Determination method of flavonoid components in haemorrhoid recovery tablet and application of flavonoid components in haemorrhoid recovery tablet

Abstract

The invention discloses a determination method and application of flavonoid components in Zhikang tablets, and relates to the technical field of traditional Chinese medicine analysis and identification. In the determination method, a test solution is determined and analyzed by adopting a high performance liquid chromatography, and the conditions of the high performance liquid chromatography are as follows: a chromatographic column is a C18 chromatographic column; the column temperature is 25-30 DEG C; the detection wavelength is 210-220 nm, the sample size is 8-12 [mu] L, and the operation is carried out for 55-80 min; the flow velocity is 0.6 to 0.8 mL / min; mobile phases: an A phase acetonitrile and a B phase 0.05-0.5% phosphoric acid solution; and eluting by adopting a gradient elution procedure. According to the determination method disclosed by the invention, most impurities in the prepared test solution are removed, so that the interference of other components on a chromatographic peak is reduced; the separation degree under the chromatographic condition is good, the base line is stable, the precision is high, the stability is high, the repeatability is good, and the RSD value is low.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine analysis and identification technology, specifically to a method for determining flavonoid components in hemorrhoid tablets and its application. Background Technology

[0002] Hemorrhoid Relief Tablets are composed of six traditional Chinese medicines: Siegesbeckia orientalis, honeysuckle, charred Sanguisorba officinalis, Sophora japonica, Scutellaria baicalensis, and rhubarb. They have the effects of clearing heat and cooling blood, purging heat and promoting bowel movements, reducing swelling and relieving pain. They are mainly suitable for symptoms such as rectal bleeding, anal swelling and pain, and a feeling of heaviness caused by excessive heat and wind or damp-heat accumulation in the lower body. They are particularly effective for patients with stage I and II internal hemorrhoids. Flavonoids are natural polyphenols widely found in plants and possess various biological activities. Many flavonoid components have anti-inflammatory, heat-clearing, asthma-relieving, and antibacterial activities. Related literature reports that the herbs in Hemorrhoid Relief Tablets, such as Siegesbeckia orientalis, honeysuckle, charred Sanguisorba officinalis, Sophora japonica, and Scutellaria baicalensis, contain abundant flavonoids, mainly quercetin and its glycosides. Flavonoids are one of the main active ingredients in Hemorrhoid Relief Tablets and have significant effects in clearing heat and reducing inflammation.

[0003] The current quality standard for Zhikang tablets only includes thin-layer chromatography identification of three medicinal materials and determination of rutin content. The identification of these components is limited and lacks identification of other major active ingredients. In addition, thin-layer chromatography identification is a qualitative judgment of a single or a few components. Its main purpose is to confirm the presence or absence of the target component. It cannot reflect the quality and content of other effective components and cannot assess the overall quality of the drug.

[0004] Due to the complexity of the components of traditional Chinese medicine (TCM), simply measuring and confirming a single component is insufficient for quality control and evaluation. Therefore, TCM testing needs to incorporate multi-component characteristic chromatograms to comprehensively evaluate the relationship between the effective components and the overall drug.

[0005] Traditional Chinese medicine fingerprinting technology, targeting the multi-component and multi-target effects of traditional Chinese medicine, has been widely used in the quality control research of Chinese medicinal materials and has been gradually promoted and applied in Chinese medicine injections. However, there are few research reports on its application in the quality standards of Chinese medicine solid dosage forms. Compared with injections, the active ingredients in solid dosage forms are complex and diverse, and the current quality standards for solid dosage forms are difficult to comprehensively and effectively control their quality.

[0006] Chinese invention patent CN120214145A discloses a method for determining the content of components of Tianmingjing (a type of medicinal ingredient). The method involves ultrasonic extraction of Tianmingjing components using 50%-80% methanol or 50%-80% ethanol. Using isochlorogenic acid A as an internal reference, the relative correction factors for chlorogenic acid, caffeic acid, luteolin, 11(13)-dehydroadenosylpyridin, isochlorogenic acid C, 2,3,4,5-tetracaffeoyl-D-gluconic acid, 2-deoxy-4-epio-tianrenjulin, Tianmingjing lactone, and telerolactone were calculated to rapidly and accurately detect the content of these 10 components. However, this invention still does not record a technical solution for the quality testing of hemorrhoid tablets, and the extract contains other interfering components that affect the separation of chromatographic peaks.

[0007] Chinese invention patent CN101513432A discloses the preparation and new uses of Selaginella tamariscina biflavonoids. The method involves extracting Selaginella tamariscina using 40%-95% aqueous ethanol or methanol under reflux for 2-3 times, each time for 1-3 hours. The extract is then purified by adsorption on a polyamide column, eluting the column with 60%-90% ethanol or methanol. The 60%-90% ethanol or methanol eluent is collected, the solvent is recovered, and the extract is dried to obtain total Selaginella tamariscina flavonoids. Using Taxodium biflavonoids as a reference standard, the content of total Selaginella tamariscina flavonoids is determined to be above 85%. The total Selaginella tamariscina flavonoids are then dissolved and filtered with 50%-60% ethanol or methanol, purified by adsorption on a polyamide column, and eluted with 80% ethanol or methanol in the polyamide adsorption portion. The ethanol or methanol eluent is collected, decolorized with an appropriate amount of activated carbon, filtered, and the solvent is recovered to obtain Taxodium biflavonoids. Although this invention describes a method of alcohol extraction and purification, it does not explain the impact of the extraction method on the quality testing of hemorrhoid tablets.

