Method for detecting multiple flavonoid components in radix folii abelmoschus

Abstract

The present application belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a detection method for multiple flavonoid components in leaf of Hibiscus mutabilis L. The present application detects the test sample by HPLC to obtain the high performance liquid chromatography detection result of the flavonoid components in the test sample; the test sample is an extraction solution of the leaf of Hibiscus mutabilis L., and the flavonoid components include at least four of rutin, isoquercitrin, kaempferol-3-O-beta-acacia disaccharide, kaempferol-3-O-oleandrin, kaempferol-3-O-beta-D-glucoside and aglycone. The present application can simultaneously determine the contents of the six flavonoid components of rutin, isoquercitrin, kaempferol-3-O-beta-acacia disaccharide, kaempferol-3-O-oleandrin, kaempferol-3-O-beta-D-glucoside and aglycone by reasonably optimizing the HPLC chromatographic detection conditions, and has the characteristics of simple operation, stability and reliability, high accuracy, good reproducibility and strong practicability.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine analysis technology, specifically relating to a method for detecting multiple flavonoid components in Hibiscus mutabilis leaves. Background Technology

[0002] Hibiscus mutabilis leaves, originally named ground hibiscus leaves, were first recorded in the "Illustrated Materia Medica" and are the dried leaves of Hibiscus mutabilis L., a plant of the Malvaceae family. They are neutral in nature and pungent in taste, and have the effects of cooling blood and detoxifying, reducing swelling and relieving pain (see the National Pharmacopoeia Commission, Pharmacopoeia of the People's Republic of China, Volume 1, 2020 edition, Beijing: China Medical Science and Technology Press, p. 63). Studies have shown that the main components of Hibiscus mutabilis leaves are flavonoids, and more than 50 compounds have been isolated so far, which have pharmacological effects such as anti-inflammatory, antioxidant, antifungal, antiviral, antitumor, and immunomodulatory effects (see Eric W.C.Chan et al. A Review on the Phytochemistry and Pharmacology of two Hibiscus Species with Spectacular Flower Colour Change: H. tiliaceus and H. mutabilis, International Journal of Pharmacognosy and Phytochemical Research, 2016, 8(7): 1200-1208). Hibiscus leaves have significant clinical efficacy. Internally, they can be used for respiratory illnesses such as coughs due to lung heat and viral colds, as seen in products like Hibiscus Anti-Influenza Granules, Hibiscus Anti-Influenza Capsules, and Hibiscus Cold Capsules. Externally, they are a key medicine for treating carbuncles and boils, such as Compound Hibiscus Leaf Tincture. Due to their widespread clinical application, the quality control of this medicinal material has received considerable attention.

[0003] Hibiscus mutabilis is widely distributed, mainly produced in Sichuan, Hunan, Jiangsu, Zhejiang, Hebei and other places. Due to the influence of various factors such as place of origin, harvesting time and growing environment, the quality of medicinal materials varies (see Wang Xilin et al., Determination of Rutin in Hibiscus mutabilis Leaves from 17 Origin Areas, Chinese Patent Medicine, 2018, 40(4):886-890; Diangang Liu et al. Determination of Rutin and Isoquercetin Contents in Hibisci mutabilis Folium in Different Collection Periods by HPLC, Journal of Chromatographic Science, 2015, 53(10):1680-1684). Therefore, establishing a scientific, reasonable, simple and feasible multi-index content determination method is crucial for effectively controlling the quality of Hibiscus mutabilis leaf medicinal materials.

[0004] Among the flavonoids in Hibiscus mutabilis leaves, rutin, isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutin, kaempferol-3-O-β-D-glucoside, and cypermethrin are the components that differentiate the quality of Hibiscus mutabilis leaves from different sources. These components are stable and can be selected as indicators for quality control of the medicinal material (see Huang Lilu et al., Study on the Spectral Relationship of Antioxidant Active Components in Hibiscus mutabilis Leaves, Chinese Journal of Modern Applied Pharmacy, 2022, 39(19):2489-2497). The 2020 edition of the Chinese Pharmacopoeia only specifies rutin as the content determination indicator for Hibiscus mutabilis leaves, using tetrahydrofuran-0.3% phosphoric acid solution (15:85) as the mobile phase and detecting at a wavelength of 359 nm. This method has only one indicator and no detection capability for cypermethrin. Furthermore, using tetrahydrofuran as the mobile phase results in poor stability and can easily damage the chromatographic column.

[0005] Feng Liping et al. established a method for determining four components: rutin, isoquercitrin, kaempferol-3-O-rutin, and kaempferol glycoside (see Feng Liping et al., Research on the Improvement of Quality Standards of Hibiscus mutabilis Leaves, Chinese Traditional Medicine, 2023, 45(8):2622-2627). This method cannot completely separate kaempferol-3-O-β-robinin and kaempferol-3-O-β-D-glucoside. There are many impurity peaks in the chromatogram. The separation of the analytes rutin, isoquercitrin, and kaempferol-3-O-rutin is poor. The peak shape of the chromatogram needs further improvement. Summary of the Invention

[0006] The purpose of this invention is to provide a method for detecting multiple flavonoid components in Hibiscus mutabilis leaves. The detection method provided by this invention can simultaneously determine the content of six flavonoid components: rutin, isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and kaempferol glycoside. It is characterized by simple operation, stability and reliability, high accuracy, good reproducibility, and strong practicality.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for detecting multiple flavonoid components in Hibiscus mutabilis leaves, comprising the following steps:

[0009] The test sample was analyzed by high performance liquid chromatography (HPLC) to obtain the HPLC results of flavonoid components in the test sample; the test sample was an extract solution of Hibiscus mutabilis leaf, and the flavonoid components included at least four of the following: rutin, isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and cypermethrin.

[0010] The detection conditions of the high-performance liquid chromatography (HPLC) method include: gradient elution; the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of phosphoric acid or an aqueous solution of formic acid, the phosphoric acid aqueous solution contains 0.1-0.3% phosphoric acid by mass, the formic acid aqueous solution contains 0.1% formic acid by mass, and mobile phase B is acetonitrile; the gradient elution program is: 0-10 min, 5-12% (v:v) mobile phase B; 10-40 min, 12-18% (v:v) mobile phase B; 40-50 min, 18-25% (v:v) mobile phase B; 50-60 min, 25-27% (v:v) mobile phase B; 60-70 min, 27-40% (v:v) mobile phase B; or: 0-10 min, 5-12% (v:v) mobile phase B; 10- 20 min, 12–16% (v:v) mobile phase B; 20–35 min, 16% (v:v) mobile phase B; 35–42 min, 16–25% (v:v) mobile phase B; 42–52 min, 25–26% (v:v) mobile phase B; 52–65 min, 26–42% (v:v) mobile phase B; or: 0–12 min, 5–12% (v:v) mobile phase B. Mobile phase B; 12–30 min, 12–16% (v:v) mobile phase B; 30–40 min, 16% (v:v) mobile phase B; 40–50 min, 16–25% (v:v) mobile phase B; 50–60 min, 25–26% (v:v) mobile phase B; 60–70 min, 26–35% (v:v) mobile phase B; detection wavelength: 254–265 nm.

[0011] Preferably, the high performance liquid chromatography detection results are obtained using the external standard method or the one-test-multiple-evaluation method;

[0012] The one-test-multiple-evaluation method uses rutin as an internal reference and determines the relative correction factors of the remaining flavonoid components according to Formula 1.

[0013] f s / i =(A s / C s ) / (A i / C i ) Formula 1;

[0014] In Formula 1: A s C represents the peak area of ​​the internal reference material. s For internal reference concentration, A i C represents the peak area of ​​the flavonoid component to be tested. i f represents the concentration of the flavonoid component to be measured. s / i This is the relative correction factor for the flavonoid components to be tested.

[0015] Preferably, the packing material in the chromatographic column used in the high-performance liquid chromatography is octadecylsilane-bonded silica gel.

[0016] Preferably, the volumetric flow rate of the mobile phase is 0.8 to 1.2 mL / min.

[0017] Preferably, the column temperature of the chromatographic column used in the high performance liquid chromatography method is 25-35°C.

[0018] Preferably, the preparation method of the Hibiscus mutabilis leaf extract solution includes the following steps:

[0019] The leaves of Hibiscus mutabilis were mixed with an ethanol aqueous solvent for extraction to obtain the Hibiscus mutabilis leaf extract solution; the ethanol aqueous solvent contained 50-70% by mass.

[0020] Preferably, the ratio of the hibiscus leaf medicinal material to the ethanol aqueous solvent is 1:10 to 1:40;

[0021] The extraction is performed by heating and reflux extraction or ultrasonic extraction; the ultrasonic extraction power is 600-800W, the frequency is 40-50kHz, and the time is 30-60min.

