Method for detecting various flavonoid components in cottonrose hibiscus leaf medicinal material

Abstract

The invention belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a method for detecting various flavonoid components in cottonrose hibiscus leaf medicinal materials. The method comprises the following steps: detecting a test sample by adopting HPLC (High Performance Liquid Chromatography) to obtain a high performance liquid chromatography detection result of flavonoid components in the test sample; the test sample is a cotton rose hibiscus leaf medicinal material extraction solution, and the flavonoid components comprise at least four of rutin, isoquercitrin, kaempferol-3-O-beta-robinia bioside, kaempferol-3-O-rutinoside, kaempferol-3-O-beta-D-glucoside and silver forging glycoside. According to the method, HPLC chromatographic detection conditions are reasonably optimized, the contents of six flavonoid components including rutin, isoquercitrin, kaempferol-3-O-beta-robinia bioside, kaempferol-3-O-rutinoside, kaempferol-3-O-beta-D-glucoside and silver forging glycoside can be simultaneously determined, and the method has the advantages of being easy to operate, stable, reliable, high in accuracy, good in reproducibility, high in sensitivity, high in accuracy, high in accuracy, high in accuracy, high in sensitivity, high in accuracy and the like. And the practicability is high.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine analysis, and particularly relates to a method for detecting multiple flavonoid components in Hibiscus mutabilis leaf medicinal materials. Background Art

[0002] Hibiscus mutabilis L., originally named Hibiscus tiliaceus L., was first recorded in the Illustrated Compendium of Materia Medica. It is the dried leaves of Hibiscus mutabilis L., a plant of the Malvaceae family. It is mild in nature and pungent in taste, and has the effects of cooling blood, detoxifying, reducing swelling and relieving pain (see the Pharmacopoeia of the People's Republic of China, Part I, 2020 edition, compiled by the National Pharmacopoeia Committee, Beijing: China Medical Science and Technology Press, p63). Studies have shown that the main component of Hibiscus mutabilis L. is flavonoids, and more than 50 compounds have been isolated, with pharmacological effects such as anti-inflammatory, antioxidant, antifungal, antiviral, antitumor, and immunomodulatory (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 mutabilis leaves have significant clinical efficacy. They are taken internally for respiratory ailments such as lung-heat coughs and viral colds, as exemplified by Hibiscus Anti-Influenza Granules, Hibiscus Anti-Influenza Capsules, and Fupu Cold Capsules. Topically, they are a key treatment for carbuncles, sores, and boils, as exemplified by Compound Hibiscus Leaf Tincture. Due to its widespread clinical application, the quality control of these medicinal materials has drawn considerable attention.

[0003] Hibiscus mutabilis is widely distributed and is mainly produced in Sichuan, Hunan, Jiangsu, Zhejiang, Hebei and other places. Due to the influence of various factors such as production area, harvest period, and growth environment, the quality of the medicinal material varies (see Wang Xilin et al. Determination of Rutin in Hibiscus mutabilis Leaves from 17 Production Areas, Chinese Patent Medicine, 2018, 40(4):886-890; Diangang Liu et al. Determination of Rutin and Isoquercetin Contents in Hibisci mutabilis Foliumin Different Collection Periods by HPLC, Journal of Chromatographic Science, 2015, 53(10):1680-1684). Therefore, it is crucial to establish a scientific, reasonable, simple and feasible multi-index content determination method to effectively control the quality of Hibiscus mutabilis leaves.

[0004] Among the flavonoids in Hibiscus mutabilis leaves, six components, namely rutin, isoquercitrin, kaempferol-3-O-β-robinobioside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and argentin, are the quality-differentiating components of Hibiscus mutabilis leaves from different sources. They are stable in nature and can be selected as indicators for quality control of medicinal materials (see Huang Lilu et al., Study on the spectrum-activity relationship of antioxidant active ingredients in Hibiscus mutabilis leaves, Chinese Journal of Modern Applied Pharmacy, 2022, 39(19): 2489-2497). The 2020 edition of the Chinese Pharmacopoeia only stipulates that rutin is the content determination indicator of Hibiscus mutabilis leaves, using tetrahydrofuran-0.3% phosphoric acid solution (15:85) as the mobile phase and detection at a wavelength of 359nm. This method has a single indicator and has no detection ability for argentin. At the same time, using tetrahydrofuran as the mobile phase has poor stability and is prone to damage to the chromatographic column.

[0005] Feng Liping et al. established a determination method for rutin, isoquercetin, kaempferol-3-O-rutinoside, and argentin (see Feng Liping et al., Research on the improvement of the quality standard of Hibiscus mutabilis leaves, Traditional Chinese Medicine, 2023, 45(8): 2622-2627). This method cannot completely separate kaempferol-3-O-β-robinia disaccharide and kaempferol-3-O-β-D-glucoside. There are many impurity peaks in the chromatogram. The separation of the test components rutin, isoquercetin, and kaempferol-3-O-rutinoside is poor, and the chromatographic peak shape needs to be further improved. Summary of the Invention

[0006] The present invention aims to provide a method for detecting multiple flavonoid components in Hibiscus mutabilis leaf medicinal materials. The detection method provided by the present invention can realize the simultaneous determination of the contents of six flavonoid components, namely rutin, isoquercetin, kaempferol-3-O-β-robiniabiside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside and argentin, and has the characteristics of simple operation, stability and reliability, high accuracy, good reproducibility and strong practicality.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

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

[0009] A high performance liquid chromatography method is used to detect the test sample to obtain a high performance liquid chromatography detection result of flavonoid components in the test sample; the test sample is a Hibiscus mutabilis leaf medicinal material extract solution, and the flavonoid components include at least four of rutin, isoquercetin, kaempferol-3-O-β-robinobioside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and argentin;

[0010] The detection conditions of the high performance liquid chromatography method include: the elution mode is gradient elution; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is a phosphoric acid aqueous solution or a formic acid aqueous solution, the mass content of phosphoric acid in the phosphoric acid aqueous solution is 0.1-0.3%, the mass content of formic acid in the formic acid aqueous solution is 0.1%, and the mobile phase B is acetonitrile; the elution program of 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; 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 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.

[0011] Preferably, the HPLC test results are obtained using an external standard method or a one-measurement-multiple-evaluation method;

[0012] The one-measurement-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 is the peak area of ​​the internal reference, C s is the internal reference concentration, A i is the peak area of ​​the flavonoid components to be tested; C i is the concentration of the flavonoids to be tested, f s / i is the relative correction factor of the flavonoid components to be measured.