[0008] Currently, there are no records of using fingerprinting technology for the quality testing of hemorrhoid tablets. Furthermore, different extraction and purification methods and chromatographic conditions all affect the test results. Therefore, further research is needed on the quality determination of hemorrhoid tablets to develop a method that can accurately, efficiently, precisely, densely, and reproducibly determine the quality of multiple active ingredients. Summary of the Invention

[0009] This invention conducts fingerprint chromatographic research on hemorrhoid tablets. By establishing standardized chromatographic characteristic spectra (high performance liquid chromatography), adulteration behavior of flavonoid products can be quickly identified, and the similarity of fingerprint spectra of different batches of products can be dynamically monitored to ensure the stability of the production process and controllable product quality. By quantifying the efficacy contribution of key components through grey relational analysis or artificial neural networks, the source medicinal materials of characteristic peaks in traditional Chinese medicine compound prescriptions can be located, the synergistic mechanism of multi-component action can be elucidated, and the modernization and improvement of classic prescriptions can be supported. This invention replaces traditional single-indicator detection, using overall chromatographic similarity as the core quality control indicator.

[0010] This invention establishes a correlation model between the characteristic peak groups of the effective components of Zhikang tablets and their efficacy, thus overcoming the deficiency of the disconnect between component research and efficacy. It also links the fingerprint spectrum of flavonoids with their biological activity through the digitization of chemical characteristics, achieving a leap from "experience-based quality control" to "quantitative regulation," thereby enabling good control of the quality of solid dosage forms. At the same time, it provides a reference for research on improving the quality standards of solid dosage forms.

[0011] This invention provides a method for determining flavonoid components in hemorrhoid tablets and its application.

[0012] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows: On one hand, this invention provides a method for determining flavonoid components in hemorrhoid tablets. The test solution is analyzed by high-performance liquid chromatography (HPLC), and the HPLC conditions are as follows: Chromatographic column: C18 column; Column temperature: 25℃-30℃; Detection wavelength: 210-220nm, injection volume: 8-12μL, running time: 55-80min; Flow rate: 0.6-0.8 mL / min; Mobile phase: Phase A is acetonitrile, and Phase B is 0.05-0.5% phosphoric acid solution; Gradient elution, the elution procedure is as follows: .

[0013] Preferably, the chromatographic column is a SunFire®-C18 (250mm × 4.6mm, 5μm).

[0014] Preferably, the column temperature is 25°C.

[0015] Preferably, the detection wavelength is 216 nm, the injection volume is 10 μL, and the running time is 60 min.

[0016] Preferably, the flow rate is 0.8 mL / min.

[0017] Preferably, the mobile phase is: phase A acetonitrile, phase B a 0.1% volume concentration phosphoric acid solution; specifically, the phosphoric acid solution is an aqueous phosphoric acid solution.

[0018] Preferably, the gradient elution process is as follows: .

[0019] Preferably, the method for preparing the test solution includes the following steps: grinding hemorrhoid tablets, extracting them with ethanol using ultrasound, adsorbing them with polyamide, evaporating the ethanol, transferring them to a chromatography column, eluting them with petroleum ether, discarding the eluent, eluting them with methanol, and making up to volume to obtain the hemorrhoid tablet test solution.

[0020] More preferably, the preparation method of the test solution includes the following steps: taking 0.5-1.5g of ground hemorrhoid tablets and mixing them with 7.5-25mL of ethanol, and ultrasonically extracting for 20-40min; filtering, adding 0.75-2.5g of 100-300 mesh polyamide powder for adsorption, evaporating the ethanol on a water bath, and then transferring it to a chromatography column, eluting twice with 10-40mL of petroleum ether (30℃-60℃), 5-20mL each time, discarding the eluent, and then eluting with 15-25mL of methanol, and making up to 20-30mL.

[0021] More preferably, the preparation method of the test solution includes the following steps: take 0.6g of ground hemorrhoid tablets and 15mL of ethanol, mix them, and extract by ultrasonication for 30min; filter, add 1.5g of 200-mesh polyamide powder for adsorption, evaporate the ethanol on a water bath, and then transfer it to a chromatography column, elute twice with 20mL of petroleum ether (30℃-60℃), 10mL each time, discard the eluent, then elute with 20mL of methanol, and make up to 25mL.

[0022] Furthermore, the present invention provides a method for preparing a test solution of hemorrhoid tablets, comprising the following steps: grinding hemorrhoid tablets, extracting them with ethanol using ultrasound, adsorbing them with polyamide powder, evaporating the ethanol, transferring the solution to a glass chromatography column, eluting with petroleum ether, discarding the eluent, eluting with methanol, and adjusting the volume to obtain the test solution of hemorrhoid tablets.

[0023] Preferably, the preparation method of the hemorrhoid tablet test solution includes the following steps: taking 0.5-1.5g of ground hemorrhoid tablets and mixing them with 7.5-25mL of ethanol, and ultrasonically extracting for 20-40min; filtering, adding 0.75-2.5g of 100-300 mesh polyamide powder for adsorption, evaporating the ethanol on a water bath, and then transferring it to a chromatography column, eluting twice with 10-40mL of petroleum ether (30℃-60℃), 5-20mL each time, discarding the eluent, and then eluting with 15-25mL of methanol, and making up to 20-30mL.

[0024] More preferably, the preparation method of the hemorrhoid tablet test solution includes the following steps: take 0.6g of ground hemorrhoid tablets and 15mL of ethanol, mix them, and extract by ultrasonication for 30min; filter, add 1.5g of 200-mesh polyamide powder for adsorption, evaporate the ethanol on a water bath, and then transfer it to a chromatography column, elute twice with 20mL of petroleum ether (30℃-60℃), 10mL each time, discard the eluent, then elute with 20mL of methanol, and make up to 25mL.

[0025] Preferably, the amount of polyamide powder used is 2-5 times the mass of the hemorrhoid tablets; more preferably, the amount of polyamide powder used is 2.5 times the mass of the hemorrhoid tablets.

[0026] Preferably, the chromatography column is packed with polyamide powder; the inner diameter of the chromatography column is 1-2 cm, the column length is 5-15 cm, and it is dry-packed.

[0027] More preferably, the chromatography column is a glass column with an inner diameter of 1.5 cm and a length of 10 cm, and is dry-packed.