[0022] Preferably, the relative correction factors for isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutin, kaempferol-3-O-β-D-glucoside, and kaempferol glycoside determined by the one-test-multiple-evaluation method are 1.3593–1.3708, 0.4242–0.4265, 1.0852–1.1005, 0.6118–0.6167, and 1.1524–1.1596, respectively.

[0023] Preferably, the injection volume of the high performance liquid chromatography is 10 μL.

[0024] Preferably, the chromatographic column used in the high-performance liquid chromatography method has a column length of 250 mm, an inner diameter of 4.6 mm, and a packing particle diameter of 5 μm.

[0025] This invention provides a method for detecting multiple flavonoid components in Hibiscus mutabilis leaf extract, comprising the following steps: detecting the test sample using high-performance liquid chromatography (HPLC) to obtain the HPLC detection results of the flavonoid components in the test sample; wherein the test sample is an extract solution of Hibiscus mutabilis leaf extract, and the flavonoid components include at least four of the following: rutin, isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutin, kaempferol-3-O-β-D-glucoside, and cypermethrin; and the detection method using HPLC... The conditions include: gradient elution; the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of phosphoric acid or an aqueous solution of formic acid, wherein the phosphoric acid aqueous solution contains 0.1-0.3% phosphoric acid by mass, and the formic acid aqueous solution contains 0.1% formic acid by mass, and mobile phase B is acetonitrile; the elution program for the gradient elution is: 0-10 min, 5-12% (v:v) mobile phase B; 10-40 min, 12-18% (v:v) mobile phase B; 40-50 min, 18-25% (v:v) mobile phase B. Mobile phase B; 50–60 min, 25–27% (v:v) mobile phase B; 60–70 min, 27–40% (v:v) mobile phase B; or: 0–10 min, 5–12% (v:v) mobile phase B; 10–20 min, 12–16% (v:v) mobile phase B; 20–35 min, 16% (v:v) mobile phase B; 35–42 min, 16–25% (v:v) mobile phase B; 42–52 min, 25–26% (v:v) mobile phase B; 52–6 5 min, 26–42% (v:v) mobile phase B; or: 0–12 min, 5–12% (v:v) mobile phase B; 12–30 min, 12–16% (v:v) mobile phase B; 30–40 min, 16% (v:v) mobile phase B; 40–50 min, 16–25% (v:v) mobile phase B; 50–60 min, 25–26% (v:v) mobile phase B; 60–70 min, 26–35% (v:v) mobile phase B; detection wavelength is 254–265 nm. This invention, through rational optimization of HPLC chromatographic detection conditions, constructs a method for simultaneously determining the content of six flavonoid components: rutin, isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutin glycoside, kaempferol-3-O-β-D-glucoside, and cynomolpicrin. Compared with existing technologies, this invention has the following significant advantages:

[0026] The chromatographic detection method constructed in this invention has a stable chromatographic baseline, high peak resolution, and good symmetry. It can completely separate six components: rutin, isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutin, kaempferol-3-O-β-D-glucoside, and cypermethrin, thus overcoming the shortcomings of existing single-index and multi-index content determination methods.

[0027] The content determination method constructed in this invention has good specificity, accuracy and reliability, and can simultaneously determine the content of six components: rutin, isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutin, kaempferol-3-O-β-D-glucoside, and cypermethrin. It has good durability and practicality, and improves the quality evaluation system of Hibiscus mutabilis leaf medicinal material.

[0028] Furthermore, in this invention, the high-performance liquid chromatography (HPLC) detection results are obtained using the quality-assay-multiple-analysis (QAMS) method. The QAMS content determination method constructed in this invention is highly efficient, rapid, and easy to operate. Using readily available and inexpensive rutin as a single internal reference, it achieves simultaneous determination of the contents of six flavonoid components, improving detection efficiency and reducing detection costs. Attached Figure Description

[0029] Figure 1 HPLC chromatograms of the test solution under different mobile phase gradient elution conditions;

[0030] Figure 2 HPLC chromatograms of the test sample solutions under different mobile phase systems;

[0031] Figure 3 HPLC chromatograms of the test sample solution at different detection wavelengths;

[0032] Figure 4 HPLC chromatogram of Hibiscus mutabilis leaf test solution ( Figure 4 HPLC chromatograms of A and reference solution (in the text) Figure 4 (B) in the middle;

[0033] Figure 5 Linearity graph of the reference standard;

[0034] Figure 6 The spectral data (carbon spectrum) of kaempferol-3-O-β-robinin;

[0035] Figure 7 The spectroscopic spectrum (H2N) of kaempferol-3-O-β-D-glucoside is shown.

[0036] Figure 8 The spectral pattern (carbon spectrum) of kaempferol-3-O-β-D-glucoside. Detailed Implementation

[0037] This invention provides a method for detecting multiple flavonoid components in Hibiscus mutabilis leaves, comprising the following steps:

[0038] The test sample was analyzed by high performance liquid chromatography (HPLC) to obtain the HPLC results of flavonoid components in the test sample; the test sample was an extract solution of Hibiscus mutabilis leaf, and the flavonoid components included at least four of the following: rutin, isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and cypermethrin.

[0039] The detection conditions of the high-performance liquid chromatography (HPLC) method include: gradient elution; the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of phosphoric acid or an aqueous solution of formic acid, the phosphoric acid aqueous solution contains 0.1-0.3% phosphoric acid by mass, the formic acid aqueous solution contains 0.1% formic acid by mass, and mobile phase B is acetonitrile; the gradient elution program is: 0-10 min, 5-12% (v:v) mobile phase B; 10-40 min, 12-18% (v:v) mobile phase B; 40-50 min, 18-25% (v:v) mobile phase B; 50-60 min, 25-27% (v:v) mobile phase B; 60-70 min, 27-40% (v:v) mobile phase B; or: 0-10 min, 5-12% (v:v) mobile phase B; 10- 20 min, 12–16% (v:v) mobile phase B; 20–35 min, 16% (v:v) mobile phase B; 35–42 min, 16–25% (v:v) mobile phase B; 42–52 min, 25–26% (v:v) mobile phase B; 52–65 min, 26–42% (v:v) mobile phase B; or: 0–12 min, 5–12% (v:v) mobile phase B. Mobile phase B; 12–30 min, 12–16% (v:v) mobile phase B; 30–40 min, 16% (v:v) mobile phase B; 40–50 min, 16–25% (v:v) mobile phase B; 50–60 min, 25–26% (v:v) mobile phase B; 60–70 min, 26–35% (v:v) mobile phase B; detection wavelength: 254–265 nm.

[0040] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0041] In this invention, the method for preparing the extract solution of Hibiscus mutabilis leaves preferably includes the following steps:

[0042] The leaves of Hibiscus mutabilis were mixed with an ethanol aqueous solvent for extraction to obtain the Hibiscus mutabilis leaf extract solution; the ethanol aqueous solvent contained 50-70% by mass.

[0043] In this invention, the Hibiscus mutabilis leaf medicinal material is preferably Hibiscus mutabilis leaf medicinal material powder. The Hibiscus mutabilis leaf medicinal material powder is preferably the material passing through a 60-mesh sieve. The ethanol-water solvent preferably contains 70% ethanol by mass. The material-to-liquid ratio of the Hibiscus mutabilis leaf medicinal material to the ethanol-water solvent is preferably 1:10 to 1:40, more preferably 1:10. The material-to-liquid ratio is the ratio of the mass of the Hibiscus mutabilis leaf medicinal material to the volume of the ethanol-water solvent. In this invention, the extraction is preferably performed by reflux extraction or ultrasonic extraction. The reflux extraction time is preferably 60 minutes. After the reflux extraction is completed, the reflux extract is obtained. This invention preferably further includes: placing the reflux extract at room temperature, replenishing the weight loss from the reflux extraction with the corresponding ethanol-water solvent used during extraction, and then performing solid-liquid separation. The liquid product obtained from the solid-liquid separation is the Hibiscus mutabilis leaf medicinal material extract solution. The solid-liquid separation is preferably performed by filtration.

[0044] In this invention, the extraction is more preferably ultrasonic extraction, with a preferred power of 600-800W, a preferred frequency of 40-50kHz, and a preferred time of 30-60min. After ultrasonic extraction, an ultrasonic extract is obtained. Preferably, this invention further includes: placing the ultrasonic extract at room temperature, replenishing the weight loss from the ultrasonic extraction with the corresponding ethanol-water solvent used during extraction, and then performing solid-liquid separation. The liquid product obtained from the solid-liquid separation is the extract solution of the Hibiscus mutabilis leaf medicinal material. The solid-liquid separation is preferably filtration.