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

[0016] Preferably, the volume 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 method for preparing the Hibiscus mutabilis leaf medicinal material extract solution comprises the following steps:

[0019] The Hibiscus mutabilis leaf medicinal material and an ethanol-water solvent are mixed for extraction to obtain the Hibiscus mutabilis leaf medicinal material extraction solution; the mass content of ethanol in the ethanol-water solvent is 50-70%.

[0020] Preferably, the material-liquid ratio of the Hibiscus mutabilis leaf medicinal material and the ethanol-water solvent is 1:10 to 1:40;

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

[0022] Preferably, the relative correction factors of isoquercetin, kaempferol-3-O-β-acaciabioside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and argentin determined by the one-measurement-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 filler particle diameter of 5 μm.

[0025] The invention provides a method for detecting multiple flavonoid components in Hibiscus mutabilis leaf medicinal materials, comprising the following steps: using high performance liquid chromatography (HPLC) to detect a sample, and obtaining a high performance liquid chromatography detection result of the flavonoid components in the sample; the sample is a Hibiscus mutabilis leaf medicinal material extract solution, and the flavonoid components include at least four of rutin, isoquercetin, kaempferol-3-O-β-robiniabiside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside and argentin; the high performance liquid chromatography detection method The conditions include: the elution mode is gradient elution; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is a phosphoric acid aqueous solution or a formic acid aqueous solution, the mass content of phosphoric acid in the phosphoric acid aqueous solution is 0.1-0.3%, the mass content of formic acid in the formic acid aqueous solution is 0.1%, and the mobile phase B is acetonitrile; the elution program of 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; the detection wavelength is 254-265 nm. By rationally optimizing HPLC chromatographic detection conditions, the present invention constructs a detection method capable of simultaneously determining the contents of six flavonoid components: rutin, isoquercetin, kaempferol-3-O-β-robinobioside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and arganin. Compared with the existing technology, the present invention has the following significant advantages:

[0026] The chromatographic detection method constructed by the present invention has a stable chromatogram baseline, high chromatographic peak separation and good symmetry, and can achieve complete separation of six components: rutin, isoquercetin, kaempferol-3-O-β-robiniabiside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and argentin, thus making up for the shortcomings of existing single-index and multi-index content determination methods.

[0027] The content determination method constructed by the present invention has good specificity, is accurate and reliable, can simultaneously determine the contents of six components including rutin, isoquercetin, kaempferol-3-O-β-robinobioside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside and argininoside, has good durability and strong practicality, and improves the quality evaluation system of Hibiscus mutabilis leaf medicinal materials.

[0028] Furthermore, in the present invention, the HPLC test results are obtained using a single-measurement, multiple-evaluation method (QAMS). The QAMS content determination method constructed in the present invention is efficient, fast, and easy to operate. It uses a single, inexpensive, and readily available rutin reference substance as an internal reference substance to achieve simultaneous determination of the contents of six flavonoid components, thereby improving detection efficiency and reducing detection costs. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0030] Figure 2 HPLC spectra of the test solution under different mobile phase systems;

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

[0032] Figure 4 is the HPLC spectrum of the test solution of Hibiscus mutabilis leaf ( Figure 4 A) and HPLC spectrum of reference solution ( Figure 4 B in the);

[0033] Figure 5 is the linear relationship diagram of the reference substance;

[0034] Figure 6 This is the spectrum test spectrum of kaempferol-3-O-β-acacia disaccharide (carbon spectrum);

[0035] Figure 7 This is the spectrum test pattern of kaempferol-3-O-β-D-glucoside (hydrogen spectrum);

[0036] Figure 8 This is the spectrum test pattern of kaempferol-3-O-β-D-glucoside (carbon spectrum). DETAILED DESCRIPTION

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

[0038] A high performance liquid chromatography method is used to detect the test sample to obtain a high performance liquid chromatography detection result of flavonoid components in the test sample; the test sample is a Hibiscus mutabilis leaf medicinal material extract solution, and the flavonoid components include at least four of rutin, isoquercetin, kaempferol-3-O-β-robinobioside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and argentin;

[0039] The detection conditions of the high performance liquid chromatography method include: the elution mode is gradient elution; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is a phosphoric acid aqueous solution or a formic acid aqueous solution, the mass content of phosphoric acid in the phosphoric acid aqueous solution is 0.1-0.3%, the mass content of formic acid in the formic acid aqueous solution is 0.1%, and the mobile phase B is acetonitrile; the elution program of 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; 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 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.

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

[0041] In the present invention, the method for preparing the Hibiscus mutabilis leaf medicinal material extract solution preferably comprises the following steps:

[0042] The Hibiscus mutabilis leaf medicinal material and an ethanol-water solvent are mixed for extraction to obtain the Hibiscus mutabilis leaf medicinal material extraction solution; the mass content of ethanol in the ethanol-water solvent is 50-70%.

[0043] In the present invention, the Hibiscus mutabilis leaf medicinal material is preferably Hibiscus mutabilis leaf medicinal material powder. The Hibiscus mutabilis leaf medicinal material powder is preferably a 60-mesh sieve. The mass content of ethanol in the ethanol-water solvent is preferably 70%. The solid-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 solid-liquid ratio is the ratio of the mass of the Hibiscus mutabilis leaf medicinal material to the volume of the ethanol-water solvent. In the present invention, the extraction is preferably reflux extraction or ultrasonic extraction. The reflux extraction time is preferably 60 minutes. After the reflux extraction, a reflux extract material is obtained. The present invention preferably further comprises: placing the reflux extract material at room temperature, replenishing the weight loss of the reflux extraction with the corresponding ethanol-water solvent used during the extraction, and then performing solid-liquid separation. The liquid phase product obtained by the solid-liquid separation is the Hibiscus mutabilis leaf medicinal material extract solution. The solid-liquid separation is preferably filtration.

[0044] In the present invention, the extraction is preferably ultrasonic extraction, preferably at a power of 600-800W, a frequency of 40-50kHz, and a duration of 30-60 minutes. After completion of the ultrasonic extraction, an ultrasonically extracted material is obtained. The present invention preferably further comprises: allowing the ultrasonically extracted material to stand at room temperature, replenishing the weight loss from the ultrasonic extraction with the corresponding ethanol-water solvent used during the 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.