[0028] Preferably, the sample loading rate of the transfer column is 1-2 mL / min.

[0029] On the other hand, the present invention provides the application of the above-mentioned determination method in the quality testing of hemorrhoid tablets.

[0030] The beneficial effects of this invention are as follows: 1. This invention studies the HPLC fingerprint of flavonoid components in Zhikang tablets. Due to the complexity of the compound preparation, direct solvent extraction to prepare the test solution results in significant interference in the HPLC chromatogram, making it difficult to separate the peaks. Therefore, this invention uses polyamide powder to purify and enrich the flavonoid components in the formulation. Using the HPLC fingerprint conditions of this invention, an HPLC fingerprint analysis method for flavonoid components in different batches of Zhikang tablets is established. Rutin is used as a reference peak to determine the relative retention times of other common fingerprint peaks and calculate the ratio of their areas. Simultaneously, the similarity of the HPLC fingerprint spectra of flavonoid components in different batches of products is calculated, providing a scientific basis for the quality control of Zhikang tablets.

[0031] 2. In the preparation method of the test solution of the present invention, polyamide powder adsorbent is used to purify the sample, and the elution solvent is clearly defined, so that the flavonoid components are highly enriched. Compared with direct extraction and purification with methanol, most impurities are effectively removed, and the interference of other components on the chromatographic peaks is reduced, resulting in better separation effect.

[0032] 3. The determination method under specific conditions of the present invention enables the effective separation of each peak of the flavonoid active ingredients in hemorrhoid tablets, with good separation degree and stable baseline. The methodological investigation results show that the determination method of the present invention has high precision, stability, good repeatability, low RSD value, and the fingerprint spectrum similarity after testing multiple batches of drugs is as high as 0.92 or above.

[0033] 4. This invention conducts a comparative study on the fingerprint spectra of hemorrhoid tablets and medicinal materials, and identifies the specific absorption peaks of five medicinal materials, namely Siegesbeckia orientalis, Scutellaria baicalensis, Lonicera japonica, Sanguisorba officinalis charcoal, and Scutellaria baicalensis, in the fingerprint spectra. The established fingerprint spectra also put forward higher requirements for the quality control of medicinal materials.

[0034] 5. This invention establishes a method for quantitative determination of common peaks in hemorrhoid tablets by locating the common peaks in different batches of hemorrhoid tablets and using the standard curve method. After multiple verifications, it can be seen that the accuracy and repeatability meet the requirements. The determination method of this invention provides a reliable basis for the quality control of hemorrhoid tablets. Attached Figure Description

[0035] Figure 1 This is the HPLC chromatogram of the determination method in Example 1.

[0036] Figure 2 This is the HPLC chromatogram of the determination method in Example 2.

[0037] Figure 3 This is the HPLC chromatogram of the determination method in Example 3.

[0038] Figure 4 This is the HPLC chromatogram of the determination method in Comparative Example 1.

[0039] Figure 5 This is the HPLC chromatogram of the determination method in Comparative Example 2.

[0040] Figure 6 This is the HPLC chromatogram of the determination method in Comparative Example 3.

[0041] Figure 7 This is the HPLC chromatogram of the determination method in Comparative Example 4.

[0042] Figure 8 This is the HPLC chromatogram of the determination method in Comparative Example 5.

[0043] Figure 9 This is the HPLC chromatogram of the determination method in Comparative Example 6.

[0044] Figure 10 This is the standard curve of gallic acid.

[0045] Figure 11 This is a standard curve of chlorogenic acid.

[0046] Figure 12 This is the standard curve of baicalin.

[0047] Figure 13 This is the standard curve of chinonyl alcohol.

[0048] Figure 14 This is a standard curve graph of rutin.

[0049] Figure 15 This is the standard curve of rhein.

[0050] Figure 16 This is the HPLC chromatogram of the mixed standard solution.

[0051] Figure 17 This is a chromatogram of a elution process where petroleum ether is replaced with methanol.

[0052] Figure 18 This relates the rutin desorption rate to the methanol concentration in the eluent.

[0053] Figure 19 These are HPLC chromatograms of the herbal medicine *Siegesbeckia orientalis*, the hemorrhoid tablets made from *Siegesbeckia orientalis*, and a sample of the hemorrhoid tablets.

[0054] Figure 20 These are HPLC chromatograms of honeysuckle medicinal material, hemorrhoid tablets lacking honeysuckle, and hemorrhoid tablet samples.

[0055] Figure 21 These are HPLC chromatograms of Sophora japonica flower medicinal material, Hemorrhoid Relief Tablets (lacking Sophora japonica flower), and Hemorrhoid Relief Tablets samples.

[0056] Figure 22 These are HPLC chromatograms of the medicinal material *Sanguisorba officinalis* charcoal, *Hemorrhoid Relief Tablets* without *Sanguisorba officinalis* charcoal, and *Hemorrhoid Relief Tablets* samples.

[0057] Figure 23 These are HPLC chromatograms of Scutellaria baicalensis, Zhikang tablets (containing Scutellaria baicalensis), and Zhikang tablet samples.

[0058] Figure 24 These are HPLC chromatograms of rhubarb medicinal material, hemorrhoid tablets lacking rhubarb, and hemorrhoid tablet samples.

[0059] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Detailed Implementation

[0060] To make the technical means, creative features, and achieved objectives and effects of this invention easier to understand, the invention is further illustrated below with specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the protection scope of this invention. Unless otherwise specified, the operating methods and equipment used in the following embodiments are conventional operating methods, and the materials and equipment used in each embodiment are the same.

