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

Abstract

The application provides a quality detection method of an anti-pharyngitis traditional Chinese medicine compound extract, adopts high performance liquid chromatography to qualitatively and quantitatively analyze iriflorend and arctiin, adopts ultraviolet-visible spectrophotometry to quantitatively analyze total flavones, further comprehensively evaluates indexes of the contents of iriflorend and arctiin, the content of total flavones and the yield of the extract, investigates extraction time and solvent amount which have great influence on water extraction effect, and optimizes the extraction process of the anti-pharyngitis traditional Chinese medicine compound extract; the high performance liquid chromatography and the ultraviolet-visible spectrophotometry have good linear relationship, high precision, good repeatability and accurate determination results, and the combination of qualitative and quantitative analysis lays a foundation for the establishment of the quality standard of the anti-pharyngitis traditional Chinese medicine compound extract.

Description

TECHNICAL FIELD

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

[0002] Acute pharyngitis is a common and frequently-occurring disease in clinical otolaryngology, and its incidence accounts for 7-17% of laryngeal diseases. Clinically, it is an acute inflammation of the pharynx, which is mainly manifested by sore throat, pharyngeal redness, pharyngeal granular protrusion, non-obvious posterior nucleus swelling, and systemic wind-heat symptoms. If acute pharyngitis cannot be treated in time and effectively, it may cause chronic pharyngitis, chronic trachea and bronchitis, nephritis, heart disease, etc. Clinically, antibiotics or hormones are often used to treat acute pharyngitis. However, due to problems such as antibiotic resistance and side effects of hormone treatment, the current clinical treatment drugs are still insufficient, and there is an urgent need to research treatment drugs with good clinical efficacy and small side effects.

[0003] Traditional Chinese medicine, especially traditional Chinese medicine compound, has unique advantages in the prevention and treatment of acute pharyngitis. Traditional Chinese medicine compound has become an important drug for preventing and treating acute pharyngitis because it can regulate the acute pharyngitis pathogenesis network through multiple pathways, multiple targets and mutual influence of multiple components. Golden lamp mountain root soup: Jin Denglong, Shegan, Fried Niubangzi, Shandougen, Gancao and Jiegeng, has the effects of dispelling wind, reducing phlegm, clearing heat and detoxifying, and relieving swelling and soothing the throat. As a classic anti-pharyngitis traditional Chinese medicine compound, it has high development and application value. However, there is currently a lack of quality control method. In order to ensure the safety, stability, effectiveness and controllability of the compound drug extract, a simple, accurate and reliable method for quality detection needs to be found. SUMMARY

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

[0005] To achieve the above-mentioned purposes and other related purposes, the present application provides a quality detection method of an anti-pharyngitis traditional Chinese medicine compound extract, wherein the anti-pharyngitis traditional Chinese medicine compound comprises the following components: Jin Denglong, Shegan, Fried Niubangzi, Shandougen, Gancao and Jiegeng; and the quality detection method is as follows:

[0006] Step one, qualitative and quantitative analysis is carried out by high performance liquid chromatography, which specifically includes the following steps:

[0007] 11) Preparation of test solution: each component of the anti-pharyngitis traditional Chinese medicine compound is weighed, mixed, and then a traditional Chinese medicine compound extract is prepared by water extraction method; the traditional Chinese medicine compound extract is dissolved in a solvent, centrifuged, and the supernatant is diluted to obtain a test solution;

[0008] 12) Preparation of the control solution: the standard samples of tectorigenin and arctiin were weighed and dissolved respectively, then mixed and diluted to prepare the first control solution;

[0009] 13) Preparation of the negative control solution: the traditional Chinese medicine compound extract without Caulis Sargentodoxae was prepared by the water extraction method in step 11), and then dissolved in solvent, centrifuged, and the supernatant was diluted to obtain the negative control solution of Caulis Sargentodoxae; the traditional Chinese medicine compound extract without Fructus Arctii was prepared by the water extraction method in step 11), and then dissolved in solvent, centrifuged, and the supernatant was diluted to obtain the negative control solution of Fructus Arctii;

[0010] 14) Detection: the test sample solution, the first control solution and the negative control solution were determined by high performance liquid chromatography, the retention time was compared for qualitative analysis, and the tectorigenin and arctiin in the test sample solution were quantitatively analyzed by the standard curve method;

[0011] Step two, quantitative analysis by ultraviolet-visible spectrophotometry, specifically including the following steps:

[0012] 21) Preparation of the test sample solution: the traditional Chinese medicine compound extract was prepared by the water extraction method in step 11), and then the traditional Chinese medicine compound extract was weighed and dissolved in solvent, ultrasonically treated, centrifuged, and the supernatant was diluted to obtain the test sample solution;

[0013] 22) Preparation of the control solution: the standard sample of rutin was weighed to prepare the second control solution;

[0014] 23) The test sample solution and the second control solution were determined by ultraviolet-visible spectrophotometry, and the content of total flavonoids in the test sample solution was quantitatively analyzed by the standard curve method.