[0045] In the present invention, before the Hibiscus mutabilis leaf medicinal material extract solution is sampled, the present invention preferably filters the Hibiscus mutabilis leaf medicinal material extract solution to obtain a test solution, and the filtration is preferably performed using a 0.45 μm microporous filter membrane.

[0046] The present invention prepares the test solution by adopting the method of ultrasonic extraction, and further realizes the accurate detection of rutin, isoquercetin, kaempferol-3-O-β-robinobioside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside and arginine.

[0047] In the present invention, the flavonoid components preferably include rutin, isoquercetin, kaempferol-3-O-β-robinobioside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside and argentin.

[0048] In the present invention, the filler in the chromatographic column used in the HPLC method is preferably octadecylsilane bonded silica gel. The chromatographic column used in the HPLC method preferably has a column length of 250 mm, an inner diameter of 4.6 mm, and a filler particle diameter of 5 μm.

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

[0050] In the present invention, the column temperature of the chromatographic column used in the HPLC 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 the present invention, the injection volume of the high performance liquid chromatography method is preferably 10 μL.

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

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

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

[0055] In the present invention, the HPLC test results are obtained using an external standard method (ESM) or a one-measurement-multiple-evaluation method.

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

[0057] In the present invention, the one-measurement-multiple-evaluation method preferably uses rutin as an internal reference, and determines the relative correction factors (f s / i );

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

[0059] In formula 1: A s is the peak area of ​​the internal reference, C s is the internal reference concentration, A i is the peak area of ​​the flavonoid components to be tested; C i is the concentration of the flavonoids to be tested, f s / i is the relative correction factor of the flavonoid components to be measured.

[0060] In the present invention, the relative correction factors of isoquercetin, kaempferol-3-O-β-acaciabioside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and argentin determined by the one-measurement-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.

[0061] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with 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 the present invention are as follows:

[0063] Instruments: Agilent 1260 high performance liquid chromatograph (Agilent Technologies); Waters e2695 high performance liquid chromatograph (Waters, USA). In Example 16, an Agilent 1260 high performance liquid chromatograph and a Waters e2695 high performance liquid chromatograph were used, and in the remaining examples, an Agilent 1260 high performance liquid chromatograph was used; XA105DU electronic balance (METTLER TOLEDO); KDM temperature-controlled electric heating mantle (Shandong Juancheng Guangming Instrument Co., Ltd.); KQ-600E ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.); Agilent ZORBAX Eclipse XDB-C18 chromatographic column (250 mm × 4.6 mm, 5 μm, Agilent Technologies Co., Ltd.); Waters C18 chromatographic column (250 mm×4.6 mm, 5 μm, Waters Company, USA); GL Sciences-InertSustain C18 chromatographic column (250 mm×4.6 mm, 5 μm, Tech-Tech (Shanghai) Trading Co., Ltd.).

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

[0065] Reference substances and samples: rutin (batch number 100080-202012, purity 91.6%) and isoquercetin (batch number 111809-202205) were purchased from the China Food and Drug Administration; kaempferol-3-O-rutinoside (batch number C15563189, purity 98%) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; arginine (batch number HS20605B1, purity 98%) was purchased from Baoji Chenguang Biotechnology Co., Ltd.; kaempferol-3-O-β-acacia disaccharide (purity 98.6%) and kaempferol-3-O-β-D-glucoside (purity 92.4%) were homemade reference substances, and the purity was calibrated by the peak area normalization method.

[0066] The above instruments and reagents, kaempferol-3-O-β-acacia disaccharide and kaempferol-3-O-β-D-glucoside are separated and purified by the present invention, and the spectrum test spectrum is shown in Figure 6 、 Figure 7 and Figure 8 The rest are commercially available products.

[0067] In the present invention, the preparation method of kaempferol-3-O-β-robinia pseudopyranoside and kaempferol-3-O-β-D-glucoside is prepared by adopting the extraction and separation procedure of the chemical components of hibiscus leaves described in the experimental section 2.3 (pages 29 and 30) of Chapter 2 of "Study on the Chemical Components of Two Ethnic Medicines [D]" (Chen Lingfang, South-Central University for Nationalities, 2013.5, pages 29-30), wherein compound 4 is kaempferol-3-β-D-glucoside (i.e., kaempferol-3-O-β-D-glucoside in the present invention), and compound 6 is kaempferol-3-O-β-D-[α-L-pyranosyl rhamnosyl (1→6)]-galactopyranoside (i.e., kaempferol-3-O-β-robinia pseudopyranoside in the present invention).

[0068] Example 1

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

[0070] 1.1 Preparation of test solution

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

[0072] 1.2HPLC detection

[0073] The test solution was subjected to HPLC detection, and the chromatographic detection conditions were as follows:

[0074] Chromatographic 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; elution program is shown in Table 1. Volume flow rate: 1.0 mL / min; Column temperature: 30°C; Detection wavelength: 265 nm; Injection volume: 10 μL.

[0075] Table 1 Mobile phase elution conditions for Example 1

[0076] Time (Tmin) Mobile phase (A) Mobile phase (B) 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 chart of the test solution was obtained as follows: Figure 1 In the Figure 1-1 As shown in FIG. 3 , it can be seen that the chromatogram has a stable baseline, uniform distribution of chromatographic peaks, good peak shape, and the separation degrees 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, and can be well separated from adjacent chromatographic peaks. Therefore, the chromatographic conditions of this embodiment can be used to determine the content of each component to be measured.

[0079] Example 2

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

[0081] 2.1 Preparation of test solution

[0082] Same as item 1.1 in Example 1.

[0083] 2.2HPLC detection

[0084] The test solution was subjected to HPLC detection. Among the chromatographic conditions, the mobile phase elution program is shown in Table 2, and other conditions are the same as those in 1.2 of Example 1.

[0085] Table 2 Mobile phase elution conditions for Example 2

[0086]

[0087]

[0088] 2.3 Test results

[0089] Using the chromatographic conditions of this embodiment, the HPLC chart of the test solution was obtained as follows: Figure 1 In the Figure 1-2 As shown in the figure, it can be seen that the baseline of the chromatogram is stable, the chromatographic peaks are evenly distributed, and the separation degrees 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, and can be well separated from adjacent chromatographic peaks. Therefore, the chromatographic conditions of this example can be used for the content determination of the components to be tested.

[0090] Example 3

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

[0092] 3.1 Preparation of test solution

[0093] Same as item 1.1 in Example 1.

