 mg / mL, for example, 0.8-0.9 mg / mL, 0.9-1.0 mg / mL, 1.0-1.1 mg / mL, or 1.1-1.2 mg / mL. Preferably, the concentration of the second reference substance solution is 1.0 mg / mL.

[0070] In some embodiments of the present invention, the UV-visible spectrophotometry in step 23) includes a sample pretreatment step, and the sample pretreatment specifically includes: taking the test solution, adding 50-70%, 50-60% or 60-70% ethanol, 4-6%, 4-5% or 5-6% sodium nitrite solution in sequence, mixing, letting stand, adding 8-12%, 8-9%, 9-10%, 10-11% or 11-12% aluminum nitrate solution, mixing, letting stand, adding 3-5%, 3-4% or 4-5% sodium hydroxide solution, adding 50-70%, 50-60% or 60-70% ethanol to make up the volume, mixing, letting stand, and measuring the absorbance at 510 nm using a UV-visible spectrophotometer.

[0071] Preferably, the test solution is sequentially added with 60% ethanol and 5% sodium nitrite solution, mixed and allowed to stand, 10% aluminum nitrate solution is added, mixed and allowed to stand, 4% sodium hydroxide solution is added, 60% ethanol is added to the volume, mixed and allowed to stand, and the absorbance is measured at 510 nm using a UV-visible spectrophotometer.

[0072] In some embodiments of the present invention, the quantitative determination of the total flavonoid content in the test solution using a standard curve method in step 23) comprises the following steps:

[0073] a) preparing a series of second reference solutions with different concentrations of a rutin standard;

[0074] b) measuring a series of second reference substance solutions of different concentrations obtained in step a) using a UV-visible spectrophotometer to obtain a linear relationship between the absorbance of rutin and its concentration, plotting a corresponding standard working curve, and calculating a regression equation for the standard working curve of rutin;

[0075] c) measuring the absorbance of the test solution using a UV-visible spectrophotometer, substituting the obtained absorbance into the regression equation of the standard working curve in step b) to calculate the total flavonoid content in the test solution.

[0076] The second aspect of the present invention provides a method for preparing an anti-pharyngitis Chinese medicine compound extract, wherein the Chinese medicine compound extract is prepared by a double water extraction method, and the content of irisin, arctiin, and total flavonoids in the obtained Chinese medicine compound extract and the extract yield are detected by the quality detection method of the anti-pharyngitis Chinese medicine compound extract according to any one of claims 1 to 9. The double water extraction method is finally determined to specifically include: the first water extraction is to soak in 8 to 9 times the amount of water for 30 to 40 minutes, 30 to 35 minutes, or 35 to 40 minutes, and then decoct for 1 to 2 hours, 1 to 1.5 hours, or 1.5 to 2 hours; the second water extraction is to soak in 5 to 7 times, 5 to 6 times, or 6 to 7 times the amount of water for 1 to 2 hours, 1 to 1.5 hours, or 1.5 to 2 hours. In a preferred embodiment of the present invention, the double water extraction method specifically includes: the first water extraction is to soak in 8 times the amount of water for 30 minutes and then decoct for 2 hours; the second water extraction is to decoct in 6 times the amount of water for 1.5 hours.

[0077] The extract yield is the ratio of the weight of the extract to the total weight of the components of the traditional Chinese medicine compound.

[0078] Example 1 Qualitative and quantitative analysis by high performance liquid chromatography

[0079] 1. Preparation of the test solution: Weigh 100 ml of ginseng root, 100 ml of iris root, 100 ml of burdock root, 100 ml of radix schoenophorae, 100 ml of licorice root, and 100 ml of platycodon grandiflorum in a mass ratio of 9:6:9:6:3:4.5. Add 8 times the total mass of the compound components in water. Soak at room temperature for 30 minutes, then decoct for 2 hours. Filter through gauze, add 6 times the total mass of water to the residue, decoct for 2 hours, filter, and combine the filtrates. Concentrate under reduced pressure to obtain the compound extract. Dilute 200 μL of the compound extract with 1800 μL of 50% methanol. Centrifuge at 14,000 rpm for 5 minutes. Aspirate the supernatant to obtain the test solution and set aside.