[0045] In this invention, before the hibiscus leaf extract solution is injected, the hibiscus leaf extract solution is preferably filtered to obtain a test solution, and the filtration is preferably performed using a 0.45μm microporous membrane.

[0046] This invention utilizes ultrasonic extraction to prepare test solutions, thereby enabling the accurate detection of rutin, isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutin, kaempferol-3-O-β-D-glucoside, and cypermethrin.

[0047] In this invention, the flavonoid components preferably include rutin, isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutin, kaempferol-3-O-β-D-glucoside, and kaempferol glycoside.

[0048] In this invention, the packing material in the chromatographic column used in the high-performance liquid chromatography (HPLC) method is preferably octadecylsilane-bonded silica gel. The column length is preferably 250 mm, the inner diameter is preferably 4.6 mm, and the particle diameter of the packing material is preferably 5 μm.

[0049] In this embodiment of the invention, the chromatographic column can be an Agilent ZORBAX Eclipse XDB-C18 column (250mm × 4.6mm, 5μm) or a Waters column. C18 column (250 mm × 4.6 mm, 5 μm) or GL Sciences-InertSustain C18 column (250 mm × 4.6 mm, 5 μm).

[0050] In this invention, the column temperature of the chromatographic column used in the high performance liquid chromatography method is preferably 25-35°C, more preferably 30°C, and in the embodiments it can be 25°C, 30°C or 35°C.

[0051] In this invention, the injection volume of the high performance liquid chromatography is preferably 10 μL.

[0052] In this invention, the volumetric flow rate of the mobile phase is preferably 0.8 to 1.2 mL / min, more preferably 1.0 mL / min, and in the embodiments it can be 0.8 mL / min, 1.0 mL / min or 1.2 mL / min.

[0053] In this invention, the water in the phosphoric acid aqueous solution and the water in the formic acid aqueous solution are preferably purified water. The mass content of phosphoric acid in the phosphoric acid aqueous solution is preferably 0.1%, but in the examples it can be 0.1%, 0.2%, or 0.3%.

[0054] In this invention, the detection wavelength is preferably 265nm, and in the embodiments it can be 254nm, 260nm or 265nm.

[0055] In this invention, the high performance liquid chromatography detection results are obtained using the external standard method (ESM) or the one-test-multiple-evaluation method.

[0056] In this invention, the external standard method is preferably the standard curve method.

[0057] In this invention, the one-measure-multiple-evaluation method preferably uses rutin as an internal reference, and determines the relative correction factor (f) of the remaining flavonoid components according to Formula 1. s / i );

[0058] f s / i =(A s / C s ) / (A i / Ci ) Formula 1;

[0059] In Formula 1: A s C represents the peak area of ​​the internal reference substance. s For internal reference concentration, A i C represents the peak area of ​​the flavonoid component to be tested. i f represents the concentration of the flavonoid component to be measured. s / i This is the relative correction factor for the flavonoid components to be tested.

[0060] In this invention, the relative correction factors for isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutin, kaempferol-3-O-β-D-glucoside, and kaempferol glycoside determined by the one-test-multiple-evaluation method are preferably 1.3593–1.3708, 0.4242–0.4265, 1.0852–1.1005, 0.6118–0.6167, and 1.1524–1.1596, respectively; more preferably 1.3648, 0.4251, 1.0942, 0.6147, and 1.1542, respectively.

[0061] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0062] The instruments and reagents used in the embodiments of this invention are as follows:

[0063] Instruments: Agilent 1260 HPLC system (Agilent Technologies); Waters e2695 HPLC system (Waters Corporation, USA), with both Agilent 1260 and Waters e2695 HPLC systems used in Example 16, and Agilent 1260 HPLC system used in all other examples; XA105DU electronic balance (METTLER TOLEDO); KDM temperature-controlled heating mantle (Shandong Juancheng Guangming Instrument Co., Ltd.); KQ-600E ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Agilent ZORBAX Eclipse XDB-C18 column (250mm × 4.6mm, 5μm, Agilent Technologies); Waters C18 column (250mm×4.6mm, 5μm, Waters Corporation, USA); GL Sciences-InertSustain C18 column (250mm×4.6mm, 5μm, JIER (Shanghai) Trading Co., Ltd.).

[0064] Reagents: Acetonitrile (HPLC grade, TEDIA Reagents, USA); phosphoric acid, 95% ethanol (AR, Sinopharm Chemical Reagent Co., Ltd.); purified water (Hangzhou Wahaha Company).

[0065] Reference standards and samples: Rutin (batch number 100080-202012, purity 91.6%) and isoquercitrin (batch number 111809-202205) were purchased from the National Institutes for Food and Drug Control; Kaempferol-3-O-rutin (batch number C15563189, purity 98%) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; Ginkgoside (batch number HS20605B1, purity 98%) was purchased from Baoji Chenguang Biotechnology Co., Ltd.; Kaempferol-3-O-β-robinin (purity 98.6%) and Kaempferol-3-O-β-D-glucoside (purity 92.4%) were self-prepared reference standards, and their purity was determined by peak area normalization method.

[0066] The instruments and reagents mentioned above, including kaempferol-3-O-β-robinin and kaempferol-3-O-β-D-glucoside, were isolated and purified using this invention. The spectroscopic spectral results are shown below. Figure 6 , Figure 7 and Figure 8 The rest are commercially available products.

[0067] In this invention, the preparation methods of kaempferol-3-O-β-robinin and kaempferol-3-O-β-D-glucoside are prepared according to the extraction and separation procedure of Hibiscus leaf chemical components recorded in Chapter 2.3 Experimental Section (pages 29 and 30) of "Study on Chemical Components of Two Ethnic Medicines [D]" (Chen Lingfang, South-Central University for Nationalities, 2013.5, pp. 29-30). Compound 4 is kaempferol-3-β-D-glucoside (i.e., kaempferol-3-O-β-D-glucoside in this invention), and compound 6 is kaempferol-3-O-β-D-[α-L-rhamnopyranose(1→6)]-galactopyranoside (i.e., kaempferol-3-O-β-robinin in this invention).

[0068] Example 1

[0069] This embodiment provides an HPLC detection method for Hibiscus mutabilis leaves, including the following steps:

[0070] 1.1 Preparation of the test solution

[0071] Take 2.5g of Hibiscus mutabilis leaf powder (S1, passed through a 60-mesh sieve), accurately weigh it, place it in a round-bottom flask, accurately add 25mL of 70% ethanol, heat under reflux for 60min, cool to room temperature, make up the weight loss, filter, and collect the filtrate to obtain the final product.

[0072] 1.2 HPLC detection

[0073] The test solution was analyzed by HPLC under the following chromatographic conditions:

[0074] Chromatographic column: Agilent ZORBAX Eclipse XDB-C18 column (250×4.6mm, 5μm); mobile phase: 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B) gradient elution, elution program shown in Table 1. Flow rate: 1.0 mL / min; column temperature: 30℃; detection wavelength: 265 nm; injection volume: 10 μL.

[0075] Table 1. Elution conditions of mobile phase in Example 1

[0076] 0～10 95→88 5→12 10～40 88→80 12→18 40～50 80→75 18→25 50～60 75→73 25→27 60～70 73→60 27→40

[0077] 1.3 Test Results

[0078] Using the chromatographic conditions of this embodiment, the HPLC chromatogram of the test solution was obtained as follows: Figure 1 "in Figure 1-1 As shown in the figure, the chromatogram baseline is stable, the chromatographic peaks are evenly distributed and have good peak shapes. The resolutions of components 1 to 6 are 1.83, 2.53, 3.29, 6.20, 1.87 and 3.21, respectively, all greater than 1.5, which means they can be well separated from adjacent chromatographic peaks. Therefore, the chromatographic conditions of this embodiment can be used to determine the content of each analyte.

[0079] Example 2

[0080] This embodiment provides an HPLC detection method for Hibiscus mutabilis leaves, including the following steps:

[0081] 2.1 Preparation of the test solution

[0082] Same as item 1.1 in Example 1.

[0083] 2.2 HPLC detection

[0084] The test solution was analyzed by HPLC. The elution procedure of the mobile phase is shown in Table 2. Other conditions are the same as those in item 1.2 of Example 1.

[0085] Table 2. Elution conditions of mobile phase in Example 2

[0086]

[0087]

[0088] 2.3 Test Results

[0089] Using the chromatographic conditions of this embodiment, the HPLC chromatogram of the test solution was obtained as follows: Figure 1 "in Figure 1-2 As shown in the figure, the chromatogram baseline is stable, the chromatographic peaks are relatively uniformly distributed, and the resolutions of components 1 to 6 are 1.62, 2.20, 2.94, 6.25, 4.54, and 1.68, respectively, all greater than 1.5, indicating good separation from adjacent chromatographic peaks. Therefore, the chromatographic conditions of this embodiment can be used for the determination of the content of the analyte.