[0015] The application also provides a preparation method of the anti-pharyngitis traditional Chinese medicine compound extract, characterized in that the traditional Chinese medicine compound extract is prepared by twice water extraction, and the content and transfer rate of tectorigenin, arctiin and total flavonoids in the obtained traditional Chinese medicine compound extract are detected by the quality detection method of the anti-pharyngitis traditional Chinese medicine compound extract according to any one of claims 1-9, and the twice water extraction specifically includes: the first water extraction is performed by soaking in 8-9 times the amount of water for 30-40 min and then decocting for 1-2 h; and the second water extraction is performed by decocting with 5-7 times the amount of water for 1-2 h.

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

[0017] This invention provides a method for quality testing of extracts from traditional Chinese medicine compound formulas. It can accurately determine the content of irisin, arctiin, and total flavonoids in the extracts to control their quality. The method is simple, accurate, and rapid, and has good application prospects.

[0018] The high-performance liquid chromatography method used in this invention can simultaneously determine the contents of irisin and arctiin at a single wavelength. The total flavonoid content can be quantitatively determined using ultraviolet-visible spectrophotometry with rutin as a standard. The determination method is simple, highly sensitive, and reproducible, and can be used for its quality control. Attached Figure Description

[0019] Figure 1 The image shows the specific chromatogram of Jin Deng Shan Gen Tang determined by HPLC in Example 1 of this invention; wherein, A is a mixed reference solution; B is the test solution; C is the Belamcanda chinensis negative reference solution; and D is the stir-fried Arctium lappa negative reference solution. Detailed Implementation

[0020] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed 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 only schematic representations of the basic concept of the present invention. Therefore, the drawings only show the components related to the present invention and are not drawn according to the actual number, shape and size of the components in the actual implementation. In the actual implementation, the form, quantity and proportion of each component can be arbitrarily changed, and the layout of the components may also be more complex.

[0022] The first aspect of this invention provides a method for quality testing of an extract from a traditional Chinese medicine compound for treating pharyngitis. The traditional Chinese medicine compound for treating pharyngitis comprises the following components: *Physalis alkekengi*, *Belamcanda chinensis*, stir-fried *Arctium lappa*, *Sophora tonkinensis*, licorice, and *Platycodon grandiflorus*. The quality testing method is as follows:

[0023] Step 1: Qualitative and quantitative analysis is performed using high-performance liquid chromatography (HPLC), specifically including the following steps:

[0024] 11) Preparation of test solution: Weigh each component of the anti-pharyngitis traditional Chinese medicine compound, mix them and prepare the traditional Chinese medicine compound extract by water extraction; take the traditional Chinese medicine compound extract, add solvent to dissolve, centrifuge, take the supernatant and dilute to obtain the test solution;

[0025] 12) Preparation of reference solution: Weigh the standards of irisin and arctiin, dissolve them separately, mix and dilute the two solutions to prepare the first reference solution;

[0026] 13) Preparation of negative control solution: Prepare a traditional Chinese medicine compound extract without Belamcanda chinensis using the water extraction method in step 11), add solvent to dissolve, centrifuge, take the supernatant, and dilute to obtain the Belamcanda chinensis negative control solution; Prepare a traditional Chinese medicine compound extract without fried burdock seed using the water extraction method in step 11), add solvent to dissolve, centrifuge, take the supernatant, and dilute to obtain the fried burdock seed negative control solution;

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

[0028] Step 2: Quantitative analysis is performed using ultraviolet-visible spectrophotometry, which includes the following steps:

[0029] 21) Preparation of test solution: The traditional Chinese medicine compound extract was prepared by water extraction in step 11); the traditional Chinese medicine compound extract was weighed, dissolved in solvent, sonicated, centrifuged, and the supernatant was diluted to obtain the test solution;

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

[0031] 23) The total flavonoid content in the test solution and the second reference solution were determined by ultraviolet-visible spectrophotometry and quantified using the standard curve method.