[0080] 2. Preparation of reference solution: Accurately weigh irisin and arctiin, and prepare a first reference solution with 50% methanol to a concentration of 0.3 mg irisin and 2.5 mg arctiin per 1 mL of solution, and set aside.

[0081] 3. Preparation of negative control solution: Prepare a Chinese herbal compound extract without Belamcanda chinensis by the same method as in step 1 above, take 200 μL of the compound extract and add 1800 μL of 50% methanol to dilute it, centrifuge it at 14000 r / min for 5 minutes after dilution, and absorb the supernatant to obtain a Belamcanda chinensis negative control solution; prepare a Chinese herbal compound extract without stir-fried Burdock Fruit by the same method as in step 1 above, take 200 μL of the compound extract and add 1800 μL of 50% methanol to dilute it, centrifuge it at 14000 r / min for 5 minutes after dilution, and absorb the supernatant to obtain a stir-fried Burdock Fruit negative control solution.

[0082] 4. Detection: Separately aspirate the above test solution, reference solution, and negative control solution and perform determination according to the following HPLC conditions and gradient elution procedure:

[0083] The chromatographic column is Discovery C 18 , 25 cm × 4.6 mm, 5 μm; injection volume 5 μL, column temperature 30 ° C, flow rate 1.0 mL / min; acetonitrile as mobile phase A, 2 mmol / L ammonium acetate / 0.1% acetic acid aqueous solution as mobile phase B;

[0084] Gradient elution program:

[0085] 0-14 min, 80% → 80% mobile phase B, 20% → 20% mobile phase A;

[0086] 14-18 min, 80% → 75% mobile phase B, 20% → 25% mobile phase A;

[0087] 18-23 min, 75% → 70% mobile phase B, 25% → 30% mobile phase A;

[0088] 23-33 min, 70% → 70% mobile phase B, 30% → 30% mobile phase A;

[0089] 33-33.1 min, 70% → 10% mobile phase B, 30% → 90% mobile phase A;

[0090] 33.1-38 min, 10% → 10% mobile phase B, 90% → 90% mobile phase A;

[0091] 38-38.1 min, 10% → 80% mobile phase B, 90% → 20% mobile phase A;

[0092] 38.1~48min, 80%→80% mobile phase B, 20%→20% mobile phase A.

[0093] Depend on Figure 1The data show that the negative control solution contains no impurities at the corresponding positions of the mixed control solution, indicating that other ingredients in the compound do not interfere with the determination of irisin and arctiin. In the figure: A is the mixed control solution; B is the test solution; C is the Belamcanda chinensis negative control solution; D is the stir-fried Arctium lappa negative control solution.

[0094] Example 2HPLC linear relationship experiment

[0095] Take the first reference solution prepared in Example 1 and dilute it in equal proportion to obtain a series of concentrations (the concentrations of irisin are 4.69 μg / mL, 9.38 μg / mL, 18.75 μg / mL, 37.5 μg / mL, 75 μg / mL, 150 μg / mL, and 300 μg / mL, respectively; the concentrations of irisin are 39.06 μg / mL, 78.13 μg / mL, 156.25 μg / mL, 312.5 μg / mL, 625 μg / mL, 1250 μg / mL, and 2500 μg / mL, respectively). The peak areas of irisin and arctiin were determined according to the liquid chromatography conditions and gradient elution procedure of Example 1. The standard curves were drawn with the concentrations of irisin and arctiin as the abscissa (X) and the peak area of ​​the chromatographic peak as the ordinate (Y). As shown in Tables 1 and 2:

[0096] Table 1 Results of linear relationship investigation of irisin

[0097]

[0098] Table 2 Results of investigation on the linear relationship of arctiin

[0099]

[0100] The data in Tables 1 and 2 show that irisin has a good linear relationship in the range of 4.69 μg / mL to 300 μg / mL (Y=9.34X+15.52, r=0.999), and arctiin has a good linear relationship in the range of 39.06 μg / mL to 2500 μg / mL (Y=2.42X+12.13, r=0.999).