[0090] Example 3

[0091] This embodiment provides an HPLC detection method for Hibiscus mutabilis leaves, including the following steps:

[0092] 3.1 Preparation of the test solution

[0093] Same as item 1.1 in Example 1.

[0094] 3.2 HPLC detection

[0095] The test solution was analyzed by HPLC. The elution procedure of the mobile phase is shown in Table 3. Other conditions are the same as those in item 1.2 of Example 1.

[0096] Table 3. Elution conditions of mobile phase in Example 3

[0097] 0～12 95→88 5→12 12～30 88→84 12→16 30～40 84 16 40～50 84→75 16→25 50～60 75→74 25→26 60～70 74→65 26→35

[0098] 3.3 Test Results

[0099] Using the chromatographic conditions of this embodiment, the HPLC chromatogram of the test solution was obtained as follows: Figure 1 "in Figure 1-3 As shown in the figure, the chromatogram baseline is stable, the chromatographic peaks are evenly distributed, and the resolutions of components 1 to 6 are 1.80, 2.42, 3.09, 5.99, 3.84, and 2.57, respectively, all greater than 1.5, indicating complete separation from other components. Therefore, the chromatographic conditions of this embodiment can be used to determine the content of each analyte. Furthermore, compared with the chromatographic conditions of Examples 1 and 2, components 5 and 6 are better separated in this embodiment.

[0100] In summary, under the mobile phase gradient elution conditions of Examples 1 to 3, the resolution of each analyte peak is greater than 1.5, and it can be well separated from other chromatographic peaks. Based on the peak shape and distribution, the mobile phase elution conditions of Example 3 are preferred.

[0101] Example 4

[0102] This embodiment provides an HPLC detection method for Hibiscus mutabilis leaves, including the following steps:

[0103] 4.1 Preparation of the test solution

[0104] Same as item 1.1 in Example 1.

[0105] 4.2 HPLC detection

[0106] The test solution was analyzed by HPLC. Gradient elution was performed using 0.1% formic acid aqueous solution (A)-acetonitrile (B) as the mobile phase. The elution conditions are shown in Table 1. Other conditions are the same as those in item 1.2 of Example 1.

[0107] 4.3 Test Results

[0108] Using the mobile phase system of this embodiment, the HPLC chromatogram of the test solution was obtained as follows: Figure 2 "in Figure 2-1 As shown in the figure, the chromatographic peaks of the analyte can be completely separated, and the chromatographic peak symmetry factor is in the range of 0.95 to 1.05, with good peak shape. Therefore, the 0.1% formic acid aqueous solution (A)-acetonitrile (B) mobile phase system can be used for the elution separation of the analyte.

[0109] Example 5

[0110] 5.1 Preparation of the test solution

[0111] Same as item 1.1 in Example 1.

[0112] 5.2 HPLC detection

[0113] The test solution was analyzed by HPLC. Gradient elution was performed using 0.2% phosphoric acid aqueous solution (A)-acetonitrile (B) as the mobile phase. The elution conditions are shown in Table 1. Other conditions are the same as those in item 1.2 of Example 1.

[0114] 5.3 Test Results

[0115] Using the mobile phase system of this embodiment, the HPLC chromatogram of the test solution was obtained as follows: Figure 2 "in Figure 2-2 As shown in the figure, the chromatographic peaks of the analyte can be completely separated with good peak shape. Therefore, the 0.2% phosphoric acid aqueous solution (A)-acetonitrile (B) mobile phase system can be used for the elution separation of the analyte.

[0116] Example 6

[0117] This embodiment provides an HPLC detection method for Hibiscus mutabilis leaves, including the following steps:

[0118] 6.1 Preparation of the test solution

[0119] Same as item 1.1 in Example 1.

[0120] 6.2 HPLC detection

[0121] The test solution was analyzed by HPLC. Gradient elution was performed using 0.3% phosphoric acid aqueous solution (A)-acetonitrile (B) as the mobile phase. The elution conditions are shown in Table 1. Other conditions are the same as those in item 1.2 of Example 1.

[0122] 6.3 Test Results

[0123] Using the mobile phase system of this embodiment, the HPLC chromatogram of the test solution was obtained as follows: Figure 2 "in Figure 2-3 As shown in the figure, the chromatographic peaks of the analyte can be well separated and the peak shapes are good. Therefore, the 0.3% phosphoric acid aqueous solution (A)-acetonitrile (B) mobile phase system can be used for the elution separation of the analyte.

[0124] In summary (Examples 1, 4-6), based on the separation effect and peak shape improvement of each chromatographic peak, both the acetonitrile-phosphoric acid aqueous solution (0.1%-0.3%) system and the acetonitrile-formic acid aqueous solution (0.1%) system can achieve good separation of the analyte and are suitable for the elution separation of the analyte chromatographic peaks. Considering factors such as the pH stability of the mobile phase and the lifespan of the chromatographic column, the preferred mobile phase system is acetonitrile-0.1% phosphoric acid aqueous solution.

[0125] Example 7

[0126] This embodiment investigated the effect of different wavelengths on the chromatographic peak response values ​​of the analyte, including the following steps:

[0127] 7.1 Preparation of the test solution

[0128] Same as item 1.1 in Example 1.

[0129] 7.2 HPLC detection

[0130] The test solution was analyzed by HPLC. The mobile phase elution program is shown in Table 2. Based on the 3D spectrum, the effects of detection wavelengths of 254 nm, 260 nm, 265 nm, 330 nm, and 360 nm on the chromatographic peak response values ​​were compared. Other conditions were the same as those in item 1.2 of Example 1. The corresponding peak areas of each chromatographic peak were recorded.

[0131] 7.3 Test Results

[0132] HPLC chromatograms measured at different wavelengths are shown below Figure 3 The peak areas of each chromatographic peak are shown in Table 4. The results show that the chromatographic baseline is stable in the wavelength range of 254 nm to 265 nm, and each chromatographic peak responds. Based on the response intensity of each chromatographic peak, the detection wavelength is preferably 265 nm.

[0133] Table 4. Peak areas of each component at different wavelengths

[0134]

[0135]

[0136] Example 8

[0137] This embodiment investigated the effect of different column temperatures on the chromatographic peak resolution of the analyte, including the following steps:

[0138] 8.1 Preparation of the test solution

[0139] Same as item 1.1 in Example 1.

[0140] 8.2 HPLC detection

[0141] The test solution was analyzed by HPLC. The mobile phase elution program is shown in Table 3. The effects of column temperatures of 25℃, 30℃, 35℃ and 40℃ on the chromatographic peak resolution were compared. Other conditions were the same as those in item 1.2 of Example 1.

[0142] 8.3 Test Results

[0143] Table 5 shows the peak resolution of the analytes at different column temperatures. The results indicate that different column temperatures significantly affect peak resolution. Within the column temperature range of 25℃ to 35℃, the peak resolution of each component is greater than 1.5, indicating good separation from adjacent peaks. However, at a column temperature of 40℃, component 1 (rutin) is difficult to separate completely. Furthermore, at a column temperature of 25℃, the retention times of each peak are significantly delayed. Therefore, the optimal column temperature for chromatographic conditions is 30℃.

[0144] Table 5. Resolution of each chromatographic peak at different column temperatures

[0145]

[0146] Example 9

[0147] This embodiment investigated the effect of different volumetric flow rates on the chromatographic peak resolution of the analyte, including the following steps:

[0148] 9.1 Preparation of the test solution

[0149] Same as item 1.1 in Example 1.

[0150] 9.2 HPLC detection

[0151] The test solution was analyzed by HPLC. The mobile phase elution program is shown in Table 3. The effects of flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min on the chromatographic peak resolution were compared. Other conditions were the same as those in item 1.2 of Example 1.

[0152] 9.3 Test Results

[0153] Table 6 shows the peak resolution of the analytes at different flow rates. The results indicate that the peak resolution decreases with increasing flow rate. Within the flow rate range of 0.8 mL / min to 1.2 mL / min, the peak resolution of each component is greater than 1.5, indicating good separation from adjacent peaks. Considering factors such as peak resolution and retention time, a flow rate of 1.0 mL / min is optimal.

[0154] Table 6. Resolution of each chromatographic peak at different flow rates

[0155]

[0156] Example 10

[0157] This embodiment examines the effect of different chromatographic columns on the resolution of the analyte peaks, including the following steps:

[0158] 10.1 Preparation of the test solution

[0159] Same as item 1.1 in Example 1.