[0061] Example 1 A method for determining flavonoid components in hemorrhoid tablets (1) Preparation of the test solution of hemorrhoid tablets: Take 10 tablets of Zhikang tablets, grind them into a fine powder, accurately weigh 1.0g into a conical flask, add 25mL of ethanol, shake well, and extract by sonication for 30min. Set aside for later use. Accurately measure 15mL of the filtrate (equivalent to 0.6g of Zhikang tablets), place it in an evaporating dish, add 1.5g of 200-mesh polyamide powder for adsorption, evaporate the ethanol on a water bath, and then transfer it to a chromatography column (filled with 200-mesh polyamide powder, glass column, inner diameter of the chromatography column is 1.5cm, column length is 10cm) at a sample loading rate of 2mL / min. Elute twice with 10mL of 20mL petroleum ether (30℃-60℃) each time, discard the eluent, and then elute with 20mL of methanol. Make up to 25mL to obtain the test solution.

[0062] (2) The test solution was analyzed by high performance liquid chromatography (HPLC). The HPLC conditions were as follows: Column: SunFire®-C18 (250mm × 4.6mm, 5μm); Column temperature: 25℃; Detection wavelength: 216nm; Injection volume: 10μL; Run time: 60min; Flow rate: 0.8 ml / min; Mobile phase: Phase A (acetonitrile) - Phase B (0.1% phosphoric acid aqueous solution); Linear gradient elution, the elution procedure is as follows:

[0063] The HPLC chromatogram of this embodiment is shown below. Figure 1 .from Figure 1 As can be seen from the results, the determination method of the present invention can effectively separate chromatographic peaks.

[0064] Example 2 The difference from Example 1 is that the column temperature in the high-performance liquid chromatography (HPLC) conditions is 30°C. All other conditions are the same as in Example 1.

[0065] The HPLC chromatogram of this embodiment is shown below. Figure 2 .from Figure 2 As can be seen from the results, the determination method of the present invention can effectively separate chromatographic peaks.

[0066] Example 3 Unlike Example 1, the flow rate in the high-performance liquid chromatography (HPLC) conditions was 0.8 mL / min. All other conditions were the same as in Example 1.

[0067] The HPLC chromatogram of this embodiment is shown below. Figure 3 .from Figure 3 As can be seen from the results, the determination method of the present invention can effectively separate chromatographic peaks.

[0068] Comparative Example 1 The difference from Example 1 is that the mobile phase is different; the mobile phase is replaced by: Phase A (acetonitrile) - Phase B (0.5% aqueous acetic acid solution). Everything else is the same as in Example 1.

[0069] The HPLC chromatogram of Comparative Example 1 is shown in Figure 1. Figure 4 .from Figure 4 As can be seen, the measurement method of changing the mobile phase results in an unstable baseline of the peaking system, poor separation, and a small number of peaks.

[0070] Comparative Example 2 The difference from Example 1 is that the mobile phase is different; the mobile phase is replaced by: Phase A (acetonitrile) - Phase B (0.1% formic acid aqueous solution). Everything else is the same as in Example 1.

[0071] The HPLC chromatogram of Comparative Example 2 is shown in Figure 2. Figure 5 .from Figure 5 As can be seen, the measurement method of changing the mobile phase results in an unstable baseline of the peaking system, poor separation, and a small number of peaks.

[0072] Comparative Example 3 The difference from Example 1 is that the column temperature in the high-performance liquid chromatography (HPLC) conditions was set to 40°C. All other conditions were the same as in Example 1.

[0073] The HPLC chromatogram of Comparative Example 3 is shown in Figure 3. Figure 6 .from Figure 6 As can be seen, changing the column temperature in high performance liquid chromatography resulted in earlier peak elution and poorer peak resolution.

[0074] Comparative Example 4 Unlike Example 1, the flow rate in the high-performance liquid chromatography (HPLC) conditions was set to 1.0 mL / min. All other conditions were the same as in Example 1.

[0075] The HPLC chromatogram of Comparative Example 4 is shown in [reference needed]. Figure 7 .from Figure 7 As can be seen, changing the flow rate under high performance liquid chromatography conditions leads to excessive concentration of chromatographic peaks, making separation difficult.

[0076] Comparative Example 5 Unlike Example 1, the elution procedure in the high-performance liquid chromatography conditions is different, specifically:

[0077] Everything else is the same as in Example 1.

[0078] The HPLC chromatogram of Comparative Example 5 is shown below. Figure 8 .from Figure 8As can be seen, after changing the elution program, the gradients at 0-14 minutes and 24-34 minutes were too fast, the chromatographic peaks were too concentrated, and the peaks could not be separated efficiently.

[0079] Comparative Example 6 Unlike Example 1, the elution procedure in the high-performance liquid chromatography conditions is different, specifically:

[0080] Everything else is the same as in Example 1.

[0081] The HPLC chromatogram of Comparative Example 6 is shown in Figure 6. Figure 9 .from Figure 9 As can be seen, after changing the elution program, there was no chromatographic peak information after 35-60 minutes, and the chromatographic peaks could not be separated efficiently.

[0082] Example 4 Study on the preparation method of test solution 1. Preparation of reference solution Take appropriate amounts of reference standards gallic acid, chlorogenic acid, baicalin, kirunol, rutin, and rhein, weigh them accurately, and add methanol to prepare a mixed standard solution containing 0.1 mg of each reference standard per 1 mL (the concentration of each reference standard in the mixed standard solution is 0.1 mg / mL).

[0083] 2. Preparation of the test solution Take 10 tablets of Zhikang tablets, grind them into a fine powder, accurately weigh 1.0g into a conical flask, add 25mL of ethanol, shake well, and extract by sonication for 30min. Set aside for later use. Accurately measure 15mL of the filtrate (equivalent to 0.6g of Zhikang tablets), place it in an evaporating dish, add 1.5g of 200-mesh polyamide powder for adsorption, evaporate the ethanol on a water bath, and then transfer it to a chromatography column (filled with 200-mesh polyamide powder, glass column, inner diameter of the chromatography column is 1.5cm, column length is 10cm) at a sample loading rate of 2mL / min. Elute twice with 10mL of 20mL petroleum ether (30℃-60℃) each time, discard the eluent, and then elute with 20mL of methanol. Make up to 25mL to obtain the test solution.