[0101] Example 3 HPLC precision experiment

[0102] The precision of the instrument was evaluated by testing intra-day and inter-day precision. The first reference solution prepared according to Example 1 was diluted to 75 μg / mL irisin and 625 μg / mL arctiin. Samples were injected for determination. Intra-day precision was achieved by injecting the same concentration of the mixed reference solution six times on the same day. Inter-day precision was achieved by injecting the same concentration of the reference solution five times over three consecutive batches. Liquid chromatography conditions were the same as in Example 1. Peak areas were used to calculate the relative standard deviations (RSDs) of the peak areas of irisin and arctiin, respectively. The results are shown in Tables 3 and 4.

[0103] Table 3 Intra-day precision test results

[0104]

[0105] Table 4 Results of inter-day precision experiments

[0106]

[0107] The above results show that the intra-day RSDs of irisin and arctiin are 0.16% and 0.14%, respectively, and the inter-day RSDs of irisin and arctiin are 2.22% and 2.33%, respectively, indicating that the instrument has good precision.

[0108] Example 4 HPLC stability test

[0109] The test solution was prepared according to the method of Example 1, and the samples were injected and analyzed at different times. The liquid chromatography conditions were the same as those of Example 1. The peak areas of irisin and arctiin at different times were recorded, and the RSDs were calculated. The results are shown in Table 5.

[0110] Table 5 Stability test results

[0111]

[0112]

[0113] The results in Table 5 show that the RSDs of irisin and arctiin were 2.51% and 2.27%, respectively, indicating that the test solution had good stability within 12 h.

[0114] Example 5 HPLC repeatability experiment

[0115] According to the preparation method of the test solution in Example 1, 5 test solutions were prepared in parallel, and the samples were injected and analyzed separately. The liquid chromatography determination conditions were the same as in Example 1. The peak areas of irisin and arctiin were recorded, and the RSDs were calculated. The results are shown in Table 6.

[0116] Table 6 Repeatability test results

[0117]

[0118] The data in Table 6 showed that the RSDs of irisin and arctiin were 2.39% and 1.86%, respectively, indicating that the method had good repeatability.

[0119] Example 6 Sample recovery experiment

[0120] Take 6 parts of the traditional Chinese medicine compound extract with known irisin and arctiin content, add a certain amount of irisin and arctiin respectively within the linear range, a total of 6 parts, the liquid chromatography determination conditions are the same as in Example 1, the liquid chromatography determination conditions are the same as in Example 1, record the peak area, and calculate the sample recovery rate. The results are shown in Table 7.

[0121] Table 7 Sample recovery experimental results

[0122]

[0123]

[0124] The results showed that the recoveries of irisin and arctiin were 97.76% and 99.24% respectively, and the RSDs were 0.46% and 0.26% respectively, indicating that the quality detection method of the present invention has a good recovery rate.

[0125] Example 7 UV-spectrophotometry sample processing

[0126] 1. Preparation of the test solution: The same method as in step 1 of Example 1 was used to prepare the Chinese herbal compound extract, and the Chinese herbal compound extract was further dried in an oven at 55°C to constant weight. 5.0 g of the extract powder dried to constant weight was accurately weighed and placed in a 50 mL conical flask. 20 mL of 60% ethanol was added to dissolve the mixture, ultrasonicated for 30 min, and centrifuged at 10,000 rpm for 10 min. 0.8 mL of the supernatant was accurately transferred to a 5 mL volumetric flask and the volume was adjusted to the mark to obtain the test solution.