[0160] 10.2 HPLC detection

[0161] The test solution was analyzed by HPLC. The elution program of the mobile phase is shown in Table 3. Comparisons were made between different brands of columns: Agilent ZORBAX Eclipse XDB-C18 column (250 mm × 4.6 mm, 5 μm); Waters... The effect of C18 column (250mm×4.6mm, 5μm) and GL Sciences-InertSustain C18 column (250mm×4.6mm, 5μm) on chromatographic peak resolution was the same as that in item 1.2 of Example 1.

[0162] 10.3 Test Results

[0163] The peak resolution of the analytes under different chromatographic columns is shown in Table 7. The results indicate that the column packed with octadecylsilane-bonded silica gel (250 mm × 4.6 mm, 5 μm) exhibits good robustness in peak separation. All three columns effectively separated the peaks with good peak shapes. Considering factors such as peak resolution and retention time, the Agilent ZORBAX Eclipse XDB-C18 column (250 mm × 4.6 mm, 5 μm) is the preferred choice for this invention.

[0164] Table 7. Peak resolution under different chromatographic columns

[0165]

[0166] In summary (Examples 1-10), the preferred chromatographic conditions for the Hibiscus mutabilis leaf test solution are as follows: Column: Agilent ZORBAX Eclipse XDB-C18 column (250 × 4.6 mm, 5 μm); Mobile phase: 0.1% phosphoric acid aqueous solution (A) - acetonitrile (B) gradient elution, with the elution program as follows: 0-12 min, 5%-12% B; 12-30 min, 12%-16% B; 30-40 min, 16% B; 40-50 min, 16%-25% B; 50-60 min, 25%-26% B; 60-70 min, 26%-35% B. Flow rate: 1.0 mL / min; Column temperature: 30℃; Detection wavelength: 265 nm; Injection volume: 10 μL.

[0167] Example 11

[0168] This embodiment investigated the effect of different extraction methods on the extraction rate of the analytes in Hibiscus mutabilis leaves, including the following steps:

[0169] 11.1 Preparation of the test solution

[0170] Method 1: Take 2.5g of Hibiscus mutabilis leaf powder (S1, passed through a 60-mesh sieve), accurately weigh it, place it in a round-bottom flask, accurately add 25mL of 70% ethanol, heat under reflux for 60min, cool to room temperature, make up the weight loss, filter, and take the filtrate to obtain test solution 1.

[0171] Method 2: Take 2.5g of Hibiscus mutabilis leaf powder (S1, passed through a 60-mesh sieve), accurately weigh it, place it in an Erlenmeyer flask, accurately add 25mL of 70% ethanol, sonicate for 60min, cool to room temperature, make up the weight loss, filter, and take the filtrate to obtain test solution 2.

[0172] Method 3: Accurately weigh 2.5g of Hibiscus mutabilis leaf powder (S1, passed through a 60-mesh sieve), place it in a round-bottom flask, accurately add 25mL of 70% ethanol, heat under reflux for 60min, cool to room temperature, make up the weight loss, filter, and evaporate the filtrate to dryness. Dissolve in 25mL of distilled water, extract three times with an equal volume of petroleum ether, collect the aqueous layer, and extract three times with an equal volume of ethyl acetate, collect the ethyl acetate layer, evaporate to dryness, dissolve in 70% ethanol, and dilute to 25mL in a volumetric flask to obtain organic phase test solution 3-1. Separately, evaporate the aqueous layer to dryness, dissolve in 70% ethanol, and dilute to 25mL in a volumetric flask to obtain aqueous phase test solution 3-2.

[0173] 11.2 Preparation of reference solution

[0174] Take appropriate amounts of each reference standard, weigh them accurately, and dissolve them in methanol to prepare a mixed reference solution containing 160.57 μg / mL rutin, 25.25 μg / mL isoquercitrin, 139.32 μg / mL kaempferol-3-O-β-robinin, 59.09 μg / mL kaempferol-3-O-rutin, 34.82 μg / mL kaempferol-3-O-β-D-glucoside, and 82.17 μg / mL cypermethrin.

[0175] 11.3 HPLC detection

[0176] The test solution and reference solution prepared in this embodiment were analyzed by HPLC. The chromatographic conditions were the same as those in item 3.2 of Example 3. The peak areas were recorded and the contents of each component were calculated.

[0177] 11.4 Test Results

[0178] The results of the content determination of each component under different extraction methods are shown in Table 8. The results of Method 1 and Method 2 show that the content of each analyte is higher under ultrasonic extraction conditions. The results of Method 3 show that after extraction with petroleum ether and ethyl acetate, components such as rutin and kaempferol-3-O-β-robinin are still largely retained in the aqueous phase and cannot be completely extracted. In conclusion, Method 2, ultrasonic treatment, is the preferred method.

[0179] Table 8. Results of component content determination (mg / g) under different extraction methods

[0180]

[0181] Example 12

[0182] This embodiment investigates the effect of different extraction solvents on the extraction rate of the analytes in Hibiscus mutabilis leaves, including the following steps:

[0183] 12.1 Preparation of the test solution

[0184] Accurately weigh 2.5g of Hibiscus mutabilis leaf powder (passed through a 60-mesh sieve), place it in an Erlenmeyer flask, add 30% ethanol, 50% ethanol, 70% ethanol, 90% ethanol and 25mL of water respectively, seal tightly, weigh, extract by sonication for 60min, cool to room temperature, replenish the lost weight with the corresponding solvent, shake well, filter, and collect the filtrate to obtain the final product.

[0185] 12.2 Preparation of reference solution

[0186] Same as item 11.2 in Example 11.

[0187] 12.3 HPLC detection

[0188] The test solution and reference solution prepared in this embodiment were analyzed by HPLC. The chromatographic conditions were the same as those in item 3.2 of Example 3. The peak areas were recorded and the contents of each component were calculated.

[0189] 12.4 Test Results

[0190] The results of the content determination of each component under different solvent conditions are detailed in Table 9. It can be seen that different extraction solvents have a significant impact on the content determination results of each component. 50% ethanol and 70% ethanol extract each component relatively completely, and there is no significant difference in the content results. Therefore, the extraction solvent can be 50% to 70% ethanol, with 70% ethanol being preferred.

[0191] Table 9. Results of component content determination (mg / g) using different extraction solvents.

[0192]

[0193] Example 13

[0194] This embodiment investigated the effect of different extraction times on the extraction rate of the analytes in Hibiscus mutabilis leaves, including the following steps:

[0195] 13.1 Preparation of the test solution

[0196] Accurately weigh 2.5g of Hibiscus mutabilis leaf powder (S1, passed through a 60-mesh sieve), place it in an Erlenmeyer flask, add 25mL of 70% ethanol, seal tightly, weigh, and extract by ultrasonication for 15min, 30min, 45min, and 60min respectively. Cool to room temperature, replenish the lost weight with the appropriate solvent, shake well, filter, and collect the filtrate to obtain the final product.

[0197] 13.2 Preparation of reference solution

[0198] Same as item 11.2 in Example 11.

[0199] 13.3 HPLC detection

[0200] The test solution and reference solution prepared in this embodiment were analyzed by HPLC. The chromatographic conditions were the same as those in item 3.2 of Example 3. The peak areas were recorded and the contents of each component were calculated.

[0201] 13.4 Test Results

[0202] The results of the content determination of each component at different extraction times are shown in Table 10. The results show that there is no significant difference in the content determination of each component when ultrasonically extracted for 30–60 min, so ultrasonic treatment for 30 min is preferred.

[0203] Table 10. Results of component content determination at different extraction times (mg / g)

[0204]

[0205] Example 14

[0206] This embodiment investigated the effect of different material-to-liquid ratios on the extraction rate of the analyte components from Hibiscus mutabilis leaves, including the following steps:

[0207] 14.1 Preparation of the test solution

[0208] Accurately weigh 2.5g of Hibiscus mutabilis leaf powder (passed through a 60-mesh sieve) and place it in an Erlenmeyer flask. Add 25mL, 50mL, and 100mL of 70% ethanol at ratios of 1:10, 1:20, and 1:40, respectively. Seal the flask tightly, weigh the flask, and extract by sonication for 30min. Cool to room temperature, replenish the lost weight with the appropriate solvent, shake well, filter, and evaporate the filtrate to dryness. Dissolve the filtrate in 70% ethanol and dilute to a volumetric flask of 25mL.

[0209] 14.2 Preparation of reference solution

[0210] Same as item 11.2 in Example 11.

[0211] 14.3 HPLC detection

[0212] The test solution and reference solution prepared in this embodiment were analyzed by HPLC. The chromatographic conditions were the same as those in item 3.2 of Example 3. The peak areas were recorded and the contents of each component were calculated.