[0084] 3. Plotting the standard curve Accurately pipette 5 μL, 10 μL, 15 μL, 20 μL, and 25 μL of the above mixed reference solution, respectively, and inject them into the high-performance liquid chromatograph. Measure the peak area using the method described in Example 1. Plot a standard curve with peak area as the ordinate and injection volume as the abscissa. The results of the standard curve are shown below. Figures 10-15 (The horizontal axis is in μg, and the vertical axis is the peak area), thus obtaining the linear regression equations for each reference standard.

[0085] The results of the standard curves show that the concentrations of gallic acid (5.17µg-25.85µg), chlorogenic acid (5.26µg-26.26µg), rutin (5.56µg-27.78µg), baicalin (5.40µg-26.98µg), chrysenoyl alcohol (5.48µg-27.40µg), and emodin (5.09µg-25.45µg) all exhibit good linearity with peak area (R² > 0.995).

[0086] The HPLC chromatogram of the mixed standard solution is shown in [reference needed]. Figure 16 . Figure 16 In the middle, peak 4: baicalin; peak 1: gallic acid; peak 2: chlorogenic acid; peak 3: rutin; peak 5: nonyl alcohol; peak 6: aloe-emodin; peak 7: rhein; peak 8: emodin; peak 9: chrysophanol; peak 10: emodin methyl ether.

[0087] 4. Investigation of the preparation method of the test solution 4.1 Investigation on the adsorption capacity of polyamide powder Unlike the preparation of the test solution in Example 1, the amount of polyamide powder used was different. The specific amount and adsorption effect are shown in Table 1.

[0088] Table 1

[0089] As can be seen from Table 1, the highest adsorption effect is achieved with 1.5-2.5g of polyamide powder. However, when the amount of polyamide powder added is greater than 1.5g, the adsorption effect tends to be gradual. Therefore, 1.5g of polyamide powder (2.5 times the mass of the hemorrhoid tablets) was selected as the adsorption amount.

[0090] 4.2 Investigation of sample loading flow rate Unlike the preparation of the test solution in Example 1, the loading flow rate was different. The specific loading flow rate and adsorption rate are shown in Table 2.

[0091] Table 2

[0092] As can be seen from Table 2, when the loading flow rate is 1.0 mL / min and 2.0 mL / min, the adsorption rate of rutin is relatively high and the difference is not significant; when the loading flow rate is 3.0 mL / min and 4.0 mL / min, the adsorption rate decreases.

[0093] 4.3 Determination of elution solvent Unlike the preparation of the test solution in Example 1, petroleum ether was replaced with methanol.

[0094] Take 10 tablets of Zhikang tablets, grind them into a fine powder, accurately weigh 1.0g into a conical flask, add 25mL of ethanol, shake well, and extract by sonication for 30min. Set aside for later use. Accurately measure 15mL of the filtrate (equivalent to 0.6g of Zhikang tablets), place it in an evaporating dish, add 1.5g of 200-mesh polyamide powder for adsorption, evaporate the ethanol on a water bath, and then transfer it to a chromatography column (filled with 200-mesh polyamide powder, glass column, inner diameter of the column is 1.5cm, column length is 10cm) at a sample loading rate of 2mL / min. Elute with 20mL of methanol, and dilute to 25mL to obtain the test solution.

[0095] The obtained test solution was separated and detected using the high-performance liquid chromatography (HPLC) conditions described in Example 1. Since no peaks were observed in the chromatogram after 50 minutes, chromatograms within the first 50 minutes were selected. (See chromatogram for details.) Figure 17 ,from Figure 17 As can be seen, there are many chromatographic peaks, making effective separation impossible.

[0096] 4.4 Investigation of eluent solvent concentration The difference between this and the preparation of the test solution in Example 1 is the concentration of methanol. Specifically, the methanol concentration and the effect on rutin desorption rate are shown in [link to example]. Figure 18 .

[0097] Rutin desorption rate (%) = Measured amount of rutin / Theoretical amount of rutin added × 100%.

[0098] from Figure 18 As can be seen, the desorption rate of rutin is highest when 100% methanol is used. The desorption rate of rutin is significantly reduced when methanol concentrations of 10%, 30%, 50%, 70%, and 90% are used.

[0099] I. Methodological Examination The fingerprint spectrum of flavonoids in Zhikang tablets was validated using the determination method of Example 1 of this invention. The selected evaluation indicators mainly included the following three aspects: precision, stability, and repeatability.

[0100] 1. Precision test Using the determination method described in Example 1, the same test sample solution was injected 6 times consecutively. The number of peaks in the obtained HPLC chromatograms was the same, the RSD of retention time of all peaks was less than 0.3%, and the RSD of peak area was less than 2.3%, which met the requirements of fingerprint chromatogram. The results are shown in Table 3.

[0101] Table 3

[0102] 2. Repeatability test For the same batch of hemorrhoid tablets, six test solutions were prepared according to the preparation method of the test solution described in Example 1. The solutions were measured under the high performance liquid chromatography conditions described in Example 1. The number of peaks in the obtained HPLC chromatograms were the same, the RSD of the retention time of all peaks was less than 0.2%, and the RSD of the peak area was less than 4.7%, which met the requirements of fingerprint chromatogram. The results are shown in Table 4.

[0103] Table 4

[0104] 3. Stability test The test solution was prepared according to the preparation method of the test solution in Example 1. After being sealed and placed at room temperature in the dark for 0h, 3h, 6h, 28h, 35h, 39h, and 44h, the solution was measured using the high performance liquid chromatography conditions described in Example 1. The number of peaks in the obtained HPLC chromatograms was basically the same, the RSD of the retention time of all peaks was less than 0.2%, and the RSD of the peak area was less than 4.2%, which met the requirements of fingerprint chromatogram. The results are shown in Table 5.