[0127] 2. Preparation of reference solution: Accurately weigh 5.00 mg of rutin reference substance into a 5 mL volumetric flask and dissolve it in 60% ethanol to make up to volume to prepare a 1 mg / mL reference solution.

[0128] 3. Take 500 μL of the test solution in a 5 mL volumetric flask, add 1.5 mL of 60% ethanol and 0.5 mL of 5% sodium nitrite solution, mix well, let stand for 6 minutes, add 0.5 mL of 10% aluminum nitrate solution, mix well, let stand for 6 minutes, add 2.0 mL of 4% sodium hydroxide solution, add 60% ethanol to make up to volume, mix well, let stand for 15 minutes, and measure the absorbance at 510 nm using a UV-visible spectrophotometer.

[0129] Example 8 UV-spectrophotometry linear relationship experiment

[0130] Accurately measure 0.10, 0.15, 0.20, 0.30, and 0.40 mL of the rutin reference solution prepared in Example 7 into a 5 mL volumetric flask. Measure the absorbance according to the experimental method in step 3 of Example 7. A standard curve is plotted with rutin concentration as the abscissa (X) and absorbance as the ordinate (Y). The results are shown in Table 8.

[0131] Table 8 Linear relationship investigation results of rutin

[0132]

[0133] The results in Table 8 show that the standard rutin is linearly correlated in the range of 20 μg / mL to 80 μg / mL, and the standard curve is Y=0.006X+0.002 (r=0.999)

[0134] Example 9 UV-Vis Spectrophotometry Precision Experiment

[0135] The absorbance of the reference solution prepared in Example 7 was measured according to the experimental method in step 3 of Example 7. The absorbance was measured six times in a row. The results are shown in Table 9.

[0136] Table 9 Precision test results

[0137]

[0138] The RSD of the total flavonoids content calculated from the data in Table 9 was 1.08%, which showed that the instrument had good precision.

[0139] Example 10 UV-Visible Spectrophotometry Stability Test

[0140] The test solution was prepared according to the method of Example 7, pretreated according to the experimental method of step 3 of Example 7, and then placed in the dark for 0, 20, 40, 60, and 80 minutes before measuring the absorbance. The results are shown in Table 10.

[0141] Table 10 Stability test results

[0142]

[0143] The RSD of the total flavonoids content calculated from the data in Table 10 was 2.39%, which showed that the method had good stability.

[0144] Example 11 UV-Visible Spectrophotometry Repeatability Experiment

[0145] The test solution was prepared according to the method of Example 7 and pretreated according to the experimental method of step 3 of Example 7. Six samples were prepared in parallel and the absorbance value of the total flavonoid content was measured. The results are shown in Table 11.

[0146] Table 11 Repeatability test results

[0147]

[0148] From the data in Table 11, we can see that the RSD of the total flavonoids content is 2.75%, which shows that the method has good repeatability.

[0149] Example 12 Optimization of preparation process

[0150] Single-factor experiment

[0151] Weigh each piece of Chinese lantern (9g), belamcanda (6g), fried burdock fruit (9g), sophora flavescens (6g), liquorice (3g), and platycodon (4.5g), add 8 times the amount of water, soak for 0.5h, and decoct once, twice, and three times (in triplicate), each time for 1h with 8 times the amount of water. Filter through gauze, combine the decoctions, and concentrate to 1g crude drug / mL (1g crude drug / mL means that the concentrate is concentrated to the same volume as the crude drug, for example, 30g crude drug, and finally concentrated to 30mL of extract). The contents of irisin and arctiin were determined by HPLC. The contents of irisin and arctiin per gram of crude drug in the Chinese herbal compound extract and the transfer rate are shown in Table 12 (the transfer rate is the ratio of the contents of irisin and arctiin per gram of crude drug in the Chinese herbal compound extract to the contents of irisin and arctiin per gram of crude drug in the original medicinal material. The contents of irisin and arctiin in the original medicinal material are 73.80 mg / g and 701.88 mg / g, respectively).