[0213] 14.4 Test Results

[0214] The results of the component content determination under different material-to-liquid ratios are shown in Table 11. The results indicate that the component content determination results are higher when the material-to-liquid ratio is 1:10.

[0215] Table 11. Results of component content determination (mg / g) under different feed-to-liquid ratios.

[0216]

[0217] In summary (Examples 11-14), in the detection method of the present invention, preferably, the test solution is prepared as follows: 2.5g of Hibiscus mutabilis leaf powder (passed through a 60-mesh sieve) is accurately weighed, placed in an Erlenmeyer flask, 25mL of 70% ethanol is accurately added, the flask is sealed, the weight is weighed, and the solution is sonicated (600W, 40kHz) for 30min. After cooling to room temperature, the weight loss is replenished with 70% ethanol, the solution is shaken well, filtered, and the filtrate is filtered through a 0.45μm microporous membrane to obtain the test solution.

[0218] Example 15

[0219] This embodiment validates the preferred method for determining the content of analytes in Hibiscus mutabilis leaves, including the following steps:

[0220] 15.1 Preparation of the test solution

[0221] Accurately weigh 2.5g of Hibiscus mutabilis leaf powder (S1, passed through a 60-mesh sieve), place it in an Erlenmeyer flask, accurately add 25mL of 70% ethanol, seal tightly, weigh, sonicate (600W, 40kHz) for 30min, cool to room temperature, replenish the lost weight with 70% ethanol, shake well, filter, and take the filtrate, filter through a 0.45μm microporous membrane to obtain the final product.

[0222] 15.2 Preparation of reference solution

[0223] Take appropriate amounts of each reference standard, weigh them accurately, dissolve them in methanol, and dilute them equally to form 6 mixed reference standard solutions of different concentrations. The concentrations of rutin, isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutin, kaempferol-3-O-rutin, kaempferol-3-O-β-D-glucoside, kaempferol-3-O-rutin, kaempferol-3-O-β-D-glucoside, and kaempferol-3-O-rutin, respectively, are 10.27–328.69 μg / mL.

[0224] 15.3 HPLC Detection

[0225] The chromatographic conditions were as follows: Column: Agilent ZORBAX Eclipse XDB-C18 column (250 × 4.6 mm, 5 μm); Mobile phase: 0.1% phosphoric acid aqueous solution (A) - acetonitrile (B) gradient elution, with the elution program as follows: 0–12 min, 5%–12% B; 12–30 min, 12%–16% B; 30–40 min, 16% B; 40–50 min, 16%–25% B; 50–60 min, 25%–26% B; 60–70 min, 26%–35% B. Flow rate: 1.0 mL / min; Column temperature: 30℃; Detection wavelength: 265 nm; Injection volume: 10 μL.

[0226] 15.4 Specificity Test

[0227] The reference solution and the test solution were injected and analyzed under the chromatographic conditions specified in section 15.3, and the chromatograms were recorded. The results are shown in [Figure 1]. Figure 4 . Figure 4 In the figure, A represents the HPLC chromatogram of the Hibiscus mutabilis leaf test sample solution. Figure 4B in the figure represents the HPLC chromatogram of the reference solution; the HPLC chromatogram of the test solution contains: 1. Rutin; 2. Isoquercetin; 3. Kaempferol-3-O-β-robinin; 4. Kaempferol-3-O-rutin; 5. Kaempferol-3-O-β-D-glucoside; 6. Acanthocyanin. The results show that the retention time of component 1 (rutin) is 33.6 min, component 2 (isoquercetin) is 35.9 min, component 3 (kaempferol-3-O-β-robinin) is 38.1 min, component 4 (kaempferol-3-O-rutin) is 43.2 min, component 5 (kaempferol-3-O-β-D-glucoside) is 46.3 min, and component 6 (acanthocyanin) is 61.2 min. The peak elution time is stable, and the resolution with adjacent chromatographic peaks is greater than 1.5, indicating good separation effect. The theoretical plate number of the chromatographic column is greater than 5000, and the method specificity is good.

[0228] 15.5 Precision Test

[0229] A sample solution of Hibiscus mutabilis leaves was injected six times consecutively, and the peak areas of each component were recorded. The calculated RSD values ​​of the peak areas of rutin, isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutin glycoside, kaempferol-3-O-β-D-glucoside, and cypermethrin were 1.8%, 0.83%, 0.69%, 0.40%, 0.68%, and 0.28%, respectively, indicating good instrument precision.

[0230] 15.6 Stability Test

[0231] The sample solution of Hibiscus mutabilis leaves was injected and analyzed at 0, 4, 8, 12, 16, 20, and 24 h after sample preparation. The peak areas of each component were recorded. The RSD values ​​of the peak areas of rutin, isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutin, kaempferol-3-O-β-D-glucoside, and kaempferol glycoside were 2.0%, 1.2%, 1.2%, 1.4%, 0.65%, and 0.23%, respectively, indicating that the sample solution had good stability within 24 h.

[0232] 15.7 Repeatability Test

[0233] Six test solutions were prepared from hibiscus leaf powder (passed through a 60-mesh sieve). Each solution was injected and analyzed, and the peak areas were recorded. The RSD values ​​of the concentrations of rutin, isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and cypermethrin were calculated to be 2.2%, 3.0%, 2.2%, 2.5%, 0.70%, and 1.9%, respectively, indicating good reproducibility of the method.

[0234] 15.8 Recovery Test

[0235] Six portions of 1.25 g of Hibiscus mutabilis (S1) powder with known content were accurately weighed and added to the reference standard at 100% of the content of each component in the sample to prepare test solutions. The solutions were analyzed by HPLC, and the recovery rates were calculated. The results are shown in Table 12. The average recoveries of rutin, isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutin, kaempferol-3-O-β-D-glucoside, and kaempferol glycoside were 99.59%, 100.09%, 99.28%, 98.43%, 98.61%, and 100.99%, respectively, with RSDs of 1.3%, 2.6%, 1.1%, 1.4%, 2.0%, and 1.9%, respectively, indicating that the method has good accuracy.

[0236] Table 12 Recovery rates of various components in Hibiscus mutabilis medicinal material

[0237]

[0238] 15.9 Examination of Linear Relationships

[0239] The reference solutions prepared in section 15.2 were injected and detected under the chromatographic conditions described in section 15.3, and the peak areas were recorded. A standard curve was plotted with the concentration (μg / mL) of each reference standard on the x-axis (X) and the peak area on the y-axis (Y). Figure 5 The linear regression equation was obtained. The limit of quantitation (LOQ) was determined based on a signal-to-noise ratio of 10:1, and the limit of detection (LOD) was determined based on a signal-to-noise ratio of 3:1. The results are shown in Table 13. The results show that the six components exhibit good linearity within the specified concentration range.

[0240] Table 13 Results of linear relationship investigation of 6 components in Hibiscus mutabilis medicinal material

[0241]

[0242] Example 16

[0243] This embodiment applies the preferred method for determining the content of analytes in Hibiscus mutabilis leaves using the external standard method (ESM) and the quality assessment method (QAMS), including the following steps:

[0244] 16.1 Preparation of the test solution

[0245] Sixteen batches of Hibiscus mutabilis leaves were collected and identified as dried leaves of Hibiscus mutabilis L., a plant of the Malvaceae family. Sample information is shown in Table 14. The preparation method of the test solution is the same as that in item 15.1 of Example 15.

[0246] Table 1416 Information on Hibiscus mutabilis Leaf Samples

[0247] S1 Yichang, Hubei 230721 S9 Kunming (Shilin), Yunnan 230803 S2 Yichang, Hubei 220420 S10 Jinhua, Zhejiang 2309003 S3 Yiyang, Hunan 20230802 S11 Jinhua, Zhejiang 2307001 S4 Chengdu, Sichuan 2308003 S12 Wenzhou, Zhejiang 2309007 S5 Yulin, Guangxi 220615 S13 Suqian, Jiangsu 230805 S6 Meizhou, Guangxi 20230724 S14 Baoding (Anguo), Hebei 20231005 S7 Nanning, Guangxi 230820 S15 Baoding, Hebei (Lianchi) 2307002 S8 Zunyi, Guizhou 20230703 S16 Xianyang, Shaanxi 231025

[0248] 16.2 Preparation of reference solution

[0249] Same as item 15.2 in Example 15.

[0250] 16.3 HPLC detection

[0251] Same as item 15.3 in Example 15.