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

[0033] Physalis alkekengi L. var. franchetii (Mast.) Makino, a plant belonging to the genus Physalis in the Solanaceae family, is a dried persistent calyx or persistent calyx with fruit. It is bitter and cold in nature; it enters the Lung meridian. It has the effects of clearing heat and detoxifying, relieving sore throat and resolving phlegm, and promoting urination. It is mainly used to treat sore throat and hoarseness, cough with phlegm and heat, difficulty urinating, and painful urination due to heat.

[0034] Fried burdock seeds are the dried, ripe fruits of *Arctium lappa* L., a plant in the Asteraceae family, prepared by stir-frying. They are pungent, bitter, and cold in nature; and enter the lung and stomach meridians. They have the effects of dispersing wind-heat, promoting lung function and relieving rashes, and detoxifying and reducing swelling. They are mainly used to treat wind-heat colds, cough with excessive phlegm, measles, rubella, sore throat, mumps, erysipelas, carbuncles, and boils.

[0035] Belamcanda chinensis (L.) DC., a plant in the Iridaceae family, is a dried rhizome. It is bitter and cold in nature, and enters the Lung meridian. It clears heat and detoxifies, resolves phlegm, and soothes the throat. It is used for conditions such as heat-toxin phlegm accumulation, sore throat, excessive phlegm, cough, and asthma.

[0036] Sophora tonkinensis Gagnep., a plant belonging to the genus Sophora in the legume family, is the root and rhizome of the plant. It is bitter and cold in nature; it enters the lung and stomach meridians. It has the effects of clearing heat and detoxifying, relieving sore throat and reducing swelling. It is mainly used to treat sore throat and swollen gums.

[0037] Platycodon grandiflorum (Jacq.) A.DC., a plant in the Campanulaceae family, is a dried root. It is bitter, pungent, and neutral in nature. It enters the Lung meridian. Its functions include clearing the lungs, resolving 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 legume family. It is neutral in nature and sweet in taste. It enters the heart, stomach, spleen, and lung meridians. Its functions include tonifying the spleen and replenishing qi, relieving cough and phlegm, alleviating spasms and pain, and harmonizing the properties of other herbs.

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

[0040] Weigh the following traditional Chinese medicine compound components according to a mass ratio of 8-10:5-7:8-10:5-7:2-4:3.5-5.5: Physalis alkekengi, Belamcanda chinensis, stir-fried Arctium lappa, Sophora tonkinensis, Glycyrrhiza uralensis, and Platycodon grandiflorus. The preferred mass ratio of each component is 9:6:9:6:3:4.5. Add 7-9 times, 7-8 times, or 8-9 times the total mass of the compound components of water, and soak at room temperature for 20-40 minutes, 20-25 minutes, or 25 minutes respectively. After decocting for 30 min, 30-35 min, or 35-40 min for 1-2 h, 1-1.5 h, or 1.5-2 h, filter with gauze, add 5-7 times, 5-6 times, or 6-7 times the amount of water (i.e., 5-7 times the total mass of the Chinese herbal compound components) to the residue, decoct for 1-2 h, 1-1.5 h, or 1.5-2 h, filter, combine the filtrates, concentrate under reduced pressure, and dry to constant weight to obtain the Chinese herbal 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, which is a 50% (v / v) aqueous methanol solution.

[0042] In some embodiments of the present invention, the centrifugation speed in step 11) is 12,000–16,000 r / min. For example, it is 12,000–13,000 r / min, 13,000–14,000 r / min, 14,000–15,000 r / min, or 15,000–16,000 r / min. Preferably, the centrifugation 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, it is 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, it is 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, it is 40-45% methanol, 45-50% methanol, 50-55% methanol, or 55-60% methanol. Preferably, it is 50% methanol, which is a 50% (v / v) aqueous methanol 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, it is 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, it is 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, it is 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 high-performance liquid chromatography method described in step 14) uses a Discovery C10 column. 18The column dimensions are: 25 cm × 4.6 mm, 5 μm; injection volume is 4–6 μL, preferably 5 μL; column temperature is 25–40℃, 25–30℃, 30–35℃, or 35–40℃, preferably 40℃; flow rate is 0.8–1.3 mL / min, 0.8–0.9 mL / min, 0.9–1.0 mL / min, 1.0–1.1 mL / min, 1.1–1.2 mL / min, or 1.2–1.3 mL / min, preferably 1.0 mL / min; mobile phase A is acetonitrile, and mobile phase B is an aqueous solution containing 1–3 mmol / L ammonium acetate / 0.05–0.2% acetic acid. Mobile phase B can be 1-2 mmol / L ammonium acetate / 0.05-0.2% aqueous acetic acid solution, 2-3 mmol / L ammonium acetate / 0.05-0.2% aqueous acetic acid solution, 1-3 mmol / L ammonium acetate / 0.05-0.1% aqueous acetic acid solution, 1-3 mmol / L ammonium acetate / 0.1-0.15% aqueous acetic acid solution, or 1-3 mmol / L ammonium acetate / 0.15-0.2% aqueous acetic acid solution; in a preferred embodiment of this invention, mobile phase B is 2 mmol / L ammonium acetate / 0.1% aqueous acetic acid solution, wherein the 0.1% acetic acid is an aqueous acetic acid solution with a volume percentage of 0.1%.