[0105] Table 5

[0106] II. Establishment of fingerprint patterns 1. Sample determination Seven batches of hemorrhoid tablets (batch numbers: 22031001(S1), 22031002(S2), 23041001(S3), 23071001(S4), 24021001(S5), 24071001(S6), 24081001(S7)) were taken and prepared according to the preparation method of the test solution in Example 1. The fingerprint chromatograms were determined and recorded according to the high performance liquid chromatography conditions in Example 1. The baicalin peak (peak 4) was compared with the reference peak (peak 5) by comparing the retention times. Ten common peaks were identified. The relative retention time and relative peak area of ​​each common peak in the fingerprint chromatograms of the seven batches of samples were calculated.

[0107] The relative retention times (min) of each common peak in the fingerprint spectrum are shown in Table 6, and the relative peak areas (mAU.s) of each common peak in the fingerprint spectrum are shown in Table 7.

[0108] Table 6

[0109] Table 7

[0110] 2. Similarity Calculation The fingerprint spectra of flavonoid components in the seven batches of Zhikang tablets (S1, S2, S3, S4, S5, S6, and S7) were analyzed using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system. The "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software System (Version 2012A)" was used for comprehensive evaluation. Using the generated sample chromatograms as reference fingerprint spectra, the average relative peak area of ​​the fingerprint spectra of the seven batches of samples was used as the reference fingerprint (R) template. Based on the peak matching results, the similarity of each sample was calculated by comparing the peak area of ​​the test fingerprint spectra with the peak area on the reference template. The results are shown in Table 8.

[0111] Table 8

[0112] As shown in Table 8, the similarity values ​​of all seven batches of samples were above 0.96, indicating that they were similar overall. When analyzing unknown samples using this method, it is only necessary to measure the chromatographic fingerprint of the sample and calculate the similarity between the unknown sample and the common pattern to identify and evaluate the sample.

[0113] III. The Relationship Between Common Peaks and Medicinal Herbs Following the test solution preparation method in Example 1, test solutions for hemorrhoid tablets, raw medicinal materials, and negative samples lacking a single medicinal material were prepared. The flavonoid components of the hemorrhoid tablets, raw medicinal materials, and negative samples lacking a single medicinal material were compared and analyzed under the high-performance liquid chromatography (HPLC) conditions of Example 1. The correlation between the common peaks in the fingerprint chromatogram of hemorrhoid tablets and the peak assignments in the chromatogram of the raw medicinal material was studied.

[0114] 1. Correlation study between the common peaks of the fingerprint spectrum of flavonoid components in Zhikang tablets and the fingerprint spectrum of corresponding parts of Siegesbeckia orientalis. Take 1.0g each of Zhikang tablets, negative samples lacking Siegesbeckia orientalis (Zhikang tablets lacking Siegesbeckia orientalis), and 0.3g of Siegesbeckia orientalis (equivalent to the amount of Siegesbeckia orientalis in 1.0g tablets). Prepare Zhikang tablet test solutions, negative sample test solutions lacking Siegesbeckia orientalis test solutions, and Siegesbeckia orientalis test solutions according to the test solution preparation method in Example 1.

[0115] Fingerprint chromatograms were determined according to the high-performance liquid chromatography (HPLC) conditions of Example 1. The chromatograms and corresponding UV absorption spectra of the hemorrhoid tablets, *Siegesbeckia orientalis* herbal material, and *Siegesbeckia orientalis* negative solution were compared to determine the *Siegesbeckia orientalis* herbal material with common fingerprint peaks in each fingerprint spectrum. The results are shown in [Figure 1]. Figure 19 . Figure 19 A, B, and C are the HPLC chromatograms of Siegesbeckia orientalis, the negative sample lacking Siegesbeckia orientalis, and the HPLC chromatogram of Zhikang tablets, respectively.

[0116] from Figure 19As can be seen from the chromatogram, peaks 1 and 2 of the herbal medicine Siegesbeckia orientalis appeared at the corresponding positions in the hemorrhoid tablets. Peak 5 did not appear at the corresponding position in the negative chromatogram of Siegesbeckia orientalis. It can be basically determined that peak 5 originated from the herbal medicine Siegesbeckia orientalis and is a unique absorption peak of Siegesbeckia orientalis.

[0117] 2. Correlation study between the common fingerprint peaks of flavonoid components in Zhikang tablets and the fingerprint spectra of corresponding parts of honeysuckle. Take 1.0g each of Zhikang tablets and negative samples lacking honeysuckle (Zhikang tablets lacking honeysuckle), and 0.2g of honeysuckle (equivalent to the amount of honeysuckle in 1.0g tablets). Prepare Zhikang tablet test solutions, negative sample test solutions lacking honeysuckle, and honeysuckle test solutions according to the test solution preparation method in Example 1.

[0118] Fingerprint chromatograms were determined according to the high-performance liquid chromatography (HPLC) conditions and methods described in Example 1. The chromatograms and UV absorption spectra of the corresponding chromatographic peaks of Zhikang tablets, honeysuckle medicinal materials, and negative samples lacking honeysuckle were compared to determine the honeysuckle medicinal materials with common fingerprint peaks in each fingerprint spectrum. The results are shown in [Figure 1]. Figure 20 . Figure 20 A, B, and C are the HPLC chromatograms of honeysuckle medicinal material, the HPLC chromatogram of a negative sample lacking honeysuckle medicinal material, and the HPLC chromatogram of hemorrhoid tablets, respectively.

[0119] From above Figure 20 The results show that the common peaks 1 and 2 in the chromatogram of honeysuckle appeared at the corresponding positions in the hemorrhoid tablets. However, these two peaks also appeared at the corresponding positions in the negative chromatogram of honeysuckle. Therefore, it can be basically determined that honeysuckle does not have a specific absorption peak.