[0152] Table 12 Results of investigation on decoction times

[0153]

[0154] Combined with Table 12, taking into account the transfer rate of each indicator component and production cost, the subsequent experiments finally chose to decoct twice.

[0155] Example 13 Optimization of preparation process

[0156] Comprehensive experiment

[0157] The extraction time and solvent amount, which have a great influence on the water extraction effect, were taken as the investigation factors of the comprehensive experiment. 9 (3 4 ) Comprehensive experimental table, such as Table 13.

[0158] Table 13 Comprehensive experimental factor level table

[0159]

[0160]

[0161] Weigh 9 portions of each slice in parallel according to the ratio, and9 (3 4 ) Arrange the experiment using the comprehensive experimental table. After two decoctions, combine the filtrates and concentrate them to 1 g crude drug / mL to obtain each sample solution.

[0162] The extract sample of 1 g crude drug / mL was dried in an oven at 80°C to obtain an extract, and the extract yield was calculated as the weight of the extract / the total amount of the crude drug; the content of irisin and arctiin was determined by the HPLC method in Example 1, and the transfer rate of irisin and arctiin extraction was calculated according to the method in Example 12; the content of total flavonoids was determined by UV-visible spectrophotometry.

[0163] The calculation was based on the formula D = (I × 0.2) + (II × 0.3) + (III × 0.5), where D represents the evaluation index, and I, II, and III represent the extract yield, total flavonoid content, and the transfer rates of irisin and arctiin, respectively. The transfer rates of irisin and arctiin were weighted, using the formula: W = (a × 0.5) + (b × 0.5), where W represents the final active ingredient transfer rate, a represents the transfer rate of irisin, and b represents the transfer rate of arctiin. The results of the visual analysis are shown in Table 14; the results of the variance analysis are shown in Table 15.

[0164] Table 14L9(3 4 ) Comprehensive experimental intuitive analysis table

[0165]

[0166] Table 15 Variance analysis table

[0167]

[0168] Note F 0.05(2,2) =19

[0169] Taking the evaluation index as the calculated value, it can be seen from the intuitive analysis results in Table 14 that the primary and secondary relationship (R) of the three factors is A>B>A*B, that is, solvent amount>extraction time>interaction between extraction time and solvent amount; from the variance analysis results in Table 15, it can be seen that the two factors have no significant effect on the extraction effect. Therefore, according to the intuitive analysis results and combined with the actual production situation, the optimal extraction process is A2B2A*B1, that is, extraction twice, the first time is soaked in 8 times the amount of water for half an hour and then decocted for 2 hours, and the second time is decocted in 6 times the amount of water for 1.5 hours.

[0170] Example 14 Optimization of preparation process

[0171] Extraction process validation experiment

[0172] The verification experiment was carried out with the optimal process conditions, and the results are shown in 16.

[0173] Table 16 Verification experimental results

[0174]

[0175] From the data in Table 16, we can see that the optimized process has good repeatability, is stable and reliable, and can achieve good extraction effects.

[0176] In summary, the quality detection method of the anti-pharyngitis Chinese medicine compound extract of the present invention established an analytical method for qualitatively and quantitatively determining irisin and arctiin by high performance liquid chromatography (HPLC) and quantitatively determining total flavonoids by ultraviolet-visible spectrophotometry; further, the extraction time and solvent amount, which have a greater impact on the water extraction effect by high performance liquid chromatography and ultraviolet-visible spectrophotometry, were used as the investigation factors of the comprehensive experiment, and L9(3 4 ) experimental table, using the extract yield, total flavonoid content in the aqueous extract, and the transfer rate of irisin and arctiin as indicators, to optimize the extraction process of an anti-pharyngitis Chinese herbal compound extract. The experimental method of the present invention is simple, feasible, highly specific, and reproducible, and can also be used to optimize the extraction process of an anti-pharyngitis Chinese herbal compound extract. Therefore, the present invention fills a gap in existing quality evaluation methods and has high industrial application value.