[0049] The gradient elution procedure 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] 38.1–48 min, 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 standard curve method in step 14) includes the following steps:

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

[0060] B) Perform high performance liquid chromatography on a series of first reference solutions of different concentrations obtained in step A) to obtain the linear relationship between the chromatographic peak area of ​​at least one component of irisin and arctiin and the concentration of the corresponding component, plot the corresponding standard working curves, and calculate the regression equations of the standard working curves of at least one component of irisin and arctiin respectively.

[0061] C) Perform high-performance liquid chromatography on the test solution, and substitute the chromatographic peak area of ​​at least one of the obtained 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% (v / v) aqueous solution of ethanol.

[0063] In some embodiments of the present invention, the ultrasound time in step 21) is 20–40 min. For example, it is 20–25 min, 25–30 min, 30–35 min, or 35–40 min. Preferably, the ultrasound time is 30 min.

[0064] In some embodiments of the present invention, the centrifugation speed in step 21) is 8000–12000 r / min. For example, it is 8000–9000 r / min, 9000–10000 r / min, 10000–11000 r / min, or 11000–12000 r / min. Preferably, the centrifugation 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, it is 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, it is 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 the traditional Chinese medicine compound extract by water extraction in step 11), weighing 4-6g of the traditional Chinese medicine compound extract, placing it in an Erlenmeyer flask, adding 15-30mL of 50-70% ethanol solution, ultrasonically extracting for 20-40min, centrifuging at 8000-12000r / min, taking the supernatant in a volumetric flask, adding 50-70% ethanol solution to make up to the mark, and obtaining the test solution.

[0068] In some embodiments of the present invention, the solvent of the second reference 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, which is a 60% (v / v) aqueous solution of ethanol.

[0069] In some embodiments of the present invention, the concentration of the second reference solution in step 22) is 0.8–1.2 mg / mL. For example, it is 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 solution is 1.0 mg / mL.

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

[0071] Preferably, the test sample solution is added sequentially to 60% ethanol and 5% sodium nitrite solution, mixed well, and allowed to stand. Then, 10% aluminum nitrate solution is added, mixed well, and allowed to stand. Finally, 4% sodium hydroxide solution is added, and 60% ethanol is added to make up to volume. The mixture is mixed well, allowed to stand, and the absorbance is measured at 510 nm using a UV-Vis spectrophotometer.

[0072] In some embodiments of the present invention, step 23) involves using a standard curve method to quantify and determine the total flavonoid content in the test solution, including the following steps:

[0073] a) Prepare a series of second reference solutions of different concentrations from the rutin standard;

[0074] b) The second reference solutions of different concentrations obtained in step a) were measured by a UV-Vis spectrophotometer to obtain the linear relationship between the absorbance of rutin and its concentration, the corresponding standard working curve was plotted, and the regression equation of the standard working curve of rutin was calculated.

[0075] c) Measure the absorbance of the test solution using a UV-Vis spectrophotometer, and substitute 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 this invention provides a method for preparing a compound extract of traditional Chinese medicine for pharyngitis. The method employs a two-stage water extraction process to prepare the extract, and uses the quality testing method described in any one of claims 1-9 to detect the content of irisin, arctiin, and total flavonoids, as well as the extract yield, in the obtained compound extract. The two-stage water extraction process specifically includes: a first water extraction using 8-9 times the amount of water for soaking for 30-40 min, 30-35 min, or 35-40 min, followed by decoction for 1-2 h, 1-1.5 h, or 1.5-2 h; and a second water extraction using 5-7 times, 5-6 times, or 6-7 times the amount of water, followed by decoction for 1-2 h, 1-1.5 h, or 1.5-2 h. In a preferred embodiment of this invention, the two-stage water extraction process specifically includes: a first water extraction using 8 times the amount of water for soaking for 30 min, followed by decoction for 2 h; and a second water extraction using 6 times the amount of water, followed by decoction for 1.5 h.