[0120] 3. Correlation study between the common fingerprint peaks of flavonoid components in Zhikang tablets and the fingerprint spectra of corresponding parts of Sophora japonica flower. Take 1.0g each of Zhikang tablets and negative samples lacking Sophora japonica (Zhikang tablets lacking Sophora japonica), and 0.2g of Sophora japonica (equivalent to the amount of Sophora japonica in 1.0g tablets). Prepare Zhikang tablet test solutions, negative sample test solutions lacking Sophora japonica, and Sophora japonica test solutions according to the test solution preparation method in Example 1.

[0121] Fingerprint chromatograms were determined according to the high-performance liquid chromatography (HPLC) conditions and methods described in Example 1. The chromatograms and UV absorption spectra of the corresponding chromatographic peaks of the hemorrhoid tablets, Sophora japonica flower extract, and negative samples lacking Sophora japonica flower were compared to determine the Sophora japonica flower extracts with shared fingerprint peaks. The results are shown in [Figure 1]. Figure 21 . Figure 21 A, B, and C are the HPLC chromatograms of Sophora japonica flower, the negative sample lacking Sophora japonica flower, and the HPLC chromatogram of Zhikang tablets, respectively.

[0122] from Figure 21As can be seen from the chromatogram, peaks 1, 2, and 3 in the Sophora japonica flower chromatogram appeared at the corresponding positions in the hemorrhoid tablets. Peak 3 did not have a significant chromatographic peak at the corresponding position in the negative chromatogram of Sophora japonica flower. Therefore, it can be basically determined that peak 3 originated from Sophora japonica flower chromatogram and is a unique absorption peak of Sophora japonica flower.

[0123] 4. Correlation study between the common fingerprint peaks of flavonoid components in Zhikang tablets and the corresponding fingerprint spectra of Sanguisorba officinalis charcoal. Take 1.0g each of Zhikang tablets and negative samples of Sanguisorba officinalis charcoal (Zhikang tablets without Sanguisorba officinalis charcoal), and 0.2g of Sanguisorba officinalis charcoal (equivalent to the amount of Sanguisorba officinalis charcoal in 1.0g tablets). Prepare Zhikang tablet test solutions, negative sample test solutions of Sanguisorba officinalis charcoal, and Sanguisorba officinalis charcoal test solutions according to the test solution preparation method in Example 1.

[0124] Fingerprint chromatograms were determined according to the high-performance liquid chromatography (HPLC) conditions and methods described in Example 1. The chromatograms and UV absorption spectra of the corresponding chromatographic peaks of Zhikang tablets, Sanguisorba officinalis charcoal, and negative samples lacking Sanguisorba officinalis charcoal were compared to determine the attribution of Sanguisorba officinalis charcoal to which each fingerprint chromatogram shared a common fingerprint peak. The results are shown in [Figure 1]. Figure 22 . Figure 22 A, B, and C are the HPLC chromatograms of charred Sanguisorba officinalis, negative HPLC chromatograms of charred Sanguisorba officinalis, and HPLC chromatograms of Zhikang tablets, respectively.

[0125] from Figure 22 The results show that the common peaks 1 and 3 in the chromatogram of charred Sanguisorba officinalis appeared at the corresponding positions in the hemorrhoid tablets. However, these two peaks also appeared at the corresponding positions in the negative chromatogram of charred Sanguisorba officinalis. Therefore, it can be basically determined that charred Sanguisorba officinalis has no specific absorption peak.

[0126] 5. Correlation study between the common fingerprint peaks of flavonoid components in Zhikang tablets and the fingerprint spectra of corresponding parts of Scutellaria baicalensis. Take 1.0g each of Zhikang tablets and negative samples lacking Scutellaria baicalensis (Zhikang tablets lacking Scutellaria baicalensis), and 0.2g of Scutellaria baicalensis (equivalent to the amount of Scutellaria baicalensis in 1.0g tablets). Prepare test solutions for Zhikang tablets, negative samples lacking Scutellaria baicalensis, and Scutellaria baicalensis according to the test solution preparation method in Example 1.

[0127] Fingerprint chromatograms were determined according to the high-performance liquid chromatography (HPLC) conditions and methods described in Example 1. The chromatograms and UV absorption spectra of the corresponding chromatographic peaks of Zhikang tablets, Scutellaria baicalensis root, and negative samples lacking Scutellaria baicalensis were compared to determine the Scutellaria baicalensis root species with common fingerprint peaks in each fingerprint spectrum. The results are shown in [Figure 1]. Figure 23 . Figure 23 A, B, and C are the HPLC chromatograms of Scutellaria baicalensis, the HPLC chromatograms of negative samples lacking Scutellaria baicalensis, and the HPLC chromatogram of Zhikang tablets, respectively.

[0128] from Figure 23The results showed that peaks 3, 4, and 5 in the chromatogram of Scutellaria baicalensis appeared at the corresponding positions in the hemorrhoid tablets. Peak 4 did not appear at the corresponding position in the negative chromatogram of Scutellaria baicalensis. Therefore, it can be basically determined that peak 4 originated from Scutellaria baicalensis and is a unique absorption peak of Scutellaria baicalensis.

[0129] 6. Correlation study of fingerprint spectra of corresponding parts of rhubarb medicinal material Take 1.0g each of Zhikang tablets and negative samples lacking rhubarb (Zhikang tablets lacking rhubarb), and 0.1g of rhubarb (equivalent to the amount of rhubarb in 1.0g tablets). Prepare Zhikang tablet test solutions, negative sample test solutions lacking rhubarb, and rhubarb test solutions according to the test solution preparation method in Example 1.

[0130] Fingerprint chromatograms were determined according to the high-performance liquid chromatography (HPLC) conditions and methods of Example 1. The chromatograms and UV absorption spectra of the corresponding chromatographic peaks of Zhikang tablets, rhubarb medicinal material, and negative samples lacking rhubarb were compared to determine the rhubarb medicinal material to which each fingerprint chromatogram shared a common fingerprint peak. The results are shown in [Figure 1]. Figure 24 . Figure 24 A, B, and C are the HPLC chromatograms of rhubarb, the negative sample lacking rhubarb, and the hemorrhoid tablets, respectively.