[0094] 3.2HPLC detection

[0095] The test solution was subjected to HPLC detection. Among the chromatographic conditions, the mobile phase elution program is shown in Table 3, and other conditions are the same as those in 1.2 of Example 1.

[0096] Table 3 Mobile phase elution conditions for Example 3

[0097] Time (Tmin) Mobile phase (A) Mobile phase (B) 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 chart of the test solution was measured as follows Figure 1 In the Figure 1-3 As shown in the figure, it can be seen that the baseline of the chromatogram is stable, the chromatographic peaks are evenly distributed, and the separation degrees 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, and can be completely separated from other components. Therefore, the chromatographic conditions of this embodiment can be used to determine the content of each component to be tested. In addition, compared with the chromatographic conditions of Examples 1 and 2, in this embodiment, component 5 and component 6 can be better separated.

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

[0101] Example 4

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

[0103] 4.1 Preparation of test solution

[0104] Same as item 1.1 in Example 1.

[0105] 4.2HPLC detection

[0106] The test solution was subjected to HPLC detection, with 0.1% formic acid aqueous solution (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 1.2 of Example 1.

[0107] 4.3 Test results

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

[0109] Example 5

[0110] 5.1 Preparation of test solution

[0111] Same as item 1.1 in Example 1.

[0112] 5.2HPLC detection

[0113] The test solution was subjected to HPLC detection, with 0.2% phosphoric acid aqueous solution (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 1.2 of Example 1.

[0114] 5.3 Test results

[0115] Using the mobile phase system of this embodiment, the HPLC chart of the test solution was measured as follows: Figure 2 In the Figure 2-2 As shown in the figure, the chromatographic peaks of the components to be analyzed can be completely separated and the peak shape is good, so the 0.2% phosphoric acid aqueous solution (A)-acetonitrile (B) mobile phase system can be used for the elution and separation of the components to be analyzed.

[0116] Example 6

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

[0118] 6.1 Preparation of test solution

[0119] Same as item 1.1 in Example 1.

[0120] 6.2HPLC detection

[0121] The test solution was subjected to HPLC detection, with 0.3% phosphoric acid aqueous solution (A)-acetonitrile (B) as the mobile phase gradient elution. The elution conditions are shown in Table 1. Other conditions are the same as those in 1.2 of Example 1.

[0122] 6.3 Test results

[0123] Using the mobile phase system of this embodiment, the HPLC chart of the test solution was measured as follows: Figure 2 In the Figure 2-3 As shown in the figure, the chromatographic peaks of the components to be measured can be well separated and the peak shape is good, so the 0.3% phosphoric acid aqueous solution (A)-acetonitrile (B) mobile phase system can be used for the elution and separation of the components to be measured.

[0124] In summary (Examples 1, 4 to 6), based on the separation effect of each chromatographic peak and the improvement of the peak shape, the acetonitrile-phosphoric acid aqueous solution (0.1% to 0.3%) system and the acetonitrile-formic acid aqueous solution (0.1%) system can better separate the components to be measured and are suitable for the elution and separation of the chromatographic peaks of the components to be measured. Considering factors such as the pH stability of the mobile phase and the life of the chromatographic column, the mobile phase system is preferably acetonitrile-0.1% phosphoric acid aqueous solution.

[0125] Example 7

[0126] This example investigates the effect of different wavelengths on the chromatographic peak response value of the component to be measured, including the following steps:

[0127] 7.1 Preparation of test solution

[0128] Same as item 1.1 in Example 1.

[0129] 7.2 HPLC detection

[0130] The test solution was subjected to HPLC detection. Among the chromatographic conditions, the mobile phase elution program is shown in Table 2. According to the 3D spectrogram, 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 1.2 of Example 1. The corresponding peak area of ​​each chromatographic peak was recorded.

[0131] 7.3 Test results

[0132] HPLC images measured at different wavelengths are shown in Figure 3 The peak areas of each chromatographic peak are shown in Table 4. The results show that within the wavelength range of 254nm to 265nm, the chromatogram baseline is stable and each chromatographic peak has a response. According to the response intensity of each chromatographic peak, the detection wavelength is preferably 265nm.

[0133] Table 4 Chromatographic peak areas of each component at different wavelengths

[0134]

[0135]

[0136] Example 8

[0137] This example investigates the effect of different column temperatures on the chromatographic peak separation of the components to be measured, including the following steps:

[0138] 8.1 Preparation of test solution

[0139] Same as item 1.1 in Example 1.

[0140] 8.2HPLC detection

[0141] The test solution was subjected to HPLC detection. Among the chromatographic conditions, the mobile phase elution program is shown in Table 3. The effects of column temperatures of 25°C, 30°C, 35°C, and 40°C on the chromatographic peak separation were compared. Other conditions were the same as those in 1.2 of Example 1.

[0142] 8.3 Test results

[0143] Table 5 shows the peak resolution of the components under different column temperatures. The results show that column temperature significantly affects peak resolution. Within the 25°C to 35°C column temperature range, the peak resolution of each component was greater than 1.5, indicating good separation from adjacent peaks. However, at 40°C, component 1 (rutin) was difficult to completely separate. Furthermore, at 25°C, the retention times of the peaks were significantly delayed. Therefore, a column temperature of 30°C is the optimal chromatographic condition.

[0144] Table 5 Separation of chromatographic peaks at different column temperatures

[0145]

[0146] Example 9

[0147] This example investigates the effect of different volume flow rates on the chromatographic peak separation of the components to be measured, including the following steps:

[0148] 9.1 Preparation of test solution

[0149] Same as item 1.1 in Example 1.

[0150] 9.2HPLC detection

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

[0152] 9.3 Test results

[0153] Table 6 shows the peak resolution of the components at different flow rates. The results show that 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. Taking into account factors such as peak resolution and retention time, a flow rate of 1.0 mL / min is optimal.

[0154] Table 6 Separation of chromatographic peaks at different volume flow rates

[0155]

[0156] Example 10

[0157] This example investigates the effects of different chromatographic columns on the chromatographic peak separation of the components to be measured, including the following steps:

[0158] 10.1 Preparation of test solution

[0159] Same as item 1.1 in Example 1.