[0252] 16.4 QAMS method f s / i The determination

[0253] (1) Relative correction factor (f) s / i ) Measurement

[0254] Using rutin as an internal reference, based on the linearity study results in Example 15, section 15.9, according to formula f s / i = (As / Cs) / (Ai / Ci) (As is the peak area of ​​the internal reference, Cs is the concentration of the internal reference, Ai is the peak area of ​​a certain analyte, and Ci is the concentration of a certain analyte). Calculate the f values ​​of isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutin, kaempferol-3-O-β-D-glucoside, and kaempferol glycoside relative to the internal reference at different concentrations. s / i The results are shown in Table 15. The f-values ​​of isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutin, kaempferol-3-O-β-D-glucoside, and kaempferol glycoside were... s / i The ranges were 1.3593–1.3708, 0.4242–0.4265, 1.0852–1.1005, 0.6118–0.6167, and 1.1524–1.1596, respectively, with average values ​​of 1.3648, 0.4251, 1.0942, 0.6147, and 1.1542, respectively.

[0255] Table 15 f of each component s / i Measurement results

[0256]

[0257]

[0258] (2) The effect of different HPLC instruments and chromatographic columns on f s / i Impact

[0259] Two high-performance liquid chromatographs (HPLC) were tested: the Agilent ZORBAX Eclipse XDB-C18 column (250 mm × 4.6 mm, 5 μm) and the Waters e2695. Three different chromatographic columns, namely the C18 column (250 mm × 4.6 mm, 5 μm) and the GL Sciences-InertSustain C18 column (250 mm × 4.6 mm, 5 μm), were compared to f. s / i The effects of different instruments and columns are shown in Table 16. s / i The RSD values ​​were all less than 2.0%, indicating that f in different chromatographic systems s / i It has good durability.

[0260] Table 16 Effects of different instruments and chromatographic columns on f s / i Impact

[0261]

[0262] (3) The effect of different column temperatures on f s / i Impact

[0263] An Agilent 1260 high-performance liquid chromatograph and an Agilent ZORBAX Eclipse XDB-C18 column (250 mm × 4.6 mm, 5 μm) were used to investigate the effects of different column temperatures (25, 30, 35 °C) on f s / i The effects of different column temperatures are shown in Table 17. s / i The RSD values ​​were all less than 1.0%, indicating that the column temperature change between 25℃ and 35℃ had little effect on f. s / i No significant impact; good durability.

[0264] Table 17 Effects of different column temperatures on f s / i Impact

[0265]

[0266] (4) The effect of different volumetric flow rates on f s / i Impact

[0267] An Agilent 1260 high-performance liquid chromatograph and an Agilent ZORBAX Eclipse XDB-C18 column (250 mm × 4.6 mm, 5 μm) were used to investigate the effects of different flow rates (0.8, 1.0, 1.2 mL / min) on f s / i The effects of different column temperatures are shown in Table 18. s / i The RSD values ​​were all less than 1.0%, indicating that changes in volumetric flow rate between 0.8 and 1.2 mL / min had a significant impact on f. s / i No significant impact.

[0268] Table 18 Effects of different volumetric flow rates on f s / i Impact

[0269]

[0270]

[0271] (5) Peak localization of the analyte

[0272] Using rutin as a reference, the relative retention time (R0) of six components—isoquercetin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutin, kaempferol-3-O-β-D-glucoside, and kaempferol glycoside—was calculated using the relative retention time method. s / i The results are shown in Table 19. The R values ​​for each component are... s / i The RSD values ​​were all less than 3.0%, indicating that the relative retention time method used in this experiment to locate the chromatographic peaks of each component was stable and reliable.

[0273] Table 19 Results of relative retention time determination using different instruments and chromatographic columns

[0274]

[0275] 16.5 Content determination results

[0276] The contents of the analytes in each batch were calculated using both ESM and QAMS methods: isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutin, kaempferol-3-O-β-D-glucoside, and cypermethrin. s / i The results are shown in Table 20 for the values ​​of 1.3648, 0.4251, 1.0942, 0.6147, and 1.1542, respectively; the values ​​of each component f s / i The results are shown in Table 21 for the values ​​of 1.3593, 0.4242, 1.0852, 0.6118, and 1.1524, respectively; the values ​​of each component f s / i The results are shown in Table 22 when the values ​​are 1.3708, 0.4265, 1.1005, 0.6167, and 1.1596 respectively. The results show that there is no significant difference in the content of each component obtained by the two methods (RE% ± 5%, P > 0.05), indicating that the chromatographic detection method constructed in this invention is suitable for content determination by ESM and QAMS methods. The results also show that the f values ​​of isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutin, kaempferol-3-O-β-D-glucoside, and cypermethrin are... s / i Within the ranges of 1.3593–1.3708, 0.4242–0.4265, 1.0852–1.1005, 0.6118–0.6167, and 1.1524–1.1596, the QAMS method is accurate, reliable, and robust, and can be used for the determination of multiple components in Hibiscus mutabilis leaves. Furthermore, comparing the content results in Tables 20, 21, and 22, the optimal values ​​for each component (f) were selected. s / iThe values ​​are 1.3648, 0.4251, 1.0942, 0.6147, and 1.1542, respectively.

[0277] Table 20 Results of QAMS and ESM content determination (mg / g)

[0278]

[0279]

[0280] Table 21 Results of QAMS and ESM content determination (mg / g)

[0281]

[0282] Table 22 Results of QAMS and ESM content determination (mg / g)

[0283]

[0284]

[0285] Comparative Example 1

[0286] This comparative example provides an HPLC detection method for Hibiscus mutabilis leaves, including the following steps:

[0287] 1.1 Preparation of the test solution

[0288] Same as item 1.1 in Example 1.

[0289] 1.2 HPLC detection

[0290] The test solution was analyzed by HPLC. The mobile phase elution procedure is shown in Table 23. Other conditions are the same as those in item 1.2 of Example 1.

[0291] Table 23 Elution conditions of mobile phase in Comparative Example 1

[0292] 0～10 95→88 5→12 10～45 88→80 12→20 45～65 80→55 20→45

[0293] 1.3 Test Results

[0294] Using the chromatographic conditions of this comparative example, the HPLC chromatogram of the test solution is as follows: Figure 1 "in Figure 1-4 As shown in the image. Figure 1 "in Figure 1-4 It can be seen that the resolution of component 6 (silver glycoside) is 0.91, which is difficult to completely separate from the adjacent chromatographic peaks. Therefore, the content of this component cannot be accurately determined under these chromatographic conditions.

[0295] Comparative Example 2

[0296] This comparative example provides an HPLC detection method for Hibiscus mutabilis leaves, including the following steps:

[0297] 2.1 Preparation of the test solution

[0298] Same as item 1.1 in Example 1.

[0299] 2.2 HPLC detection

[0300] The test solution was analyzed by HPLC. The mobile phase elution procedure is shown in Table 24. Other conditions are the same as those in item 1.2 of Example 1.

[0301] Table 24 Elution conditions of mobile phase in Comparative Example 2

[0302]

[0303]

[0304] 2.3 Test Results

[0305] The HPLC chromatogram of the test solution obtained using the chromatographic conditions of this comparative example is shown below. Figure 1 "in Figure 1-5 As shown in the figure, the resolution of component 1 (rutin) is 1.49 and the resolution of component 6 (silver glycoside) is 1.37. They cannot be completely separated from adjacent chromatographic peaks. Therefore, the content of the analyte cannot be accurately determined using these chromatographic conditions.

[0306] Comparative Example 3

[0307] This comparative example provides an HPLC detection method for Hibiscus mutabilis leaves, including the following steps:

[0308] 3.1 Preparation of the test solution

[0309] Same as item 1.1 in Example 1.

[0310] 3.2 HPLC detection

[0311] The test solution was analyzed by HPLC. The mobile phase elution procedure is shown in Table 25. Other conditions are the same as those in item 1.2 of Example 1.

[0312] Table 25 Elution conditions of mobile phase in Comparative Example 3

[0313] 0～10 90→85 10→15 10～35 85→83 15→17 35～40 83→68 17→32 40～50 68 32 50～60 68→65 32→35

[0314] 3.3 Test Results

[0315] The HPLC chromatogram of the test solution obtained using the chromatographic conditions of this comparative example is shown below. Figure 1 "in Figure 1-6As shown in the figure, the chromatographic peaks accumulate during the 40-50 minute time period, making it impossible to accurately locate component 5 (kaempferol-3-O-β-D-glucoside) and difficult to separate component 6 (silver glycoside). Therefore, under these chromatographic conditions, the content of the analyte cannot be accurately determined.

[0316] Comparative Example 4

[0317] This comparative example provides an HPLC detection method for Hibiscus mutabilis leaves, including the following steps:

[0318] 4.1 Preparation of the test solution

[0319] Same as item 1.1 in Example 1.