[0177] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A quality detection method for an anti-pharyngitis Chinese medicine compound extract, characterized in that: The anti-pharyngitis Chinese medicine compound comprises the following components: Chinese lantern, Belamcanda chinensis, stir-fried burdock fruit, Sophora flavescens root, Licorice root, and Platycodon grandiflorum; the quality inspection method is as follows: Step 1: Use high performance liquid chromatography to perform qualitative and quantitative analysis, specifically including the following steps: 11) Preparation of test solution: Weigh the components of the anti-pharyngitis Chinese herbal compound, mix them, and prepare a Chinese herbal compound extract using a water extraction method; dissolve the Chinese herbal compound extract in a solvent, centrifuge, and dilute the supernatant to obtain a test solution; 12) Preparation of Reference Solution: Weigh and dissolve the standard solutions of irisin and arctiin, respectively, then mix and dilute the two solutions to prepare the first reference solution; 13) Preparation of negative control solution: Prepare a Chinese herbal compound extract not containing Belamcanda chinensis by the water extraction method in step 11), add a solvent to dissolve, centrifuge, and dilute the supernatant to obtain a Belamcanda chinensis negative control solution; prepare a Chinese herbal compound extract not containing stir-fried Arctium lappa by the water extraction method in step 11), add a solvent to dissolve, centrifuge, and dilute the supernatant to obtain a stir-fried Arctium lappa negative control solution; 14) Detection: The test solution, the first reference solution, and the negative control solution were assayed by high performance liquid chromatography (HPLC). The retention times were compared for qualitative analysis, and the irisin and arctiin in the test solution were quantified using a standard curve method. Step 2: Quantitative analysis is performed using UV-visible spectrophotometry, specifically comprising the following steps: 21) Preparation of test solution: Prepare a Chinese herbal compound extract using the water extraction method in step 11); weigh the Chinese herbal compound extract, dissolve it in a solvent, sonicate, centrifuge, and dilute the supernatant to obtain a test solution; 22) Preparation of reference solution: Weigh rutin standard and prepare a second reference solution; 23) The test solution and the second reference solution were measured by UV-visible spectrophotometry, and the standard curve method was used for quantitative determination to determine the content of total flavonoids in the test solution.

2. The quality detection method of the anti-pharyngitis Chinese medicine compound extract according to claim 1, characterized in that: The water extraction method in step 11) specifically comprises the following steps: The Chinese medicinal compound components of Jin Deng Zhang, Belamcanda chinensis, stir-fried Arctium lappa, Sophora flavescens, Licorice root and Platycodon grandiflorum were weighed in a mass ratio of 8-10:5-7:8-10:5-7:2-4:3.5-5.5, and 7-9 times the total mass of the Chinese medicinal compound components of water were added. The mixture was soaked at room temperature for 20-40 minutes and then decocted for 1-2 hours. After filtering with gauze, 5-7 times the amount of water was added to the residue, and the mixture was decocted for 1-2 hours. The mixture was filtered, the filtrate was combined, concentrated under reduced pressure, and dried to constant weight to obtain a Chinese medicinal compound extract.

3. The quality detection method of the anti-pharyngitis Chinese medicine compound extract according to claim 1, characterized in that: The solvent in step 11) is 40-60% methanol; And / or, the centrifugal speed in step 11) is 12000-16000 r / min; And / or, the centrifugation time in step 11) is 4 to 7 minutes; And / or, the dilution multiple in step 11) is 8 to 13 times.

4. The quality detection method of the anti-pharyngitis Chinese medicine compound extract according to claim 1, wherein The solvent of the first reference solution is 40-60% methanol; And / or, the concentration of the first reference solution is 0.2-0.4 mg of irisin and 2.3-2.7 mg of arctiin per 1 mL of solution.