[0160] 10.2HPLC detection

[0161] The test solution was subjected to HPLC detection. The chromatographic conditions and mobile phase elution program were shown in Table 3. The chromatographic columns of different brands were compared: Agilent ZORBAX Eclipse XDB-C18 column (250 mm × 4.6 mm, 5 μm); Waters C18 chromatographic column (250 mm × 4.6 mm, 5 μm); GL Sciences-InertSustain C18 chromatographic column (250 mm × 4.6 mm, 5 μm) on the effect of chromatographic peak separation. Other conditions are the same as those in 1.2 of Example 1.

[0162] 10.3 Test Results

[0163] Table 7 shows the peak resolution of the analytes using different columns. The results show that the octadecylsilane bonded silica column (250 mm × 4.6 mm, 5 μm) provides excellent peak resolution and durability. All three columns provide excellent peak resolution and good peak shape. Considering factors such as peak resolution and retention time, the Agilent ZORBAX Eclipse XDB-C18 column (250 mm × 4.6 mm, 5 μm) is preferred.

[0164] Table 7 Separation of chromatographic peaks under different chromatographic columns

[0165]

[0166] In summary (Examples 1 to 10), preferably, the chromatographic detection conditions for the Hibiscus mutabilis leaf sample solution are as follows: chromatographic 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, elution program: 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. Volume flow rate: 1.0 mL / min; column temperature: 30°C; detection wavelength: 265 nm; injection volume: 10 μL.

[0167] Example 11

[0168] This example investigates the effects of different extraction methods on the extraction rate of the test components in Hibiscus mutabilis leaves, comprising the following steps:

[0169] 11.1 Preparation of test solution

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

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

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

[0173] 11.2 Preparation of reference solution

[0174] An appropriate amount of each reference substance was taken, accurately weighed, and dissolved in methanol to prepare a mixed reference substance solution containing 160.57 μg / mL of rutin, 25.25 μg / mL of isoquercetin, 139.32 μg / mL of kaempferol-3-O-β-robinobioside, 59.09 μg / mL of kaempferol-3-O-rutinoside, 34.82 μg / mL of kaempferol-3-O-β-D-glucoside, and 82.17 μg / mL of argentoside.

[0175] 11.3HPLC detection

[0176] The test solution and reference solution prepared in this example were subjected to HPLC detection. The chromatographic conditions were the same as those in Example 3, item 3.2. The chromatographic 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 Methods 1 and 2 show that the content of each component is higher under ultrasonic extraction. The results of Method 3 show that after the extract is extracted with petroleum ether and ethyl acetate, components such as rutin and kaempferol-3-O-β-robiniabiside remain largely in the aqueous phase, preventing complete extraction. In summary, Method 2, the ultrasonic treatment method, is preferred.

[0179] Table 8 Determination results of the content of each component under different extraction methods (mg / g)

[0180]

[0181] Example 12

[0182] This example investigates the effects of different extraction solvents on the extraction rate of the test components in Hibiscus mutabilis leaves, including the following steps:

[0183] 12.1 Preparation of test solution

[0184] Take 2.5 g of Hibiscus mutabilis leaf powder (passed through a 60-mesh sieve), accurately weigh it, place it in a conical flask, add 30% ethanol, 50% ethanol, 70% ethanol, 90% ethanol, and 25 mL of water, respectively, plug it tightly, weigh it, and perform ultrasonic extraction for 60 minutes. Cool it to room temperature, make up the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate to obtain the product.

[0185] 12.2 Preparation of reference solution

[0186] Same as item 11.2 in Example 11.

[0187] 12.3HPLC detection

[0188] The test solution and reference solution prepared in this example were subjected to HPLC detection. The chromatographic conditions were the same as those in Example 3, item 3.2. The chromatographic 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 great influence 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, preferably 70% ethanol.

[0191] Table 9 Determination results of the content of each component in different extraction solvents (mg / g)

[0192]

[0193] Example 13

[0194] This example investigates the effects of different extraction times on the extraction rate of the test components in Hibiscus mutabilis leaves, comprising the following steps:

[0195] 13.1 Preparation of test solution

[0196] Take 2.5 g of Hibiscus mutabilis leaf powder (S1, passed through a 60-mesh sieve), accurately weigh it, place it in a conical flask, add 25 mL of 70% ethanol, plug it tightly, weigh it, and ultrasonically extract it for 15 min, 30 min, 45 min, and 60 min respectively. Cool it to room temperature, make up the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate to obtain it.

[0197] 13.2 Preparation of Reference Solution

[0198] Same as item 11.2 in Example 11.

[0199] 13.3HPLC detection

[0200] The test solution and reference solution prepared in this example were subjected to HPLC detection. The chromatographic conditions were the same as those in Example 3, item 3.2. The chromatographic 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 results of each component when ultrasonic extraction is performed for 30 to 60 minutes, so ultrasonic treatment for 30 minutes is preferred.

[0203] Table 10 Determination results of the content of each component at different extraction times (mg / g)

[0204]

[0205] Example 14

[0206] This example investigates the effects of different material-liquid ratios on the extraction rate of the test components in Hibiscus mutabilis leaves, comprising the following steps:

[0207] 14.1 Preparation of test solution

[0208] Take 2.5 g of Hibiscus mutabilis leaf powder (passed through a 60-mesh sieve), accurately weigh it, place it in a conical flask, add 25 mL, 50 mL, and 100 mL of 70% ethanol in a ratio of 1:10, 1:20, and 1:40, respectively, plug it, weigh it, and extract it by ultrasonic for 30 minutes. Cool it to room temperature, make up the loss with the corresponding solvent, shake it well, filter it, take the filtrate and spin it dry, dissolve it with 70% ethanol, and make up the volume in a 25 mL volumetric flask.

[0209] 14.2 Preparation of Reference Solution

[0210] Same as item 11.2 in Example 11.

[0211] 14.3HPLC detection

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

[0213] 14.4 Test Results

[0214] The results of the determination of the content of each component under different material-liquid ratios are shown in Table 11. The results show that when the material-liquid ratio is 1:10, the content of each component is too high.

[0215] Table 11 Determination results of the content of each component under different material-liquid ratio conditions (mg / g)

[0216]

[0217] In summary (Examples 11 to 14), in the detection method of the present invention, preferably, the test solution preparation method is: take 2.5 g of Hibiscus mutabilis leaf powder (passed through a 60-mesh sieve), accurately weighed, placed in a conical flask, accurately added 25 mL of 70% ethanol, plugged, weighed, ultrasonically treated (600 W, 40 kHz) for 30 min, cooled to room temperature, supplemented with 70% ethanol to make up for the loss in weight, shaken well, filtered, and the filtrate was taken and filtered with a 0.45 μm microporous membrane to obtain.