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

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

[0079] 1. Preparation of the test solution: Weigh out Physalis alkekengi, Belamcanda chinensis, stir-fried Arctium lappa, Sophora tonkinensis, Glycyrrhiza uralensis, and Platycodon grandiflorus in a mass ratio of 9:6:9:6:3:4.5. Add 8 times the total mass of the compound components to water, soak at room temperature for 30 minutes, then decoct for 2 hours. Filter with gauze, add 6 times the amount of water to the residue, decoct for 2 hours, filter again, combine the filtrates, and concentrate under reduced pressure to obtain the herbal compound extract. Take 200 μL of the herbal compound extract and dilute it with 1800 μL of 50% methanol. After dilution, centrifuge at 14000 r / min for 5 minutes, and collect the supernatant to obtain the test solution for later use.

[0080] 2. Preparation of reference solution: Accurately weigh iridoside and arctiin, and prepare a first reference solution with a concentration of 0.3 mg iridoside and 2.5 mg arctiin per 1 mL of 50% methanol for later use.

[0081] 3. Preparation of negative control solutions: Prepare the traditional Chinese medicine compound extract without Belamcanda chinensis using the same method as in step 1 above. Take 200 μL of the compound extract and dilute it with 1800 μL of 50% methanol. After dilution, centrifuge at 14000 r / min for 5 min and collect the supernatant to obtain the Belamcanda chinensis negative control solution. Prepare the traditional Chinese medicine compound extract without fried burdock seeds using the same method as in step 1 above. Take 200 μL of the compound extract and dilute it with 1800 μL of 50% methanol. After dilution, centrifuge at 14000 r / min for 5 min and collect the supernatant to obtain the fried burdock seeds negative control solution.

[0082] 4. Detection: Take the above-mentioned test solution, reference solution, and negative control solution separately and determine them according to the following high-performance liquid chromatography (HPLC) conditions and gradient elution procedure:

[0083] The chromatographic column was a Discovery C1000 chromatographic column. 18 The column diameter was 25 cm × 4.6 mm and the column depth was 5 μm. The injection volume was 5 μL, the column temperature was 30 °C, and the flow rate was 1.0 mL / min. Acetonitrile was used as the mobile phase A, and 2 mmol / L ammonium acetate / 0.1% acetic acid aqueous solution was used as the mobile phase B.

[0084] Gradient elution procedure:

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

[0093] Depend on Figure 1The data shows that the negative control solution had no impurities at the corresponding positions in the mixed control, indicating that other components in the compound did 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 burdock seed negative control solution.

[0094] Example 2: HPLC Linearity Experiment

[0095] The first reference solution prepared in Example 1 was serially diluted to obtain a series of concentrations (the concentrations of irisin were 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; the concentrations of irisin were 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). The concentrations of irisin and arctiin were determined according to the liquid chromatography conditions and gradient elution program described in Example 1. The peak areas of irisin and arctiin were measured. Standard curves were plotted with the concentrations of irisin and arctiin as the x-axis (X) and the peak areas as the y-axis (Y), as shown in Tables 1 and 2.

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

[0097]

[0098] Table 2. Results of linearity analysis of arctiin.

[0099]

[0100] The data in Tables 1 and 2 show that iridoside exhibits good linearity in the range of 4.69 μg / mL to 300 μg / mL (Y = 9.34X + 15.52, r = 0.999), and arctiin exhibits good linearity 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 instrument's precision was evaluated by testing intra-day and inter-day precision. The first reference solution prepared according to Example 1 was diluted to concentrations of 75 μg / mL for iridoside and 625 μg / mL for arctiin, and then injected for determination. Intra-day precision: six consecutive injections of the same concentration of the mixed reference solution were performed on the same day; inter-day precision: five consecutive injections of the same concentration of the reference solution were performed on three consecutive batches. The liquid chromatography conditions were the same as in Example 1. The relative standard deviations (RSDs) of the peak areas of iridoside and arctiin were calculated based on the peak areas, respectively. The results are shown in Tables 3 and 4.

[0103] Table 3 Results of Precision Experiment within 3 Days

[0104]

[0105] Table 4 Results of Daytime Precision Experiment

[0106]

[0107] The results show that the intra-day precision RSDs of iridoside and arctiin are 0.16% and 0.14%, respectively, and the inter-day precision RSDs are 2.22% and 2.33%, respectively. This indicates that the instrument precision is good.

[0108] Example 4: HPLC Stability Test

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

[0110] Table 5. Results of the stability experiment

[0111]

[0112]

[0113] Table 5 shows that the RSDs of irisin and arctiin were 2.51% and 2.27%, respectively, indicating that the test solution had good stability within 12 hours.