[0131] from Figure 24 The results showed that peaks 1, 2, 6, 7, 8, 9, and 10 in the chromatogram of rhubarb appeared at the corresponding positions in the hemorrhoid tablets. However, peaks 6, 7, 8, 9, and 10 did not appear at the corresponding positions in the negative chromatogram of rhubarb. It can be basically determined that peaks 6, 7, 8, 9, and 10 originated from rhubarb and are exclusive absorption peaks of rhubarb.

[0132] IV. Conclusion This invention develops a fingerprint spectroscopy detection method for flavonoid components in hemorrhoid tablets, achieving a perfect combination of fingerprint spectroscopy and quality control of traditional Chinese medicine solid dosage forms, filling the gap in the original quality standards for multi-component quality control. The determination method of this invention provides good quality control for solid dosage forms with complex formulations of six traditional Chinese medicines. Compared with single-component quality control methods, it is more scientific and comprehensive, providing a certain reference for the application of fingerprint spectroscopy technology in the quality standards of traditional Chinese medicine solid dosage forms.

[0133] 1. A standard fingerprint spectrum of flavonoid components in Zhikang tablets was established. (1) The preparation method of the test sample was studied. A specific amount of polyamide powder adsorbent was used to purify the sample, the elution solvent was identified, and the preparation method of the test sample solution was determined. This method can highly enrich the flavonoid components. Compared with direct extraction with methanol, it can effectively remove most of the impurities and reduce the interference of other components on the chromatographic peaks, resulting in better separation effect.

[0134] (2) The column temperature, mobile phase and wavelength were studied, and the gradient of the mobile phase was explored in depth. The detection conditions of high performance liquid chromatography were determined. Under these chromatographic conditions, each peak can be effectively separated.

[0135] (3) The HPLC fingerprint spectra of flavonoid components of 7 batches of Zhikang tablets were determined, and the similarity of the fingerprint spectra of flavonoid components of 7 batches of Zhikang tablets reached 0.92 or above.

[0136] (4) Through comparative study of the fingerprint spectrum of hemorrhoid tablets and medicinal materials, the exclusive absorption peaks of five medicinal materials, namely Siegesbeckia orientalis, Scutellaria baicalensis, Lonicera japonica, Sanguisorba officinalis charcoal and Scutellaria baicalensis, were determined in the fingerprint spectrum. The established fingerprint spectrum also puts forward higher requirements for the quality control of medicinal materials.

[0137] 2. A method for quantitative determination of common peaks in hemorrhoid tablets was established: By locating the common peaks and using the standard curve method, a quantitative determination method for the common peaks in Zhikang tablets was established. This method has been verified multiple times, and its accuracy and repeatability meet the requirements, providing a reliable basis for the quality control of Zhikang tablets.

[0138] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for determining flavonoid components in hemorrhoid tablets, characterized in that, The test solution was analyzed by high-performance liquid chromatography (HPLC). The HPLC conditions were as follows: Chromatographic column: C18 column; Column temperature: 25℃-30℃; Detection wavelength: 210-220nm, injection volume: 8-12μL, running time: 55-80min; Flow rate: 0.6-0.8 mL / min; Mobile phase: Phase A is acetonitrile, and Phase B is 0.05-0.5% phosphoric acid solution; Gradient elution, the elution procedure is as follows: 。 2. The determination method according to claim 1, characterized in that, The chromatographic column is a SunFire®-C18.

3. The determination method according to claim 1, characterized in that, The column temperature is 25℃.

4. The determination method according to claim 1, characterized in that, The detection wavelength is 216 nm, the injection volume is 10 μL, and the running time is 60 min; the flow rate is 0.8 mL / min.

5. The determination method according to claim 1, characterized in that, The mobile phase is: phase A acetonitrile, phase B a 0.1% volume concentration phosphoric acid solution; the phosphoric acid solution is specifically an aqueous phosphoric acid solution.

6. The determination method according to claim 1, characterized in that, The gradient elution process is as follows: 。 7. The determination method according to claim 1, characterized in that, The preparation method of the test solution includes the following steps: grinding hemorrhoid tablets, extracting them with ethanol using ultrasound, adsorbing them with polyamide, evaporating the ethanol, transferring them to a chromatography column, eluting them with petroleum ether, discarding the eluent, eluting them with methanol, and making up to volume to obtain the hemorrhoid tablet test solution.

8. A method for preparing a test solution of hemorrhoid tablets, characterized in that, The steps include: grinding hemorrhoid tablets, extracting them with ethanol using ultrasound, adsorbing them with polyamide, evaporating the ethanol, transferring them to a chromatography column, eluting with petroleum ether, discarding the eluent, eluting with methanol, and making up to volume to obtain the hemorrhoid tablet test solution.

9. The preparation method according to claim 8, characterized in that, The steps include: take 0.5-1.5g of the ground hemorrhoid tablets and mix them with 7.5-25mL of ethanol, and extract by ultrasonication for 20-40min; filter, add 0.75-2.5g of polyamide powder for adsorption, evaporate the ethanol on a water bath, then transfer to a chromatography column, elute twice with 10-40mL of petroleum ether, 5-20mL each time, discard the eluent, then elute with methanol, and make up to 20-30mL; The amount of polyamide used is 2-5 times the mass of the hemorrhoid tablets; The chromatography column is packed with polyamide powder; the inner diameter of the chromatography column is 1-2 cm, the column length is 5-15 cm, and it is dry-packed. The sample loading rate into the chromatography column is 1-2 mL / min.

10. The application of the determination method according to any one of claims 1-7 in the quality testing of hemorrhoid tablets.

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