[0218] Example 15

[0219] This example conducts methodological verification on the preferred method for determining the content of the test components in Hibiscus mutabilis leaves, comprising the following steps:

[0220] 15.1 Preparation of test solution

[0221] Take 2.5 g of Hibiscus mutabilis leaf powder (S1, passed through a 60-mesh sieve), accurately weigh it, place it in a conical flask, accurately add 25 mL of 70% ethanol, seal it tightly, weigh it, and ultrasonically treat it (600 W, 40 kHz) for 30 min. Let it cool to room temperature, make up the loss with 70% ethanol, shake it well, filter it, take the filtrate, and filter it with a 0.45 μm microporous membrane to obtain the product.

[0222] 15.2 Preparation of reference solution

[0223] Take an appropriate amount of each reference substance, accurately weigh it, dissolve it in methanol, and dilute 6 mixed reference substance solutions of different concentrations in equal multiples. The concentration of rutin is 20.07-642.30 μg / mL, the concentration of isoquercetin is 3.16-101.00 μg / mL, the concentration of kaempferol-3-O-β-robinobioside is 17.42-557.29 μg / mL, the concentration of kaempferol-3-O-rutinoside is 7.39-236.38 μg / mL, the concentration of kaempferol-3-O-β-D-glucoside is 4.35-139.27 μg / mL, and the concentration of argininoside is 10.27-328.69 μg / mL.

[0224] 15.3HPLC detection

[0225] Chromatographic detection conditions were as follows: chromatographic 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, elution program: 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. Volume flow rate: 1.0 mL / min; column temperature: 30°C; detection wavelength: 265 nm; injection volume: 10 μL.

[0226] 15.4 Specificity test

[0227] Inject the reference solution and the test solution under the chromatographic conditions of 15.3 respectively and record the chromatograms. Figure 4 . Figure 4 A in FIG is the HPLC spectrum of the test solution of Hibiscus mutabilis leaves, Figure 4B is the HPLC spectrum of the reference solution; the HPLC spectrum of the test solution includes: 1. rutin; 2. isoquercetin; 3. kaempferol-3-O-β-robinoside; 4. kaempferol-3-O-rutinoside; 5. kaempferol-3-O-β-D-glucoside; 6. arginine. The results showed that the retention time of component 1 (rutin) was 33.6 min, component 2 (isoquercetin) 35.9 min, component 3 (kaempferol-3-O-β-robinoside) 38.1 min, component 4 (kaempferol-3-O-rutinoside) 43.2 min, component 5 (kaempferol-3-O-β-D-glucoside) 46.3 min, and component 6 (arginine) 61.2 min. The peak elution time was stable, the separation degree between adjacent chromatographic peaks was greater than 1.5, the separation effect was good, the theoretical plate number of the chromatographic column was greater than 5000, and the method specificity was good.

[0228] 15.5 Precision test

[0229] A sample solution of Hibiscus mutabilis leaf was injected six times continuously, and the chromatographic peak area of ​​each component was recorded. The calculated RSD values ​​for the peak areas of rutin, isoquercetin, kaempferol-3-O-β-robinobioside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and argentin 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 test solution of Hibiscus mutabilis leaf was taken and sampled and analyzed at 0, 4, 8, 12, 16, 20, and 24 hours after sample preparation. The chromatographic peak area of ​​each component was recorded, and the RSD values ​​of the peak areas of rutin, isoquercetin, kaempferol-3-O-β-robinobioside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and arginine were 2.0%, 1.2%, 1.2%, 1.4%, 0.65%, and 0.23%, respectively, indicating that the test solution had good stability within 24 hours.

[0232] 15.7 Repeatability test

[0233] Take Hibiscus mutabilis leaf powder (passed through a 60-mesh sieve) to prepare 6 test solutions, which were sampled and analyzed separately. The chromatographic peak areas were recorded, and the RSD values ​​of rutin, isoquercetin, kaempferol-3-O-β-robinobioside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and arginine concentrations were calculated to be 2.2%, 3.0%, 2.2%, 2.5%, 0.70%, and 1.9%, respectively, indicating that the method has good repeatability.

[0234] 15.8 Sample recovery test

[0235] 1.25 g of powdered Hibiscus mutabilis (S1) with a known content was accurately weighed into six portions. A reference substance was added to each sample at 100% of the content of each component to prepare a test solution. The solution was analyzed by HPLC injection, and the recovery rate was calculated. The results are shown in Table 12. The average recoveries of rutin, isoquercitrin, kaempferol-3-O-β-robinobioside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and argentin 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 good accuracy of the method.

[0236] Table 12 Recovery of each component of Hibiscus mutabilis

[0237]

[0238] 15.9 Investigation of Linear Relationships

[0239] Inject the reference solution prepared in 15.2 under the chromatographic conditions of 15.3 and record the chromatographic peak area. Draw a standard curve ( Figure 5 ) to obtain a linear regression equation. The limit of quantification (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 showed good linear relationships for the six components within the specified concentration ranges.

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

[0241]

[0242] Example 16

[0243] This embodiment adopts the external standard method (ESM) and the one-measurement-multiple-evaluation method (QAMS) to implement and apply the preferred method for determining the content of the test component of Hibiscus mutabilis leaves, including the following steps:

[0244] 16.1 Preparation of test solution

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

[0246] Table 1416 Batch of Hibiscus mutabilis Leaf Sample Information

[0247] serial number Origin batch number serial number Origin batch number S1 Yichang, Hubei 230721 S9 Kunming, Yunnan (Stone Forest) 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, Hebei (Anguo) 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] 16.3HPLC detection

[0251] Same as item 15.3 in Example 15.

[0252] 16.4 QAMS method f s / i Determination

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

[0254] With rutin as the internal reference, according to the linear investigation results of 15.9 in Example 15, according to the 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 component to be measured; Ci is the concentration of a certain component to be measured), and the f of isoquercetin, kaempferol-3-O-β-robinobioside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and argentin at different concentrations were calculated respectively. s / i The results are shown in Table 15. The f of isoquercetin, kaempferol-3-O-β-robinoside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and silver forging glycoside 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, and the average values ​​were 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) Effects of different HPLC instruments and columns on f s / i Impact

[0259] Two high performance liquid chromatographs, Agilent 1260 and Waters e2695, were investigated, with Agilent ZORBAX Eclipse XDB-C18 columns (250 mm × 4.6 mm, 5 μm) and Waters The three different chromatographic columns, C18 column (250 mm × 4.6 mm, 5 μm) and GL Sciences-InertSustain C18 column (250 mm × 4.6 mm, 5 μm), were used to analyze the effect of different chromatographic columns on the f s / i The results are shown in Table 16. s / i The RSD values ​​of the two groups were all less than 2.0%, indicating that the s / i Has good durability.