[0114] Example 5: HPLC Repeatability Test

[0115] Five test solutions were prepared in parallel according to the preparation method of the test solution in Example 1, and each solution was injected for analysis. The liquid chromatography determination conditions were the same as in Example 1. The peak areas of irisin and arctiin were recorded, and the RSD was calculated. The results are shown in Table 6.

[0116] Table 6 Results of Repeatability Experiments

[0117]

[0118] Table 6 shows that the RSDs of irisin and arctiin were 2.39% and 1.86%, respectively, indicating that the method has good reproducibility.

[0119] Example 6: Recovery Experiment

[0120] Six portions of traditional Chinese medicine compound extracts with known contents of irisin and arctiin were taken, and a certain amount of irisin and arctiin were added to each portion within the linear range. The liquid chromatography determination conditions were the same as in Example 1. The peak areas were recorded, and the recovery rate was calculated. The results are shown in Table 7.

[0121] Table 7. Results of the sample recovery rate experiment.

[0122]

[0123]

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

[0125] Example 7: Sample Processing for Ultraviolet-Spectrophotometry

[0126] 1. Preparation of the test solution: The traditional Chinese medicine compound extract was prepared using the same method as in step 1 of Example 1. The traditional Chinese medicine compound extract was further dried in an oven at 55°C to constant weight. 5.0 g of the dried extract powder was accurately weighed and placed in a 50 mL Erlenmeyer flask. 20 mL of 60% ethanol was added to dissolve the extract. The mixture was sonicated for 30 min and centrifuged at 10000 r / min for 10 min. 0.8 mL of the supernatant was accurately transferred to a 5 mL volumetric flask and diluted to the mark to obtain the test solution.

[0127] 2. Preparation of reference solution: Accurately weigh 5.00 mg of rutin reference standard into a 5 mL volumetric flask, dissolve and dilute to volume with 60% ethanol to prepare a 1 mg / mL reference solution.

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

[0129] Example 8: Linearity Experiment of Ultraviolet-Spectrophotometry

[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 5 mL volumetric flasks. Measure the absorbance according to the experimental method in step 3 of Example 7. Plot a standard curve with rutin concentration as the x-axis and absorbance as the y-axis. The results are shown in Table 8.

[0131] Table 8 Results of the linear relationship investigation of rutin

[0132]

[0133] Table 8 shows that the standard rutin showed a linear correlation in the range of 20 μg / mL to 80 μg / mL, and the standard curve was Y = 0.006X + 0.002 (r = 0.999).

[0134] Example 9: Precision Experiment of Ultraviolet-Vis Spectrophotometry

[0135] Take the reference solution prepared in Example 7 and measure the absorbance according to the experimental method in step 3 of Example 7. The measurement was repeated 6 times, and the results are shown in Table 9.

[0136] Table 9 Precision Experiment Results

[0137]

[0138] The RSD of the total flavonoid content calculated from the data in Table 9 is 1.08%, indicating that the instrument has good precision.

[0139] Example 10: Stability Test of Ultraviolet-Vis Spectrophotometry

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

[0141] Table 10 Stability test results

[0142]

[0143] The RSD of the total flavonoid content calculated from the data in Table 10 was 2.39%, indicating that the method has good stability.

[0144] Example 11: Repeatability Experiment of Ultraviolet-Vis Spectrophotometry

[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 Results of Repeatability Experiments

[0147]

[0148] The data in Table 11 show that the RSD of the total flavonoid content is 2.75%, indicating that the method has good reproducibility.

[0149] Example 12 Optimization of the preparation process

[0150] Single-factor experiment

[0151] Weigh out the following ingredients: Physalis alkekengi (9g), Belamcanda chinensis (6g), stir-fried Arctium lappa (9g), Sophora tonkinensis (6g), Glycyrrhiza uralensis (3g), and Platycodon grandiflorus (4.5g). Add 8 times the amount of water and soak for 0.5 hours. Decoct once, twice, and three times (three parallel decoctions), 1 hour each time with 8 times the amount of water. Filter the decoctions through gauze and combine them. Concentrate to 1g crude drug / mL (1g crude drug / mL means concentrating the concentrate to the same volume as the amount of crude drug, for example, 30g of crude drug to 30mL of extract). The contents of iridin and arctiin were determined by HPLC. Table 12 shows the content and transfer rate of iridin and arctiin per gram of raw medicinal material in the extract of traditional Chinese medicine compound (the transfer rate is the ratio of the content of iridin and arctiin per gram of raw medicinal material in the extract of traditional Chinese medicine compound to the content of iridin and arctiin per gram of raw medicinal material. The content of iridin and arctiin in the raw medicinal material is 73.80 mg / g and 701.88 mg / g, respectively).