[0320] 4.2 HPLC detection

[0321] The test solution was analyzed by HPLC. The mobile phase elution procedure is shown in Table 26. Other conditions are the same as those in item 1.2 of Example 1.

[0322] Table 26 Elution conditions of mobile phase in Comparative Example 4

[0323] 0～15 90→85 10→15 15～35 85→83 15→17 35～50 83→68 17→32 50～60 68 32

[0324] 4.3 Test Results

[0325] The HPLC chromatogram of the test solution obtained using the chromatographic conditions of this comparative example is shown below. Figure 1 "in Figure 1-7 As shown in the figure, component 5 (kaempferol-3-O-β-D-glucoside) could not be accurately located, and there were basically no peaks in the 35-50 minute time period. The chromatographic peak distribution was uneven. Therefore, the chromatographic conditions could not completely determine the content of the analyte.

[0326] Comparative Example 5

[0327] This comparative example provides an HPLC detection method for Hibiscus mutabilis leaves, including the following steps:

[0328] 5.1 Preparation of the test solution

[0329] Same as item 1.1 in Example 1.

[0330] 5.2 HPLC detection

[0331] The test solution was analyzed by HPLC. Gradient elution was performed using 0.1% acetic acid aqueous solution (A)-acetonitrile (B) as the mobile phase. The elution conditions are shown in Table 1. Other conditions are the same as those in item 1.2 of Example 1.

[0332] 5.3 Test Results

[0333] Using the mobile phase system of this comparative example, the HPLC chromatogram of the test solution was obtained as follows: Figure 2 "in Figure 2-4 As shown in the figure, the chromatographic peaks of the analyte can be separated well, but the peak widths have broadened, and the resolution has decreased. The elution time of analyte 4 (kaempferol-3-O-rutin) has drifted, which is presumably due to the decrease in the pH of the mobile phase. Therefore, the 0.1% acetic acid aqueous solution (A)-acetonitrile (B) mobile phase system is not suitable for the elution and separation of the analyte.

[0334] Comparative Example 6

[0335] This comparative example provides an HPLC detection method for Hibiscus mutabilis leaves, including the following steps:

[0336] 6.1 Preparation of the test solution

[0337] Same as item 1.1 in Example 1.

[0338] 6.2 HPLC detection

[0339] The test solution was analyzed by HPLC with water (A)-acetonitrile (B) as the mobile phase for gradient elution. The elution conditions are shown in Table 1. Other conditions are the same as those in item 1.2 of Example 1.

[0340] 6.3 Test Results

[0341] Using the mobile phase system of this comparative example, the HPLC chromatogram of the test solution was obtained as follows: Figure 2 "in Figure 2-5 As shown in the figure, the chromatographic peaks are broadened and their symmetry is reduced, with leading peaks appearing. The peak shape needs further improvement.

[0342] Comparative Example 7

[0343] This comparative example provides an HPLC detection method for Hibiscus mutabilis leaves, including the following steps:

[0344] 7.1 Preparation of the test solution

[0345] Same as item 1.1 in Example 1.

[0346] 7.2 HPLC detection

[0347] The test solution was analyzed by HPLC, with water (A)-methanol (B) as the mobile phase for gradient elution. The elution conditions are shown in Table 1. Other conditions are the same as those in item 1.2 of Example 1.

[0348] 7.3 Test Results

[0349] Using the mobile phase system of this comparative example, the HPLC chromatogram of the test solution was obtained as follows: Figure 2 "in Figure 2-6As shown in the figure, the chromatographic peaks are sparse and the analyte cannot be located. Therefore, the water (A)-methanol (B) mobile phase system is not suitable for the elution and separation of the analyte.

[0350] As demonstrated by the above embodiments, the method for detecting multiple flavonoid components in Hibiscus mutabilis leaves provided by this invention, through reasonable optimization of HPLC chromatographic detection conditions and sample solution preparation methods, has constructed a method capable of simultaneously determining the content of six flavonoid components: rutin, isoquercitrin, kaempferol-3-O-β-robinin, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and cynomolpicrin. Furthermore, the combination of multi-analysis technology improves detection efficiency and reduces detection costs. This invention's method is simple to operate, stable and reliable, highly accurate, reproducible, and highly practical, providing a new analytical tool for the quality control of Hibiscus mutabilis leaves.

[0351] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative intent, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for detecting multiple flavonoid components in Hibiscus mutabilis leaves, characterized in that, Includes the following steps: High performance liquid chromatography (HPLC) was used to detect the flavonoid components in the test sample, and the HPLC detection results were obtained. The test sample is an extract solution of Hibiscus mutabilis leaves. The preparation method of the Hibiscus mutabilis leaf extract solution includes the following steps: mixing Hibiscus mutabilis leaves with an ethanol-water solvent for extraction to obtain the Hibiscus mutabilis leaf extract solution; the ethanol-water solvent contains 50-70% ethanol by mass; the flavonoid components include rutin, isoquercitrin, and kaempferol-3- O-β -Robinin, Kaempferol-3- O -Rutin, Kaempferol-3- O-β- D-glucoside and silver calcinate; The detection conditions of the high-performance liquid chromatography (HPLC) method include: gradient elution; the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of phosphoric acid or an aqueous solution of formic acid, the phosphoric acid aqueous solution contains 0.1-0.3% phosphoric acid by mass, the formic acid aqueous solution contains 0.1% formic acid by mass, and mobile phase B is acetonitrile; the gradient elution program is: 0-10 min, 5-12% (v:v) mobile phase B; 10-40 min, 12-18% (v:v) mobile phase B; 40-50 min, 18-25% (v:v) mobile phase B; 50-60 min, 25-27% (v:v) mobile phase B; 60-70 min, 27-40% (v:v) mobile phase B; or: 0-10 min, 5-12% (v:v) mobile phase B; 10-20 min, 5-12% (v:v) mobile phase B; 10 ... 0-12 min, 12-16% (v:v) mobile phase B; 20-35 min, 16% (v:v) mobile phase B; 35-42 min, 16-25% (v:v) mobile phase B; 42-52 min, 25-26% (v:v) mobile phase B; 52-65 min, 26-42% (v:v) mobile phase B; or: 0-12 min, 5-12% (v:v) mobile phase B; 12-30 min, 12-16% (v:v) mobile phase B; 30-40 min, 16% (v:v) mobile phase B; 40-50 min, 16-25% (v:v) mobile phase B; 50-60 min, 25-26% (v:v) mobile phase B; 60-70 min, 26-35% (v:v) mobile phase B; detection wavelength 254-265 nm. nm; The packing material in the chromatographic column used in the high performance liquid chromatography method is octadecylsilane-bonded silica gel.

2. The detection method according to claim 1, characterized in that, The high-performance liquid chromatography (HPLC) detection results were obtained using the external standard method or the one-test-multiple-evaluation method. The one-test-multiple-evaluation method uses rutin as an internal reference and determines the relative correction factors of the remaining flavonoid components according to Formula 1. f s / i = (A s / C s ) / (A i / C i ) Formula 1; In Formula 1: A s C represents the peak area of ​​the internal reference substance. s For internal reference concentration, A i C represents the peak area of ​​the flavonoid component to be tested. i The concentration of the flavonoid component to be tested. f s / i This is the relative correction factor for the flavonoid components to be tested.

3. The detection method according to claim 1, characterized in that, The volumetric flow rate of the mobile phase is 0.8~1.2 mL / min.

4. The detection method according to claim 1, characterized in that, The column temperature of the chromatographic column used in the high performance liquid chromatography method is 25~35 ℃.

5. The detection method according to claim 1, characterized in that, The ratio of the hibiscus leaf medicinal material to the ethanol aqueous solvent is 1:10 to 1:40; The extraction is performed by heating and reflux extraction or ultrasonic extraction; the ultrasonic extraction power is 600~800 W, the frequency is 40~50 kHz, and the time is 30~60 min.

6. The detection method according to claim 2, characterized in that, The isoquercitrin and kaempferol-3- determined by the one-test-multiple-evaluation method O-β -Robinin, Kaempferol-3- O -Rutin, Kaempferol-3- O-β- The relative correction factors for D-glucoside and ginsenoside are 1.3593~1.3708, 0.4242~0.4265, 1.0852~1.1005, 0.6118~0.6167, and 1.1524~1.1596, respectively.

7. The detection method according to claim 1, characterized in that, The injection volume for the high-performance liquid chromatography method is 10 μL.

8. The detection method according to claim 1, characterized in that, The high-performance liquid chromatography (HPLC) method uses a column with a length of 250 mm, an inner diameter of 4.6 mm, and a packing particle diameter of 5 μm.

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