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

[0261]

[0262] (3) 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 effect of different column temperatures (25, 30, and 35 °C) on the f s / i The results are shown in Table 17. The results show that the f s / i The RSD values ​​of the two groups were all less than 1.0%, indicating that the change of column temperature between 25℃ and 35℃ had a significant effect on the f s / i No noticeable impact, good durability.

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

[0265]

[0266] (4) Effect of different volume 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, and 1.2 mL / min) on the f s / i The results are shown in Table 18. The results show that the f s / i The RSD values ​​of the two groups were all less than 1.0%, indicating that the change of volume flow rate between 0.8 and 1.2 mL / min had a significant effect on f s / i No significant impact.

[0268] Table 18 Effect of different volume flow rates on f s / i Impact

[0269]

[0270]

[0271] (5) Positioning of chromatographic peaks of the components to be measured

[0272] The relative retention time method was used to calculate the relative retention times (R) of the six components, including isoquercetin, kaempferol-3-O-β-robinobioside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside and argentin. s / i ), the results are shown in Table 19. Results R of each component 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 Relative retention time determination results of different instruments and chromatographic columns

[0274]

[0275] 16.5 Assay results

[0276] The contents of the tested components in each batch were calculated by ESM and QAMS methods: the contents of isoquercetin, kaempferol-3-O-β-robinobioside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and argentin. s / i The results are shown in Table 20 when the values ​​of f are 1.3648, 0.4251, 1.0942, 0.6147 and 1.1542, respectively; s / i The results are shown in Table 21 when the values ​​of f are 1.3593, 0.4242, 1.0852, 0.6118 and 1.1524, respectively; s / i The results are shown in Table 22. The results showed that there was 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 by the present invention is suitable for the content determination of ESM method and QAMS method; the results showed that the f of isoquercitrin, kaempferol-3-O-β-robinobioside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and argentin were 1.3708, 0.4265, 1.1005, 0.6167, and 1.1596, respectively. s / i The results were in the range of 1.3593-1.3708, 0.4242-0.4265, 1.0852-1.1005, 0.6118-0.6167, and 1.1524-1.1596, respectively. The QAMS method is accurate, reliable, and durable, and can be used for the determination of multiple components in Hibiscus mutabilis leaves. In addition, comparing the content results in Table 20, Table 21, and Table 22, the preferred content of each component f s / iThey are 1.3648, 0.4251, 1.0942, 0.6147, and 1.1542 respectively.

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

[0278]

[0279]

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

[0281]

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

[0283]

[0284]

[0285] Comparative Example 1

[0286] This comparative example provides an HPLC detection method for Hibiscus mutabilis leaf medicinal material, comprising the following steps:

[0287] 1.1 Preparation of test solution

[0288] Same as the content of 1.1 in Example 1.

[0289] 1.2HPLC detection

[0290] The test solution was subjected to HPLC detection. Among the chromatographic conditions, the mobile phase elution program is shown in Table 23, and other conditions are the same as those in 1.2 of Example 1.

[0291] Table 23 Comparative Example 1 Mobile Phase Elution Conditions

[0292] Time (Tmin) Mobile phase (A) Mobile phase (B) 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 diagram of the test solution is as follows: Figure 1 In the Figure 1-4 "As shown. Figure 1 In the Figure 1-4 It can be seen that the separation degree of component 6 (argentin) is 0.91, which is difficult to be completely separated from the adjacent chromatographic peaks. Therefore, the chromatographic conditions cannot accurately determine its content.

[0295] Comparative Example 2

[0296] This comparative example provides an HPLC detection method for Hibiscus mutabilis leaf medicinal material, comprising the following steps:

[0297] 2.1 Preparation of test solution

[0298] Same as item 1.1 in Example 1.

[0299] 2.2HPLC detection

[0300] The test solution was subjected to HPLC detection. Among the chromatographic conditions, the mobile phase elution program is shown in Table 24, and other conditions are the same as those in 1.2 of Example 1.

[0301] Table 24 Comparative Example 2 Mobile Phase Elution Conditions

[0302]

[0303]

[0304] 2.3 Test results

[0305] Using the chromatographic conditions of this comparative example, the HPLC chart of the test solution was as follows: Figure 1 In the Figure 1-5 As shown in the figure, it can be seen that the separation degree of component 1 (rutin) is 1.49, and the separation degree of component 6 (argentoside) is 1.37, which cannot be completely separated from the adjacent chromatographic peaks. Therefore, the content of the components to be measured cannot be accurately determined using this chromatographic condition.

[0306] Comparative Example 3

[0307] This comparative example provides an HPLC detection method for Hibiscus mutabilis leaf medicinal material, comprising the following steps:

[0308] 3.1 Preparation of test solution

[0309] Same as item 1.1 in Example 1.

[0310] 3.2HPLC detection

[0311] The test solution was subjected to HPLC detection. Among the chromatographic conditions, the mobile phase elution program is shown in Table 25, and other conditions are the same as those in 1.2 of Example 1.

[0312] Table 25 Comparative Example 3 Mobile Phase Elution Conditions

[0313] Time (Tmin) Mobile phase (A) Mobile phase (B) 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] Using the chromatographic conditions of this comparative example, the HPLC chart of the test solution was as follows: Figure 1 In the Figure 1-6As shown in the figure, it can be seen that the chromatographic peaks are stacked during the period of 40 to 50 minutes, component 5 (kaempferol-3-O-β-D-glucoside) cannot be accurately located, and component 6 (argentoside) is difficult to separate. Therefore, the content of the components to be tested cannot be accurately determined using this chromatographic condition.

[0316] Comparative Example 4

[0317] This comparative example provides an HPLC detection method for Hibiscus mutabilis leaf medicinal material, comprising the following steps:

[0318] 4.1 Preparation of test solution

[0319] Same as item 1.1 in Example 1.

[0320] 4.2HPLC detection

[0321] The test solution was subjected to HPLC detection. Among the chromatographic conditions, the mobile phase elution program is shown in Table 26, and other conditions are the same as those in 1.2 of Example 1.