[0152] Table 12 Results of the investigation on the number of times the food was boiled

[0153]

[0154] Based on Table 12, and taking into account the transfer rate of each component and production cost, the final subsequent experiment selected to decoct the ingredients twice.

[0155] Example 13 Optimization of the preparation process

[0156] Full-scale experiment

[0157] Extraction time and solvent volume, which have a significant impact on water extraction efficiency, were considered as factors in a comprehensive experiment, and an L... 9 (3 4 The complete experimental results are shown in Table 13.

[0158] Table 13 Comprehensive Experimental Factor Level Table

[0159]

[0160]

[0161] Weigh out nine equal portions of each medicinal slice according to the proportions, and follow the L...9 (3 4 The experiments were arranged according to the comprehensive experimental table. After two decoctions, the filtrates were combined and concentrated to 1g of raw drug / mL to obtain the solutions for each sample.

[0162] The extract sample of 1g crude drug / mL was dried in an oven at 80℃ to obtain an extract. The yield of the extract was calculated by dividing the weight of the extract by the amount of the total crude drug. The contents of iridin and arctiin were determined by HPLC in Example 1. The transfer rate of iridin and arctiin was calculated by the method in Example 12. The content of total flavonoids was determined by UV-Vis spectrophotometry.

[0163] The calculation was performed using 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 transfer rates of irisin and arctiin, respectively. The transfer rates of irisin and arctiin were weighted and calculated using the formula W = (a × 0.5) + (b × 0.5), where W represents the final effective component transfer rate, a represents the irisin transfer rate, and b represents the arctiin transfer rate. The results of the intuitive analysis are shown in Table 14; the results of the variance analysis are shown in Table 15.

[0164] Table 14L9(3) 4 Comprehensive Experimental Visual Analysis Table

[0165]

[0166] Table 15 Analysis of Variance Table

[0167]

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

[0169] Using the evaluation index as the calculated value, the intuitive analysis results in Table 14 show that the order of importance (R) of the three factors is A > B > A*B, which means solvent volume > extraction time > the interaction between extraction time and solvent volume. The variance analysis results in Table 15 show that the two factors have no significant impact on the extraction effect. Therefore, based on the intuitive analysis results and combined with the actual production situation, the optimal extraction process is A2B2A*B1, which means two extractions: the first extraction is soaking in 8 times the amount of water for half an hour and then decocting for 2 hours, and the second extraction is decocting in 6 times the amount of water for 1.5 hours.

[0170] Example 14 Optimization of the preparation process

[0171] Extraction process verification experiment

[0172] Verification experiments were conducted using the optimized process conditions, and the results are shown in 16.

[0173] Table 16 Validation Experiment Results

[0174]

[0175] As shown in Table 16, the optimized process exhibits good repeatability, is stable and reliable, and achieves good extraction results.