5. The quality detection method of the anti-pharyngitis Chinese medicine compound extract according to claim 1, characterized in that: The high performance liquid chromatography method in step 14) is carried out using a Discovery C 18 , 25cm×4.6mm, 5μm; injection volume 4-6μL, column temperature 25-40℃, flow rate 0.8-1.3mL / min; acetonitrile as mobile phase A, 1-3mmol / L ammonium acetate / 0.05-0.2% acetic acid aqueous solution as mobile phase B, gradient elution program as follows: 0-14 min, 80% → 80% mobile phase B, 20% → 20% mobile phase A; 14-18 min, 80% → 75% mobile phase B, 20% → 25% mobile phase A; 18-23 min, 75% → 70% mobile phase B, 25% → 30% mobile phase A; 23-33 min, 70% → 70% mobile phase B, 30% → 30% mobile phase A; 33-33.1 min, 70% → 10% mobile phase B, 30% → 90% mobile phase A; 33.1-38 min, 10% → 10% mobile phase B, 90% → 90% mobile phase A; 38-38.1 min, 10% → 80% mobile phase B, 90% → 20% mobile phase A; 38.1~48min, 80%→80% mobile phase B, 20%→20% mobile phase A.

6. The quality detection method of the anti-pharyngitis Chinese medicine compound extract according to claim 1, characterized in that: The step 14) of quantifying irisin and arctiin by the standard curve method comprises the following steps: A) preparing a series of first reference solutions of different concentrations of irisin and arctiin standards; B) subjecting a series of first reference substance solutions of different concentrations obtained in step A) to high performance liquid chromatography to obtain a linear relationship between the chromatographic peak area of ​​at least one component of irisin and arctiin and the concentration of the corresponding component, plotting a corresponding standard working curve, and calculating the regression equation of the standard working curve of at least one component of irisin and arctiin; C) subjecting the test solution to high performance liquid chromatography (HPLC) and substituting the obtained chromatographic peak area of ​​at least one component of irisin and arctiin into the regression equation of the standard working curve of the corresponding component in step B) to calculate the content of the corresponding component in the test solution.

7. The quality detection method of the anti-pharyngitis Chinese medicine compound extract according to claim 1, characterized in that: The solvent in step 21) is 50-70% ethanol; And / or, the ultrasonication time in step 21) is 20 to 40 minutes; And / or, the centrifugal speed in step 21) is 8000-12000 r / min; And / or, the centrifugation time in step 21) is 8 to 12 minutes; And / or, the dilution multiple in step 21) is 9 to 13 times.

8. The quality detection method of the anti-pharyngitis Chinese medicine compound extract according to claim 1, characterized in that: In step 22), the solvent of the second reference substance solution is 50-70% ethanol; And / or, in step 22), the concentration of the second reference substance solution is 0.8-1.2 mg / mL.

9. The quality detection method of the anti-pharyngitis Chinese medicine compound extract according to claim 1, characterized in that: The UV-visible spectrophotometry method in step 23) includes a sample pretreatment step, and the sample pretreatment specifically includes: taking the test sample solution, adding 50-70% ethanol and 4-6% sodium nitrite solution in sequence, mixing, letting it stand, adding 8-12% aluminum nitrate solution, mixing, letting it stand, adding 3-5% sodium hydroxide solution, adding 50-70% ethanol to make up the volume, mixing, letting it stand, and measuring the absorbance at 510 nm using a UV-visible spectrophotometer.

10. A method for preparing an anti-pharyngitis Chinese medicine compound extract, characterized in that: A two-water extraction method is used to prepare a Chinese herbal compound extract, and the quality detection method of the anti-pharyngitis Chinese herbal compound extract as described in any one of claims 1 to 9 is used to detect the content of irisin, arctiin, and total flavonoids in the obtained Chinese herbal compound extract and the extract yield. It is finally determined that the two-water extraction method specifically includes: the first water extraction uses 8 to 9 times the amount of water to soak for 30 to 40 minutes and then boil for 1 to 2 hours; the second water extraction uses 5 to 7 times the amount of water to boil for 1 to 2 hours.

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