[0322] Table 26 Comparative Example 4 Mobile Phase Elution Conditions

[0323] Time (Tmin) Mobile phase (A) Mobile phase (B) 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] Using the chromatographic conditions of this comparative example, the HPLC chart of the test solution was as follows: Figure 1 In the Figure 1-7 As shown in the figure, it can be seen that component 5 (kaempferol-3-O-β-D-glucoside) cannot be accurately located, and there is basically no peak in the time period of 35 to 50 minutes, and the chromatographic peak distribution is uneven. Therefore, this chromatographic condition cannot completely determine the content of the component to be tested.

[0326] Comparative Example 5

[0327] This comparative example provides an HPLC detection method for Hibiscus mutabilis leaf medicinal material, comprising the following steps:

[0328] 5.1 Preparation of test solution

[0329] Same as item 1.1 in Example 1.

[0330] 5.2HPLC detection

[0331] The test solution was subjected to HPLC detection, with 0.1% acetic acid aqueous solution (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 1.2 of Example 1.

[0332] 5.3 Test results

[0333] Using the mobile phase system of this comparative example, the HPLC chart of the test solution was measured as follows: Figure 2 In the Figure 2-4 As shown in the figure, the chromatographic peaks of the test components can be well separated, but the peak width is broadened, the resolution is reduced, and the elution time of the test component 4 (kaempferol-3-O-rutinoside) drifts. It is speculated that this may be caused by 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 test components.

[0334] Comparative Example 6

[0335] This comparative example provides an HPLC detection method for Hibiscus mutabilis leaf medicinal material, comprising the following steps:

[0336] 6.1 Preparation of test solution

[0337] Same as item 1.1 in Example 1.

[0338] 6.2HPLC detection

[0339] The test solution was subjected to HPLC detection 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 1.2 of Example 1.

[0340] 6.3 Test results

[0341] Using the mobile phase system of this comparative example, the HPLC chart of the test solution was measured as follows: Figure 2 In the Figure 2-5 "As shown in the figure, the chromatographic peak is broadened, the symmetry is reduced, a front peak appears, and the peak shape needs to be further improved.

[0342] Comparative Example 7

[0343] This comparative example provides an HPLC detection method for Hibiscus mutabilis leaf medicinal material, comprising the following steps:

[0344] 7.1 Preparation of test solution

[0345] Same as item 1.1 in Example 1.

[0346] 7.2 HPLC detection

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

[0348] 7.3 Test results

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

[0350] From the above examples, it can be seen that the detection method of various flavonoid components in the medicinal material of Hibiscus mutabilis leaves provided by the present invention, through the reasonable optimization of HPLC chromatographic detection conditions and the preparation method of the test solution, is constructed to realize the simultaneous determination of the contents of 6 flavonoid components, namely rutin, isoquercetin, kaempferol-3-O-β-robinia disaccharide, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside, and silver forging glycoside. At the same time, combined with the one-test-multiple-evaluation technology, the detection efficiency is improved and the detection cost is reduced. The method of the present invention is simple to operate, stable and reliable, with high accuracy, good reproducibility, and strong practicality, and provides a new analytical means for the quality control of Hibiscus mutabilis leaves.

[0351] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for detecting multiple flavonoid components in Hibiscus mutabilis leaves, characterized in that: The following steps are involved: The test sample is detected by high performance liquid chromatography to obtain high performance liquid chromatography detection results of flavonoid components in the test sample; the test sample is a Hibiscus mutabilis leaf medicinal material extract solution, and the flavonoid components include at least four of rutin, isoquercetin, kaempferol-3-O-β-robiniacacia disaccharide, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside and arginine; The detection conditions of the high performance liquid chromatography method include: the elution mode is gradient elution; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is a phosphoric acid aqueous solution or a formic acid aqueous solution, the mass content of phosphoric acid in the phosphoric acid aqueous solution is 0.1-0.3%, the mass content of formic acid in the formic acid aqueous solution is 0.1%, and the mobile phase B is acetonitrile; the elution program of the gradient elution is: 0-10min, 5-12% (v:v) mobile phase B; 10-40min, 12-18% (v:v) mobile phase B; 40-50min, 18-25% (v:v) mobile phase B; 50-60min, 25-27% (v:v) mobile phase B; 60-70min, 27-40% (v:v) mobile phase B; or: 0-10min, 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; 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; the detection wavelength is 254-265 nm.

2. The detection method according to claim 1, characterized in that: The high performance liquid chromatography test results are obtained by using an external standard method or a one-measurement-multiple-evaluation method; The one-measurement-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 is the peak area of ​​the internal reference, C s is the internal reference concentration, A i is the peak area of ​​the flavonoid component to be tested; C i is the concentration of the flavonoid component to be tested, f s / i is the relative correction factor of the flavonoid components to be tested.

3. The detection method according to claim 1, characterized in that: The filler in the chromatographic column used in the high performance liquid chromatography is octadecylsilane bonded silica gel.

4. The detection method according to claim 1, characterized in that: The volume flow rate of the mobile phase is 0.8-1.2 mL / min.

5. The detection method according to claim 1 or 3, characterized in that: The column temperature of the chromatographic column used in the high performance liquid chromatography is 25-35°C.

6. The detection method according to claim 1, characterized in that: The method for preparing the Hibiscus mutabilis leaf medicinal material extract solution comprises the following steps: The Hibiscus mutabilis leaf medicinal material and ethanol water solvent are mixed for extraction to obtain the Hibiscus mutabilis leaf medicinal material extraction solution; the mass content of ethanol in the ethanol water solvent is 50-70%.

7. The detection method according to claim 6, characterized in that: The solid-liquid ratio of the Hibiscus mutabilis leaf medicinal material to the ethanol-water solvent is 1:10 to 1:40; The extraction is heating reflux extraction or ultrasonic extraction; the power of the ultrasonic extraction is 600-800W, the frequency is 40-50kHz, and the time is 30-60min.

8. The detection method according to claim 2, characterized in that: The relative correction factors of isoquercetin, kaempferol-3-O-β-robinia pseudoglucoside, kaempferol-3-O-rutinoside, kaempferol-3-O-β-D-glucoside and argentoside determined by the one-measurement-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.

9. The detection method according to claim 1 or 3, characterized in that: The injection volume of the HPLC method was 10 μL.

10. The detection method according to claim 1 or 3, characterized in that: 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 particle diameter of the filler of 5 μm.

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