[0176] In summary, the present invention establishes a high-performance liquid chromatography (HPLC) method for the qualitative and quantitative determination of irisin and arctiin, and a UV-Vis spectrophotometric method for the quantitative determination of total flavonoids from the extract of a traditional Chinese medicine compound for pharyngitis. Furthermore, the extraction time and solvent volume, which significantly affect the water extraction effect, are used as factors in a comprehensive experiment, and an L9(3) method is designed. 4 The experimental results, using the extract yield, total flavonoid content in the aqueous extract, and transfer rates of irisin and arctiin as indicators, optimized the extraction process of the compound extract of traditional Chinese medicine for pharyngitis. The experimental method of this invention is simple, feasible, highly specific, and reproducible, and can also be used to optimize the extraction process of the compound extract of traditional Chinese medicine for pharyngitis. Therefore, this 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 invention. Any person skilled in the art can 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 those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for quality testing of a traditional Chinese medicine compound extract for pharyngitis, characterized in that, The traditional Chinese medicine compound for treating pharyngitis includes the following components: Physalis alkekengi, Belamcanda chinensis, stir-fried Arctium lappa, Sophora tonkinensis, Glycyrrhiza uralensis, and Platycodon grandiflorus; the quality testing method is as follows: Step 1: Qualitative and quantitative analysis is performed using high-performance liquid chromatography (HPLC), specifically including the following steps: 11) Preparation of test solution: Weigh each component of the anti-pharyngitis traditional Chinese medicine compound, mix them, and prepare the traditional Chinese medicine compound extract by water extraction; dissolve the traditional Chinese medicine compound extract in solvent, centrifuge, and dilute the supernatant to obtain the test solution; the solvent is 40-60% methanol; 12) Preparation of reference solution: Weigh the standards of irisin and arctiin, dissolve them separately, mix and dilute the two solutions to prepare the first reference solution; 13) Preparation of negative control solution: The Chinese herbal medicine compound extract without Belamcanda chinensis was prepared by water extraction in step 11). After dissolving in solvent, centrifuging, and diluting the supernatant, the Belamcanda chinensis negative control solution was obtained. The Chinese herbal medicine compound extract without fried burdock seed was prepared by water extraction in step 11). After dissolving in solvent, centrifuging, and diluting the supernatant, the fried burdock seed negative control solution was obtained. 14) Detection: The test solution, first reference solution, and negative control solution are determined by high-performance liquid chromatography (HPLC). Qualitative analysis is performed by comparing retention times, and quantitative analysis is performed using a standard curve method to determine the content of irisin and arctiin in the test solution. The contents of irisin and arctiin are simultaneously determined at one wavelength. The HPLC method uses a Discovery C10 column. 18 The column diameter was 25 cm × 4.6 mm, and the column depth was 5 μm. The injection volume was 4–6 μL, the column temperature was 25–40 °C, and the flow rate was 0.8–1.0 mL / min. Acetonitrile was used as the mobile phase A, and an aqueous solution containing 1–3 mmol / L ammonium acetate / 0.05–0.2% acetic acid was used as the mobile phase B. The gradient elution program was 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; 38.1–48 min, 80%→80% mobile phase B, 20%→20% mobile phase A; Step 2: Quantitative analysis is performed using ultraviolet-visible spectrophotometry, which includes the following steps: 21) Preparation of test solution: The traditional Chinese medicine compound extract was prepared by water extraction in step 11); the traditional Chinese medicine compound extract was weighed, dissolved in solvent, sonicated, centrifuged, and the supernatant was diluted to obtain the test solution; 22) Preparation of reference solution: Weigh rutin standard and prepare second reference solution; 23) The total flavonoid content in the test solution and the second reference solution were determined by ultraviolet-visible spectrophotometry and quantified using the standard curve method.

2. The quality testing method for the compound extract of traditional Chinese medicine for pharyngitis according to claim 1, characterized in that, The water extraction method described in step 11) specifically includes the following steps: Weigh the following Chinese herbal compound components according to a mass ratio of 8~10:5~7:8~10:5~7:2~4:3.5~5.5: Physalis alkekengi, Belamcanda chinensis, stir-fried Arctium lappa, Sophora tonkinensis, Glycyrrhiza uralensis, and Platycodon grandiflorus. Add 7~9 times the total mass of the Chinese herbal compound components to water, soak at room temperature for 20~40 minutes, then decoct for 1~2 hours. Filter with gauze, add 5~7 times the amount of water to the residue, decoct for 1~2 hours, filter again, combine the filtrates, concentrate under reduced pressure, and dry to constant weight to obtain the Chinese herbal compound extract.

3. The quality testing method for the compound extract of traditional Chinese medicine for pharyngitis according to claim 1, characterized in that, The centrifugation speed in step 11) is 12000~16000 r / min; And / or, the centrifugation time described in step 11) is 4~7 min; And / or, the dilution factor described in step 11) is 8 to 13 times.

4. The quality testing method for the compound extract of traditional Chinese medicine for pharyngitis according to claim 1, characterized in that, The solvent for 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 testing method for the compound extract of traditional Chinese medicine for pharyngitis according to claim 1, characterized in that, Step 14) describes the quantification of irisin and arctiin using a standard curve method, which includes the following steps: A) Prepare a series of first reference solutions of different concentrations using irisin and arctiin standards; B) Perform high performance liquid chromatography on a series of first reference solutions of different concentrations obtained in step A) to obtain the linear relationship between the chromatographic peak area of ​​at least one component of irisin and arctiin and the concentration of the corresponding component, plot the corresponding standard working curves, and calculate the regression equations of the standard working curves of at least one component of irisin and arctiin respectively. C) Perform high-performance liquid chromatography on the test solution, and substitute the chromatographic peak area of ​​at least one of the components, iridoidin 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.

6. The quality testing method for the compound extract of traditional Chinese medicine for pharyngitis according to claim 1, characterized in that, The solvent mentioned in step 21) is 50-70% ethanol; And / or, the ultrasound time described in step 21) is 20~40 min; And / or, the centrifugation speed in step 21) is 8000~12000 r / min; And / or, the centrifugation time in step 21) is 8~12 min; And / or, the dilution factor described in step 21) is 9 to 13 times.

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

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

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